Activatable cytokine construct and related compositions and methods

Abstract

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

Description

[0001] Cross-reference to related applications

[0002] 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.

[0003] 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

[0004] This disclosure relates to the field of biotechnology, and more specifically, to constructs of activatable cytokines. Background Technology

[0005] 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.

[0006] 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.

[0007] Interleukins are another subclass of cytokines. Interleukins regulate cell growth, differentiation, and motility. They are particularly important in stimulating immune responses, such as inflammation. Interleukins have been used to treat cancer, autoimmune diseases, and other 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.

[0008] 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

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] The ACC of this disclosure is characterized in that CP1 and CP2 are not linked to peptide masking agents, such as affinity masking moieties.

[0015] 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-anchored 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.

[0016] 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).

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] In some embodiments, CM1 and / or CM2 comprise a sequence selected from the group consisting of: LSGRSDNH (SEQ ID NO:5), TGRGPSWV (SEQ ID NO:6), PLTGRSGG (SEQ ID NO:7), TARGPSFK (SEQ ID NO:8), NTLSGRSENHSG (SEQ ID NO:9), NTLSGRSGNHGS (SEQ ID NO:10), TSTSGRSANPRG (SEQ ID NO:11), TSGRSANP (SEQ ID NO:12), VHMPLGFLGP (SEQ ID NO:13), AVGLLAPP (SEQ ID NO:14), AQNLLGMV (SEQ ID NO:15), QNQALRMA (SEQ ID NO:16), LAAPLGLL (SEQ ID NO:17), STFPFGMF (SEQ ID NO:18), ISSGLLSS (SEQ ID NO:19), PAGLWLDP (SEQ ID NO:20), VAGRSMRP (SEQ ID NO:21), VVPEGRRS (SEQ ID NO:22), ILPRSPAF (SEQ ID NO:23), MVLGRSLL (SEQ ID NO:24), QGRAITFI (SEQ ID NO:25), SPRSIMLA (SEQ ID NO:26), SMLRSMPL (SEQ ID NO:27), ISSGLLSGRSDNH (SEQ ID NO:28), AVGLLAPPGGLSGRSDNH (SEQ ID NO:29), ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO:30), LSGRSGNH (SEQ ID NO:31), SGRSANPRG (SEQ ID NO:32), LSGRSDDH (SEQ ID NO:33), LSGRSDIH (SEQ ID NO:34), LSGRSDQH (SEQ ID NO:35), LSGRSDTH (SEQ ID NO:36), LSGRSDYH (SEQ ID NO:37), LSGRSDNP (SEQ ID NO:38), LSGRSANP (SEQ ID NO:39), LSGRSANI (SEQ ID NO:40), LSGRSDNI (SEQ ID NO:41), MIAPVAYR (SEQ ID NO:42), RPSPMWAY (SEQ ID NO:43), WATPRPMR (SEQ ID NO:44), FRLLDWQW (SEQID 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)、ISSGLLSGRSDH(SEQ ID NO:53)、ISSGLLSGRSDQH(SEQ ID NO:54)、ISSGLLSGRSDTH(SEQ ID NO:55)、ISSGLLSGRSDYH(SEQ ID NO:56)、ISSGLLSGRSDNP(SEQ ID NO:57), ISSGLLSGRSANP(SEQ IDNO: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 ISSGLSS(SEQ ID NO:73)、PVGYTSSL(SEQ ID NO:74)、DWLYWPGI(SEQ ID NO:75)、LKAAPRWA(SEQ ID NO:76)、GPSHLVLT(SEQ ID NO:77)、LPGGLSPW(SEQ ID NO:78)、MGLFSEAG(SEQ IDNO:79)、SPLPLRVP(SEQ IDNO:80), RMHLRSLG (SEQ ID NO:81), LLAPSHRA (SEQ ID NO:82), GPRSFGL (SEQ ID NO:83), GPRSFG (SEQ ID NO:84), SARGPSRW (SEQ ID NO:85), GGWHTGRN (SEQ ID NO:86), HTGRSGAL (SEQ ID NO:87), AARGPAIH (SEQ ID NO:88), RGPAFNPM (SEQ ID NO:89), SSRGPAYL (SEQ ID NO:90), RGPATPIM (SEQ ID NO:91), RGPA (SEQ ID NO:92), GGQPSGMWGW (SEQ ID NO:93), FPRPLGITGL (SEQ ID NO:94), SPLTGRSG (SEQ ID NO:95), SAGFSLPA (SEQ ID NO:96), LAPLGLQRR (SEQ ID NO:97), SGGPLGVR (SEQ ID NO:98), PLGL (SEQ ID NO:99) and SGRSDNI (SEQ ID In some embodiments, the CM comprises a sequence selected from the group consisting of: ISSGLLSGRSDNH (SEQ ID NO:28), LSGRSDDH (SEQ ID NO:33), ISSGLLSGRSDQH (SEQ ID NO:54), 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.

