Activatable cytokine constructs and related compositions and methods

Abstract

Provided herein are activatable cytokine constructs comprising: (a) a first monomeric construct comprising a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerization domain (DD1) wherein the CM1 is located between the CP1 and the DD1; and (b) a second monomeric construct comprising a second mature cytokine protein (CP2), a second cleavable moiety (CM2) and a second dimerization domain (DD2) wherein said CM2 is located between said CP2 and said DD2 wherein: said CM1 and said CM2 act as substrates for a protease; the DD1 and the DD2 are combined with each other; and wherein said ACC is characterized in that said at least one activity of said CP1 and / or said CP2 is reduced compared to a control level of said at least one activity of said CP1 and / or said CP2.

Description

[0001] This application is a divisional application of Chinese patent application No. 202180030934.8, filed on April 9, 2021, entitled "Active Cytokine Constructs and Related Compositions and Methods".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 63 / 008,542, filed April 10, 2020; U.S. Provisional Application No. 63 / 161,889, filed March 16, 2021; and U.S. Provisional Application No. 63 / 164,849, filed March 23, 2021, the entire contents of which are incorporated herein by reference.

[0004] This application includes a sequence list submitted electronically. The sequence list, named “CYTX-071-PCT_ST25.txt”, was created on April 7, 2021, and is 379,000 bytes in size. The information in the electronic sequence list is a part of this application and is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure relates to the field of biotechnology, and more specifically, to constructs of activatable cytokines. Background Technology

[0006] Cytokines are a family of naturally occurring small proteins and glycoproteins produced and secreted by most nucleated cells in response to viral infection and / or other antigenic stimulation. Interferons are a subclass of cytokines. Interferons are currently classified into three main classes: type I interferons, type II interferons, and type III interferons. Interferons exert their cellular activity by binding to specific membrane receptors on the cell surface.

[0007] Interferon therapy offers numerous clinical benefits. For example, interferon is known to upregulate the immune system and also possesses antiviral and antiproliferative properties. These biological properties lead to its clinical use as a therapeutic agent for viral infections and malignancies. Furthermore, interferon is effective in recruiting a 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, hairy cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, and other disease states. However, systemic administration of interferon is associated with dose-dependent toxicities, including severe flu-like symptoms, neurological symptoms, hepatotoxicity, bone marrow suppression, and arrhythmias. In a melanoma patient study, the combination of pembrolizumab and pegylated IFNa resulted in an objective response rate (ORR) of 60.5%. Combination therapy was also associated with 49% of G3 / G4 adverse events, which required a reduction in the dose of pegylated IFNa (Davar et al., J. Clin. Oncol., 2018). These undesirable side effects limit the dosage of interferon therapy and sometimes lead to interruption or delay of interferon treatment.

[0008] Interleukins are another subclass of cytokines. Interleukins regulate cell growth, differentiation, and movement. They are particularly important in stimulating immune responses, such as inflammation. Interleukins have been used to treat cancer, autoimmune diseases, and other conditions. For example, interleukin-2 (IL2) has been indicated for the treatment of melanoma, graft-versus-host disease (GVHD), neuroblastoma, and renal cell carcinoma (RCC), and is also considered suitable for treating conditions including: acute coronary syndrome, acute myeloid syndrome, atopic dermatitis, autoimmune liver disease, basal cell carcinoma, bladder cancer, breast cancer, candidiasis, colorectal cancer, cutaneous T-cell lymphoma, endometriosis, HIV infection, ischemic heart disease, rheumatoid arthritis, nasopharyngeal carcinoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, systemic lupus erythematosus, tuberculosis, and other conditions. Other interleukins, such as IL-6, IL-7, IL-12, and IL-21, are potential therapeutic agents for cancer and other conditions. Interleukin therapy is often accompanied by undesirable side effects, including flu-like symptoms, nausea, vomiting, diarrhea, low blood pressure, and arrhythmias.

[0009] Therefore, the need and expectation for improved specificity and selectivity of cytokine therapy for desired targets is of great interest. Increased targeting of disease sites by cytokine therapeutics can reduce systemic toxicity and lead to broader therapeutic efficacy. Summary of the Invention

[0010] 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 dimerizing domain (DD1), wherein CM1 is located between CP1 and DD1; and (b) a second monomer comprising a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2), wherein CM2 is located between CP2 and DD2, wherein: CM1 and CM2 act as substrates for a protease; DD1 and DD2 bind to each other; and wherein the ACC is characterized by reduced activity of at least one of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2. The protease that cleaves CM1 and CM2 may be overexpressed in diseased tissues (e.g., tumor tissues) relative to healthy tissues. The ACC can be activated upon cleavage of CM1 and / or CM2, allowing the cytokines to exert their activity in diseased tissues (e.g., in the tumor microenvironment), while cytokine activity is reduced in healthy tissues. Therefore, the ACC presented in this article can provide reduced toxicity compared to traditional cytokine therapeutics, enabling higher effective doses of cytokines, and / or increasing the therapeutic window of cytokines.

[0011] This article provides an activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct, wherein: (a) the first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1), wherein CM1 is located 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 dimerizing domain (DD2), wherein CM2 is located between CP2 and DD2; wherein DD1 and DD2 bind to each other to form a dimer of the first monomer construct and the second monomer construct; and wherein the ACC is characterized by having at least one CP1 and / or CP2 activity level reduced compared to a control level of at least one CP1 and / or CP2 activity.

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

[0013] This disclosure provides an activatable cytokine construct (ACC) comprising: (a) a first monomer comprising a first mature cytokine protein (CP1), a cleavable portion (CM), and a first dimerizing domain (DD1), wherein CM is located between CP1 and DD1; and (b) a second monomer comprising a second mature cytokine protein (CP2) and a second dimerizing domain (DD2), wherein: CM acts as a substrate for a protease; DD1 and DD2 bind to each other; and wherein the ACC is characterized in that at least one activity of CP1 and / or CP2 is reduced compared to a control level of at least one activity of CP1 and / or CP2.

[0014] This disclosure provides an activatable cytokine construct (ACC) comprising: (a) a first monomer comprising a first mature cytokine protein (CP1) and a first dimerizing domain (DD1); and (b) a second monomer comprising a second mature cytokine protein (CP2) and a second dimerizing domain (DD2), wherein CP1, CP2, or both CP1 and CP2 comprise an amino acid sequence serving as a substrate for a protease; DD1 and DD2 are bound to each other; and wherein the ACC is characterized in that at least one activity of CP1 and / or CP2 is reduced compared to a control level of at least one activity of CP1 and / or CP2.

[0015] The ACC of this disclosure is characterized in that CP1 and CP2 are not linked to peptide masking agents, such as affinity masking moieties.

[0016] In some embodiments, the first monomeric construct comprises a first polypeptide comprising CP1, CM1, and DD1. In some embodiments, the second monomeric construct comprises a second polypeptide comprising CP2, CM2, and DD2. In some embodiments, DD1 and DD2 are a pair selected from the group consisting of: a pair of Fc domains, a sushi domain derived from the human IL-15 receptor α chain (IL15Rα) and soluble IL-15; barnase and barnstar; protein kinase A (PKA) and A kinase anchoring protein (AKAP); an adaptor / docking tag module based on a mutated RNase I fragment; an epitope and single-domain antibody (sdAb); an epitope and single-chain variable fragment (scFv); and a soluble N-ethylmaleimide sensitive factor attachment protein (SNARE) module, antigen-binding domain, and epitope based on the interaction of protein synaptin, synaptotagmin, synaptobrevin, and SNAP25.

[0017] In some embodiments, DD1 and DD2 are a pair of Fc domains. In some embodiments, the pair of Fc domains is a pair of human Fc domains. In some embodiments, the human Fc domain is a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain. In some embodiments, the human Fc domain is a human IgG4 Fc domain. In some embodiments, the human Fc domain contains a sequence having at least 80% identity with SEQ ID NO: 3. In some embodiments, each of the human Fc domains contains a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. In some embodiments, each of the human Fc domains contains SEQ ID NO: 3. In some embodiments, DD1 and DD2 are identical. For example, DD1 and DD2 can be a pair of identical human IgG4 Fc domains. In some embodiments, the dimerizing domains have the amino acid sequences of SEQ ID NO: 315 and 316, respectively. In some embodiments, the human Fc domain includes mutations that eliminate glycosylation and / or reduce Fc-γ receptor binding. In some embodiments, the human Fc domain contains mutations N297Q, N297A, or N297G; in some embodiments, the human Fc domain contains mutations at positions 234 and / or 235, such as L235E, or L234A and L235A (in IgG1), or F234A and L235A (in IgG4); in some embodiments, the human Fc domain is an IgG2 Fc domain containing mutations V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or combinations thereof (all according to EU designations).

[0018] Other examples of engineered human Fc domains are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids whose mutations in at least one amino acid result in reduced Fc function include, but are not limited to, mutations in amino acids 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330, and 331 (all according to EU designations). Examples of combinations of mutated amino acids are also known in the art, such as, but not limited to, combinations of mutations in amino acids 234, 235, and 331, such as L234F, L235E, and P331S, or combinations of amino acids 318, 320, and 322, such as E318A, K320A, and K322A.

[0019] Other examples of engineered 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 the opposite heavy chain, D270E / K326D, A330M / K334EIgG; G236A / S239D / I332E IgG1; K326W / E333S IgG1; S267E / H268F / S324T IgG1; E345R / E430G / S440Y IgG1; N297A or N297Q or N297G IgG1; L235E IgG1; L234A / L235A IgG1; F234A / L235A IgG4; H268Q / V309L / A330S / P331S IgG2; V234A / G237A / P238S / H268A / V309L / A330S / P331S IgG2; M252Y / S254T / T256E IgG1; M428L / N434S IgG1; S267E / L328F IgG1; N325S / L328F IgG1, etc. In some implementations, the engineered Fc domain includes one or more substitutions selected from the group consisting of N297A IgG1, N297Q IgG1 and S228P IgG4.

[0020] 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 wherein 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 dimerizing domain substituent selected from the group consisting of non-peptide polymers and small molecules. In some embodiments, DD1 and DD2 comprise non-peptide polymers covalently bonded to each other. In some embodiments, the non-peptide polymer is sulfur-containing polyethylene glycol, and wherein 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.

[0021] In some embodiments, CP1 and CP2 are mature cytokines. In some embodiments, each of CP1 and CP2 contains a mature cytokine sequence and also contains a signal peptide (also referred to herein as a “signal sequence”). In some embodiments, CP1 and / or CP2 are each 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 can be wild-type human or non-human animal sequences, mutant sequences, truncated sequences, heterozygous sequences, or sequences containing insertions. In some embodiments, CP1 and CP2 are identical. In some embodiments, CP1 and CP2 are different, and this disclosure includes any two selections and combinations of the cytokine proteins listed 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, CP1 or CP2 is an interferon. In some embodiments, one of CP1 and CP2 is an interferon, while the other of CP1 or CP2 is a cytokine other than an interferon. In some aspects, one or both cytokines are monomeric cytokines. In some aspects, one or both interferons are monomeric interferons. In some aspects, CP1 or CP2 is a monomeric interferon, while the other CP1 or CP2 is a different cytokine. In some aspects, CP1 and / or CP2 include mutant cytokine sequences. In some aspects, CP1 and / or CP2 include universal cytokine sequences. In some aspects, CP1 and / or CP2 include truncated sequences that retain cytokine activity.

[0022] In some embodiments, the interferon is a mature, wild-type human interferon. In some embodiments, the interferon can be type I and type II interferons, such as, 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 α-2a, interferon α-2b, and interferon α-n3. In some embodiments, the interferon is interferon α-2b. 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 has been defunctionalized by substitution, deletion, or insertion of one or more amino acids. In some embodiments, the interferon is a universal cytokine molecule, such as a heterozygous sequence or chimeric cytokine sequence or humanized cytokine sequence with different cytokine subtypes. In some embodiments, the interferon is a universal interferon molecule. In some embodiments, the interferon is universal interferon α, such as a hybrid of interferon α1 and interferon α2b. In some embodiments, CP1 and / or CP2 comprise a sequence having at least 80% identity with SEQ ID NO: 1. In some embodiments, CP1 and / or CP2 comprise a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In some embodiments, CP1 and / or CP2 comprise 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 comprise an IFab domain. In some embodiments, CP1 and / or CP2 comprise interleukin. In some implementations, 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.

[0023] In some embodiments, CM1 and / or CM2 comprise a total of about 3 amino acids to about 15 amino acids. In some embodiments, CM1 and CM2 comprise substrates of different proteases. In some embodiments, CM1 and CM2 have the same length and comprise the same amino acid sequence. In some embodiments, CM1 and CM2 comprise substrates of the same protease. In some embodiments, the protease is selected from the group consisting of: ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, etc. Cathepsin 14, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Cruzipain, Legumin, Otubain-2, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Meprin, Neprilysin, PSMA, BMP-1, Matrix Gold Proteases (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), activator protein C, cathepsin A, cathepsin G, chymase, FVIIa, FIXa, FXa, FXIa, FXIIa, Elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil lyase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, DESC1, DPP-4, FAP, heparin, Matriptase-2, MT-SP1 / Matriptase, TMPRSS2, TMPRSS3, and TMPRSS4.In some embodiments, the protease is selected from the group consisting of: uPA, asparagine endopeptidase, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-2, MMP-9, MMP-12, MMP-13, and MMP-14.

[0024] Suitable pyrolytic portions have been disclosed in WO 2010 / 081173, WO 2015 / 048329, WO 2015 / 116933, WO2016 / 118629 and WO 2020 / 118109, the disclosures of which are incorporated herein by reference in their entirety.

[0025] In some embodiments, CM1 and / or CM2 comprise 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), QNQALRMA (SEQ ID NO: 16), LAAPLGLL (SEQ ID NO: 17), STFPFGMF (SEQ ID NO: 18), ISSGLLSS (SEQ ID NO: 19), PAGLWLDP (SEQ ID NO: 20), VAGRSMRP (SEQ ID NO: 20), LSGRSDNH (SEQ ID NO: 19), TGRGPSWV (SEQ ID NO: 10), PLTGRSGG (SEQ ID NO: 11), TARGPSFK (SEQ ID NO: 12), VHMPLGFLGP (SEQ ID NO: 13), AVGLLAPP (SEQ ID NO: 14), AQNLLGMV (SEQ ID NO: 15), QNQALRMA (SEQ ID NO: 16), LAAPLGLL (SEQ ID NO: 17), STFPFGMF (SEQ ID NO: 18), ISSGLLSS (SEQ ID NO: 19), PAGLWLDP (SEQ ID NO: 20), VAGRSMRP ... 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), ISSGLLSSGGGGSLSGRSDNH (SEQ ID NO: 30), LSGRSGNH (SEQ ID NO: 31), SGRSANPRG (SEQ ID NO: 32), LSGRSDDH (SEQ ID NO: 33), LSGRSDIH (SEQ ID NO: 34), LSGRSDQH (SEQ ID NO: 35), LSGRSDTH (SEQ ID NO:36), LSGRSDYH (SEQ ID NO: 37), LSGRSDNP (SEQ ID NO: 38), LSGRSANP (SEQ ID NO: 39), LSGRSANI (SEQ ID NO: 40), LSGRSDNI (SEQ ID NO: 41), MIAPVAYR (SEQID NO:42)、RPSPMWAY (SEQ ID NO: 43)、WATPRPMR (SEQ ID NO: 44)、FRLLDWQW (SEQ ID NO:45)、ISSGL (SEQ ID NO: 46)、ISSGLLS (SEQ ID NO: 47)、ISSGLL (SEQ ID NO: 48)、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: 53) 54) ISSGLLSGRSDTH (SEQ ID NO: 55) ISSGLLSGRSDYH (SEQ ID NO: 56) ISSGLLSGRSDNP (SEQ ID NO: 57) ISSGLLSGRSANP (SEQ ID NO: 58) ISSGLLSGRSANI (SEQ ID NO: 59) AVGLLAPPGGLSGRSDDH (SEQ ID NO: 60) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 61) AVGLLAPPGGLSGRSDQH (SEQ ID NO: 62) AVGLLAPPGGLSGRSDTH (SEQ ID NO: 63) AVGLLAPPGGLSGRSDYH (SEQ ID NO: 64) AVGLLAPPGGLSGRSDNP (SEQ ID NO: 65)、AVGLLAPPGGLSGRSANP (SEQ ID NO: 66)、AVGLLAPPGGLSGRSANI (SEQ ID NO: 67)、ISSGLLSGRSDNI (SEQ ID NO: 68)、AVGLLAPPGGLSGRSDNI (SEQ ID NO: 69)、GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 69) 70)、GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 71)、LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 72)、ISSGLSS (SEQ ID NO: 73)、PVGYTSSL (SEQ ID NO: 70)74), DWLYWPGI (SEQ ID NO: 75), LKAAPRWA (SEQ ID NO: 76), GPSHLVLT (SEQ ID NO: 77), LPGGLSPW (SEQ ID NO: 78), MGLFSEAG (SEQ ID NO: 79), SPLPLRVP (SEQ ID NO: 80), RMHLRSLG (SEQ ID NO: 81), LLAPSHRA (SEQ ID NO: 82), GPRSFGL (SEQ ID NO: 83), GPRSFG (SEQ ID NO: 84), SARGPSRW (SEQ ID NO: 85), GGWHTGRN (SEQ ID NO: 86), HTGRSGAL (SEQ ID NO: 87), AARGPAIH (SEQ ID NO: 88), RGPAFNPM (SEQ ID NO: 89), SSRGPAYL (SEQ ID NO: 90), RGPATPIM (SEQ ID NO: 91), RGPA (SEQ ID NO: 92), GGQPSGMWGW (SEQ ID NO: 93), FPRLGITGL (SEQ ID NO: 94), SPLTGRSG (SEQ ID NO: 95), SAGFSLPA (SEQ ID NO: 96), LAPLGLQRR (SEQ ID NO: 97), SGGPLGVR (SEQ ID NO: 98), PLGL (SEQ ID NO: 99), and SGRSDNI (SEQ ID NO: 100). In some embodiments, CM comprises 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). In some embodiments, the protease is produced by the subject's tumor, for example, the amount of protease produced in the tumor is greater than the amount produced in the subject's healthy tissue. In some embodiments, the subject has been diagnosed or identified as having cancer.

[0026] 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 comprises CP1 directly adjacent to CM1 and CM1 directly adjacent to DD1, wherein CM1 comprises a sequence selected from the group consisting of SEQ ID Nos. 5-100. In some embodiments, the second monomer construct comprises CP2 directly adjacent to CM2 and CM2 directly adjacent to DD2, wherein CM2 comprises a sequence selected from the group consisting of SEQ ID Nos. 5-100. In some embodiments, the first monomer construct comprises CP1 directly adjacent to CM1 and CM1 directly adjacent to DD1, wherein CM1 comprises a sequence of no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids. In some embodiments, the second monomer construct comprises CP2 directly adjacent to CM2 and CM2 directly adjacent to DD2, wherein CM2 comprises a sequence of no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids. In some embodiments, the first and second monomer constructs are each configured such that the cytokines (CM1 and CM2, respectively) are directly adjacent to cleavable portions (CM1 and CM2, respectively) of no more than 10, 9, 8, 7, 6, 5, or 4 amino acids, and the cleavable portions are directly adjacent to dimerizing domains (DD1 and DD2, respectively) that are the Fc region of human IgG, wherein the N-terminus of the Fc region is the first cysteine ​​residue read in the N-to-C direction of the hinge region (e.g., cysteine ​​226 of human IgG1, using EU numbering). In some aspects, the dimerizing domain is an IgG Fc region in which the upstream hinge residue has been deleted. For example, Fc is a variant in which the N-terminal sequence EPKSCDKTHT (SEQ ID NO: 516), ERK, ELKTPLGDTTHT (SEQ ID NO: 517), or ESKYGPP (SEQ ID NO: 518) is missing.

[0027] In some embodiments, the first monomer construct includes at least one linker. In some embodiments, the at least one linker is linker L1 positioned between CP1 and CM1 and / or linker L2 positioned 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 positioned between CP2 and CM2 and / or linker L4 positioned 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 amino acid to about 15 amino acids. In some embodiments, each linker has a total length of at least 5 amino acids. As used herein, the term "linker" refers to a peptide whose amino acid sequence is not a substrate of a protease.

[0028] In some embodiments, the first monomeric construct includes at least one connector, wherein each connector is independently selected from the group consisting of: 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); GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 214); GGGGSGGGGS (SEQ ID NO: 215); GGGGS (SEQ ID NO: 216); GS; GGGGSGS (SEQ ID NO: 217); GGGGSGGGGSGGGGSGS (SEQ ID NO: 218); GGSLDPKGGGGS (SEQ ID NO: 219). 219); PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 220); SKYGPPCPPCPAPEFLG (SEQ ID NO: 221); GKSGSGSESKS (SEQ ID NO: 222); 224); GSTGSSGKPGSGEGSTKG (SEQ ID NO: 225); 216), wherein each n is an integer of at least one; GGSG (SEQ ID NO: 229); GGSGG (SEQ ID (SEQ ID NO: 230); GGSSG (SEQ ID NO: 231); GSGGG (SEQ ID NO: 232); GGGSG (SEQ ID NO: 233); GSSSG (SEQ ID NO: 234); GGGGSGGGGGSGGGGS (SEQ ID NO: 213); GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 214); and GSTGSGSGKPGSSEGST (SEQ ID NO: 226). In some embodiments, the connector comprises the sequence GGGS (SEQ ID NO: 2).

[0029] As used herein, the term "spacer" refers to an amino acid residue or peptide incorporated into the free terminus of a mature ACC (e.g., between the signal peptide and the N-terminus of the mature ACC). In some respects, the spacer (or "head") may contain a glutamine (Q) residue. In some respects, residues in the spacer minimize the activity of aminopeptidases and / or exopeptidases to prevent the cleavage of the N-terminal amino acid. The illustrative and non-restrictive spacer amino acid sequence may comprise 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 sequence may be omitted.