[0025] 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.

[0026] 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.

[0027] In some embodiments, the first monomeric construct comprises 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); PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 219). NO:220); SKYGPPCPPCPAPEFLG (SEQ ID NO:221); GKSSGGSESKS (SEQ ID NO:222); GSTSGSGKSSEGKG (SEQ ID NO:223); GSTGSSGKSSEGSGSTKG (SEQ ID NO:224); IDNO:225); GSTGSSGKPGSSEGST (SEQ ID NO:226); (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO:227), (GGGS)n (SEQ ID NO:228), (GGGGS)n (SEQ ID NO:216), where each n is an integer of at least one; GGSG (SEQ ID NO:229); GGSGG (SEQ ID NO:230); GGSSG (SEQ ID NO:231); GSGGG (SEQ ID (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 includes the sequence GGGS (SEQ ID NO:2).

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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 that of the wild-type mature cytokines, demonstrating that the activities of CP1 and / or CP2 are fully or almost fully restored after lysis.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Other features and advantages of the invention will become apparent from the following detailed description and drawings, as well as from the claims.

[0042] 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.

[0043] 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.

[0044] 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 the breadth of the range or the characteristics described in any case. Additionally, it should be noted that all percentages are by weight unless otherwise stated.

[0045] 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 basic 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[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 "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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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).

[0055] 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.

[0056] 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

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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).

[0062] 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 a human IgG4 Fc domain (not italic, bold, or underlined text).

[0063] 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.

[0064] 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 (1204MT-SP1); (3) a product of an ACC IFNα-2b-hIgG4 Fc with a cleavable moiety 1204 and a protease uPA (1204uPA); (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 an IFNα-2b-hIgG4 Fc 1204+1 and MT-SP1 (1204+1MT-SP1); (6) an ACC IFNα-2b-hIgG4 Fc with a cleavable moiety 1490. Fc; (7) MT-SP1 and the product of ACC IFNα-2b-hIgG4 Fc with a cleavable portion of 1490; uPA and the product of ACC IFNα-2b-hIgG4 Fc with a cleavable portion of 1490 (1490uPA).

[0065] Figure 7 Results of a reporter gene assay based on HEK293 cells were provided, which evaluated 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).

[0066] 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 8B The 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.

[0067] Figure 9 Results of a Daudi cell apoptosis assay were provided, which determined... 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).

[0068] 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.

[0069] Figure 11 Results based on Daudi lymphoma cells were provided, which measured the antiproliferative activity of ACC (IFNa2b12O4DNIdL NhG4); protease-treated ACC (IFNa2b12O4DNIdL 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.

[0070] Figure 12 Results of a reporter gene assay based on HEK293 cells were described, which evaluated ACC (IFNa2b1204DNIdL NhG4); protease-treated (activated) ACC (IFNα-2b1204DNIdL NhG4+uPA); The activity of recombinant parental cytokines (IFNa2b) was also investigated. The results showed that after treatment of ACC with protease, the activity of cytokines in ACC could be restored to levels comparable to those of the recombinant parental cytokines.

[0071] Figure 13The results of Daudi lymphoma cell-based assays (top figure) for measuring the antiproliferative activity of ACC (ProC440), protease-treated ACC (ProC440+uPA), and stem cell IFNa2b are depicted, along with reporter gene assays (bottom figure) based on HEK293 cells for measuring their activities. 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.

[0072] 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.

[0073] 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.

[0074] 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) depicts a tumor model using Daudi xenograft. Antiproliferative effect in vivo.

[0075] Figure 17A The structure of ProC286 and its activity in Daudi cell apoptosis assays were described. A comparison of their activities. ProC286 and The similar activity levels suggest that ProC286 can be used as... A control alternative was used to evaluate tolerance to IFNα-2b in hamster studies. Figure 17B The structure of ProC291 and its activity in Daudi cell apoptosis assays were described. A comparison of their activities. (Comparison with...) Compared to ProC286, ProC291 showed significantly reduced activity.

[0076] Figure 18 The specific activity and expected toxicity doses of IFNa-con (recombinant interferon α, a non-natural type I interferon), ProC440+uPA, PEG-IFNa2b (Sylatron), and ProC440 were depicted in in vivo dose escalation studies (e.g., at escalating doses of 0.08, 0.4, 2, 10, and 15 mg / kg (“mpk”)).