[0030] In some embodiments, the first monomer construct includes CP1, CM1, and DD1 directly or indirectly linked to the C-terminus of CM1 in the N-terminal to C-terminal direction. In some embodiments, the first polypeptide includes CP1, CM1, and DD1 directly or indirectly linked to the N-terminus of CM1 in the C-terminal to N-terminal direction. In some embodiments, the second polypeptide includes CP2, CM2, and DD2 directly or indirectly linked to the C-terminus of CM2 in the N-terminal to C-terminal direction. In some embodiments, the second polypeptide includes CP2, CM2, and DD2 directly or indirectly linked to CM2 in the C-terminal to N-terminal direction.

[0031] In some embodiments, the first monomer construct comprises CP1, optional linker, CM1, optional linker, and DD1 in the N-to-C direction, wherein DD1 is the Fc region of IgG, wherein the N-terminus of the Fc region is the first cysteine ​​residue read in the N-to-C direction in the hinge region (e.g., cysteine ​​226 of human IgG1 or IgG4, using EU number), and wherein CM1 and any linker inserted between the N-terminal cysteine ​​residues of CP1 and DD1 have a total length of no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids, preferably no more than 10 amino acids, and particularly preferably no more than 7 amino acids. In some embodiments, the second monomer construct comprises CP2, optional linker, CM2, optional linker, and DD2 in the N-to-C direction, wherein DD2 is the Fc region of IgG, wherein the N-terminus of the Fc region is the first cysteine ​​residue read in the N-to-C direction in the hinge region (e.g., cysteine ​​226 of human IgG1 or IgG4, using EU number), and wherein CM2 and any linker inserted between the N-terminal cysteine ​​residues of CP2 and DD2 have a total length of no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids, preferably no more than 10 amino acids, and particularly preferably no more than 7 amino acids.

[0032] In some embodiments, the ACC is a homodimer, wherein 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 having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 313. In some embodiments, the first monomer construct and the second monomer construct each contain, in the N-terminal to C-terminal direction, SEQ ID NO: 1; a CM containing an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 68, and SEQ ID NO: 100; and a dimerizing domain.

[0033] In some embodiments, at least one CP1 and / or CP2 activity is the binding affinity (K0) of CP1 and / or CP2 to its homologous receptor, as determined using surface plasmon resonance. DFor example, when CP1 or CP2 is interferon, the homologous receptor may be the interferon-α / β receptor (IFNAR). In some embodiments, the activity of at least one CP1 and / or CP2 is the level of lymphoma cell proliferation. In some embodiments, the activity of at least one CP1 and / or CP2 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 the protease) is characterized by at least a 2-fold reduction in the activity of at least one CP1 and / or CP2 compared to a control level. In some embodiments, ACC is characterized by at least a 5-fold reduction in the activity of at least one CP1 and / or CP2 compared to a control level. In some embodiments, ACC is characterized by at least a 10-fold reduction in the activity of at least one CP1 and / or CP2 compared to a control level. In some embodiments, the ACC is characterized by a reduction in the activity of at least one CP1 and / or CP2 by at least 20-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, or 2000-fold compared to a control level. In some embodiments, the control level for at least one CP1 and / or CP2 activity is the activity of CP1 and / or CP2 in the ACC after exposure to the protease. In some embodiments, the control level for at least one CP1 and / or CP2 is the corresponding CP1 and / or CP2 activity of the corresponding wild-type mature cytokine.

[0034] In some embodiments, ACC is characterized by producing lysis products upon exposure to the protease, wherein the lysis products contain at least one activity of CP1 and / or CP2. In some embodiments, at least one activity of CP1 and / or CP2 is antiproliferative activity. In some embodiments, the control level is the EC50 value of wild-type mature cytokines, and wherein the ratio of EC50 (lysis products) to EC50 (wild-type control level) is less than about 10, or less than about 9, or less than about 8, or less than about 7, or less than about 6, or less than about 5, or less than about 4, or less than about 3, or less than about 2, or less than about 1.5, or equal to about 1. In some embodiments, the EC50 of the lysis products is substantially the same as the EC50 of wild-type mature cytokines, demonstrating that the activities of CP1 and / or CP2 are fully or almost fully restored after lysis.

[0035] This document provides compositions comprising any of the ACCs described herein. In some embodiments, the composition is a pharmaceutical composition. This document also provides a kit comprising at least one dose of any of the compositions described herein.

[0036] This document provides a method of treating a subject in need, comprising administering to the subject a therapeutically effective amount of any of the ACCs described herein or any of the compositions described herein. In some embodiments, the subject has been identified or diagnosed with cancer. In some non-limiting embodiments, the cancer is Kaposi's sarcoma, hairy 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.

[0037] This document provides nucleic acids encoding polypeptides comprising CP1 and CM1 of any of the ACCs described herein. In some embodiments, the polypeptide also comprises any of the DD1s described herein. This document also provides nucleic acids encoding polypeptides comprising CP2 and CM2 of any of the ACCs described herein. When the monomers are identical, this disclosure provides a single nucleic acid encoding a monomer dimerized to form an ACC. In some embodiments, the polypeptide also comprises any of the DD2s described herein. This document also provides vectors comprising any of the nucleic acids described herein. In some embodiments, the vector is an expression vector. This document also provides cells comprising any of the nucleic acids described herein or any of the vectors described herein.

[0038] This document provides nucleic acid pairs that together encode polypeptides comprising CP1 and CM1 of a first monomeric construct comprising any of the ACCs described herein, and polypeptides comprising CP2 and CM2 of a second monomeric construct. This document also provides vector pairs that together comprise any of the nucleic acid pairs described herein. In some embodiments, the vector pair is a pair of expression vectors. This document also provides cells comprising any of the nucleic acid pairs described herein or any of the vector pairs described herein. In other embodiments, the invention provides vectors comprising the vector pairs.

[0039] This document provides a method for generating ACC, comprising: culturing any of the cells described herein in a liquid culture medium under conditions sufficient for generating ACC; and recovering ACC from the cells or the liquid culture medium. In some embodiments, the method further comprises: isolating the ACC recovered from the cells or the liquid culture medium. In some embodiments, the method further comprises: formulating the isolated ACC into a pharmaceutical composition.

[0040] This document provides ACC produced by any of the methods described herein. This document also provides compositions comprising any of the ACCs described herein. This document further provides compositions comprising any of the compositions described herein, wherein said compositions are pharmaceutical compositions. This document also provides a kit comprising at least one dose of any of the compositions described herein.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification (including definitions) shall prevail.

[0042] Other features and advantages of the invention will become apparent from the following detailed description and drawings, as well as from the claims.

[0043] The term "a / an" refers to one or more (i.e., at least one) grammatical objects of an article. For example, "a cell" encompasses one or more cells.

[0044] As used herein, the terms “about” and “approximately” when used to modify a numerical value or a quantity specified within a range indicate the numerical value and a reasonable deviation from a value known to those skilled in the art. For example, where appropriate, ±20%, ±10%, or ±5% may be within the intended meaning of the value.

[0045] Concentration, amount, and other numerical data may be expressed or presented in range format herein. It should be understood that this range format is used merely for convenience and brevity, and therefore should be interpreted flexibly to include not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, the numerical range “about 0.01 to 2.0” should be interpreted to include not only the explicitly listed values ​​of about 0.01 to about 2.0, but also the individual values ​​and subranges within the specified range. Thus, included within the numerical range are 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, etc. Furthermore, this interpretation should apply to any extent the range is broad or the characteristics described. Additionally, it should be noted that all percentages are by weight unless otherwise stated.

[0046] In understanding the scope of this disclosure, as used herein, the terms “comprising” or “including” and their derivatives are intended to be open-ended terms specifying the presence of stated features, elements, components, groups, integers, and / or steps, but not excluding the presence of other unstated features, elements, components, groups, integers, and / or steps. The foregoing also applies to words with similar meanings, such as the terms “comprising,” “having,” and their derivatives. As used herein, the term “consisting of” and its derivatives are intended to be closed-ended terms specifying the presence of stated features, elements, components, groups, integers, and / or steps, but excluding the presence of other unstated features, elements, components, groups, integers, and / or steps. As used herein, the term “substantially constitutes” is intended to specify the presence of stated features, elements, components, groups, integers, and / or steps, as well as those features, elements, components, groups, integers, and / or steps that do not substantially affect one or more fundamental and novel features of the features, elements, components, groups, integers, and / or steps. It should be understood that reference to any of these transitional terms (i.e., "comprising," "consisting of," or "substantially constitutes") provides direct support for replacing any of other transitional terms not specifically used. For example, modifying the term from "comprising" to "substantially constitutes" or "consisting of" would provide direct support because the definition applies to any element disclosed throughout this disclosure. Based on the definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0047] As used herein, for convenience, multiple compounds, elements, or steps may be presented in a common list. However, these lists should be interpreted as if each member of the list were individually identified as a separate and unique member. Therefore, without indication to the contrary, no individual member of the list should be construed as being substantially equivalent to any other member of the same list solely based on its presentation in a common group.

[0048] Furthermore, certain molecules, constructs, compositions, elements, portions, excipients, symptoms, properties, steps, etc., may be discussed in the context of a particular embodiment or aspect or in a separate paragraph or section of this disclosure. It should be understood that this is merely for convenience and brevity, and any such disclosure is equally applicable to and intended to be combined with any other embodiment or aspect found anywhere in this disclosure and the claims, all of which, as of the date of filing, constitute the application and claimed invention. For example, the list of constructs, molecules, method steps, kits, or compositions described with respect to constructs, compositions, or methods is intended to and does find direct support for embodiments relating to constructs, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not restated in the context or section of said embodiment or aspect.

[0049] Unless otherwise stated, “nucleic acid sequence encoding protein” includes all nucleotide sequences that are degenerate to each other and therefore encode the same amino acid sequence.

[0050] When referring to the position of a first domain or sequence relative to a second domain or sequence in the primary amino acid sequence of a polypeptide, the term "N-terminal positioning" means that the first domain or sequence is closer to the N-terminus of the primary amino acid sequence of the polypeptide than the second domain or sequence. In some embodiments, additional sequences and / or domains may exist between the first and second domains or sequences.

[0051] When referring to the position of a first domain or sequence relative to a second domain or sequence in the primary amino acid sequence of a polypeptide, the term "C-terminal positioning" means that the first domain or sequence is closer to the C-terminus of the primary amino acid sequence of the polypeptide than the second domain or sequence. In some embodiments, additional sequences and / or domains may exist between the first and second domains or sequences.

[0052] The term “exogenous” means any material introduced from or derived from outside a cell, tissue, or organism, which is not produced by or derived from the same cell, tissue, or organism to which it is introduced.

[0053] The terms “transduced,” “transfected,” or “transformed” refer to the process of introducing or transferring exogenous nucleic acids into or into cells. “Transduced,” “transfected,” or “transformed” cells (e.g., mammalian cells) are cells that have been transduced, transfected, or transformed with exogenous nucleic acids (e.g., vectors), including exogenous nucleic acids encoding any of the cytokine constructs described herein.

[0054] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, or combinations thereof. Unless specifically defined, the term encompasses nucleic acids containing known natural nucleotide analogs having binding properties similar to a reference nucleotide. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses complementary sequences as well as explicitly specified sequences. In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.

[0055] Modifications can be introduced into nucleotide sequences using standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conserved amino acid substitutions are amino acid substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, 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 amino acid families include: aliphatic-hydroxy amino 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).

[0056] As used herein, the phrases “specific binding” or “immune response to” mean that an activated antigen-binding protein complex reacts with one or more antigenic determinants of a desired target antigen and does not react with other peptides, or with a much lower affinity (e.g., about or greater than 10). -6 M) combination.

[0057] The term "treatment" refers to improving at least one symptom of a condition. In some implementations, the condition being treated is cancer and at least one symptom of the cancer is being improved. Attached Figure Description

[0058] Figure 1A This is a schematic diagram of an illustrative activatable cytokine construct comprising a first monomer construct and a second monomer construct covalently or non-covalently linked to each other via first and second dimerizing domains DD1 140 and DD2 190, respectively. The first monomer construct, from its N-terminus to its C-terminus, comprises a first mature cytokine protein CP1 100, a first optional linker 110, a first cleavable portion CM1 120, a second optional linker 130, and a first dimerizing domain DD1 140. The second monomer construct, from its N-terminus to its C-terminus, comprises a second mature cytokine protein CP2 150, a third optional linker 160, a second cleavable portion CM2 170, a fourth optional linker 180, and a second dimerizing domain DD2 190.

[0059] Figure 1B This is a schematic diagram of an illustrative activatable cytokine construct comprising a first monomer construct and a second monomer construct covalently or non-covalently linked to each other via first and second dimerizing domains DD1 200 and DD2 250, respectively. The first monomer construct, from its N-terminus to its C-terminus, comprises a first dimerizing domain DD1 200, a second optional linker 210, a first cleavable portion CM1 220, a first optional linker 230, and a first mature cytokine protein CP1 240. The second monomer construct, from its N-terminus to its C-terminus, comprises a second dimerizing domain DD2 250, a fourth optional linker 260, a second cleavable portion CM2 270, a third optional linker 280, and a second mature cytokine protein CP2 290.

[0060] Figure 2A This is a schematic diagram of an illustrative activatable cytokine construct comprising a first monomer construct and a second monomer construct nonvalently linked to each other via first and second dimerizing domains DD1 340 and DD2 390, respectively. The first monomer construct, from its N-terminus to its C-terminus, comprises a first mature cytokine protein CP1 300, a first optional linker 310, a first cleavable portion CM1 320, a second optional linker 330, and a first dimerizing domain DD1 340. The second monomer construct, from its N-terminus to its C-terminus, comprises a second mature cytokine protein CP2 350, a third optional linker 360, a second cleavable portion CM2 370, a fourth optional linker 380, and a second dimerizing domain DD2 390.

[0061] Figure 2BThis is a schematic diagram of an illustrative activatable cytokine construct comprising a first monomer construct and a second monomer construct nonvalently linked to each other via first and second dimerizing domains DD1 400 and DD2 450, respectively. The first monomer construct, from its N-terminus to its C-terminus, comprises a first dimerizing domain DD1 400, a second optional linker 410, a first cleavable portion CM1 420, a first optional linker 430, and a first mature cytokine protein CP1 440. The second monomer construct, from its N-terminus to its C-terminus, comprises a second dimerizing domain DD2 450, a fourth optional linker 460, a second cleavable portion CM2 470, a third optional linker 480, and a second mature cytokine protein CP2 490.

[0062] Figure 3 The amino acid sequence of an illustrative activatable cytokine construct IFN-α2b-1204dL-hIgG4 (SEQ ID NO:309) is provided, wherein the first monomer construct and the second monomer construct have the same amino acid sequence. From the N-terminus to the C-terminus, the amino acid sequences of the first monomer construct and the second monomer construct encode: a mouse signal peptide (italic text, not bold); mature human interferon-α2b (underlined text); the cleavable moiety 1204dL (bold text); a linker (italic and bold text); and a human IgG4 Fc domain (not italic, bold, or underlined text).

[0063] Figure 4 The amino acid sequence of an illustrative activatable cytokine construct IFN-α2b-1490DNI-hIgG4 (SEQ ID NO:311) is provided, wherein the first and second monomeric constructs have the same amino acid sequence. From the N-terminus to the C-terminus, the amino acid sequence encodes: a mouse signal peptide (italic text, not bold); mature human interferon-α2b (underlined text); the cleavable portion 1490DNI (bold text, not italic); a linker (italic and bold text); and the human IgG4 Fc domain (not italic, bold, or underlined text).

[0064] Figure 5 The cleavage reaction of the activatable cytokine construct IFNα-2b-hIgG4 Fc (with a cleavable portion of 1204dL or 1490dL) and a protease (uPA or MT-SP1) was described, which produced monomeric mature IFNα-2b.

[0065] Figure 6Images of gels containing the following: (1) ACC (1204) with a cleavable moiety 1204; (2) a product of a membrane-type serine protease 1 (MT-SP1) and an ACC IFNα-2b-hIgG4 Fc with a cleavable moiety 1204 (1204 MT-SP1); (3) a product of an ACC IFNα-2b-hIgG4 Fc with a cleavable moiety 1204 and a protease uPA (1204 uPA); (4) an ACC IFNα-2b-hIgG4 Fc (1204+1) with a cleavable moiety 1204 fused with a linker of 5 amino acids; (5) a product of IFNα-2b-hIgG4 Fc 1204+1 and MT-SP1 (1204+1 MT-SP1); (6) an ACC IFNα-2b-hIgG4 Fc with a cleavable moiety 1490; (7) The product of MT-SP1 and ACC IFNα-2b-hIgG4 Fc with a cleavable 1490 moiety; uPA and the product of ACC IFNα-2b-hIgG4 Fc with a cleavable 1490 moiety (1490 uPA).

[0066] Figure 7 Results of a reporter gene assay based on HEK293 cells were provided, which evaluated Sylatron. ® Interferon-α2b activity of (pegylated interferon α-2b) and various interferon α-2b (IFNa2b) fusions: human IgG4 fused to IFNa2b at the N-terminus (IFNa2b NhG4); human IgG4 fused to IFNa2b at the N-terminus via a five-amino acid linker (IFNa2b 5AA NhG4); cytokine-activating construct IFN-α2b-1204dL-hIgG4 (IFNa2b 1204DNIdLNhG4); cytokine-activating construct IFN-α2b-1204dL-hIgG4 containing the same components as IFN-α2b-1204dL-hIgG4, but also having a five-amino acid linker located between the mature cytokine protein components and the cleavable portion (IFNa2b 5AA 1204DNIdLNhG4); and cytokine-activating construct IFN-α2b-1490DNI-hIgG4 (IFNa2b 1490DNI NhG4).

[0067] Figure 8A The effect of the length of the flexible linker in the interferon-α2b-Fc fusion on EC50 was depicted, as determined by reporter gene assays based on HEK293 cells. Figure 8BThe effect of the length of the linker region (LR) in the interferon-α2b-Fc fusion on EC50 was depicted, as determined by reporter gene assays based on HEK293 cells.

[0068] Figure 9 Results of a Daudi cell apoptosis assay were provided, which identified Sylatron. ® Antiproliferative activity with various IFNa2b fusion compounds: human IgG4 fused to IFNa2b at the N-terminus (IFNa2b NhG4); human IgG4 fused to IFNa2b at the N-terminus via a five-amino acid linker (IFNa2b 5AA NhG4); cytokine-activating construct IFN-α2b-1204dL-hIgG4 (IFNa2b 1204DNIdL NhG4); cytokine-activating construct IFNa2b 5AA 1204DNIdL NhG4, which includes the same components as IFN-α2b-1204dL-hIgG4 but also has a five-amino acid linker located between the mature cytokine protein components and the cleavable portion; and cytokine-activating construct IFN-α2b-1490DNI-hIgG4 (IFNa2b 1490DNI NhG4).

[0069] Figure 10A The effect of the linker length in the interferon-α2b-Fc fusion protein on EC50 was depicted, as determined by the Daudi apoptosis assay. Figure 10B The effect of the length of the linker region (LR) in the interferon-α2b-Fc fusion on EC50 was depicted, as determined by the Daudi apoptosis assay.

[0070] Figure 11 Results based on Daudi lymphoma cells were provided, which measured the antiproliferative activity of ACC (IFNa2b1204DNIdL NhG4); protease-treated ACC (IFNa2b1204DNIdL NhG4 + uPA); and recombinant parental cytokine (IFNa2b). The results showed that after protease treatment of ACC, the activity of cytokines in ACC could be restored to levels comparable to those of the recombinant parental cytokine.

[0071] Figure 12Results of a reporter gene assay based on HEK293 cells were presented, which assessed the activities of ACC (IFNa2b 1204DNIdL NhG4); protease-treated (activated) ACC (IFNα-2b 1204DNIdL NhG4 + uPA); Sylatron®; and recombinant parental cytokine (IFNa2b). The results showed that cytokine activity in ACC was restored to levels comparable to those of the recombinant parental cytokine after protease treatment.

[0072] Figure 13 The results of Daudi lymphoma cell-based assays (top panel) for measuring the antiproliferative activity of ACC (ProC440), protease-treated ACC (ProC440 + uPA), and stem cell IFNa2b are depicted, along with the results of HEK293 cell-based reporter gene assays for measuring their activities (bottom panel). The results indicate that by preparing the ACC structure disclosed herein, the activity was reduced by 1000-fold, and that after protease treatment of ACC, the activity of cytokines in ACC was restored to levels comparable to those of the recombinant parental cytokines.

[0073] Figure 14A The structure of ProC440 was depicted, and the expected site in CM was confirmed by mass spectrometry analysis using uPa cleavage. In addition to being sensitive to uPa activation, ProC440 was also cleaved by MMP4. Figure 14B The MMP14 cleavage site at the C-terminus (L161) of IFNa near the cleavable portion was identified by mass spectrometry analysis. Protease activation with MMP14 restored activity to levels comparable to recombinant cytokines.

[0074] Figure 15 The structures of ProC440 and ProC657 (N IFNa2b 0AA 1204DNIdL 0AA IgG4 KiHSS) were depicted. The activities of ACC ProC440 and ProC657, protease-treated ACC (ProC440 + uPA), and stem cell IFNa2b were tested using IFN-reactive HEK293 cells. The results showed that ProC657 activity was decreased compared to stem cell IFNα-2b or uPA-activated ProC440, but increased compared to ProC440.

[0075] Figure 16(Above figure) illustrates the antiproliferative effect of ACC ProC440 in vivo using the Daudi xenograft tumor model. ACC ProC440 induced complete tumor regression at doses as low as 0.1 mg / kg and slowed tumor growth at a dose of 0.02 mg / kg. Figure 16 (The figure below) illustrates the antiproliferative effect of Sylatron® in vivo using a Daudi xenograft tumor model.

[0076] Figure 17A The structure of ProC286 was depicted, and its activity was compared with that of Sylatron® in a Daudi cell apoptosis assay. ProC286 and Sylatron® showed similar activity levels, suggesting that ProC286 can be used as an alternative to Sylatron® control to evaluate IFNα-2b tolerance in hamster studies. Figure 17B The structure of ProC291 was depicted, and its activity was compared with that of Sylatron® in a Daudi cell apoptosis assay. The activity of ProC291 was significantly reduced compared to Sylatron® and ProC286.