[0077] 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.

[0078] Figure 20A 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 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.

[0079] Figure 20B 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 600 and DD2, respectively. The first monomer construct, from its N-terminus to its C-terminus, comprises the 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 the 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.

[0080] Figure 21A This is an illustrative diagram of an 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 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.

[0081] Figure 21B This is a schematic diagram of an illustrative cytokine-activating construct comprising a first monomer construct and a second monomer construct, which are nonvalently 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.

[0082] 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.

[0083] Figure 23Clinical 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.

[0084] 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.

[0085] Figure 25 An implementation scheme of an ACC representing the connection zone (LR) of an ACC is schematically shown. Detailed Implementation

[0086] 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.

[0087] 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.

[0088] 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 both 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 disposal (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 .

[0089] 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.

[0090] 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.

[0091] Cytokine activator construct

[0092] 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:

[0093] (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerizing domain (DD1).

[0094] CM1 is located between CP1 and DD1; and

[0095] (b) The second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerizing domain (DD2).

[0096] CM2 is located between CP2 and DD2;

[0097] DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct; and

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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).

[0104] 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.

[0105] 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.

[0106] 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 monomer construct having CP1 100; CM1 120 positioned relative to the C-terminus of CP1 100; an optional connector 110 (if present) positioned between the C-terminus of CP1 100 and the N-terminus of CM1 120; DD1 140; and an optional connector 130 (if present) positioned between the C-terminus of CM1 120 and DD1 140; (2) a second monomer construct having CP2 150; CM2 170 positioned relative to the C-terminus of CP2 150; an optional connector 160 (if present) positioned between the C-terminus of CP2 150 and the N-terminus of CM2 170; DD2 190; and an optional connector 180 (if present) positioned between the C-terminus of CM2 170 and DD2 190. Between 190; and (3) one or more covalent or non-covalent bonds (←→).

[0107] Figure 1B Another illustrative diagram of ACC is provided, whose components are organized with opposite orientations of ACC. (Reference) Figure 1BThe 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 (←→).

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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), and G-CSF. F, 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-β3, EPOo, TPO, Fl t-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.

[0113] 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: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, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161、SEQ IDNO: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 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, for example, interferon α2b molecules with a mutation at position L130, such as the L130P mutation, as in 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 mutants of I24Q, I60T, F64A, W76H, I116R, and L128N, or subsets thereof. In some aspects, ACC includes a truncated interferon α2b molecule lacking cytokine activity as one of CP1 and CP2. For example, 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 wild-type interferon α2b: 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.

[0114] 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.

[0115] 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.

[0116] In other embodiments, CP1 and / or CP2 exhibit interferon activity and comprise amino acid sequences 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 contain the same amino acid sequence.

[0117] 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.

[0118] 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 ...9, SEQ ID NO:129, SEQ ID NO:129, SEQ ID NO:12 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:138, SEQ ID NO:13 ... 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.

[0119] In some embodiments, CP1 and / or CP2 exhibit interleukin activity and comprise 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 amino acid sequences 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:111 (human IL-1α), SEQ ID NO:144 (human IL-12β), SEQ ID NO:111 (human IL-1α), SEQ ID NO:113 (human IL-1β), SEQ ID NO:113 ...2β), SEQ ID NO:113 (human IL-1α), SEQ ID NO:113 (human IL-12β), SEQ ID NO:113 (human IL- 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.

[0120] 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.

[0121] 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 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.

[0122] For example, in some embodiments, DD1 and DD2 are a pair of members selected from the group consisting of: a sushi domain from the human IL-15 receptor α chain (IL15Rα) and soluble IL-15; barnase and barns. tar; 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); soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) modules based on the interactions of protein synapses, synapse-binding proteins, small synaptic vesicle proteins, and SNAP25; single-domain antibodies (sdAbs) and corresponding epitopes; antigen-binding domains (e.g., single-chain antibodies (such as single-chain variable fragments (scFv)), single-domain antibodies, etc.) and corresponding epitopes; coiled-coil polypeptide structures (e.g., Fos-Jun coiled-coil structures, acid / base coiled-coil helices, Glu-Lys coiled-coil helices, leucine zipper structures), small molecule binding pairs such as biotin and avidin or 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.

[0123] 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.

[0124] 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.).

[0125] 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.

[0126] 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).

[0127] 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., in the CH3 domain). In one example, the modification comprises amino acid substitutions 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 having 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).

[0128] 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.