[0077] Figure 18 The specific activity and expected toxicity of IFNa-con (recombinant interferon α, a non-natural type I interferon), ProC440+uPA, PEG-IFNa2b (Sylatron) and ProC440 in in vivo dose escalation studies (e.g., at escalating doses of 0.08, 0.4, 2, 10, and 15 mg / kg (“mpk”)) are described.

[0078] Figure 19 The structure of ACC ProC859 universal interferon (top figure) was depicted, as well as the antiproliferative effect of ACC ProC859 in B16 mouse melanoma cell assay and the activity of ACC ProC859 in IFN-responsive HEK293 assay.

[0079] Figure 20AThis is a schematic diagram of an illustrative activatable cytokine construct comprising a first monomer construct and a second monomer construct, which are non-covalently linked to each other via first and second dimerizing domains DD1 540 and DD2, respectively. The first monomer construct, from its N-terminus to its C-terminus, comprises a first mature cytokine protein CP1 500, a first optional linker 510, a first cleavable portion CM1 520, a second optional linker 530, and a first dimerizing domain DD1 540. The second monomer construct, from its N-terminus to its C-terminus, comprises a second mature cytokine protein CP2 550, a third optional linker 560, and a second dimerizing domain DD2 590.

[0080] Figure 20B This is a schematic diagram of an illustrative activatable cytokine construct comprising a first monomer construct and a second monomer construct, which are non-covalently linked to each other via first and second dimerizing domains DD1 600 and DD2, respectively. The first monomer construct, from its N-terminus to its C-terminus, comprises a first dimerizing domain DD1 600, a first optional linker 630, and a first mature cytokine protein CP1 640. The second monomer construct, from its N-terminus to its C-terminus, comprises a second dimerizing domain DD2 650, a second optional linker 660, a cleavable portion CM 670, a third optional linker 680, and a second mature cytokine protein CP2 690.

[0081] Figure 21A This is a schematic diagram of an illustrative cytokine-activating construct comprising a first monomer construct and a second monomer construct, which are non-covalently linked to each other via first and second dimerizing domains DD1 740 and DD2, respectively. The first monomer construct, from N-terminus to C-terminus, comprises 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 dimerizing domain DD1 740. The second monomer construct, from N-terminus to C-terminus, comprises a cytokine-inactive polypeptide or protein 780 and a second dimerizing domain DD2 790. The cytokine-inactive polypeptide or protein 780 can be, for example, a truncated cytokine protein lacking cytokine activity, a mutant cytokine protein lacking cytokine activity, a stub sequence, or a polypeptide sequence that binds 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.

[0082] Figure 21BThis is a schematic diagram of an illustrative cytokine-activating construct comprising a first monomer construct and a second monomer construct, which are non-covalently linked to each other via first and second dimerizing domains DD1 800 and DD2, respectively. The first monomer construct comprises, from the N-terminus to the C-terminus, the first dimerizing domain DD1 800 and a polypeptide or protein 830 lacking cytokine activity. The second monomer construct comprises, from the N-terminus to the C-terminus, the second dimerizing domain DD2 850, a first optional linker 860, a cleavable portion CM 870, a second optional linker 880, and a mature cytokine protein CP. The polypeptide or protein 830 lacking cytokine activity can be, for example, a truncated cytokine protein lacking cytokine activity, a mutant cytokine protein lacking cytokine activity, a residual 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 800 and DD2 850 may be the same or different.

[0083] Figure 22 The study showed that when Syrian gold hamsters were administered 2 mpk, 10 mpk, and 15 mpk of control hIgG4, ProC286, or ProC440 during the treatment period, the animals experienced weight loss.

[0084] Figure 23 Clinical chemistry results (alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase) in Syrian golden hamsters administered 2 mpk, 10 mpk, and 15 mpk of control hIgG4, ProC286, or ProC440 are shown.

[0085] Figure 24 Hematological analysis results (reticulocyte, neutrophil, and white blood cell (WBC) counts) in Syrian golden hamsters administered 2 mpk, 10 mpk, and 15 mpk of control hIgG4, ProC286, or ProC440 are shown.

[0086] Figure 25 An implementation scheme of an ACC representing the connection zone (LR) of an ACC is schematically shown. Detailed Implementation

[0087] This article provides activatable cytokine constructs (ACC) that exhibit reduced levels of at least one activity of the corresponding cytokine, but produce cytokine products with substantially restored activity upon exposure to activating conditions. The activatable cytokine constructs of the present invention can be designed to selectively activate upon exposure to diseased tissues but not in normal tissues. Therefore, these compounds have the potential to confer benefits to cytokine-based therapies with potentially less toxicity associated with certain cytokine-based therapies.

[0088] This article also provides related intermediates, compositions, kits, nucleic acids and recombinant cells, as well as related methods, including methods for using any of the activatable cytokine constructs described herein and methods for producing them.

[0089] The inventors have surprisingly discovered that ACCs having the specific elements and structural orientations described herein may be effective in improving the safety and therapeutic index of cytokines in therapy, particularly for cancer treatment. While cytokines are regulators of the innate and adaptive immune systems and possess broad antitumor activity in preclinical models, their clinical success is limited by systemic toxicity and insufficient systemic exposure to target tissues. The inventors have surprisingly discovered that ACCs having the specific elements and structural orientations described herein appear to reduce systemic toxicity associated with cytokine therapeutics and improve targeting and exposure to target tissues. Therefore, this disclosure provides a method for reducing target-mediated drug disposition (TMDD) of cytokine therapeutics by administering ACCs having the specific elements and structural orientations described herein to a subject. Thus, the present invention addresses the problem that a significant portion of the administered cytokine dose is isolated by normal tissue, a problem that limits the portion of the dose available in systemic circulation to reach the target tissue (e.g., cancerous tissue) with respect to conventional cytokine therapeutics. This invention provides cytokine constructs that localize target binding to tumor tissue, thereby maintaining efficacy, reducing side effects, providing new target opportunities, improving the therapeutic window of validated targets, creating therapeutic windows for undrugable targets, and offering multiple binding modalities. This disclosure enables safe and effective systemic delivery, avoiding the dose-dependent toxicity of conventional systemic cytokine therapy and also eliminating the need for intratumoral injection. This disclosure provides a method for conferring local antiviral activity, immunomodulatory activity, antiproliferative activity, and pro-apoptotic activity. The inventors have surprisingly discovered that dimerization of the first and second monomer constructs achieves a significant reduction in cytokine activity, particularly a greater reduction compared to when a single cytokine is linked to a dimerizing domain. See also Figure 15 .

[0090] Furthermore, the inventors discovered that the degree of reduction in cytokine activity can be modulated by altering the length of the flexible linker or the linker region. Surprisingly, the inventors found that by linking cytokines via short, protease-cleavable sequences to spatially confined dimerizing domains (such as the Fc domain of human IgG truncated at the first cysteine ​​residue in the hinge region (e.g., Cys226, EU numbered), a reduction in cytokine activity of approximately 1,000-fold or more can be achieved. Surprisingly, protease cleavage still occurs despite the spatial confinement, and full cytokine activity is restored after cleavage of the cytokine from the dimerizing domain.

[0091] U.S. Provisional Application No. 63 / 008,542, filed on April 10, 2020, describes certain constructs for activating cytokines, which are incorporated herein by reference in their entirety.

[0092] Cytokine activator construct

[0093] The activatable cytokine construct of the present invention is a dimeric complex comprising a first monomeric construct and a second monomeric construct. Dimerization of the monomeric components is promoted by a pair of dimerizing domains. In one aspect, each monomeric construct comprises a cytokine protein, a cleavable moiety, and a dimerizing domain (DD). In another aspect, one monomeric construct comprises a cytokine protein, a cleavable moiety, and DD, while the other monomeric construct comprises a cytokine protein and DD, but does not include a cleavable moiety. In yet another aspect, one monomeric construct comprises a cytokine protein, a cleavable moiety, and DD, while the other monomeric construct comprises a protein or peptide lacking cytokine activity and DD, but does not include a cleavable moiety. In one specific embodiment, the present invention provides an activatable cytokine construct (ACC) comprising a first monomeric construct and a second monomeric construct, wherein:

[0094] (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1).

[0095] CM1 is located between CP1 and DD1; and

[0096] (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2).

[0097] CM2 is located between CP2 and DD2;

[0098] DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct; and

[0099] 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.

[0100] When used to refer to a cytokine construct, the term "activatable" means a cytokine construct exhibiting a first level of activity or one or more of the activities, which, upon exposure to conditions leading to the cleavage of one or more cleavable portions, results in a cytokine construct exhibiting a second level of activity or one or more of the activities, wherein 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.

[0101] The term "mature cytokine protein" herein refers to a cytokine protein lacking a signaling sequence. A cytokine protein (CP) can be a mature cytokine protein or a cytokine protein having a signaling peptide. Therefore, the ACC of this disclosure may include, in some aspects, a mature cytokine protein sequence. In some aspects, the ACC of this disclosure may include a mature cytokine protein sequence and an additional signaling sequence. In some aspects, the ACC of this disclosure may include the sequences disclosed herein, which may include or lack the signaling sequences described herein.

[0102] The terms "cleavable moiety" and "CM" are used interchangeably herein to refer to peptides whose amino acid sequence contains a substrate for a sequence-specific protease. Cleavable moieties suitable as CM1 and / or CM2 include any of the protease substrates known in the art. Exemplary cleavable moieties are described in more detail below.

[0103] The terms “dimerizing domain” and “DD” are used interchangeably herein to refer to one member of a pair of dimerizing domains, wherein each member of the pair is capable of binding to the other member through one or more covalent or non-covalent interactions. The first DD and the second DD may be the same or different. Exemplary DDs applicable as DD1 and / or DD2 are described in more detail below.

[0104] As used herein, a polypeptide (such as a cytokine or an Fc domain) can be a wild-type polypeptide (e.g., a naturally occurring polypeptide) or a variant of a wild-type polypeptide. A variant can be a polypeptide modified by substituting, inserting, deleting, and / or adding one or more amino acids of a wild-type polypeptide, provided that the variant retains the essential function or activity of the wild-type polypeptide. In some instances, a variant may have altered (e.g., increased or decreased) function or activity compared to a wild-type polypeptide. In some aspects, a variant can be a functional fragment of a wild-type polypeptide. The term "functional fragment" means that the sequence of a polypeptide (e.g., a cytokine) may include fewer amino acids than the full-length polypeptide sequence, but has sufficient polypeptide chain length to confer activity (e.g., cytokine activity).

[0105] 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 organization of the CP, CM, and DD components in each of the first and second monomer constructs may be arranged in the same order in 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, molecular weight, size, amino acid sequence, etc., of the CP and CM components (and the DD component in embodiments where the DD component is a polypeptide). Therefore, the resulting dimer may have symmetrical or asymmetrical monomeric structural components.

[0106] In some embodiments, the first monomer builder includes CP1, CM1, and DD1 directly or indirectly (through a connector) connected to the C-terminus of CM1 from the N-terminus to the C-terminus of CP and CM components. In other embodiments, the first monomer builder includes CP1, CM1, and DD1 directly or indirectly (through a connector) connected to the N-terminus of CM1 from the C-terminus to the N-terminus of CP and CM components. In some embodiments, the second monomer builder includes CP2, CM2, and DD2 directly or indirectly (through a connector) connected to the C-terminus of CM2 from the N-terminus to the C-terminus of CP and CM components. In other embodiments, the second monomer builder includes CP2, CM2, and DD2 directly or indirectly (through a connector) connected to the N-terminus of CM2 from the C-terminus to the N-terminus of CP and CM components.

[0107] In some embodiments, the first and second monomeric constructs are oriented such that the components in each member of the dimer are organized in the same order from the N-terminus to the C-terminus of the CP and CM components. Figure 1A An illustrative diagram of the ACC is provided. (See reference.) Figure 1A The ACC comprises, from the N-terminus to the C-terminus of the CP and CM components, the following: (1) a first monomeric construct having CP1 100; CM1 120, which is positioned relative to the C-terminus of CP1 100; an optional connector 110, which (if present) is positioned between the C-terminus of CP1 100 and the N-terminus of CM1 120; DD1 140; and an optional connector 130, which (if present) is positioned between the C-terminus of CM1 120 and DD1 140; (2) a second monomeric construct having CP2 150; CM2 170, which is positioned relative to the C-terminus of CP2 150; an optional connector 160, which (if present) is positioned between the C-terminus of CP2 150 and the N-terminus of CM2 170; DD2 190; and an optional connector 180, which (if present) is positioned between the C-terminus of CM2 170 and DD2 190. Between 190; and (3) one or more covalent or non-covalent bonds ( ).

[0108] Figure 1B Another illustrative diagram of ACC is provided, whose components are organized with opposite orientations of ACC. (Reference) Figure 1B The ACC comprises, from the N-terminus to the C-terminus of the CP and CM components, the following: (1) a first monomer construct having DD1 200; CM1 220; an optional connector 210, if present, positioned between the N-terminus of DD1 200 and CM1 220; CP1 240, positioned relative to the C-terminus of CM1 220; and an optional connector 230, if present, positioned between the C-terminus of CM1 220 and the N-terminus of CP1 240; (2) a second monomer construct having DD2 250; CM2 270; an optional connector 260, if present, positioned between the N-terminus of DD2 250 and CM2 270; CP2 290, positioned relative to the C-terminus of CM2 270; and an optional connector 280, if present, positioned between the C-terminus of CM2 290 and the N-terminus of CP2 240. Between the N ends of 290; and (3) one or more covalent or non-covalent bonds ( ).

[0109] Figure 2A This is a schematic diagram of an illustrative activatable cytokine construct comprising a first monomer construct and a second monomer construct nonvalently linked to each other via first and second dimerizing domains DD1 340 and DD2 390, respectively. The first monomer construct, from the N-terminus to the C-terminus of the CP and CM components, comprises a first mature cytokine protein CP1 300, a first optional linker 310, a first cleavable portion CM1 320, a second optional linker 330, and a first dimerizing domain DD1 340. The second monomer construct, from the N-terminus to the C-terminus, comprises a second mature cytokine protein CP2 350, a third optional linker 360, a second cleavable portion CM2 370, a fourth optional linker 380, and a second dimerizing domain DD2 390.

[0110] Figure 2BThis is a schematic diagram of an illustrative activatable cytokine construct comprising a first monomer construct and a second monomer construct nonvalently linked to each other via first and second dimerizing domains DD1 400 and DD2 450, respectively. The first monomer construct, from the N-terminus to the C-terminus of the CP and CM components, comprises a first dimerizing domain DD1 400, a second optional linker 410, a first cleavable portion CM1 420, a first optional linker 430, and a first mature cytokine protein CP1 440. The second monomer construct, from the N-terminus to the C-terminus of the CP and CM components, comprises a second dimerizing domain DD2 450, a fourth optional linker 460, a second cleavable portion CM2 470, a third optional linker 480, and a second mature cytokine protein CP2 490. Alternatively, one of the two portions depicted as CP1 440 and CP2 490 may be a truncated cytokine protein lacking cytokine activity. For example, CP1 or CP2 could be a truncated interferon α2b containing the first 151 amino acids of wild-type interferon α2b. Alternatively, one of the two parts described as CP1 440 and CP2 490 is a mutant cytokine protein lacking cytokine activity. For example, CP1 or CP2 could be a truncated interferon α2b with an L130P mutation. Alternatively, one of the two parts described as CP1 440 and CP2 490 is a polypeptide sequence lacking cytokine activity, such as a signaling portion and / or a residual sequence. Alternatively, the first of the two parts described as CP1 440 and CP2 490 is a polypeptide sequence that binds with high affinity to the second of the two parts described as CP1 440 and CP2 490 and reduces the cytokine activity of the second part compared to a control level of the second part.

[0111] The ACC structure was found to be highly effective in reducing the activity of mature cytokine protein components without causing significant impairment of cytokine activity after activation. The activation conditions of the ACC described herein involve exposure to at least one of the cleavable moieties (CM) in a cleavable ACC by a protease. As demonstrated in the examples, activation of the ACC results in a significant recovery of cytokine activity. The results indicate that, in the case of the ACC, the conformation of the cytokine components does not undergo irreversible changes. Importantly, the ACC does not rely on the identification and utilization of peptide masks with binding affinity to the cytokine protein components to achieve a masking effect. Therefore, the ACC does not contain peptide masks with binding affinity to the cytokine protein components. The inventors unexpectedly discovered that the ACC structure is sufficient to avoid off-target effects and undesirable activity and / or toxicity of cytokines without the use of any masking moieties with binding affinity to the cytokine protein components. Therefore, the ACCs described herein are characterized by their lack of affinity masking moieties or peptide masking moieties.

[0112] The ACC may use any one of a variety of mature cytokine proteins, cleavable moieties, and DDs as CP1, CP2, CM1, CM2, DD1, and DD2, respectively. For example, any one of a variety of mature cytokine proteins known in the art, or their sequence and / or truncated variants, may be suitable as one or both of the CP1 and CP2 components of the ACC. The mature cytokine proteins CP1 and CP2 may be the same or different. In some specific embodiments, CP1 and CP2 are the same. In other embodiments, CP1 and CP2 are different. The ACC may include additional amino acid residues at one or both of the N-terminus and / or C-terminus of CP1 and / or CP2.

[0113] In some embodiments, CP1 and / or CP2 may each independently comprise mature cytokine proteins selected from the group consisting of: interferons (such as interferon α, interferon β, interferon γ, interferon τ, and interferon ω), interleukins (such as 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, and IL-21), G-CSF, IL-12, LIF, OSM, IL-10, IL-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, the sequences of the proteins include those exemplified herein, and additional sequences are available from ncbi.nlm.nih.gov / protein. Truncated variants of ACC suitable for use in this invention include any N-terminal or C-terminal truncated cytokines that retain cytokine activity. Exemplary truncated variants used in this invention include any truncated cytokine polypeptide known in the art (see, for example, Slutzki et al., J. Mol. Biol. 360:1019-1030, 2006 and US 2009 / 0025106), and cytokine polypeptides with N-terminal and / or C-terminal truncated by 1 to 40 amino acids, 1 to 35 amino acids, 1 to 30 amino acids, 1 to 25 amino acids, 1 to 20 amino acids, 1 to 15 amino acids, 1 to 10 amino acids, 1 to 8 amino acids, 1 to 6 amino acids, or 1 to 4 amino acids, retaining cytokine activity. In some of the foregoing embodiments, the truncated CP is an N-terminal truncated CP. In other embodiments, the truncated CP is a C-terminal truncated CP. In some embodiments, the truncated CP is a C-terminal and N-terminal truncated CP.

[0114] In some embodiments, CP1 and / or CP2 each independently comprises an amino acid sequence having at least 80% identity (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 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) with a cytokine reference sequence selected from the group consisting of: SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 1 ... NO: 118, SEQ ID NO: 119, SEQ ID NO: 12, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129. SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145. SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 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: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208 and SEQ ID NO: 209. Sequence identity percentage refers to the level of amino acid sequence identity between two or more peptide sequences when aligned using a sequence alignment program (such as the BLAST program set publicly available on the NCBI website). See also Altschul et al., J. Mol. Biol. 215:403-10.1990. In some aspects, ACC includes interferon α2b mutants, such as interferon α2b molecules with a mutation at position L130, such as the L130P mutation, like CP1 or CP2. In some aspects, ACC includes interferon α2b mutants with mutations at positions I24, F64, I60, I63, F64, W76, I116, L117, F123, or L128, or combinations thereof. For example, interferon α2b mutants may include mutations in I116 to T, N, or R; L128 to N, H, or R; I24 to P or Q; L117H; or L128T; or combinations thereof. In some aspects, interferon α2b mutants may include mutations in I24Q, I60T, F64A, W76H, I116R, and L128N, or subsets thereof. In some respects, ACC includes a truncated interferon α2b molecule lacking cytokine activity as one of CP1 and CP2. For example, the truncated interferon α2b may consist of 151 or fewer amino acids of interferon α2b, such as any one of the following from the N-terminus to the C-terminus of the wild-type interferon α2b sequence: 1 to 151, 1 to 150, 1 to 149, 1 to 148…1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 or 2 to 151, 3 to 151, 4 to 151, 5 to 150, 6 to 149, 7 to 148, 8 to 147 amino acids; or any interpolated sequence of the amino acids or mutants of said interferon.

[0115] In some specific embodiments, CP1 and / or CP2 comprise interferon. Interferons suitable for use as CP1 and / or CP2 in the constructs of this invention include, for example, interferon-α, interferon-β, interferon-ω, and interferon-τ. In some embodiments, when the interferon is interferon α, it may be interferon α-2a, interferon α-2b, or interferon α-n3. Other 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 some embodiments, when 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 an IFab domain of interferon α or interferon β. The IFab domain is responsible for the cytokine release and antiviral function of interferon. Exemplary IFab sequences are provided in SEQ ID No: 325-334.

[0116] In some embodiments, CP1 and / or CP2 exhibit interferon activity and include an amino acid sequence having at least 80%, 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%, or at least 99% or 100% identity with 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 specific embodiments, the interferon α reference sequence is SEQ ID NO: 1 (human interferon α-2b). In some embodiments, CP1 and / or CP2 comprise mature alpha 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 comprise mature human alpha interferon having the amino acid sequence of SEQ ID NO: 1. In some of the above embodiments, CP1 and CP2 comprise the same amino acid sequence.

[0117] In other embodiments, CP1 and / or CP2 exhibit interferon activity and include an amino acid sequence having at least 80%, 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%, or at least 99% or 100% identity with an interferon β reference sequence selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109. In some embodiments, the interferon β reference sequence is a human interferon β reference sequence selected from the group consisting of SEQ ID NO: 106 and SEQ ID NO: 107. In some embodiments, CP1 and / or CP2 comprise mature β-interferon having an amino acid sequence selected from the group consisting of: SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109. In some of the above embodiments, CP1 and CP2 comprise the same amino acid sequence.

[0118] In some embodiments, CP1 and / or CP2 exhibit interferon activity and comprise an amino acid sequence having at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% or 100% identity with the interferon ω reference sequence corresponding to SEQ ID NO: 110 (human interferon ω). In some specific embodiments, CP1 and / or CP2 comprise mature human ω interferon having the amino acid sequence of SEQ ID NO: 110. In some of the above embodiments, CP1 and CP2 comprise the same amino acid sequence.

[0119] In some embodiments, CP1 and / or CP2 exhibit interleukin activity and include an amino acid sequence having at least 80%, 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% or 100% identity with an interleukin reference sequence selected from the group consisting of: SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 12, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127. SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 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 comprise mature interleukins having an amino acid sequence selected from the group consisting of: SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:12, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:13 ... SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160. In some of the above embodiments, CP1 and CP2 contain the same amino acid sequence.

[0120] In some embodiments, CP1 and / or CP2 exhibit interleukin activity and include an amino acid sequence having at least 80%, 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% identity with an interleukin reference sequence selected from the group consisting of: 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: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 these embodiments, CP1 and / or CP2 comprise an amino acid sequence selected from the group consisting of: 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, 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: 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 above embodiments, CP1 and CP2 contain the same amino acid sequence.

[0121] 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 comprise a total of about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 250 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 150 amino acids, and about 10 amino acids to about 10... 0 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 20 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 250 amino acids 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, about 80 amino acids to about 300 amino acids, about 80 amino acids to about 250 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, 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 to approximately 250 amino acids, approximately 150 Amino acids to about 200 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 700 amino acids, about 250 amino acids to about 650 amino acids, about 250 amino acids to about 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, 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 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 500 amino acids, approximately 350 amino acids to approximately 450 amino acids, approximately 350 amino acids to approximately 400 amino acids, Approximately 400 amino acids to approximately 700 amino acids, approximately 400 amino acids to approximately 650 amino acids, approximately 400 amino acids to approximately 600 amino acids, approximately 400 amino acids to approximately 550 amino acids, approximately 400 amino acids to approximately 500 amino acids, approximately 400 amino acids to approximately 450 amino acids, approximately 450 amino acids to approximately 700 amino acids, approximately 450 amino acids to approximately 650 amino acids, approximately 450 amino acids to approximately 600 amino acids, approximately 450 amino acids to approximately 550 amino acids, approximately 450 amino acids to Approximately 500 amino acids, approximately 500 amino acids to approximately 700 amino acids, approximately 500 amino acids to approximately 650 amino acids, approximately 500 amino acids to approximately 600 amino acids, approximately 500 amino acids to approximately 550 amino acids, approximately 550 amino acids to approximately 700 amino acids, approximately 550 amino acids to approximately 650 amino acids, approximately 550 amino acids to approximately 600 amino acids, approximately 600 amino acids to approximately 700 amino acids, approximately 600 amino acids to approximately 650 amino acids, or approximately 650 amino acids to approximately 700 amino acids. In some embodiments, CP1 and / or CP2 are mature wild-type human cytokine proteins.

[0122] Each monomeric builder of ACC can use any of a variety of dimerizing domains. Suitable DDs include polymeric (e.g., synthetic polymers, peptides, polynucleotides, etc.) and small molecule (non-polymeric moieties with a molecular weight of less than about 1,000 Daltons and sometimes less than about 800 Daltons) types. DD pairs can be any pair of moieties known in the art to bind together.

[0123] For example, in some embodiments, DD1 and DD2 are a pair selected from the group consisting of: a sushi domain and soluble IL-15 derived from the human IL-15 receptor α chain (IL15Rα); barnase and barnstar; PKA and AKAP; adaptor / docking tag molecules based on mutated RNase I fragments; a pair of antigen-binding domains (e.g., a pair of single-domain antibodies); a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) module based on the interactions of protein synapses, synapse-binding proteins, small synaptic vesicle proteins, and SNAP25; and single-domain antibodies. (sdAb) and corresponding epitopes; antigen-binding domains (e.g., single-chain antibodies (such as single-chain variable fragment (scFv)), single-domain antibodies, etc.) and corresponding epitopes; coiled-coil polypeptide structures (e.g., Fos-Jun coiled-coil structure, acid / base coiled-coil, Glu-Lys coiled-coil, leucine zipper structure), small molecule binding pairs, such as biotin and avidin or streptoavidin, amine / aldehyde, lectin / carbohydrate; a pair of polymers that can bind to each other, such as a pair of sulfur-containing polymers or thiol-containing polymers (e.g., a pair of Fc domains, a pair of thiolized human serum albumin polypeptides, etc.); and so on.

[0124] In some embodiments, DD1 and DD2 are non-peptide polymers. These non-peptide polymers can be covalently bonded to each other. In some instances, the non-peptide polymers can be sulfur-containing polymers, such as sulfur-containing polyethylene glycol. In such cases, DD1 and DD2 can be covalently bonded to each other via one or more disulfide bonds.

[0125] When the DD1 and DD2 pair are members of a pair of epitopes and antigen-binding domains, the epitopes can be native or non-native. Exemplary non-native epitopes include, for example, non-native peptides such as poly-His peptides (e.g., His tags, etc.).

[0126] In some specific implementations, DD1 and DD2 are a pair of Fc domains. As used herein, an "Fc domain" refers to the continuous amino acid sequence of a single heavy chain of an immunoglobulin. A pair of Fc domains associate together to form the Fc region of an immunoglobulin.

[0127] 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., wild-type human IgG1 Fc domain), a human IgG2 Fc domain (e.g., wild-type human IgG2 Fc domain), a human IgG3 Fc domain (e.g., wild-type human IgG3 Fc domain), or a human IgG4 Fc domain (e.g., wild-type human IgG4 Fc domain). In some embodiments, the human Fc domain comprises a sequence having at least 80% identity with SEQ 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% identity).

[0128] In some embodiments, the Fc domains are associated with club-shaped and hole-shaped mutants containing the Fc domains. The club-shaped and hole-shaped mutants may interact with each other to promote dimerization. In some embodiments, the club-shaped and hole-shaped mutants may contain one or more amino acid modifications within the interface between the two Fc domains (e.g., within the CH3 domain). In one example, the modification comprises amino acid substitution T366W and optional amino acid substitution S354C in one antibody heavy chain, and amino acid substitutions T366S, L368A, Y407V, and optional Y349C (numbered according to the EU index of the Kabat numbering system) in another antibody heavy chain. Examples of club-shaped and hole-shaped mutants include the Fc mutants of SEQ ID NO: 315 and 316, and those mutants 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 one embodiment, the dimerized domain comprises sequences that have at least 80% identity with SEQ ID NO: 315 and 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% identity).

[0129] In some embodiments, DD1 and / or DD2 may also include a serum half-life extension portion (e.g., a peptide that binds to a serum protein, such as an immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA)). Examples of half-life extension portions include hexacap 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.

[0130] In some embodiments, DD1 and / or DD2 each comprise a total of about 5 amino acids to about 250 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 250 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 20 amino acids, about 20 amino acids to about 250 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 120 amino acids. Amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 40 amino acids, about 40 amino acids to about 250 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 250 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 250 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 250 amino acids.Approximately 100 amino acids to approximately 200 amino acids, approximately 100 amino acids to approximately 180 amino acids, approximately 100 amino acids to approximately 160 amino acids, approximately 100 amino acids to approximately 140 amino acids, approximately 100 amino acids to approximately 120 amino acids, approximately 120 amino acids to approximately 250 amino acids, approximately 120 amino acids to approximately 200 amino acids, approximately 120 amino acids to approximately 180 amino acids, approximately 120 amino acids to approximately 160 amino acids, approximately 120 amino acids to approximately 140 amino acids, The range is approximately 140 amino acids to approximately 250 amino acids, approximately 140 amino acids to approximately 200 amino acids, approximately 140 amino acids to approximately 180 amino acids, approximately 140 amino acids to approximately 160 amino acids, approximately 160 amino acids to approximately 250 amino acids, approximately 160 amino acids to approximately 200 amino acids, approximately 160 amino acids to approximately 180 amino acids, approximately 180 amino acids to approximately 250 amino acids, approximately 180 amino acids to approximately 200 amino acids, or approximately 200 amino acids to approximately 250 amino acids. In some embodiments, DD1 and DD2 are each Fc domains comprising a portion of a hinge region, said hinge region comprising two cysteine ​​residues, a CH2 domain, and a CH3 domain. In some embodiments, DD1 and DD2 are each Fc domains whose N-terminus is the first cysteine ​​residue read in the N-to-C direction of the hinge region (e.g., cysteine ​​226 of human IgG1 or IgG4, using EU numbering).

[0131] In some respects, the portion containing the protease substrate is located directly or indirectly (e.g., through a linker) between the CP and DD components. In some embodiments, CM1 and CM2 may each independently contain a substrate of a protease selected from the group consisting of: ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, cathepsin A, cathepsin B, cathepsin C, cathepsin G, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, chymotrypsin, Cruzipain, DESC1, DPP-4, FAP, asparagine endopeptidase, Otubain-2, elastase, FVIIa FIXA, FXa, FXIa, FXIIa, Granzyme B, Guanidylbenzoate esterase, Heparin, HtrA1, Human neutrophil elastase, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Lactoferrin, Marapsin, Matriptase-2, Transmembrane peptidase, MT-SP1 / matriptase, Enkephalin, NS3 / 4A, PACE4, Fibrin Lysozyme, PSMA, PSA, BMP-1, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, TMPRSS2, TMPRSS3, TMPRSS4, tPA, thrombin, trypsin, and uPA.

[0132] In some embodiments of any of the ACCs described herein, the protease that cleaves any of the CMs described herein may be ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5. Caspase 6. Caspase 7. Caspase 8. Caspase 9. Caspase 10. Caspase 14. Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Cruzipain, Asparagine Endopeptidase, Otubain-2, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Penetrant Membrane peptidase, enkephalinase, 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, activating protein C, cathepsin A, cathepsin G, chymotrypsin, FVIIa, FIX a, FXa, FXIa, FXIIa, elastase, granzyme B, guanidinobenzoate esterase, HtrA1, human neutrophil lyase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, trypsin, uPA, DESC1, DPP-4, FAP, heparin, Matriptase-2, MT-SP1 / Matriptase, TMPRSS2, TMPRSS3, and TMPRSS4.

[0133] In some embodiments of any of the ACCs described herein, the protease is selected from the group consisting of: uPA, asparagine endopeptidase, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-2, MMP-9, MMP-12, MMP-13, and MMP-14.

[0134] Elevated levels of proteases with known substrates have been reported in many cancers. See, for example, La Roca et al., British J. Cancer 90(7):1414-1421, 2004. The substrates used in the CM1 and / or CM2 components herein include those more prevalent in cancer cells and tissues. Thus, in some embodiments, CM1 and / or CM2 each independently contain substrates of proteases more prevalent in cancer-associated diseased 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 implementations, the cancer is Kaposi's sarcoma, hairy 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 non-muscle-invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer, or testicular cancer, etc. In some of the above implementations, the CM component contains substrates of proteases more commonly found in tumor tissue.

[0135] In some implementations, CM1 and / or CM2 each independently comprise a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 100, and its C-terminal and N-terminal truncated variants.

[0136] In some implementations, CM includes sequences selected from the following group: ISSGLLSGRSDNH (SEQ ID NO:28), LSGRSDDH (SEQ ID NO:33), ISSGLLSGRSDQH (SEQ ID NO:54), and ISSGLLSGRSDNI (SEQ ID NO:68).

[0137] In some embodiments, CM1 and / or CM2 comprise sequences selected from the following group: 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), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 274), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 275), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 266), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 267), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 268), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 269), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 270), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 271), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 272), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 273), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 27 273), ISSGLLSGRSGNH (SEQ ID NO: 274), and its C-terminal and N-terminal truncated variants. Examples of CM also include those described in U.S. Patent Application Publications 2016 / 0289324, 2019 / 0284283 and Publications WO 2010 / 081173, WO2015 / 048329, WO 2015 / 116933, WO 2016 / 118629 and WO 2020 / 118109, which are incorporated herein by reference in their entirety.

[0138] The truncated variants of the above-described amino acid sequences applicable to CM1 and / or CM2 are any variants that retain the corresponding protease recognition site. These variants include C-terminal and / or N-terminal truncated variants that contain at least 3 consecutive amino acids of the above-described amino acid sequence retaining the protease recognition site, or at least 4, 5, 6, or 7 amino acids of the above-described amino acid sequence. In some embodiments, the truncated variants of the above-described amino acid sequences are amino acid sequences that correspond to any of the above-described amino acid sequences, but are truncated at the C-terminus and / or N-terminus by 1 to about 10 amino acids, 1 to about 9 amino acids, 1 to about 8 amino acids, 1 to about 7 amino acids, 1 to about 6 amino acids, 1 to about 5 amino acids, 1 to about 4 amino acids, or 1 to about 3 amino acids, and: (1) have at least three amino acid residues; and (2) retain the protease recognition site. In some of the foregoing embodiments, the truncated CM is an N-terminal truncated CM. In some embodiments, the truncated CM is a C-terminal truncated CM. In some implementations, the truncated C is a CM with both the C-end and N-end truncated.

[0139] In some embodiments of any of the activatable cytokine constructs described herein, CM1 and / or CM2 comprise a total of about 3 amino acids to about 25 amino acids. In some embodiments, CM1 and / or CM2 comprise a total of about 3 amino acids to about 25 amino acids, about 3 amino acids to about 20 amino acids, about 3 amino acids to about 15 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 5 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, or about 20 amino acids to about 25 amino acids.

[0140] In some embodiments, the ACC may comprise multiple CMs containing substrates of different proteases. In some embodiments, CM1 and CM2 contain substrates of different proteases. In some embodiments, CM1 and CM2 contain substrates of the same protease.

[0141] The first monomer construct and the second monomer construct may contain one or more additional components, including one or more linkers, etc. In some embodiments, the first monomer may include a linker disposed 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 contains a linker disposed between CM1 and DD1. In some embodiments, the linker has a total length of 1 amino acid to about 15 amino acids. In some embodiments, CM1 and DD1 are directly adjacent to each other in the first monomer. In some embodiments, CM and any linker disposed between CP1 and DD1 have a total length of 3 to 15 amino acids, or 3 to 10 amino acids, or a combination of 3 to 7 amino acids.

[0142] In some embodiments, the second monomer includes a linker disposed between CP2 and CM2. In some embodiments, CP2 and CM2 are directly adjacent to each other in the second monomer. In some embodiments, the second monomer includes a linker disposed between CM2 and DD2. In some embodiments, the linker has a total length of 1 amino acid to about 15 amino acids. In some embodiments, the linker includes the sequence GGGS (SEQ ID NO: 2). In some embodiments, CM2 (e.g., any of the cleavable moieties described herein) and DD2 (e.g., any of the DDs described herein) are directly adjacent to each other in the second monomer. In some embodiments, CM and any linker disposed between CP2 and DD2 have a total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0143] In some embodiments, the first monomer and / or the second monomer may comprise a total of about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 200 amino acids, or about 50 amino acids. Amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 From about 100 amino acids to about 200 amino acids, from about 100 amino acids to about 150 amino acids, from about 150 amino acids to about 800 amino acids, from about 150 amino acids to about 750 amino acids, from about 150 amino acids to about 700 amino acids, from about 150 amino acids to about 650 amino acids, from about 150 amino acids to about 600 amino acids, from about 150 amino acids to about 550 amino acids, from about 150 amino acids to about 500 amino acids, from about 150 amino acids to about 450 amino acids, from about 150 amino acids to about 400 amino acids, from about 150 amino acids to about 350 amino acids, from about 150 amino acids to about 300 amino acids, from about 150 amino acids to about 250 amino acids, from about 1 50 amino acids to about 200 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 800 amino acids.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. 1 amino acid, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 750 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to Approximately 450 amino acids, approximately 350 amino acids to approximately 400 amino acids, approximately 400 amino acids to approximately 800 amino acids, approximately 400 amino acids to approximately 750 amino acids, approximately 400 amino acids to approximately 700 amino acids, approximately 400 amino acids to approximately 650 amino acids, approximately 400 amino acids to approximately 600 amino acids, approximately 400 amino acids to approximately 550 amino acids, approximately 400 amino acids to approximately 500 amino acids, approximately 400 amino acids to approximately 450 amino acids, approximately 450 amino acids to approximately 800 amino acids, approximately 450 amino acids to approximately 750 amino acids, approximately 450 amino acids to approximately 700 amino acids, approximately 450 amino acids to approximately 650 amino acids, approximately 450 Approximately 450 amino acids to approximately 550 amino acids, approximately 450 amino acids to approximately 500 amino acids, approximately 500 amino acids to approximately 800 amino acids, approximately 500 amino acids to approximately 750 amino acids, approximately 500 amino acids to approximately 700 amino acids, approximately 500 amino acids to approximately 650 amino acids, approximately 500 amino acids to approximately 600 amino acids, approximately 500 amino acids to approximately 550 amino acids, approximately 550 amino acids to approximately 800 amino acids, approximately 550 amino acids to approximately 750 amino acids, approximately 550 amino acids to approximately 700 amino acids, approximately 550 amino acids to approximately 650 amino acids, approximately 550 amino acids to approximately 600 amino acids.Approximately 600 amino acids to approximately 800 amino acids, approximately 600 amino acids to approximately 750 amino acids, approximately 600 amino acids to approximately 700 amino acids, approximately 600 amino acids to approximately 650 amino acids, approximately 650 amino acids to approximately 800 amino acids, approximately 650 amino acids to approximately 750 amino acids, approximately 650 amino acids to approximately 700 amino acids, approximately 700 amino acids to approximately 800 amino acids, approximately 700 amino acids to approximately 750 amino acids, or approximately 750 amino acids to approximately 800 amino acids.

[0144] In some embodiments of any of the ACCs described herein, one or more adapters (e.g., flexible adapters) may be introduced into the activatable cytokine construct to provide flexibility at one or more junctions between domains, between parts, between parts and domains, or at any other junction where the adapter is beneficial. In some embodiments, where the ACC is provided as a conformationally restricted construct, flexible adapters may be inserted to facilitate the formation and maintenance of structures in the uncleaved activatable cytokine construct. Any of the adapters described herein may provide the desired flexibility to facilitate inhibition of binding to a target (e.g., a cytokine receptor) or to facilitate cleavage of CMs by proteases. In some embodiments, the adapter is included in a wholly or partially flexible ACC, such that the adapter may include a flexible adapter and one or more parts conferring less flexible structures to provide the desired ACC. Some adapters may include cysteine ​​residues, which may form disulfide bonds and reduce the flexibility of the construct. In some embodiments, reducing the length of the adapter or linker region reduces the activity of the mature cytokine protein in the ACC (see, for example...). Figures 8A to 8B and Figures 10A to 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 previous component to the C-terminus of the linker adjacent to the N-terminal amino acid of the next component in the N-to-C direction (i.e., the linker length does not include the C-terminal amino acid of the previous component or the N-terminal amino acid of the next component). In embodiments where a linker is used at the N-terminus of the DD containing the 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 previous component to the C-terminus of the linker adjacent to the first cysteine ​​residue of the Fc hinge region (i.e., the linker length does not include the C-terminal amino acid of the previous component or the first cysteine ​​residue of the Fc hinge region).

[0145] From this disclosure and Figure 25It is evident that the ACC of this disclosure comprises a segment of amino acids between the proximal points of interaction between the CP and the dimerizing domains. This segment of amino acids may be referred to as the linker region (LR). As used herein, the term "linker region" or "LR" refers to a segment of amino acid residues between the amino acid residues adjacent to the proximal points of interaction between the C-terminus and N-terminus of the cytokine's dimerizing domains (i.e., the linker region does not include the C-terminal amino acid of the cytokine or the N-terminal amino acid of the DD, which forms the proximal point of interaction with the DD of the corresponding second monomer). For example, when the DD is a pair of Fc domains, the linker region is a segment of amino acid residues between the C-terminus of the cytokine and the first N-terminal cysteine ​​residue involved in the disulfide bonding of the Fc (e.g., cysteine ​​226 of the IgG1 or IgG4 Fc domain, according to EU designation). When the dimerizing domain is not a peptide, the linker region is a segment of amino acid residues from the C-terminus of the cytokine to the last amino acid. For example, when DD is a biotin-streptavitin pair, the linker region containing the biotin monomer is a segment of amino acid residues between the C-terminus of the cytokine and the biotin molecule, while the linker region containing the streptavitin monomer is a segment of amino acid residues between the C-terminus of the cytokine and the streptavitin molecule. In some aspects, the linker region may contain no more than 24, 18, 14, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids, for example, 5 to 14, 7 to 12, or 8 to 11 amino acids.

[0146] In some embodiments, the additional amino acid sequence may be located at the N-terminus or C-terminus of any domain of either of the ACC. Examples include, but are not limited to, targeting portions (e.g., ligands of cell receptors present in target tissues) and serum half-life-extending portions (e.g., peptides that bind serum proteins, such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HSA)).

[0147] In some embodiments of any of the activatable cytokine constructs described herein, the linker may comprise a total of about 1 amino acid to about 25 amino acids (e.g., about 1 amino acid to about 24 amino acids, about 1 amino acid to about 22 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 18 amino acids, about 1 amino acid to about 16 amino acids, about 1 amino acid to about 15 amino acids, about 1 amino acid to about 14 amino acids, about 1 amino acid to about 12 amino acids, about 1 amino acid to about 10 amino acids, about 1 amino acid to about 8 amino acids, about 1 amino acid to about 6 amino acids, about 1 amino acid to about 5 amino acids, about 1 amino acid to about 4 amino acids, about 1 amino acid to about 25 amino acids). Amino acids to about 3 amino acids, about 1 amino acid to about 2 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 24 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 15 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 2 amino acids to about 3 amino acids, about 4 amino groups Acid to about 25 amino acids, about 4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 4 amino acids to about 5 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 24 amino acids, about 5 amino acids to about 22 amino acids, about 5 amino acids to about 20 amino acids, about 5 Amino acids to about 18 amino acids, about 5 amino acids to about 16 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 14 amino acids, about 5 amino acids to about 12 amino acids, about 5 amino acids to about 10 amino acids, about 5 amino acids to about 8 amino acids, about 5 amino acids to about 6 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 15 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids,About 6 amino acids to about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 24 amino acids, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 18 amino acids, about 8 amino acids to about 16 amino acids, about 8 amino acids to about 15 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 amino acids to about 22 amino groups. Acid, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 18 amino acids, about 10 amino acids to about 16 amino acids, about 10 amino acids to about 15 amino acids, about 10 amino acids to about 14 amino acids, about 10 amino acids to about 12 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 15 amino acids, about 12 amino acids to about 14 amino acids 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 16 amino acids to approximately 24 amino acids, approximately 16 amino acids to approximately 22 amino acids, approximately 16 amino acids to approximately 20 amino acids, approximately 16 amino acids to approximately 18 amino acids, approximately 18 amino acids to approximately 25 amino acids, approximately 18 amino acids to approximately 24 amino acids, approximately 18 amino acids to approximately 22 amino acids, approximately 18 amino acids to approximately 20 amino acids, approximately 20 amino acids to approximately 25 amino acids, approximately 20 amino acids to approximately 24 amino acids, approximately 20 amino acids to approximately 22 amino acids, approximately 22 amino acids to approximately 25 amino acids, approximately 22 amino acids to approximately 24 amino acids, or approximately 24 amino acids to approximately 25 amino acids.