[0129] 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).

[0130] 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, FVI Ia, FIXA, FXa, FXIa, FXI Ia, Granulase B, Guanidylbenzoate esterase, Heparin, HtrA1, Human neutrophil elastase, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Lactoferrin, Maraps In, matriptase-2, transmembrane peptidase, MT-SP1 / matriptase, enkephalinase, NS3 / 4A, PACE4, plasmin, 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.

[0131] 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, or cathepsin V. / L2, cathepsin X / Z / P, Cruzipain, asparagine endopeptidase, Otubain-2, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, transmembrane 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, FVI Ia, FIXa, FXa, FXIa, FXI Ia, 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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).

[0136] 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: 263). (SEQ ID NO: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, WO 2015 / 048329, WO2015 / 116933, WO 2016 / 118629 and WO 2020 / 118109, which are incorporated herein by reference in their entirety.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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 structure 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).

[0144] 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.

[0145] 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)).

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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).

[0150] 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:220), SKYGPPCPPCPAPEFLG (SEQ ID NO:219). NO:221), GKSSGGSESKS (SEQ ID NO:222), GSTGSSGKSSEGKG (SEQ ID NO:223), GSTSGSGKSSEGSGSTKG (SEQ ID NO:224) and GSTGSSGKPGSGEGSTKG (SEQ ID NO:225).

[0151] 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), GGGGSGGGGSGGGGSGGGS (SEQ ID NO:214), GGSLDPKGGGGS (SEQ ID NO:215), and GSTGSGKPGSSEGST (SEQ ID NO:215). NO:226) has a sequence with 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).

[0152] 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), GGGGSGGGGSGGGGSGGGS (SEQ ID NO:214), GSTGSGKPGSSEGST (SEQ ID NO:214), GSTGSGKPGSSEGST (SEQ ID NO:219), GGSLDPKGGGS (SEQ ID NO:219), GGSGGGSGGSGGGSGGGSGGSGGS (SEQ ID NO:218), GGSGGSGKPGSSEGST (SEQ ID NO:219), GGSGGSGGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO:219), G ... NO:226), (GGGGS)n (SEQ ID NO:216), where n is an integer of at least one. In some embodiments, the adapter includes a sequence selected from the group consisting of: GGSLDPKGGGGS (SEQ ID NO:219), GGGGSGGGGSGGGGSGS (SEQ ID NO:218), GGGGSGS (SEQ ID NO:217), and GS. In some embodiments of any of the ACCs described herein, the adapter includes 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 includes a sequence selected from the group consisting of: GGGGSGGGGSGGGGS (SEQ ID NO:213) or GGGGS (SEQ ID NO:216). In some implementations, the connector contains the sequence GGGS (SEQ ID NO:2).

[0153] 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.

[0154] 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 glycolated CP1 and / or CP2.

[0155] 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. Cancer Res. 14(18):5900-5906, 2008; Hilkens et al., J. Immunol. 171:5255-5263, 2003).

[0156] 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.

[0157] 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).

[0158] 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.

[0159] 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).

[0160] 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.

[0161] 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).

[0162] 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.

[0163] 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:81); LAAPLGLL (SEQ ID NO:19); CM ... NO:17); AVGLLAPP (SEQ ID NO:14); LLAPSHRA (SEQ ID NO:82); PAGLWLDP (SEQ ID NO:20); and / or ISSGLSS (SEQ ID NO:73).

[0164] 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).

[0165] In some embodiments, CM includes an amino acid sequence selected from the group consisting of: NTLSGRSENHSG (SEQ ID NO:9); NTLSGRSGNHGS (SEQ ID NO:10); TSTSGRSANPRG (SEQ ID NO:11); TSGRSANP (SEQ ID NO:12); VAGRSMRP (SEQ ID NO:21); VVPEGRRS (SEQ ID NO:22); ILPRSPAF (SEQ ID NO:23); MVLGRSLL (SEQ ID NO:24); QGRAITFI (SEQ ID NO:25); SPRSIMLA (SEQ ID NO:26); and SMLRSMPL (SEQ ID NO:27).

[0166] 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).

[0167] 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:275); NO:277); TTSSGRSANPRGGGVHMPLGFLGP (SEQ ID NO:278); AVGLLAPPGGLSGRSDNH (SEQ ID NO:29); LSGRSDNHGGAVGLLAPP (SEQ ID NO:70); VHMPLGFLGPGGLSGRSSDNH (SEQ ID NO:266); LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: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).

[0168] 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).

[0169] 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 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).

[0170] Conjugation

[0171] 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.

[0172] 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.

[0173] Non-limiting exemplary cytotoxic agents that can be conjugated with 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), aurestatin derivatives (e.g., aurestatin tyramine, aurestatin quinolone), maytansinoids (e.g., DM-1, DM-4), maytansin derivatives, duocarmycin, α-amaminine, turbostatin, phenstatin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1 Hasatatine 2, Hasatatine 3, Halocomstatin, Pyrrolebenzimidazole (PBI), Cibrostatin 6, Doxaliform, Cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, Anthracycline, Doxorubicin, Daunorubicin, Bryostatin, Camptothecin, 7-substituted Camptothecin, 10,11-Difluoromethylenedioxycamptothecin, Combretastatin, Debromoaplysiatoxin, KahaMide-F, Discodermolide, and Ecteinascidin.

[0174] 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 citric acid, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene.

[0175] 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.

[0176] 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).

[0177] Non-limiting exemplary antifungal agents that can be conjugated with any of the ACCs described herein include nystatin.

[0178] 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).

[0179] 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.

[0180] 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 cytokine-activating constructs described herein includes: an S-nitrobenzyloxycarbonyl derivative of O6-benzylguanine, a topoisomerase inhibitor, hemiasterlin, cephalotaxine, homoharringionine, or pyrrolobenzodiazepine. Dimer (PBD), Functionalized pyrrolobenzodiazepine Calcicheamicin, podophyllotoxin, taxane, and vincristine.

[0181] 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).

[0182] Non-limiting exemplary heavy metals that can be combined with any of the ACCs described herein include: barium, gold, and platinum.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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 construct is partially reduced upon contact with the 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.

[0192] 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).

[0193] 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.

[0194] 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).

[0195] The effective pH for the reduction reaction of ACC treated 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).

[0196] 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).

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] Table 1.

[0203]

[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, J.M. Curuse 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 21651G); (iv) sulfonyl-LC-SPDP (sulfosuccinimide-6-[3-(2-pyridyl-dithio)propamido]hexanoate) (Pierce Chem.Co., catalog 2165-G); and (v) sulfonyl-NHS (N-hydroxysulfonyl-succinimide: Pierce) conjugated to EDC. Chem.Co. (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 embodiments, 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, colloids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector), as well as any 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; Cancer of unknown primary site in children; 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]

[0254] Composition / Pharm Kit

[0255] 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.

[0256] 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.

[0257] 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).

[0258] 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).

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] Example

[0264] 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.

[0265] Example 1: Generation of Cytokine-Activating Constructs

[0266] 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.

[0267] An activatable cytokine construct, IFN-α-2b1490DNI-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 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: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 1204dl hIgG4.

[0268] Additional activatable cytokine constructs were prepared, which contained five additional amino acid residues in the linker.

[0269] 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 (“1204MT-SP1”); (3) uPA treated IFN-α2b-1204DNIdl-hIgG4 (“1204uPA”); (4) IFN-α2b-1204DNIdl-hIgG4 with five amino acid residues added to the linker (“1204+1”); (5) MT-SP1 treated IFN-α2b-1 (6) uPA-treated IFN-α2b-1204DNIdl-hIgG4 (“1204+1MT-SP1”); (7) uPA-treated IFN-α-2b-1490DNI-hIgG4 (“1490”); (8) uPA-treated IFN-α-2b-1490DNI-hIgG4 (“1490MT-SP1”); and (9) uPA-treated IFN-α-2b-1490DNI-hIgG4 (“1490uPA”). The results indicate that the protease effectively cleaves the cleavable portion of the activatable cytokine construct.

[0270] Example 2. IFN-α-2b activity of activating cytokine constructs

[0271] Cell-based reporter gene assays of human type I interferon were used to test the activity of ACC as described in Example 1.

[0272] IFN-responsive HEK293 cells were generated by stably transfecting the cells 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. The addition of 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 with Quant i-Blue solution. The activity of ACC containing IFNα-2b was compared with that of the reporter gene assay. The activity of (pegylated interferon α-2b) was compared. Figure 7 The data in the middle shows that, with Compared to the IFNα-2b activity of PEGylated interferon α-2b, the IFNα-2b activity of ACC was significantly reduced.

[0273] 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 a 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.

[0274] Example 3: In vitro antiproliferative effect of ACC on cancer cells

[0275] 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 it was then placed on an orbital oscillator for 10 minutes. After incubation, the luminescence signal was directly measured 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. EC50 values ​​were compared with recombinant IFNα2b or pharmaceutical grade IFNα2b. Specific activity of (pegylated interferon α-2b) was determined.