[0148] In some embodiments of any of the ACCs 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.

[0149] Surprisingly, the inventors discovered that ACC, which does not contain any linker between CP and DD, exhibits the most significant reduction in cytokine activity compared to wild-type mature cytokines. See also Figure 8A and Figure 10A Furthermore, configurations without a junction between CP and DD still allow for effective cleavage of the CM located between CP and DD. See also Figure 12 See Figure 14. Therefore, in some embodiments, the ACC does not contain any linker between the CP and DD, and the CM between the CP and DD contains no more than 10, 9, 8, 7, 6, 5, 4, or 3 amino acids. In some embodiments, the total number of amino acids in the LR contains no more than 25 amino acids, for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids, or no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids, or 3 to 10 amino acids, or 5 to 15 amino acids, or 7 to 12 amino acids, or any range or specified number of amino acids selected from the range covered by 3 to 25 amino acids.

[0150] In some embodiments of any of the ACCs described herein, the linker may be enriched with glycine (Gly or G) residues. In some embodiments, the linker may be enriched with serine (Ser or S) residues. In some embodiments, the linker may be enriched with both glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs). In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences). In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences). In some implementations, the connector 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).

[0151] In some embodiments of any of the ACCs described herein, the connector comprises any one or a combination of the following: 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), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 214), GGGGSGGGGS (SEQ ID NO: 215), GGGGS (SEQ ID NO: 216), GS, GGGGSGS (SEQ ID NO: 217), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGSLDPKGGGGS (SEQ ID NO: 219), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 219). 220), SKYGPPCPPCPAPEFLG (SEQ ID NO: 221), GKSGSGSESKS (SEQ ID NO: 222), GSTSGSGKSSEGKG (SEQ ID NO: 223), GSTGSSGKSSEGSGSTKG (SEQ ID NO: 224), and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 225).

[0152] Non-limiting examples of connectors may include those with GGGS (SEQ ID NO: 2), GSSGGSGGSGG (SEQ ID NO: 210), GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGS (SEQ ID NO: 217), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 214), GGSLDPKGGGGS (SEQ ID NO: 215), and GSTGSGSGKPGSSEGST (SEQ ID NO: 215). 226) Sequences having at least 70% identity (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% identity).

[0153] In some embodiments, the connector includes sequences 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), GGSSG (SEQ ID NO: 231), GGSGG (SEQ ID NO: 232), GGGSG (SEQ ID NO: 233), GSSSG (SEQ ID NO: 234), GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 214), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 215), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 217), GGGGSGGGGSGGGSGGGSGGGS (SEQ ID NO: 218 ...GGGS (SEQ ID NO: 218), GGGGSGGGGSGGGSGGGSGGGSGGGS (SEQ ID 214), GSTGSGSGKPGSSEGST (SEQ ID NO: 226), (GGGGS)n (SEQ ID NO: 216), where n is an integer of at least one. In some embodiments, the connector comprises a sequence selected from the group consisting of: GGSLDPKGGGGS (SEQ ID NO: 219), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGGGSGS (SEQ ID NO: 217), and GS. In some embodiments of any of the ACCs described herein, the connector comprises a sequence selected from the group consisting of: GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 214), and GSTGSGSGKPGSSEGST (SEQ ID NO: 226). In some embodiments of any of the activatable cytokine constructs described herein, the adapter comprises a sequence selected from the group consisting of GGGGSGGGGSGGGGS (SEQ ID NO: 213) or GGGGS (SEQ ID NO: 216). In some embodiments, the adapter comprises the sequence GGGS (SEQ ID NO: 2).

[0154] In some embodiments, the ACC may include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences as any of the exemplary linker sequences described herein or known in the art). In some embodiments, the linker comprises sulfon-SIAB, SMPB, and sulfon-SMPB, wherein the linker reacts with a primary amine thiol group.

[0155] In some embodiments of the 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 at least one activity 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 cleaved (activated) form of ACC. In some embodiments, the control level may be the activity level of polyethylene glycol-modified CP1 and / or CP2.

[0156] In some embodiments, at least one activity is the binding affinity (Kb) of CP1 and / or CP2 to their homologous receptors. D ), as determined using surface plasmon resonance (e.g., in phosphate-buffered saline at 25°C). In some 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 other embodiments, at least one activity of CP1 and / or CP2 is the level of gene induction by cytokine stimulation using, for example, RNAseq methods (see, for example, Zimmerer et al., Clin. CancerRes. 14(18):5900-5906, 2008; Hilkens et al., J. Immunol. 171:5255-5263, 2003).

[0157] In some embodiments, ACC is characterized by a reduction of at least 2-fold in the activity of at least one CP1 and / or CP2 compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, ACC is characterized by a reduction of at least 5-fold in the activity of at least one CP1 and / or CP2 compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, ACC is characterized by a reduction of at least 10-fold in the activity of 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, ACC is characterized by a reduction of at least 20-fold in the activity of 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, ACC is characterized by a reduction of at least 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold in the activity of 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, ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 by at least 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, or 500 times compared to a control level of at least one activity of CP1 and / or CP2. Up to 1500 times, reduced by 1000 times to 1500 times, reduced by 100 times to 1000 times, reduced by 200 times to 1000 times, reduced by 300 times to 1000 times, reduced by 400 times to 1000 times, reduced by 500 times to 1000 times, reduced by 100 times to 500 times, reduced by 20 times to 50 times, reduced by 30 times to 50 times, reduced by 40 times to 50 times, reduced by 100 times to 400 times, reduced by 200 times to 400 times or reduced by 300 times to 400 times, reduced by 100 times to 300 times, reduced by 200 times to 300 times or reduced by 100 times to 200 times.

[0158] In some embodiments, the control level for at least one activity of CP1 and / or CP2 is the activity of CP1 and / or CP2 released from the ACC after protease cleavage of CM1 and CM2 (“cleavage products”). In some embodiments, the control level for at least one activity of CP1 and / or CP2 is the activity of the corresponding wild-type mature cytokine (e.g., recombinant wild-type mature cytokine).

[0159] In some embodiments, ACC is incubated with a protease to produce an activated cytokine product, wherein the activated cytokine product has one or more activities of CP1 and / or CP2 that are greater than one or more activities of CP1 and / or CP2 in the intact ACC. In some embodiments, the activated cytokine product has one or more activities of CP1 and / or CP2 that are at least 1 times greater than one or more activities of CP1 and / or CP2 in the ACC. In some embodiments, the activated cytokine product has one or more activities of CP1 and / or CP2 that are at least 2 times greater than one or more activities of CP1 and / or CP2 in the ACC. In some embodiments, the activated cytokine product has one or more activities of CP1 and / or CP2 that are at least 5 times greater than one or more activities of CP1 and / or CP2 in the ACC. In some embodiments, the activated cytokine product has one or more activities of CP1 and / or CP2 that are at least 10 times greater than one or more activities of CP1 and / or CP2 in the ACC. In some embodiments, the activated cytokine product has one or more activities of CP1 and / or CP2 that are at least 20 times greater than one or more activities of CP1 and / or CP2 in the ACC. In some embodiments, the activity of one or more of the activated cytokine products CP1 and / or CP2 is at least 1 to 20 times, 2 to 20 times, 3 to 20 times, 4 to 20 times, 5 to 20 times, 10 to 20 times, 15 to 20 times, 1 to 15 times, 2 to 15 times, 3 to 15 times, 4 to 15 times, 5 to 15 times, 10 to 15 times, 1 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 5 to 10 times, 1 to 5 times, 2 to 5 times, 3 to 5 times, 4 to 5 times, 1 to 4 times, 2 to 4 times, 3 to 4 times, 1 to 3 times, 2 to 3 times, or 1 to 2 times that of the ACC CP1 and / or CP2.

[0160] In some embodiments, the ACC may comprise a sequence having 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%) identity with SEQ ID NO: 309 or 311. In some embodiments, the ACC may be encoded by a nucleic acid comprising a sequence having 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%) identity with SEQ ID NO: 310 or 312. In some aspects, the ACC may comprise such sequences, but not the signal sequences of those sequences. There are no particular limitations on the signal sequence. Some non-limiting examples of signal sequences include, for example, residues 1-20 of SEQ ID NO: 309 and corresponding residues and nucleotides in other sequences, or substitutions by signal sequences from other species or cell lines. Other examples of signal sequences include MRAWIFFLLCLAGRALA (SEQ ID NO: 343) and MALTFALLVALLVLSCKSSCSVG (SEQ ID NO: 344).

[0161] Various exemplary aspects of these activatable cytokine constructs are described below and can be used in any combination without limitation in the methods provided herein. Exemplary aspects of activatable cytokine constructs and methods for preparing activatable cytokine constructs are described below.

[0162] In some embodiments, CM is selected for use with a specific protease. The protease may be a protease produced by tumor cells (e.g., tumor cells may express a greater amount of protease than healthy tissue). In some embodiments, CM is a substrate of at least one protease selected from the group consisting of: ADAM 17, BMP-1, cysteine ​​proteases (such as cathepsins), HtrA1, asparagine endopeptidase, matriptase (MT-SP1), matrix metalloproteinases (MMPs), neutrophil elastase, TMPRSS (such as TMPRSS3 or TMPRSS4), thrombin, and u-type plasminogen activating factor (uPA, also known as urokinase).

[0163] In some embodiments, CM is a substrate of 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 of MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, CM is a substrate of MMP7. In some embodiments, CM is a substrate of MMP9. In some embodiments, CM is a substrate of MMP14. In some embodiments, CM is a substrate of two or more MMPs. In some embodiments, CM is a substrate of at least MMP9 and MMP14. In some embodiments, the CM includes two or more substrates of 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.

[0164] In some embodiments, CM is a substrate of MMP and includes the sequences ISSGLLSS (SEQ ID NO: 19); QNQALRMA (SEQ ID NO: 16); AQNLLGMV (SEQ ID NO: 15); STPPFGMF (SEQ ID NO: 18); PVGYTSSL (SEQ ID NO: 74); DWLYWPGI (SEQ ID NO: 75); MIAPVAYR (SEQ ID NO: 42); RPSPMWAY (SEQ ID NO: 43); WATPRPMR (SEQ ID NO: 44); FRLLDWQW (SEQ ID NO: 45); LKAAPRWA (SEQ ID NO: 76); GPSHLVLT (SEQ ID NO: 77); LPGGLSPW (SEQ ID NO: 78); MGLFSEAG (SEQ ID NO: 79); SPLPLRVP (SEQ ID NO: 80); RMHLRSLG (SEQ ID NO: 79); RMHLRSLG (SEQ ID NO: 7 ...9); RMHLRSLG (SEQ ID NO: 79); RMHLRSLG (SEQ ID NO: 79); RMHLRSLG (SEQ ID NO: 79); RMHLRSLG (SEQ ID NO: 79); RMHLRSLG (SEQ ID NO: 79); RMHLRSLG (SEQ ID NO: 79); RMHLRSLG (SEQ ID NO: 79); RMHLRSLG (SEQ ID NO: 79); RMHLRSLG 81); LAAPLGLL (SEQ ID NO: 17); AVGLLAPP (SEQ ID NO: 14); LLAPSHRA (SEQ ID NO: 82); PAGLWLDP (SEQ ID NO: 20); and / or ISSGLSS (SEQ ID NO: 73).

[0165] In some embodiments, CM is a substrate of thrombin. In some embodiments, CM is a substrate of thrombin and includes the sequence GPRSFGL (SEQ ID NO: 83) or GPRSFG (SEQ ID NO: 84).

[0166] In some embodiments, CM comprises 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).

[0167] In some embodiments, CM is a substrate of neutrophil elastase. In some embodiments, CM is a substrate of serine protease. In some embodiments, CM is a substrate of uPA. In some embodiments, CM is a substrate of asparagine endopeptidase. In some embodiments, CM is a substrate of matriptase. In some embodiments, CM is a substrate of cysteine ​​protease. In some embodiments, CM is a substrate of cysteine ​​protease (such as cathepsin).

[0168] In some embodiments, the CM includes the sequence ISSGLLSGRSDNH (SEQ ID NO: 28); ISSGLLSSSGGSGGSLSGRSDNH (SEQ ID NO: 30); AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 275); TTSSGRSANPRGGGAVGLLAPP (SEQ ID NO: 276); VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 277); 267);LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 268); LSGRSNGSGGSGGSISSGLLSS (SEQ ID NO: 279); ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 269); LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 270); QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 271); LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 271); NO: 272); QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 273) and / or ISSGLLSGRSGNH (SEQ ID NO: 274).

[0169] In some embodiments, CM1 and / or CM2 comprise sequences selected from the group consisting of: SEQ ID NO: 5 to SEQ ID NO: 100. In some embodiments, CM comprises 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).

[0170] In some aspects, the ACC includes CP1 selected from SEQ ID No: 1 and 101-209, CM1 and DD1 selected from SEQ ID No: 5-100 and 263-308, which are dimerized with CP2 selected from SEQ ID No: 1 and 101-209, and CM2 and DD2 selected from SEQ ID No: 5-100 and 263-308. In some aspects, the ACC may include a connector selected from SEQ ID No: 2 and 210-234, 245 or 250 between CP1 and CM1 and / or between CM1 and DD1, and a connector selected from SEQ ID No: 2 and 210-234, 245 or 250 between CP2 and CM2 and / or between CM2 and DD2. In some embodiments, the ACC includes DD1 and / or DD2 having an amino acid sequence having at least 80% identity with 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% identity). In some embodiments, the ACC includes DD1 having an amino acid sequence having at least 80% identity with 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% identity). 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).

[0171] Conjugation

[0172] This disclosure also provides methods and materials for including additional elements in any of the ACCs described herein, including, for example, targeted portions, agents (e.g., therapeutic agents, antitumor agents), toxins, or fragments thereof that facilitate delivery to cells or tissues of interest.

[0173] In some embodiments of any of the ACCs described herein, the ACC may be conjugated with a cytotoxic agent (including, but not limited to, toxins (e.g., bacterial, fungal, plant, or animal-derived enzyme-active toxins or fragments thereof)) or a radioisotope. In some embodiments of any of the ACCs described herein, the activatable cytokine construct may be conjugated with a cytotoxic agent (including, but not limited to, toxins (e.g., bacterial, fungal, plant, or animal-derived enzyme-active toxins or fragments thereof)) or a radioisotope.

[0174] Non-limiting exemplary cytotoxic agents that can be conjugated to any of the ACCs described herein include: dolastatin and its derivatives (e.g., auristatin E, AFP, monomethylauristatin D (MMAD), monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), normethylauristatin E (DMAE), auristatin F, normethylauristatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone), maytansinoids (e.g., DM-1, DM-4), maytansin derivatives, duocarmycin, α-amaminine, turbostatin, phenstatin, hydroxyphenstatin, and spongistatin. 5. Sponge Inhibin 7. Halistatin 1. Halistatin 2. Halistatin 3. Halocomstatin, Pyrrolobenzimidazole (PBI), Cibrostatin 6. Doxaliform, Cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthraquinone, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecin, 10,11-difluoromethylenedioxycamptothecin, comprbetastatin, debromoaplysiatoxin, KahaMide-F, discodermolide, and ecteinascidin.

[0175] Non-limiting exemplary enzymatic toxins that can be conjugated to any of the ACCs described herein include: diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuriies fordii protein, dianthin protein, Phytolacca Americana protein (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotin, and sapaonaria. Inhibitors of officinalis, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene.

[0176] Non-limiting exemplary antitumor agents that can be conjugated with any of the ACCs described herein include: adriamycin, cerubidine hydrochloride, bleomycin, alkeran, velban, oncovin sulfate, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarbazine, and cytarabine.

[0177] Non-limiting exemplary antiviral agents that can be conjugated with any of the ACCs described herein include acyclovir, vira adenosine (vira A), and amantadine (symmetrel).

[0178] Non-limiting exemplary antifungal agents that can be conjugated with any of the ACCs described herein include nystatin.

[0179] Non-limiting exemplary composable detection reagents that can be conjugated with any of the ACCs described herein include: fluorescein and its derivatives, fluorescein isothiocyanate (FITC).

[0180] Non-limiting exemplary antibacterial agents that can be conjugated to any of the activatable cytokine constructs described herein include: aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin.

[0181] Non-limiting exemplary 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranose)-(1-->3)-(2-O-acetyl-α-L-arabinopyranoside) (OSW-1) that can be conjugated to any of the activatable cytokine constructs described herein includes: an S-nitrobenzyloxycarbonyl derivative of O6-benzylguanine, a topoisomerase inhibitor, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepine dimer (PBD), functionalized pyrrolobenzodiazepine, calcicheamicin, podophyllotoxin, taxane, and vincristine.

[0182] Non-limiting exemplary radiopharmaceuticals that can be conjugated to any of the cytokine-activating 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).

[0183] Non-limiting exemplary heavy metals that can be combined with any of the ACCs described herein include: barium, gold, and platinum.

[0184] Non-limiting exemplary antimycoplasmas that can be conjugated to any of the ACCs described herein include: tylosine, spectinomycin, streptomycin B, ampicillin, sulfonamides, polymyxin, and chloramphenicol.

[0185] Those skilled in the art will recognize that a variety of possible portions can be conjugated to any of the activatable cytokine constructs described herein. Conjugation may include any chemical reaction that binds the two molecules, as long as the ACC and other portions retain their respective activity. Conjugation may include a number of chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordination binding, and complexation. In some embodiments, covalent binding is preferred. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linkers can be used to conjugate any of the activatable cytokine constructs described herein. For example, conjugates may include organic compounds such as thioesters, carbodiimides, succinimides, glutaraldehyde, diazobenzene, and hexamethylenediamine. In some embodiments, the activatable cytokine construct may include or otherwise introduce one or more non-natural amino acid residues to provide a suitable conjugation site.

[0186] In some embodiments of any of the ACCs described herein, the agent and / or conjugate is linked to the antigen-binding domain via a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Since many cancers naturally release high levels of the reducing agent glutathione, glutathione present in the cancerous tissue microenvironment can reduce the disulfide bond and subsequently release the agent and / or conjugate at the delivery site.

[0187] In some embodiments of any of the ACCs described herein, when the conjugate binds to its target in the presence of complement at the target site (e.g., diseased tissue (e.g., cancerous tissue)), the amide or ester bond connecting the conjugate and / or agent to the linker is cleaved, resulting in the release of the active form of the conjugate and / or agent. When administered to a subject, these conjugates and / or agents complete the delivery and release of the conjugate and / or agent at the target site (e.g., diseased tissue (e.g., cancerous tissue)). These conjugates and / or agents are particularly effective for in vivo delivery of any of the conjugates and / or agents described herein.

[0188] In some embodiments, the linker is not cleaved by enzymes of the complement system. For example, since complement activation ultimately cleaves target cells, the conjugate and / or agent is released in the absence of complement activation. In such embodiments, the conjugate and / or agent will be delivered to the target cells (e.g., hormones, enzymes, corticosteroids, neurotransmitters, or genes). Furthermore, the linker is slightly sensitive to cleavage by serum proteases, and the conjugate and / or agent is released slowly at the target site.

[0189] In some embodiments of any of the ACCs described herein, the conjugates and / or agents are designed such that the conjugates and / or agents are delivered to the target site (e.g., diseased tissue (e.g., cancerous tissue)) without releasing the conjugates and / or agents.

[0190] In some embodiments of any of the ACCs described herein, the conjugate and / or agent 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 may be modified to include functional groups that can subsequently be used to attach to the antigen-binding domain by the methods described herein.

[0191] In some embodiments of any of the ACCs described herein, the ACC includes at least one conjugation site of the agent. In some embodiments, all possible conjugation sites may be used for conjugation with the agent. In some embodiments, one or more conjugation sites include, but are not limited to, sulfur atoms involving disulfide bonds, sulfur atoms involving interchain disulfide bonds, sulfur atoms involving interchain disulfide bonds but not intrachain disulfide bonds, and / or sulfur atoms of cysteine ​​or other sulfur-containing amino acid residues. In such cases, the residues may be naturally present in the protein construct structure or incorporated into the protein construct using methods including, but not limited to, site-directed mutagenesis, chemical transformation, or accidental incorporation of non-natural amino acids.

[0192] This disclosure also provides methods and materials for preparing ACCs for conjugation. In some embodiments of any of the ACCs described herein, the ACC is modified to include one or more interchain disulfide bonds. For example, the disulfide bonds in the ACC may undergo reduction upon exposure to a reducing agent (such as, but not limited to, TCEP, DTT, or β-mercaptoethanol). In some cases, the reduction of the disulfide bonds is only partial. As used herein, the term partial reduction refers to a situation where the ACC is contacted with a reducing agent and a portion of all possible conjugation sites undergo reduction (e.g., not all disulfide bonds are reduced). In some embodiments, if less than 99% (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%) of all possible conjugation sites are reduced, then the cytokine-activating construct is partially reduced upon contact with a reducing agent. In some implementations, an ACC having one or more interchain disulfide bonds reduced is conjugated to a drug that is reactive with free thiols.