[0276] The antiproliferative activity of ACC containing IFNα-2b in Daudi lymphoma cells indicates that it is compatible with... Compared to pegylated interferon α-2b, uncleaved ACC showed reduced IFNα-2b activity. Figure 9 ).

[0277] 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.

[0278] Example 4: Activity of ACC after protease treatment

[0279] 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.

[0280] 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 activity testing. The results from these assays indicate that treatment of ACC containing IFNα-2b with the protease restored activity to levels comparable to recombinant cytokines. The EC50 values ​​for ACC IFNα-2b-1204DNIdl-hIgG4, ACC IFNα-2b-1204DNIdl-hIgG4+uPA, and stem cell IFNα-2b (human recombinant IFN-α2b, from StemCell Technologies, catalog number 78077.1) were calculated based on Daudi cell apoptosis assays and are provided in Table 3 below.

[0281] Table 3. EC50: Daudi cell apoptosis assay

[0282]

[0283] 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.

[0284] Table 4. EC50: IFNα / β reporter gene assay

[0285]

[0286] 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.

[0287] Example 5: General ProIFN

[0288] 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 in 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.

[0289] 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.

[0290] EC50: B16 IFNα / β reporter gene assay

[0291]

[0292] Example 6: In vitro characterization of the lead ACC ProC440:

[0293] The cytokine-activating construct ProC 440 (N IFNa2b 0 1204DNIdL 0AAFc) 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).

[0294] 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 levels of IFNα-2b shown are equivalent to those of ACC ProC440, ACC ProC440+uPA, and stem cell IFNα-2b. The EC50 values ​​were calculated based on the IFNα / β assay results and are provided in Table 5 below.

[0295] Table 5: EC50: IFNα / β reporter gene assay

[0296] ProC440(ACC) ProC440(ACC)+uPA stem cell IFNα-2b EC50 7643 4.333 10.88

[0297] The EC50 values ​​of ACC ProC440, ACC ProC440+uPA, and stem cell IFNα-2b were calculated based on Daudi apoptotic cell assay results and are provided in Table 6 below.

[0298] Table 6. EC50: Daudi cell apoptosis assay

[0299] ProC440(ACC) ProC440(ACC)+uPA stem cell IFNα-2b EC50 264.2 0.1842 0.3530

[0300] 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.

[0301] ACC ProC657 (N IFNa2b 0AA 1204DNIdL0AA 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.

[0302] 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.

[0303] Example 7: In vivo antiproliferative activity of ACC:

[0304] 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 Data from the study indicate 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 (above figure), and demonstrates… The antiproliferative effects were used for comparison (see figure below).

[0305] Example 8: In vivo tolerance activity of ACC

[0306] 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 a non-tolerance toxicity (DLT) was identified. The starting dose (0.4 mg / kg (“mpk”)) represents INFα-con (recombinant interferon α, a product of Amgen under the name...) The estimated equivalent dose of the non-naturally occurring type I interferon (INFα-con) to induce weight loss, reduced food consumption, and bone marrow suppression in hamsters (125 g) was determined. 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 125 g hamsters). If the starting dose was tolerated, the animal was moved to a “medium dose” of 2 mg / kg and given three doses of the test product unless intolerable. If tolerated, the animal was moved to a “high dose” of 10 mg / kg and given three doses of the test product unless intolerable. If tolerated, the animal was moved to a “high dose” of 15 mg / kg. At each stage, if the test dose was not tolerated, the animal was moved to the next lower dose. If the starting dose was not tolerated, the animal was 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. As a negative control, human IgG4 was administered to the animals. As expected, the negative control did not induce any toxicity in the animals.

[0307] 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).

[0308] 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).

[0309] In the Daudi cell apoptosis assay, the activities of ProC286 and ProC291 were compared with... The activity of (PEG-IFN-α2b) was compared. Figures 17A to 17B In the aforementioned measurements, ProC286 and They 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 control group was used to evaluate the tolerability of IFNα-2b in hamster studies. Compared with ProC286 and... In contrast, ProC291 exhibited reduced activity, indicating 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, positioning cytokines at the N-terminus of DD (as in ProC291) provides a greater reduction in cytokine activity compared to positioning them at the C-terminus of DD (as in ProC286).

[0310] Animals were administered an initial dose of 0.4 mg / kg on day 1. The 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.

[0311] 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.

[0312] Animals administered ProC440 at 15 mpk showed an average weight loss of 5%. Figure 22 This indicates that the ACC of this disclosure, with its 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 to inhibit cytokine activity in the ProC440 context, in conjunction with polyethylene glycol-modified IFNα-2b... It reduces the toxicity of interferon compared to ProC286.