[0193] This disclosure also provides methods and materials for conjugating therapeutic agents to specific sites on an ACC. In some embodiments of any of the ACCs described herein, the ACC is modified such that a therapeutic agent can be conjugated to the ACC at a specific site on the ACC. For example, the ACC can be partially reduced in a manner that promotes conjugation to the ACC. In such cases, 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 promote conjugation of the agent at a specific site on the protein construct. After treatment with a reducing agent, various factors can affect the “reduction level” of the ACC. For example, but not limited to, it may be necessary to optimize the ratio of reducing agent to ACC, incubation time, incubation temperature, and / or the pH of the reduction reaction solution to achieve partial reduction of the ACC using the methods and materials described herein. Any suitable combination of factors (e.g., the ratio of reducing agent to ACC, the incubation time and temperature with the reducing agent, and / or the pH of the reducing agent) can be used to achieve partial reduction of the ACC (e.g., general reduction of possible conjugation sites or reduction of specific conjugation sites).

[0194] The effective ratio of reducing agent to ACC can be any ratio that allows at least partial reduction of ACC in a manner that permits agent conjugation (e.g., general reduction of possible conjugation sites or reduction of specific conjugation sites). In some implementations, the ratio of reducing agent to ACC is in the range of about 20:1 to 1:1, about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or about 1:1 to 1:1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0195] The effective incubation time and temperature for treating ACC with a reducing agent can be any time and temperature that allows the agent to at least partially reduce ACC in a manner that allows the ACC to conjugate (e.g., general reduction of possible conjugation sites or reduction of specific conjugation sites). In some embodiments, the incubation time and temperature for treating ACC will be in the range of about 1 hour at 37°C to about 12 hours at 37°C (or any subrange thereof).

[0196] The effective pH for the reduction reaction of ACC with a reducing agent can be any pH that allows ACC to be at least partially reduced in a manner that allows ACC to conjugate with the agent (e.g., general reduction of possible conjugation sites or reduction of specific conjugation sites).

[0197] When partially reduced ACC is contacted with a thiol-containing agent, the agent can conjugate with interchain thiols in the ACC. The agent can be modified in a manner that includes thiols using a thiol-containing agent (e.g., cysteine ​​or N-acetylcysteine). For example, ACC can be partially reduced by incubating with a reducing agent (e.g., TEPC) at approximately 37°C for approximately 1 hour at a desired reducing agent to ACC ratio. An effective reducing agent to ACC ratio can be any ratio that allows for the partial reduction of at least two interchain disulfide bonds in the ACC in a manner that allows for the conjugation of the thiol-containing agent (e.g., general reduction of possible conjugation sites or reduction of specific conjugation sites).

[0198] In some embodiments of the ACC described herein, the ACC is reduced by a reducing agent in a manner that avoids the reduction of any intra-chain disulfide bonds. In some embodiments of the ACC described herein, the ACC is reduced by a reducing agent in a manner that avoids the reduction of any intra-chain disulfide bonds and reduces at least one inter-chain disulfide bond.

[0199] In some embodiments of any of the ACCs described herein, the ACC may also include an agent conjugated to the ACC. In some embodiments, the conjugated agent is a therapeutic agent.

[0200] In some embodiments, the agent (e.g., an agent conjugated to an activatable cytokine construct) is a detectable moiety, such as a label or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotin moieties detectable by labeling avidin (e.g., streptomycin containing a fluorescent marker or enzyme activity detectable by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzyme labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moieties are linked by spacer molecules.

[0201] In some implementations, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is linked to the ACC using a carbohydrate moiety, thiol group, amino group, or carboxylic acid ester group.

[0202] In some embodiments of any of the agent-conjugated ACCs described herein, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to the ACC via a linker and / or a CM (also known as a cleavable sequence). In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to a cysteine ​​or lysine residue in the ACC. In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to another residue of the ACC, such as those disclosed herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a non-cleavable linker. Table 1 provides some non-limiting examples of cleavable portions and linkers.

[0203] Table 1.

[0204]

[0205] Those skilled in the art will recognize that a variety of possible components can be coupled with the ACC of this disclosure. (See, for example, “Conjugate Vaccines,” Contributions to Microbiology and Immunology, JMCruse and RE Lewis, Jr (eds.), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference). In general, efficient conjugation of an agent (e.g., a cytotoxic agent) to an ACC can be achieved through any chemical reaction that binds the agent to the ACC while allowing both the agent and the ACC to retain their functionality.

[0206] In some embodiments of any of the agent-conjugated ACCs, a variety of bifunctional protein coupling agents may be used to conjugate the agent to the ACC, including but not limited to N-succinimide 3-(2-pyridyldimercapto)propionate (SPDP), iminothiones (IT), bifunctional derivatives of imine esters (e.g., dimethyl adipate HCl), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazid compounds (e.g., bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen derivatives (e.g., bis(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and biactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin may be prepared as described in Vitetta et al., Science 238: 1098 (1987). In some embodiments, a carbon-14 labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agent can be used to conjugate radioactive nucleotides to ACC. (See, for example, WO94 / 11026).

[0207] Suitable linkers and CMs are described in the literature. See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimide benzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of haloacetylhydrazine derivatives coupled to ACC by means of oligopeptide linkers. In some embodiments, suitable connectors include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimide-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog (21558G); (iii) SPDP (succinimide-6-[3-(2-pyridyl-dithio)propamido]hexanoate) (Pierce Chem. Co., catalog number 21651G); (iv) sulfonyl-LC-SPDP (sulfosuccinimide-6-[3-(2-pyridyl-dithio)propamido]hexanoate) (Pierce Chem. Co., catalog number 2165-G); and (v) sulfonyl-NHS (N-hydroxysulfonyl-succinimide: Pierce Chem. Co.) conjugated to EDC. (Catalogue No. 24510). Additional connectors include, but are not limited to, SMCC, sulfonated-SMCC, SPDB, or sulfonated-SPDB.

[0208] The CMs and connectors described above contain components with different properties, thus resulting in conjugates with different physicochemical properties. For example, sulfonated NHS esters of alkyl carboxylic acids are more stable than sulfonated NHS esters of aryl carboxylic acids. Connectors containing NHS esters have lower solubility than sulfonated NHS esters. Furthermore, the SMPT connector contains sterically hindered disulfide bonds and can form conjugates with increased stability. Generally, disulfide bonds are less stable than other bonds because they are cleaved in vitro, resulting in fewer usable conjugates. Sulfonated NHS can particularly enhance the stability of carbodiimide couplings. When used in combination with sulfonated NHS, carbodiimide couplings (such as EDC) form esters that are more resistant to hydrolysis compared to carbodiimide coupling reactions alone.

[0209] In some embodiments of any of the ACCs, an agent can be conjugated to the ACC using a modified amino acid sequence included in the amino acid sequence of the ACC. Protein constructs can be engineered to achieve controlled placement and / or dosing of the conjugated agent (e.g., a cytotoxic agent) by inserting conjugable amino acids at specific positions within the amino acid sequence of the ACC. For example, the ACC can be modified to include cysteine ​​residues at positions that provide reactive thiol groups on the first, second, third, and / or fourth monomers without negatively impacting protein folding and / or assembly and without altering antigen-binding properties. In some embodiments, the ACC can be modified to include one or more non-natural amino acid residues within its amino acid sequence to provide suitable conjugation sites. In some embodiments, the ACC can be modified to include an enzymatically activated peptide sequence within its amino acid sequence.

[0210] Nucleic acid

[0211] This document provides nucleic acids comprising sequences encoding a first monomeric construct (or a protein portion of a first monomeric construct) (e.g., any of the first monomeric constructs described herein) and a second monomeric construct (or a protein portion of a second monomeric construct) (e.g., any of the second monomeric constructs described herein) of any of the ACCs described herein. In some embodiments, a pair of nucleic acids together encode the first monomeric construct (or the protein portion of the first monomeric construct) and the second monomeric construct (or the protein portion of the second monomeric construct). In some implementations, the nucleic acid sequence encoding the first monomer construct (or the protein portion of the first monomer construct) has at least 70% identity with the nucleic acid sequence encoding the second monomer construct (or the protein portion of the second monomer construct) (e.g., at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, 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% identity).

[0212] 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 encodes a polypeptide comprising CP2 and CM2 portions. In some embodiments, a pair of nucleic acids together encode the protein portions of the first and second monomer constructs, wherein the protein portions are then conjugated to DD1 and DD2 portions, respectively (in a subsequent conjugation step).

[0213] 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.

[0214] carrier

[0215] This document provides vectors and vector sets including 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) to prepare any of the ACCs described herein, and to use the vector or vector set to express any of the ACCs described herein. For example, when selecting a vector or vector set, the cell must be considered, as the vector may need to be able to integrate into and / or replicate within the cell's chromosome. Exemplary vectors that can be used to generate ACCs are also described below.

[0216] 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 (such as any of the cells described herein). A "vector" is capable of delivering nucleic acids and fragments thereof into a host cell and includes regulatory sequences (e.g., promoters, enhancers, poly(A) signals). Exogenous polynucleotides can be inserted into expression vectors for expression. The term "vector" also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0217] Methods for constructing suitable vectors, including any of the nucleic acids described herein, that are suitable for transforming cells (e.g., mammalian cells), are well known in the art. See, for example, Sambrook et al., eds., “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor Press, 1989, and Ausubel et al., eds., “Current Protocols in Molecular Biology”, Current Protocols, 1993.

[0218] Non-limiting examples of vectors include plasmids, transposons, granules, and viral vectors (e.g., any adenovirus vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector), as well as any Gateway ® Vectors. For example, a vector may include sufficient cis-acting expression elements; other expression elements may be provided by host mammalian cells or in an in vitro expression system. Skilled practitioners will be able to select appropriate vectors and mammalian cells to prepare any of the ACCs described herein.

[0219] In some implementations of any of the ACCs described herein, ACCs can be prepared biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0220] In some embodiments, the vector comprises nucleic acids encoding a first and a second monomer of any of the ACCs described herein. In some embodiments, the vector is an expression vector.

[0221] In some embodiments, the vector pair together comprises a pair of nucleic acids that together encode a first and a second monomer of any of the ACCs described herein. In some embodiments, the vector pair is a pair of expression vectors.

[0222] cell

[0223] This document also provides host cells comprising any of the vectors or vector groups described herein, wherein the vectors or vector groups comprise any of the nucleic acids described herein.

[0224] Any of the ACCs described herein can be generated by any cell (e.g., mammalian cells). 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.

[0225] Methods for introducing nucleic acids and vectors (such as any of the vectors or groups of vectors described herein) into cells are known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids into cells include: lipid transfection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenovirus transduction, lentivirus transduction), nanoparticle transfection, and electroporation.

[0226] In some implementations, the introduction step includes introducing a vector (such as the vectors described herein or any of the vector group) into a cell, said vector comprising a nucleic acid encoding a monomer constituting any of the ACCs described herein.

[0227] In some embodiments of any of the methods described herein, the cell may be a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell having a distinctive membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as *Saccharomyces cerevisiae*. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells).

[0228] In some embodiments, the cell contains nucleic acids encoding a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the cell contains a nucleic acid pair that together encodes a first monomer and a second monomer of any of the ACCs described herein.

[0229] Methods for generating activatable cytokine constructs

[0230] This document provides a method for producing any of the ACCs described herein, the method comprising: (a) culturing any of the recombinant host cells described herein in a liquid culture medium under conditions sufficient to produce ACCs; and (b) recovering ACCs from the host cells and / or the liquid culture medium.

[0231] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions conducive to cell proliferation, cell differentiation, and cell growth. For example, cells can be cultured by contacting cells (such as any of the cells described herein) with a cell culture medium containing sufficient essential growth factors and supplements to support cell viability and growth.

[0232] In some embodiments of any of the methods described herein, the method further includes the separation and recovery of ACC. Non-limiting examples of separation methods include: ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand affinity chromatography, ion exchange chromatography (e.g., anion or cation), and hydrophobic interaction chromatography.

[0233] In some implementations, the cell can generate a protein portion of a first monomeric construct containing CP1 and CM1, and a protein portion of a second monomeric construct containing CP2 and CM2, which are then conjugated to portions DD1 and DD2, respectively.

[0234] The compositions and methods described herein may involve using non-reducing or partially reducing conditions that allow disulfide bonds to form between dimerizing domains to form and maintain the dimerization of ACC.

[0235] In some embodiments of any of the methods described herein, the method further includes formulating the isolated ACC into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any of the isolated ACCs described herein can be formulated for use via any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0236] This document also provides ACCs produced by any of the methods described herein. A composition (e.g., a pharmaceutical composition) comprising any of the ACCs produced by any of the methods described herein is also provided. A kit comprising at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein is also provided.

[0237] Treatment

[0238] This article provides a method for treating a subject’s disease (e.g., cancer (e.g., any of the cancers described herein)) which includes administering to the subject a therapeutically effective amount of any of the ACCs described herein.

[0239] As used herein, the term "subject" refers to any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, rat, hamster, or guinea pig), a non-human primate (e.g., an ape (e.g., a monkey (e.g., a baboon, marmoset) or an ape (e.g., a chimpanzee, gorilla, or orangutan, or gibbon)) or a human. In some embodiments, the subject is a human.

[0240] In some implementations, the subject has been previously identified or diagnosed with a disease (e.g., cancer (such as any of the cancers described herein)).

[0241] As used herein, the term "treatment" includes reducing the severity, frequency, or number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., cancer, as described herein) in a subject (e.g., any of the subjects described herein). In some implementations where the disease is cancer, the treatment results in a reduction in cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduction in the risk of cancer recurrence in a subject with cancer.

[0242] In some embodiments of any of the methods described herein, the disease is cancer. This document also provides methods for treating a subject in need (e.g., any of the exemplary subjects described herein or known in the art), comprising administering to the subject a therapeutically effective amount of any of the ACCs described herein or any of the compositions described herein (e.g., pharmaceutical compositions).

[0243] In some implementations of these methods, the subject has been identified or diagnosed with cancer. Non-limiting examples of cancer include: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell tumors, multiple myeloma, lymphomas (e.g., B-cell lymphoma, B-cell non-Hodgkin's lymphoma). (The text lists various cancers and related medical conditions, including lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma, leukemia, hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndromes (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 head and neck cancer, endometrial cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some implementations, the cancer is lymphoma. In some implementations, the lymphoma is Burkitt lymphoma.) In some respects, the subjects have been identified or diagnosed with familial cancer syndromes, such as Li Fraumeni Syndrome, familial breast-ovarian cancer (BRCA1 or BRAC2 mutation) syndrome, etc. The disclosed methods can also be used to treat non-solid tumors. Exemplary solid tumors include malignancies of various organ systems (e.g., sarcomas, adenocarcinomas, and carcinomas), such as those of the lungs, breasts, lymph nodes, gastrointestinal tract (e.g., colon), and genitourinary tracts (e.g., kidney, urothelial, or testicular tumors), pharynx, prostate, and ovaries. Exemplary adenocarcinomas include colorectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, and small bowel cancer.

[0244] Exemplary cancers described by the National Cancer Institute include: adult acute lymphoblastic leukemia; childhood acute lymphoblastic leukemia; adult acute myeloid leukemia; adrenocortical carcinoma; childhood adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; childhood cerebellar astrocytoma; childhood cerebral astrocytoma; extrahepatic bile duct cancer; bladder cancer; childhood bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma; childhood brainstem glioma; adult brain tumor; childhood brain tumor brainstem glioma. Gestational tumors; cerebellar astrocytoma in children; cerebral astrocytoma / malignant glioma in children; ependymoma in children; medulloblastoma in children; supratentorial primitive neuroectodermal tumor in children; gliomas of the visual pathway and hypothalamus in children; other brain tumors in children; breast cancer; breast cancer and pregnancy; breast cancer in children; male breast cancer; bronchial adenoma / carcinoid tumor in children; carcinoid tumor in children; gastrointestinal carcinoid tumor; adrenocortical carcinoma; insular cell carcinoma; metastatic carcinoma of unknown primary origin. Primary; Primary central nervous system lymphoma; Childhood cerebellar astrocytoma; Childhood cerebral astrocytoma / malignant glioma; Cervical cancer; Childhood cancer; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myeloproliferative disorders; Tenosynovial clear cell sarcoma; Colon cancer; Childhood colorectal cancer; Cutaneous T-cell lymphoma; Endometrial cancer; Childhood ependymoma; Ovarian epithelial cancer; Esophageal cancer; Childhood esophageal cancer; Ewing family tumors; Childhood extracranial germ cell tumors; Gonadal extragerminal tumors; Extrahepatic bile duct cancer; Ocular cancer, intraocular melanoma; Ocular cancer, retinoblastoma; Gallbladder cancer; Gastric / Stomach cancer; Childhood gastric cancer; Gastrointestinal carcinoid tumors; Childhood extracranial germ cell tumors; Gonadal extragerminal tumors; Ovarian germ cell tumors; Trophoblastic tumors; Childhood brainstem glioma; Visual pathway and hypothalamic glioma; Pilocytic leukemia; Head and neck cancer; Adult (primary) Hepatocellular carcinoma; Primary hepatocellular carcinoma in children; Hodgkin's lymphoma in adults; Hodgkin's lymphoma in children; Hodgkin's lymphoma during pregnancy; Hypopharyngeal carcinoma; Hypothalamic and optic pathway glioma in children; Intraocular melanoma; Insular cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Renal cell carcinoma; Laryngeal carcinoma; Laryngeal carcinoma in children; Acute lymphoblastic leukemia in adults; Acute lymphoblastic leukemia in children; Acute myeloid leukemia in adults; Acute myeloid leukemia in children; Chronic Lymphocytic leukemia; Chronic myeloid leukemia; Hairy cell leukemia; Lip and oral cancer; Adult (primary) liver cancer; Childhood (primary) liver cancer; Non-small cell lung cancer; Small cell lung cancer; Adult acute lymphoblastic leukemia; Childhood acute lymphoblastic leukemia; Chronic lymphocytic leukemia; AIDS-related lymphoma; Central nervous system (primary) lymphoma; Cutaneous T-cell lymphoma; Adult Hodgkin lymphoma; Childhood Hodgkin lymphoma;Hodgkin's lymphoma during pregnancy; non-Hodgkin's lymphoma in adults; non-Hodgkin's lymphoma in children; primary central nervous system lymphoma; Waldenström macroglobulinemia; male breast cancer; malignant mesothelioma in adults; malignant mesothelioma in children; malignant thymoma; medulloblastoma in children; melanoma; intraocular melanoma; Merkel cell carcinoma. Carcinoma; Malignant mesothelioma; Occult primary metastatic squamous neck cancer; Multiple endocrine tumor syndrome in children; Multiple myeloma / plasma cell tumor; Mycosis fungoides; Myelodysplastic syndrome; Chronic myeloid leukemia; Acute myeloid leukemia in children; Multiple myeloma; Chronic myeloproliferative syndrome; Nasal cavity and sinus carcinoma; Nasopharyngeal carcinoma; Nasopharyngeal carcinoma in children; Neuroblastoma; Adult non-Hodgkin lymphoma; Childhood non-Hodgkin lymphoma; Non-Hodgkin lymphoma during pregnancy; Non-small cell lung cancer; Oral cancer in children; Oral and lip cancer; Oropharyngeal cancer; Osteosarcoma / Malignant fibrous histiocytoma of bone; Ovarian cancer in children; Ovarian epithelial cancer; Ovarian germ cell tumors; Low-potential ovarian tumors; Pancreatic cancer; Pancreatic cancer in children; Insular cell pancreatic cancer; Sinus and Nasal cavity cancer; parathyroid carcinoma; penile cancer; pheochromocytoma; pediatric pineal and supratentorial primitive neuroectodermal tumors; pituitary adenoma; plasma cell tumor / multiple myeloma; pleural pulmonary blastoma; pregnancy and breast cancer; pregnancy and Hodgkin lymphoma; pregnancy and non-Hodgkin lymphoma; primary central nervous system lymphoma; adult primary liver cancer; pediatric primary liver cancer; prostate cancer; rectal cancer; renal cell (kidney) carcinoma; pediatric renal cell carcinoma; transitional cell carcinoma of the renal pelvis and ureter; retinoblastoma; pediatric rhabdomyosarcoma; salivary gland cancer; pediatric salivary gland cancer; Ewing family neoplastic sarcoma; Kaposi's sarcoma; sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; pediatric rhabdomyosarcoma; adult soft tissue sarcoma; pediatric soft tissue sarcoma; Sezary syndrome. Syndrome; Skin cancer; Childhood skin cancer; Skin cancer (melanoma); Merkel cell skin cancer; Small cell lung cancer; Small bowel cancer; Adult soft tissue sarcoma; Childhood soft tissue sarcoma; Metastatic occult primary squamous neck cancer; Stomach / Gastric cancer; Childhood gastric cancer; Childhood supratentorial primitive neuroectodermal tumor; Cutaneous T-cell lymphoma; Testicular cancer; Childhood thymoma; Malignant thymoma; Thyroid cancer; Childhood thyroid cancer; Transitional cell carcinoma of the renal pelvis and ureter; Gestational trophoblastic tumor; Childhood cancer of unknown primary site; Unusual childhood cancers; Transitional cell carcinoma of the ureter and renal pelvis; Urethral cancer; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma in children; Vulvar cancer; Waldenström macroglobulinemia; and Wilms' tumor.

[0245] Other exemplary cancers include diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).

[0246] Metastasis of the aforementioned cancers can also be treated or prevented using the methods described in this article.

[0247] In some implementations, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of a subject's cancer (e.g., compared to the number, severity, or frequency of one or more symptoms of a subject's cancer before treatment).

[0248] 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 subject.

[0249] Table 2. Additional treatments

[0250]

[0251]

[0252]

[0253] Composition / Pharm Kit

[0254] This document also provides compositions (e.g., pharmaceutical compositions) comprising any one and one or more (e.g., 1, 2, 3, 4, or 5) pharmaceutically acceptable carriers (e.g., any one of the pharmaceutically acceptable carriers described herein), diluents, or excipients.

[0255] In some embodiments, a composition comprising any of the ACCs described herein (e.g., a pharmaceutical composition) may be placed in a sterile vial or a pre-filled syringe.

[0256] 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 intratumoral).

[0257] In some embodiments, any of the pharmaceutical compositions described herein may comprise one or more buffers (e.g., neutral buffered saline, phosphate buffered saline (PBS), amino acids (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 pharmaceutically acceptable carriers (e.g., antibacterial water, PBS, or saline).