[0313] 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 N-terminal CP-CM-DD dimerization, unexpectedly limits IFNa-2b-mediated hepatotoxicity.

[0314] 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.

[0315] Example 4. In vitro characterization of additional cytokine constructs

[0316] 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 IgGκ 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 Nos:336 to 342 and SEQ ID NO:313).

[0317] Table 6: Activated cytokines with different amino acid sequence lengths between CP and cysteine ​​226 of human IgG

[0318]

[0319]

[0320] 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.

[0321] Table 7. EC50: IFNα / β reporter gene assay

[0322]

[0323] The EC50 value of ACC was calculated based on the results of Daudi cell apoptosis assay and is provided in Table 8 below.

[0324] Table 8: EC50: Daudi cell apoptosis assay

[0325]

[0326] 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.

[0327] 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.

[0328] Instance sequence

[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] Other implementation plans

[0360] 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. sequence list <110> Cytomex Therapeutics, Inc. <120> Cytokine-activating constructs and related compositions and methods <130> CYTX-071-PCT <150> 63 / 008,542 <151> 2020-04-10 <150> 63 / 161,889 <151> March 16, 2021 <150> 63 / 164,849 <151> March 23, 2021 <160> 518 <170> PatentIn version 3.5 <210> 1 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 1 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 [[ID=!41]]Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg[[ID=б]] 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 2 Gly Gly Gly Ser 1 ) <210> 3 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 3 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 35 40 45 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 50 55 60 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 85 90 95 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 115 120 125 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 <210> 4 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser 225 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 5 Leu Ser Gly Arg Ser Asp Asn His 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 6 Thr Gly Arg Gly Pro Ser Trp Val 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 7 Pro Leu Thr Gly Arg Ser Gly Gly 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 8 Thr Ala Arg Gly Pro Ser Phe Lys 1 5 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 9 Asn Thr Leu Ser Gly Arg Ser Glu Asn His Ser Gly 1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 10 Asn Thr Leu Ser Gly Arg Ser Gly Asn His Gly Ser 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 11 Thr Ser Thr Ser Gly Arg Ser Ala Asn Pro Arg Gly 1 5 10 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 12 Thr Ser Gly Arg Ser Ala Asn Pro 1 5 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 13 Val His Met Pro Leu Gly Phe Leu Gly Pro 1 5 10 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 14 Ala Val Gly Leu Leu Ala Pro Pro 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 15 Ala Gln Asn Leu Leu Gly Met Val 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 16 Gln Asn Gln Ala Leu Arg Met Ala 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 17 Leu Ala Ala Pro Leu Gly Leu Leu 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 18 Ser Thr Phe Pro Phe Gly Met Phe 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 19 Ile Ser Ser Gly Leu Leu Ser Ser 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 20 Pro Ala Gly Leu Trp Leu Asp Pro 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> twenty one Val Ala Gly Arg Ser Met Arg Pro 1 5 <210> twenty two <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> twenty two Val Val Pro Glu Gly Arg Arg Ser 1 5 <210> twenty three <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> twenty three Ile Leu Pro Arg Ser Pro Ala Phe 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> twenty four Met Val Leu Gly Arg Ser Leu Leu 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 25 Gln Gly Arg Ala Ile Thr Phe Ile 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 26 Ser Pro Arg Ser Ile Met Leu Ala 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 27 Ser Met Leu Arg Ser Met Pro Leu 1 5 <210> 28 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 28 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Asp Asn His 1 5 10 <210> 29 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 29 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Asp 1 5 10 15 Asn His <210> 30 <211> twenty two <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 30 Ile Ser Ser Gly Leu Leu Ser Ser Gly Gly Ser Gly Gly Ser Leu Ser 1 5 10 15 Gly Arg Ser Asp Asn His 20 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 31 Leu Ser Gly Arg Ser Gly Asn His 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 32 Ser Gly Arg Ser Ala Asn Pro Arg Gly 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 33 Leu Ser Gly Arg Ser Asp Asp His 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 34 Leu Ser Gly Arg Ser Asp Ile His 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 35 Leu Ser Gly Arg Ser Asp Gln His 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 36 Leu Ser Gly Arg Ser Asp Thr His 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 37 Leu Ser Gly Arg Ser Asp Tyr His 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 38 Leu Ser Gly Arg Ser Asp Asn Pro 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 39 Leu Ser Gly Arg Ser Ala Asn Pro 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 40 Leu Ser Gly Arg Ser Ala Asn Ile 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 