[0258] As used herein, the phrase “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial agents, antimicrobial agents, isotonics, and absorption delay agents that are compatible with pharmaceutical administration. Suitable carriers include, but are not limited to, water, saline, Ringer's solution, dextran solution, and approximately 5% human serum albumin.

[0259] In some embodiments of any of the pharmaceutical compositions described herein, any of the ACCs described herein are prepared using a carrier that prevents rapid elimination from the body, such as sustained-release and controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyvinyl acetate, and polylactic acid, can be used. Methods for preparing such pharmaceutical compositions and formulations will be apparent to those skilled in the art.

[0260] This document also provides a kit comprising any of the ACCs described herein, any of the compositions comprising any of the ACCs described herein, or any of the pharmaceutical compositions comprising any of the ACCs described herein. In addition to the ACCs described herein, a kit comprising one or more second therapeutic agents selected from Table 2 is also provided. The second therapeutic agent may be provided in a dosage form separate from the ACC. Alternatively, the second therapeutic agent may be formulated together with the ACC.

[0261] Any of the kits described herein may include instructions for use of any of the compositions described herein (e.g., pharmaceutical compositions) and / or any of the ACC. In some embodiments, the kit may include instructions for performing 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.

[0262] Example

[0263] The present invention will be further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0264] Example 1: Generation of Cytokine-Activating Constructs

[0265] The cytokine-activating construct IFN-α2b-1204DNIdl-hIgG4 was prepared using a recombinant method. The first and second monomeric constructs of the ACC are identical, each possessing... Figure 3The polypeptide has the amino acid sequence shown (SEQ ID NO: 309). Each of the first and second monomeric constructs contains, from the N-terminus to the C-terminus, a signal sequence from the mouse IgGκ signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human interferon α-2b (SEQ ID NO: 1), a cleavable portion having the amino acid sequence SEQ ID NO: 99, a linker having the amino acid sequence GGGS (SEQ ID NO: 2), and a DD corresponding to human IgG Fc (SEQ ID NO: 4). The polypeptide was prepared by transforming host cells with a polynucleotide having the sequence SEQ ID NO: 310, followed by culturing the resulting recombinant host cells. Dimerization of the resulting expressed polypeptide yields the cytokine-activating construct IFN-α2b 1204DNIdl hIgG4.

[0266] An activatable cytokine construct, IFN-α-2b 1490DNI-hIgG4, was also prepared via a recombinant method. The first and second monomeric constructs of the ACC are identical, each possessing... Figure 4 The polypeptide has the amino acid sequence shown (SEQ ID NO: 311). Each of the first and second monomeric constructs of the ACC contains, from the N-terminus to the C-terminus, a signal sequence from the mouse IgG κ signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human interferon α-2b (SEQ ID NO: 1), a cleavable portion having the amino acid sequence SEQ ID NO: 68, a linker having the amino acid sequence GGGS (SEQ ID NO: 2), and a DD corresponding to human IgG Fc (SEQ ID NO: 4). The polypeptide is prepared by transforming host cells with a polynucleotide having the sequence SEQ ID NO: 312, followed by culturing the resulting recombinant host cells. Dimerization of the resulting expressed polypeptide produces the cytokine-activating construct IFN-α2b 1204dlhIgG4.

[0267] Additional activatable cytokine constructs were prepared, which contained five additional amino acid residues in the linker.

[0268] Electrophoresis was performed on the activatable cytokine constructs and the protease-treated activatable cytokine constructs. Figure 6The gels were depicted, showing the following results (from left to right): (1) ACC IFN-α2b-1204DNIdl-hIgG4 (“1204”); (2) MT-SP1 treated IFN-α2b-1204DNIdl-hIgG4 (“1204 MT-SP1”); (3) uPA treated IFN-α2b-1204DNIdl-hIgG4 (“1204 uPA”); (4) IFN-α2b-1204DNIdl-hIgG4 with five amino acid residues added to the linker (“1204+1”); (5) MT-SP1 treated IFN-α2b-1204DNIdl-hIgG4 (“1204+1 MT-SP1”); (6) uPA treated IFN-α2b-1204DNIdl-hIgG4 (7) IFN-α-2b 1490DNI-hIgG4 ("1490"); (8) MT-SP1 treated IFN-α-2b 1490DNI-hIgG4 ("1490MT-SP1"); and (9) uPA treated IFN-α-2b 1490DNI-hIgG4 ("1490 uPA"). The results indicate that the protease effectively cleaves the cleavable portion of the activatable cytokine construct.

[0269] Example 2. IFN-α-2b activity of activating cytokine constructs

[0270] Cell-based reporter gene assays of human type I interferon were used to test the activity of ACC as described in Example 1.

[0271] IFN-responsive HEK293 cells were generated by stable transfection with human STAT2 and IRF9 genes to obtain a fully activated type I IFN signaling pathway. These cells also possess an inducible SEAP (secreting embryonic alkaline phosphatase) reporter gene controlled by 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 blastcin, 100 µg / mL zeocin, and 100 µg / mL normocin. Adding type I IFN to these cells activated the JAK / STAT / ISGF3 pathway and subsequently induced SEAP production, which was readily assessed in the supernatant using a colorimetric assay of alkaline phosphatase activity in Quanti-Blue solution. The activity of ACC containing IFNα-2b was compared with that of Sylatron® (pegylated interferon α-2b) using the aforementioned reporter gene assay. Figure 7Data from [the source] indicates that, compared to Sylatron ® Compared to the IFNα-2b activity of PEGylated interferon α-2b, the IFNα-2b activity of ACC was significantly reduced.

[0272] also, Figure 8A and Figure 8B The data indicate that the activity of (unfractured) ACC can be modulated by changing the length of the joint or connection region. Figures 8A to 8B The data presented here show results for IFNa-2b-hIgG4 Fc fusion constructs with different linker lengths or without a linker between IFNa-2b and hIgG4 Fc, as tested in the HEK293 reporter gene assay. The fusion proteins tested in this experiment contain a mature IFNα-2b cytokine sequence, an optional linker and / or cleavable portion, and the Fc domain of human IgG4 (SEQ ID NO: 4) in the N-to-C-terminal direction (including a complete hinge region, such that the N-terminus of the Fc sequence begins with the amino acid sequence ESKYGPPCPPC…). The first construct (linker region = 7) does not have a linker or cleavable portion; its N-to-C-terminal sequence consists of the fusions SEQ ID NO: 4-SEQ ID NO: 1. The second construct (linker region = 12) has a 5-amino acid linker SGGGG (SEQ ID NO: 335); its N-to-C-terminal sequence consists of the fusions SEQ ID NO: 1-SEQ ID NO: 335-SEQ ID NO: 4. The third construct (linker region = 18) comprises a 7-amino acid CM (SGRSDNI) and a 4-amino acid linker GGGS; its N-terminal to C-terminal sequence consists of the fusion of SEQ ID NO: 1-SEQ ID NO: 100-SEQ ID NO: 2-SEQ ID NO: 4. The fourth construct (linker region = 23) comprises a 5-amino acid linker, a 7-amino acid CM, and a 4-amino acid linker; its N-terminal to C-terminal sequence consists of the fusion of SEQ ID NO: 1-SEQ ID NO: 335-SEQ ID NO: 100-SEQ ID NO: 2-SEQ ID NO: 4. The fifth construct (linker region = 24) comprises a 13-amino acid CM (ISSGLLSGRSDNI) and a 4-amino acid linker; its N-terminal to C-terminal sequence consists of the fusion of SEQ ID NO: 1-SEQ ID NO: 68-SEQ ID NO: 2-SEQ ID NO: 4.

[0273] Example 3: In vitro antiproliferative effect of ACC on cancer cells

[0274] The antiproliferative effects of IFNα-2b and IFNα-2b-containing ACC were tested in vitro using Daudi cells, a cell line derived from human B-cell lymphoblasts. The cells were cultured in RPMI-1640 medium supplemented with 10% FBS at a concentration of 2 × 10⁻⁶ cells / mL. 5 Daudi cells were prepared at a concentration of 10 cells / mL, and 50 μL aliquots were pipetted into the wells of a white flat-bottomed 96-well plate (10K / well). The test ACC or control was diluted in RPMI 1640 medium supplemented with 10% FBS. Five-fold serial dilutions were produced, from which 50 μL was added to each well. After incubation at 37°C for 3 days, intracellular ATP levels were measured using a viability kit to indirectly estimate the number of remaining viable cells. 100 µL of cell-titer Go was added directly to the plate, and the plate was placed on an orbital oscillator for 10 minutes. Following incubation, the luminescence signal was measured directly using an Envision plate reader. Dose-response curves were generated, and EC50 values ​​were obtained using Graph Pad Prism software via sigmoid fitting and nonlinear regression. Specific activity was determined by comparing EC50 values ​​with recombinant IFNα2b or pharmaceutical-grade Sylatron® (pegylated interferon α-2b).

[0275] The antiproliferative activity of ACC containing IFNα-2b in Daudi lymphoma cells showed that, compared with Sylatron® (pegylated interferon α-2b), the IFNα-2b activity of uncleaved ACC was reduced. Figure 9 ).

[0276] Figures 10A to 10B Data also indicate that the activity of (unlyzed) ACC can be modulated by altering the length of the linker. The antiproliferative effects of IFNa-2b-hIgG4 Fc fusion protein constructs with different linker lengths or without a linker between IFNa-2b and hIgG4 Fc were tested in vitro using Daudi cells. Data showed that the length of the flexible linker and the length of the linker region (LR) between the cytokine and the Fc domain affected the activity of (unlyzed) ACC. Constructs with zero or short linkers and correspondingly short LRs showed decreased cytokine activity, while constructs with longer linkers and therefore longer LRs exhibited higher levels of cytokine activity. Results in Figures 10A to 10B As shown in the figure. The fusion protein construct is similar to that described in Example 2 above. Figure 8A and Figure 8B The same as those described.

[0277] Example 4: Activity of ACC after protease treatment

[0278] The antiproliferative response of ACC containing IFNα-2b treated with protease was tested in Daudi lymphocytes and cell-based reporter gene assays to determine whether activity could be restored.

[0279] To cleave the dimerizing domain, ACC containing IFNα-2b was treated overnight at 37°C with a recombinant human protease, such as urokinase-type plasminogen activator (uPA) or matriptase (MT-SP1). As described in Examples 2 and 3, a mixture of protease inhibitors was added to neutralize the protease before testing activity. The results from these assays indicate that treatment of ACC containing IFNα-2b with the protease restored activity to levels comparable to recombinant cytokines. EC50 values ​​for ACC IFNα-2b-1204DNIdl-hIgG4, ACC IFNα-2b-1204DNIdl-hIgG4 + uPA, and stem cell IFNα-2b (human recombinant IFN-α 2b, from StemCell Technologies, catalog number 78077.1) were calculated based on Daudi cell apoptosis assays and are provided in Table 3 below.

[0280] Table 3. EC50: Daudi cell apoptosis assay

[0281]

[0282] The EC50 values ​​of ACC IFNα-2b-1204DNIdl-hIgG4, ACC IFNα-2b-1204DNIdl-hIgG4 + uPA and stem cell IFNα-2b were calculated based on the IFNα / β assay results and are provided in Table 4 below.

[0283] Table 4. EC50: IFNα / β reporter gene assay

[0284]

[0285] These results indicate that, in the absence of activating protease, the activity of IFNα-2b-1204DNIdl-hIgG4 was significantly reduced compared to the IFNα-2b control.

[0286] Example 5: General ProIFN

[0287] The ACC according to this disclosure is prepared by a recombinant method, and the ACC has a universal interferon sequence (ProC859) (IFNaAD 0AA 1204DNIdL 0AA IgG4) that is active against both human and mouse cells. The first and second monomeric constructs of the ACC are identical, each being a polypeptide 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 the N-terminus to the C-terminus, a signal sequence, a mature cytokine protein corresponding to a universal interferon molecule as a hybrid of IFNα1 and IFNα2a (SEQ ID NO: 324), a cleavable portion having the amino acid sequence SEQ ID NO: 100, and a dimerizing domain corresponding to human IgG Fc (SEQ ID NO: 3). The activity of the universal ProIFN was tested in vitro using IFN-reactive HEK293 cells and B16 mouse melanoma cells.

[0288] Compared to mouse IFNa4, ProC859 exhibited at least a 150-fold decrease in activity. Protease activation using uPa restored the activity to levels comparable to mouse IFNa4, such as... Figure 19 As shown in the figure. The EC50 values ​​of ACC ProC859, ACC ProC859 + uPA, and mouse IFNα4 were calculated based on the measurement results and are presented in the figure. Figure 19 Provided by China.

[0289] EC50: B16 IFNα / β reporter gene assay

[0290]

[0291] Example 6: In vitro characterization of the lead ACC ProC440:

[0292] The cytokine-activating construct ProC 440 (N IFNa2b 0 1204DNIdL0AA Fc) was prepared by a recombinant method. The first and second monomeric constructs of the ACC are identical, each being a polypeptide having the amino acid sequence SEQ ID NO: 313 and a signal sequence at its N-terminus. Each of the first and second monomeric constructs contains, from the N-terminus to the C-terminus, a signal sequence, a mature cytokine protein corresponding to human interferon α-2b (SEQ ID NO: 1), a cleavable portion having the amino acid sequence SEQ ID NO: 100, and a dimerizing domain corresponding to human IgG Fc (SEQ ID NO: 3).

[0293] As previously described, the activity of ProC440 was tested in vitro using IFN-responsive HEK293 cells and Daudi cells. In both assays, the activity of ProC440 was reduced by at least 1,000-fold compared to stem cell IFNα-2b. Figure 13 Protease activation using uPa restores activity to that of recombinant cytokines (such as...). Figure 13 The corresponding levels of IFNα-2b are shown below. The EC50 values ​​of ACC ProC440, ACC ProC440 + uPA, and stem cell IFNα-2b were calculated based on the IFNα / β assay results and are provided in Table 5 below.

[0294] Table 5. EC50: IFNα / β reporter gene assay

[0295]

[0296] The EC50 values ​​of ACC ProC440, ACC ProC440 + uPA, and stem cell IFNα-2b were calculated based on Daudi apoptotic cell assays and are provided in Table 6 below.

[0297] Table 6. EC50: Daudi cell apoptosis assay

[0298]

[0299] Mass spectrometry analysis confirmed the expected sites in CM were cleaved using uPa ( Figures 14A to 14B In addition to being sensitive to uPa activation, ProC440 is also cleaved by MMP4. Figures 14A to 14B Mass spectrometry analysis identified the MMP14 cleavage site at the C-terminus of IFNa, near the cleavable portion. Figure 14B Protease activation with MMP14 restored activity to levels comparable to recombinant cytokines. In summary, this demonstrates that ACC ProC440 can recover full activity after at least uPa and MMP14 cleavage of both intrinsic and engineered cleavable portions.

[0300] ACC ProC657 (N IFNa2b 0AA 1204DNIdL 0AA IgG4KiHSS) was also prepared via a recombinant method. The first monomeric construct of the ACC is a polypeptide having the amino acid sequence SEQ ID NO: 314 and a signal sequence at its N-terminus. The first monomeric construct of the ACC, from N-terminus to C-terminus, includes a signal sequence, a mature cytokine protein corresponding to human interferon α-2b (SEQ ID NO: 1), a cleavable portion having the amino acid sequence SEQ ID NO: 68, and a dimerizing domain corresponding to human IgG Fc (SEQ ID NO: 315) with a club-shaped mutation. The second monomeric construct of the ACC is a polypeptide having the amino acid sequence SEQ ID NO: 322 and a signal sequence at its N-terminus. The second monomeric construct, from N-terminus to C-terminus, includes a signal sequence, a residual portion (SEQ ID NO: 317), and a dimerizing domain corresponding to human IgG Fc (SEQ ID NO: 316) with a club-shaped mutation.

[0301] As previously described, the activity of ProC657 was tested in vitro using IFN-responsive HEK293 cells. Compared to stem cell IFNα-2b or uPa-activated ProC440, the activity of ProC657 was decreased, but compared to ProC440, the activity was increased. Figure 15 Therefore, this disclosure provides ACCs with different structures, which make it possible to adjust the level of reduced activity in the ACC.

[0302] Example 7: In vivo antiproliferative activity of ACC:

[0303] The antiproliferative effect of ACC ProC440 containing IFNα-2b was tested in vivo using the Daudi xenograft tumor model. 10 x 10⁻⁶ cells were added to serum-free medium (1:1 Matrigel). 6 One Daudi cell was subcutaneously implanted into Beige / SCID mice. When the average tumor volume reached approximately 60-120 mm... 3 Mice were randomly assigned to groups and administered ProC440 once weekly for 5 weeks. Body weight and tumor measurements were recorded twice weekly during the study. Figure 16 The data show that ACCProC440 containing IFNα-2b induced complete tumor regression at doses as low as 0.1 mg / kg and slowed tumor growth at a dose of 0.02 mg / kg (top figure), and the antiproliferative effect of Sylatron® is shown for comparison (bottom figure).

[0304] Example 8: In vivo tolerance activity of ACC

[0305] Human IFNα-2b cross-reacts with hamster IFNα receptors and has previously been shown to be active in hamsters (Altrock et al., Journal of Interferon Research, 1986). To assess the tolerability of ACCProC440 containing IFNα-2b, Syrian golden hamsters were administered a starting dose of 0.4 mg / kg. Animals received one dose of the investigational product and continued the study for 7 days after administration unless non-tolerance toxicity (DLT) was identified. The starting dose (0.4 mg / kg (“mpk”) represents the expected induced equivalent dose of INFα-con (recombinant interferon α, a non-naturally occurring type I interferon manufactured by Amgen under the name Infergen®) to induce weight loss, reduced food consumption, and myelosuppression in hamsters (125 g). In cynomolgus monkeys, 0.1 mg / kg / day of INFα-con was associated with weight loss, reduced food consumption, and bone marrow suppression (equivalent to 1.25–2.5 x 10^7 U for a 125 g hamster). If the starting dose was tolerated, animals were moved to a “medium dose” of 2 mg / kg and given three doses of the test product unless intolerable. If tolerated, animals were moved to a “high dose” of 10 mg / kg and given three doses of the test product unless intolerable. If tolerated, animals were moved to a “high dose” of 15 mg / kg. At each stage, if the test dose was not tolerated, animals were moved to the next lower dose. If the starting dose was not tolerated, animals were moved to a “lower dose” of 0.08 mg / kg. Animals were administered ACC (ProC286) with an N-terminal to C-terminal structure of the DD-CM-CP dimer. Human IgG4 was administered to animals as a negative control. As expected, the negative control did not induce any toxicity in the animals.

[0306] ProC286 (ChIgG4 5AA 1204DNIdL IFNa2b) was also prepared via a recombinant method. The first and second monomeric constructs are identical, each being a polypeptide having the amino acid sequence SEQ ID NO: 320 and a signal sequence at its N-terminus. Each of the first and second monomeric constructs contains, from the N-terminus to the C-terminus, a signal sequence, a dimerizing domain corresponding to human IgG Fc (SEQ ID NO: 3), a linker (SEQ ID NO: 321), a cleavable portion having the amino acid sequence SEQ ID NO: 100, a linker (SEQ ID NO: 2), and a mature cytokine protein corresponding to human interferon α-2b (SEQ ID NO: 1).

[0307] ProC291 (NhIgG4 5AA 1204DNIdL IFNa2b) was also prepared via a recombinant method. The first and second monomeric constructs were identical. Each of the first and second monomeric constructs contained, from the N-terminus to the C-terminus, a mature cytokine protein corresponding to human interferon α-2b (SEQ ID NO: 1), an adaptor (SEQ ID NO: 321), a CM (SEQ ID NO: 100), an adaptor (GGGS), and a human IgG4 Fc region including a fully hinged sequence (SEQ ID NO: 4).

[0308] In the Daudi cell apoptosis assay, the activities of ProC286 and ProC291 were compared with the activity of Sylatron® (PEG-IFN-α2b). Figures 17A to 17B In the assay, ProC286 and Sylatron® exhibited similar activity levels, such as... Figure 17A As shown in the figure. This indicates that ProC286 has similar activity to commercially available polyethylene glycol-modified IFN-α2b and can be used as a Sylatron® control to evaluate the tolerability of IFNα-2b in hamster studies. Compared to ProC286 and Sylatron®, ProC291 exhibited reduced activity, suggesting that the structural orientation of the IFN N-terminus and Fc domains is important for reducing activity. That is, when DD is a pair of Fc domains, localizing the cytokine to the N-terminus of DD (as in ProC291) provides a greater reduction in cytokine activity compared to localizing the cytokine to the C-terminus of DD (as in ProC286).

[0309] Animals were administered an initial dose of 0.4 mg / kg on day 1. Animals were continued for one week unless the intolerable dose (DLT) was reached. Clinical observation was conducted, with body weight and temperature measured in each animal before administration and at 6, 24, 72, and 7 days post-administration. Blood samples were collected from each animal at 72 hours and 7 days post-administration for hematological and chemical analysis. Hematological and chemical analyses were performed immediately after sampling. For hematological analysis, blood smears, white blood cell differential count, hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular volume, platelet count, red blood cell / erythrocyte count, red blood cell distribution width, reticulocyte count, and white blood cell / 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, gamma-glutamyl transferase, globulin, glucose, inorganic phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, urea, nitrogen, and C-reactive protein. Toxicity evidence from the tolerability studies is summarized in... Figures 22 to 24 middle.

[0310] Overall, animals given the unmasked ProC286 construct showed an average weight loss of 5% when administered at 2 mpk, and a weight loss of 15% when administered at 10 mpk and 15 mpk. Figure 22 One animal administered ProC286 at 15 mpk showed a 20% weight loss 7 days after administration (end of study). This was considered a non-tolerated dose. In contrast, animals administered ProC440 at 2 mpk and 10 mpk did not show weight loss.

[0311] Animals administered ProC440 at 15 mpk showed an average weight loss of 5%. Figure 22 This indicates that the ACC of this disclosure, having a dimerized structure of CP-CM-DD starting from the N-terminus, unexpectedly restricts IFNα-2b-mediated weight loss. Not wishing to be bound by theory, it is believed that targeting interferon at the N-terminus of DD and using a relatively short LR in the case of ProC440 reduces interferon toxicity compared to PEGylated IFNα-2b (Sylatron®) or ProC286.

[0312] In clinical chemistry, animals treated 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 study). Figure 23No significant increase in ALP was measured when ProC440 was administered to animals at 10 mpk or 15 mpk. Figure 23 Elevated ALT is a marker of hepatotoxicity. IFNα-2b has been shown to induce hepatotoxicity. This suggests that the ACC of this disclosure, with its dimerized structure of CP-CM-DD originating from the N-terminus, unexpectedly limits IFNa-2b-mediated hepatotoxicity.

[0313] In hematology, animals administered ProC286 at 2 mpk, 10 mpk, and 15 mpk showed significantly reduced reticulocyte, neutrophil, and white blood cell (WBC) counts at 3 days and 7 days post-administration (end of study). Figure 24 These reductions are reminiscent of IFNa-2b-mediated myelotoxicity. Three days after administration, animals treated with ProC440 showed decreased levels of reticulocyte, neutrophil, and white blood cell (WBC) counts. Figure 24 Overall, the reduction in hematopoietic cell levels observed in animals treated with ProC440 was less pronounced than that observed in animals treated with ProC286. At 7 days post-treatment (end of study), overall levels of reticulocyte, neutrophil, and white blood cell (WBC) counts in animals treated with ProC440 returned to normal levels, or to levels similar to those observed in animals treated with the negative control IgG4. Figure 24 In animals treated with ProC286, reticulocyte, neutrophil, and white blood cell (WBC) counts remained low. This suggests that the ACC of this disclosure, with its N-terminal CP-CM-DD dimerization, unexpectedly limits IFNa-2b-mediated myelotoxicity.

[0314] Example 4. In vitro characterization of additional cytokine constructs

[0315] Additional activatable cytokine constructs were also prepared via recombinant methods. The first and second monomeric constructs of these ACCs are identical. Each of the first and second monomeric constructs contains, from the N-terminus to the C-terminus, a signal sequence from the mouse IgG κ signaling sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human interferon α-2b (SEQ ID NO: 1), a cleavable moiety (CM) having the amino acid sequence SEQ ID NO: 100, and a dimerizing domain corresponding to human IgG4 S228P Fc (containing SEQ ID NO: 3). Furthermore, these ACCs may or may not include a linker with the amino acid sequence SGGGG between the CP and CM. These ACCs may or may not include a linker with the amino acid sequence GGGS between the CM and DD. These ACCs may or may not contain a DD hinge moiety located at the N-terminus of cysteine ​​226. These additional activatable cytokine constructs are described in Table 6 (see SEQ ID No: 336 to 342 and SEQ ID NO: 313).

[0316] Table 6: Activated cytokines with different amino acid sequence lengths between CP and cysteine ​​226 of human IgG

[0317]

[0318] The activities of ProC440 (an ACC lacking a flexible linker and truncated to the Fc region of Cys226) and additional ACCs containing various linker and Fc region sequences were tested in vitro using IFN-responsive HEK293 and Daudi cells as previously described. In both assays, the activity (e.g., antiproliferative effect) of ProC440 was reduced compared to all other ACCs containing various additional sequences between the cytokine and the first amino acid (i.e., Cys226) that binds DD to the corresponding second monomer. The EC50 values ​​of the ACCs were calculated based on the IFNα / β assay results and are provided in Table 7 below.

[0319] Table 7. EC50: IFNα / β reporter gene assay

[0320]

[0321] The EC50 value of ACC was calculated based on the results of Daudi cell apoptosis assay and is provided in Table 8 below.

[0322] Table 8. EC50: Daudi cell apoptosis assay

[0323]

[0324] The data in Tables 7 and 8 also indicate that the activity of (uncleaved) ACC can be modulated by altering the length of the amino acid sequence between the cytokine and Cys226 of DD.

[0325] Not wishing to be bound by theory, based on the results presented herein, the inventors envision localizing cytokines at the N-terminus of DD and using a relatively short LR to inhibit the cytokine activity of 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 including the various cytokine proteins discussed herein. As a non-limiting example, the CP used in the ACC of the present invention can be any of those shown in SEQ ID NO: 101 to 209 and their variants. In particular, monomeric cytokines are suitable for the ACC described herein. Based on the results provided herein, it is believed that the ACC of the present invention will exhibit reduced cytokine activity relative to the corresponding wild-type cytokines, and that upon cleavage of the ACC by the relevant protease, the cleavage products will restore cytokine activity similar to that of the corresponding wild-type cytokines.

[0326] Instance sequence

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360] Other implementation plans

[0361] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct, wherein: (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1); (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2); (c) The first monomer construct is a polypeptide comprising CP1, CM1, and DD1 in the N-terminal to C-terminal direction, and is characterized in that CP1 and DD1 are linked by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM1. (d) Further among them: (i) The second monomer construct is identical to the first monomer construct, and (ii) DD1 and DD2 are a pair of human IgG Fc domains; (e) DD1 and DD2 are covalently bonded to each other through at least one disulfide bond, thereby forming a dimer of the first monomer construct and the second monomer construct; and (f) The ACC is characterized by having a level of at least one CP1 and CP2 activity that is reduced compared to the corresponding control level of at least one CP1 and CP2 activity.

2. The ACC according to claim 1, wherein CP1 is a mature human interferon or interleukin.

3. The ACC according to claim 1, wherein CP1 is mature human interferon α.

4. The ACC according to claim 3, wherein the mature interferon is mature interferon α-2b.

5. The ACC according to claim 4, wherein the mature interferon comprises a sequence having at least 95% identity with SEQ ID NO:

1.

6. The ACC according to claim 4, wherein the mature interferon α comprises the sequence of SEQ ID NO:

1.

7. The ACC according to any one of claims 1-6, wherein CM1 and CM2 each contain no more than 7 amino acids.

8. The ACC according to any one of claims 1-7, wherein CM1 and CM2 are each independently cleaved by urokinase (uPa) and / or matrix metalloproteinase (MMP).

9. The ACC according to any one of claims 1-8, wherein CM1 and CM2 each comprise a sequence having at least 85% identity with SEQ ID NO:

100.

10. The ACC according to any one of claims 1-8, wherein CM1 and CM2 each comprise a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:68 and SEQ ID NO:

100.

11. The ACC according to any one of claims 1-10, wherein DD1 and DD2 are a pair of human IgG4 Fc domains.

12. The ACC according to any one of claims 1-10, wherein DD1 and DD2 are a pair of human IgG1 or IgG4 Fc domains truncated at the N-terminus to cysteine ​​226 as numbered in the EU.

13. The ACC of claim 11, wherein the human IgG4 Fc domain comprises the S228P mutation as numbered by the EU designation.

14. The ACC according to any one of claims 1-10, wherein DD1 and DD2 each comprise a sequence having at least 95% identity with SEQ ID NO:

3.

15. The ACC according to any one of claims 1-10, wherein DD1 and DD2 each comprise the sequence of SEQ ID NO:

3.

16. The ACC according to any one of claims 1-15, wherein the first monomer builder and the second monomer builder are covalently bonded to each other by at least two disulfide bonds.

17. The ACC according to any one of claims 1-15, wherein the first monomer builder and the second monomer builder are covalently bonded to each other by at least three disulfide bonds.

18. The ACC according to any one of claims 1-15, wherein the first monomer builder and the second monomer builder are covalently bonded to each other by at least four disulfide bonds.

19. The ACC of claim 1, wherein the first monomeric construct and the second monomeric construct each comprise a sequence having at least 95% identity with SEQ ID NO:

313.

20. An activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct, wherein: (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1); (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2); (c) The first monomer construct is a polypeptide comprising CP1, CM1, and DD1 in the N-terminal to C-terminal direction, further wherein: (i) The first monomer construct and the second monomer construct each contain a linker region of no more than 18 amino acids, and (ii) CP1 is a mature interferon; (d) Further among them: (i) The second monomeric construct is identical to the first monomeric construct, and (ii) DD1 and DD2 are a pair of human IgG Fc domains; (e) DD1 and DD2 are covalently bonded to each other through at least one disulfide bond, thereby forming a dimer of the first monomer construct and the second monomer construct; and (f) The ACC is characterized by having a reduced level of interferon activity compared to the corresponding control interferon, wherein the first monomeric construct and the second monomeric construct each contain SEQ ID NO:

313.

21. An activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct, wherein: (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable portion (CM1), and a first dimerizing domain (DD1) in the direction from the N-terminus to the C-terminus, wherein CP1 and CM1 are directly adjacent to each other, and CM1 and DD1 are directly adjacent to each other. (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2) in the direction from the N-terminus to the C-terminus, wherein CP2 and CM2 are directly adjacent to each other, and CM2 and DD2 are directly adjacent to each other; (c) wherein CP1 is a mature interferon and CM1 contains a sequence that has at least 85% identity with SEQ ID NO: 100; (d) Further among them: (i) The second monomer construct is identical to the first monomer construct, and (ii) DD1 and DD2 are a pair of human IgG1 or IgG4 Fc domains; (e) DD1 and DD2 are covalently bonded to each other via at least one disulfide bond, thereby forming a dimer of the first monomer construct and the second monomer construct; and (f) The ACC is characterized by having a reduced level of interferon α activity compared to the interferon α activity of pegylated interferon α-2b.

22. An activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct, wherein: (a) The first monomeric construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1). Wherein CM1 is located between CP1 and DD1, and the first monomer construct is characterized in that CP1 and DD1 are connected by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM1; and (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2). Wherein CM2 is located between CP2 and DD2, and the second monomer construct is characterized in that CP2 and DD2 are connected by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM2; or (a) The first monomeric construct comprises a first mature cytokine protein (CP1), a first dimerizing domain (DD1); and (b) The second monomeric construct comprises a second mature cytokine protein (CP2), a cleavable moiety (CM), and a second dimerizing domain (DD2), wherein CM is located between CP2 and DD2, wherein CM acts as a substrate of the protease, and the second monomeric construct is characterized in that CP2 and DD2 are linked by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM2; or (a) The first monomer construct comprises a first mature cytokine protein (CP1), a cleavable moiety (CM), and a first dimerizing domain (DD1), wherein CM is located between CP1 and DD1, and the first monomer construct is characterized in that CP1 and DD1 are linked by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM1; and (b) The second monomer construct contains a second mature cytokine protein (CP2) and a second dimerization domain (DD2). CM acts as a substrate for the protease; or (a) The first monomeric construct comprises a first mature cytokine protein (CP1) and a first dimerizing domain (DD1); and (b) The second monomer construct comprises a second mature cytokine protein (CP2) and a second dimerization domain (DD2), wherein CP1, CP2, or both CP1 and CP2 contain an amino acid sequence that serves as a protease substrate, and the first monomer construct is characterized in that CP1 and DD1 are linked by a linker region of no more than 18 amino acids, and the second monomer construct is characterized in that CP2 and DD2 are linked by a linker region of no more than 18 amino acids; CP1 and CP2 are mature interferons; DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct; The ACC is characterized by having a reduced level of CP1 or CP2 activity compared to a control level of CP1 or CP2 activity, and The first and second monomeric constructs have the following structures, wherein in each construct, CP1 and CP2 are located at the N-terminus of DD1 and DD2, respectively.

23. The ACC according to claim 22, wherein DD1 and DD2 are a pair of Fc structural domains.

24. The ACC of claim 23, wherein the pair of Fc domains is a pair of human Fc domains.

25. The ACC of claim 24, wherein 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.

26. The ACC of claim 25, wherein the human Fc domain is the human IgG4 Fc domain.

27. The ACC of claim 24, wherein the human Fc domain comprises a sequence having at least 90% identity with SEQ ID NO: 3, SEQ ID NO: 315 or SEQ ID NO:

316.

28. The ACC of claim 24, wherein the human Fc domain comprises SEQ ID NO: 3, SEQ ID NO: 315 or SEQ ID NO:

316.

29. The ACC of claim 22, wherein CP1 and / or CP2 comprises a sequence having at least 95% identity with SEQ ID NO:

1.

30. The ACC of claim 22, wherein CP1 and / or CP2 comprise the sequence of SEQ ID NO:

1.

31. A method for manufacturing an activatable cytokine construct (ACC), said ACC comprising a first monomer construct and a second monomer construct, wherein: (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1); (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2); (c) The first monomer construct is a polypeptide containing CP1, CM1 and DD1 in the direction from the N-terminus to the C-terminus, and the first monomer construct is characterized in that CP1 and DD1 are linked by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM1. (d) Further among them: (i) The second monomeric construct is identical to the first monomeric construct, and (ii) DD1 and DD2 are a pair of human IgG Fc domains; (e) DD1 and DD2 are covalently bonded to each other through at least one disulfide bond, thereby forming a dimer of the first monomer construct and the second monomer construct; and (f) The ACC is characterized by having a level of at least one CP1 and CP2 activity that is reduced compared to a corresponding control level of at least one CP1 and CP2 activity; the method comprises: culturing cells containing nucleic acids encoding a first monomer construct and a second monomer construct in a liquid culture medium under conditions sufficient to produce ACC; and recovering ACC from the cells or the liquid culture medium.

32. The method of claim 31, wherein CP1 is mature human interferon α.

33. The method according to claim 32, wherein the mature interferon is mature interferon α-2b.

34. The method of claim 31, wherein CM1 and CM2 each comprise no more than 7 amino acids, or wherein CM1 and CM2 are each independently cleaved by urokinase (uPa) and / or matrix metalloproteinase (MMP).

35. The method of claim 31, wherein CM1 and CM2 each comprise a sequence having at least 85% identity with SEQ ID NO:

100.

36. The method of claim 31, wherein CM1 and CM2 each comprise a sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 68 and SEQ ID NO:

100.

37. The method of claim 31, wherein the first monomer construct and the second monomer construct are covalently bonded to each other by at least two, three or four disulfide bonds.

38. The method of claim 31, wherein the first monomer construct and the second monomer construct each comprise a sequence having at least 95% identity with SEQ ID NO:

313.

39. A method for manufacturing an activatable cytokine construct (ACC), said ACC comprising a first monomer construct and a second monomer construct, wherein: (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1); (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2); (c) The first monomer construct is a polypeptide comprising CP1, CM1, and DD1 in the N-terminal to C-terminal direction, further wherein: (i) The first monomer construct and the second monomer construct each contain a linker region of no more than 18 amino acids, and (ii) CP1 is a mature interferon; (d) Further among them: (i) The second monomer construct is identical to the first monomer construct, and (ii) DD1 and DD2 are a pair of human IgG Fc domains; (e) DD1 and DD2 are covalently bonded to each other via at least one disulfide bond, thereby forming a dimer of the first monomer construct and the second monomer construct; and (f) The ACC is characterized by having a reduced level of interferon activity compared to the corresponding control interferon, wherein the first monomer construct and the second monomer construct each comprise SEQ ID NO: 313; the method comprises: culturing cells containing nucleic acids encoding the first monomer construct and the second monomer construct in a liquid culture medium under conditions sufficient to produce ACC; and recovering ACC from the cells or the liquid culture medium.

40. A method for manufacturing an activatable cytokine construct (ACC), said ACC comprising a first monomer construct and a second monomer construct, wherein: (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable portion (CM1), and a first dimerizing domain (DD1) in the direction from the N-terminus to the C-terminus, wherein CP1 and CM1 are directly adjacent to each other, and CM1 and DD1 are directly adjacent to each other. (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2) in the direction from the N-terminus to the C-terminus, wherein CP2 and CM2 are directly adjacent to each other, and CM2 and DD2 are directly adjacent to each other; (c) wherein CP1 is a mature interferon and CM1 contains a sequence that has at least 85% identity with SEQ ID NO: 100; (d) Further among them: (i) The second monomer construct is identical to the first monomer construct, and (ii) DD1 and DD2 are a pair of human IgG1 or IgG4 Fc domains; (e) DD1 and DD2 are covalently bonded to each other through at least one disulfide bond, thereby forming a dimer of the first monomer construct and the second monomer construct; and (f) The ACC is characterized by having a level of reduced interferon α activity compared to the interferon α activity of pegylated interferon α-2b; the method comprises: culturing cells containing nucleic acids encoding a first monomer construct and a second monomer construct in a liquid culture medium under conditions sufficient to produce the ACC; and recovering the ACC from the cells or the liquid culture medium.

41. A method for manufacturing an activatable cytokine construct (ACC), said ACC comprising a first monomer construct and a second monomer construct, wherein: (a) The first monomeric construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1). Wherein CM1 is located between CP1 and DD1, and the first monomer construct is characterized in that CP1 and DD1 are connected by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM1; and (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2). Wherein CM2 is located between CP2 and DD2, and the second monomer construct is characterized in that CP2 and DD2 are connected by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM2; or (a) The first monomeric construct comprises a first mature cytokine protein (CP1) and a first dimerizing domain (DD1); and (b) The second monomer construct comprises a second mature cytokine protein (CP2), a cleavable portion (CM), and a second dimerizing domain (DD2), wherein CM is located between CP2 and DD2, wherein CM acts as a substrate of the protease, and the second monomer construct is characterized in that CP2 and DD2 are linked by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM2. or (a) The first monomeric construct comprises a first mature cytokine protein (CP1), a cleavable moiety (CM), and a first dimerizing domain (DD1), wherein CM is located between CP1 and DD1, and the first monomeric construct is characterized in that CP1 and DD1 are linked by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM1; and (b) The second monomer construct comprises a second mature cytokine protein (CP2) and a second dimerization domain (DD2), wherein CP2 acts as a substrate for the protease; or (a) The first monomeric construct comprises a first mature cytokine protein (CP1) and a first dimerizing domain (DD1), and (b) The second monomer construct comprises a second mature cytokine protein (CP2) and a second dimerization domain (DD2), wherein CP1, CP2, or both CP1 and CP2 contain an amino acid sequence that serves as a protease substrate, and the first monomer construct is characterized in that CP1 and DD1 are linked by a linker region of no more than 18 amino acids, and the second monomer construct is characterized in that CP2 and DD2 are linked by a linker region of no more than 18 amino acids; CP1 and CP2 are mature interferons; DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct; The characteristic of ACC is that it has a reduced level of CP1 or CP2 activity compared to the control level of CP1 or CP2 activity; and The first monomer construct and the second monomer construct have the following structures, wherein in each construct, CP1 and CP2 are located at the N-terminus of DD1 and DD2, respectively; the method includes: culturing cells containing nucleic acids encoding the first monomer construct and nucleic acids encoding the second monomer construct in a liquid culture medium under conditions sufficient to produce ACC; and recovering ACC from the cells or the liquid culture medium.

42. The method according to claim 41, wherein the DD1 and DD2 domains are human IgG1 Fc domains, human IgG2 Fc domains, human IgG3 Fc domains, or human IgG4 Fc domains.

43. The method of claim 41, wherein the DD1 and DD2 domains comprise sequences having at least 90% identity with SEQ ID NO: 3, SEQ ID NO: 315 or SEQ ID NO:

316.

44. The method of claim 41, wherein the DD1 and DD2 structural domains comprise SEQ ID NO: 3, SEQ ID NO: 315 or SEQ ID NO:

316.

45. The method according to any one of claims 31-44, wherein CP1 and / or CP2 comprises a sequence having at least 95% identity with SEQ ID NO:

1.

46. ​​A nucleic acid encoding a monomer construct comprising, in the direction from the N-terminus to the C-terminus, a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1), wherein CP1 and DD1 are linked by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM1, and wherein DD1 is a human IgG Fc domain.

47. The nucleic acid according to claim 46, wherein CP1 is mature human interferon α.

48. The nucleic acid according to claim 47, wherein the mature human interferon is mature human interferon α-2b.

49. The nucleic acid according to claim 48, wherein the mature human interferon comprises a sequence having at least 95% identity with SEQ ID NO:

1.

50. The nucleic acid according to claim 48, wherein mature human interferon α comprises the sequence of SEQ ID NO:

1.

51. The nucleic acid according to any one of claims 46-50, wherein CM1 comprises a sequence having at least 85% identity with SEQ ID NO:

100.

52. The nucleic acid according to any one of claims 46-50, wherein CM1 comprises a sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 68 and SEQ ID NO:

100.

53. The nucleic acid according to any one of claims 46-52, wherein the monomer construct comprises a sequence having at least 95% identity with SEQ ID NO:

313.

54. A pair of nucleic acids that together encode the first monomer construct and the second monomer construct, wherein: (a) The first monomeric construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1). Wherein CM1 is located between CP1 and DD1, and the first monomer construct is characterized in that CP1 and DD1 are connected by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM1; and (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2). CM2 is located between CP2 and DD2, and the second monomer construct is characterized in that CP2 and DD2 are connected by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM2. or (a) The first monomeric construct comprises a first mature cytokine protein (CP1) and a first dimerizing domain (DD1); and (b) The second monomer construct comprises a second mature cytokine protein (CP2), a cleavable portion (CM), and a second dimerizing domain (DD2), wherein CM is located between CP2 and DD2, wherein CM acts as a substrate of the protease, and the second monomer construct is characterized in that CP2 and DD2 are linked by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM2. or (a) The first monomer construct comprises a first mature cytokine protein (CP1), a cleavable moiety (CM), and a first dimerizing domain (DD1), wherein CM is located between CP1 and DD1, and the first monomer construct is characterized in that CP1 and DD1 are linked by a linker region of no more than 18 amino acids, such that the linker region of no more than 18 amino acids includes CM1; and (b) The second monomer construct contains a second mature cytokine protein (CP2) and a second dimerization domain (DD2). CM acts as a substrate for the protease; or (a) The first monomer construct comprises a first mature cytokine protein (CP1) and a first dimerizing domain (DD1), and (b) The second monomer construct comprises a second mature cytokine protein (CP2) and a second dimerization domain (DD2), wherein CP1, CP2, or both CP1 and CP2 contain an amino acid sequence that serves as a protease substrate, and the first monomer construct is characterized in that CP1 and DD1 are linked by a linker region of no more than 18 amino acids, and the second monomer construct is characterized in that CP2 and DD2 are linked by a linker region of no more than 18 amino acids.

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