41 Leu Ser Gly Arg Ser Asp Asn Ile 1 5 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 42 Met Ile Ala Pro Val Ala Tyr Arg 1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 43 Arg Pro Ser Pro Met Trp Ala Tyr 1 5 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 44 Trp Ala Thr Pro Arg Pro Met Arg 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 45 Phe Arg Leu Leu Asp Trp Gln Trp 1 5 <210> 46 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 46 Ile Ser Ser Gly Leu 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 47 Ile Ser Ser Gly Leu Leu Ser 1 5 <210> 48 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 48 Ile Ser Ser Gly Leu Leu 1 5 <210> 49 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 49 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Ala Asn Pro Arg Gly 1 5 10 15 <210> 50 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 50 Ala Val Gly Leu Leu Ala Pro Pro Thr Ser Gly Arg Ser Ala Asn Pro 1 5 10 15 Arg Gly <210> 51 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 51 Ala Val Gly Leu Leu Ala Pro Pro Ser Gly Arg Ser Ala Asn Pro Arg 1 5 10 15 Gly <210> 52 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 52 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Asp Asp His 1 5 10 <210> 53 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 53 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Asp Ile His 1 5 10 <210> 54 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 54 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Asp Gln His 1 5 10 <210> 55 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 55 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Asp Thr His 1 5 10 <210> 56 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 56 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Asp Tyr His 1 5 10 <210> 57 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 57 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Asp Asn Pro 1 5 10 <210> 58 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 58 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Ala Asn Pro 1 5 10 <210> 59 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 59 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Ala Asn Ile 1 5 10 <210> 60 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 60 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Asp 1 5 10 15 Asp His <210> 61 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 61 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Asp 1 5 10 15 Ile His <210> 62 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 62 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Asp 1 5 10 15 Gln His <210> 63 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 63 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Asp 1 5 10 15 Thr His <210> 64 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 64 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Asp 1 5 10 15 Tyr His <210> 65 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 65 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Asp 1 5 10 15 Asn Pro <210> 66 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 66 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Ala 1 5 10 15 Asn Pro <210> 67 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 67 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Ala 1 5 10 15 Asn Ile <210> 68 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 68 Ile Ser Ser Gly Leu Leu Ser Gly Arg Ser Asp Asn Ile 1 5 10 <210> 69 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 69 Ala Val Gly Leu Leu Ala Pro Pro Gly Gly Leu Ser Gly Arg Ser Asp 1 5 10 15 Asn Ile <210> 70 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 70 Gly Leu Ser Gly Arg Ser Asp Asn His Gly Gly Ala Val Gly Leu Leu 1 5 10 15 Ala Pro Pro <210> 71 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 71 Gly Leu Ser Gly Arg Ser Asp Asn His Gly Gly Val His Met Pro Leu 1 5 10 15 Gly Phe Leu Gly Pro 20 <210> 72 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 72 Leu Ser Gly Arg Ser Asp Asn His Gly Gly Val His Met Pro Leu Gly 1 5 10 15 Phe Leu Gly Pro 20 <210> 73 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 73 Ile Ser Ser Gly Leu Ser Ser 1 5 <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 74 Pro Val Gly Tyr Thr Ser Ser Leu 1 5 <210> 75 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 75 Asp Trp Leu Tyr Trp Pro Gly Ile 1 5 <210> 76 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 76 Leu Lys Ala Ala Pro Arg Trp Ala 1 5 <210> 77 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 77 Gly Pro Ser His Leu Val Leu Thr 1 5 <210> 78 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 78 Leu Pro Gly Gly Leu Ser Pro Trp 1 5 <210> 79 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 79 Met Gly Leu Phe Ser Glu Ala Gly 1 5 <210> 80 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 80 Ser Pro Leu Pro Leu Arg Val Pro 1 5 <210> 81 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 81 Arg Met His Leu Arg Ser Leu Gly 1 5 <210> 82 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 82 Leu Leu Ala Pro Ser His Arg Ala 1 5 <210> 83 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 83 Gly Pro Arg Ser Phe Gly Leu 1 5 <210> 84 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 84 Gly Pro Arg Ser Phe Gly 1 5 <210> 85 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 85 Ser Ala Arg Gly Pro Ser Arg Trp 1 5 <210> 86 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 86 Gly Gly Trp His Thr Gly Arg Asn 1 5 <210> 87 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 87 His Thr Gly Arg Ser Gly Ala Leu 1 5 <210> 88 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 88 Ala Ala Arg Gly Pro Ala Ile His 1 5 <210> 89 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 89 Arg Gly Pro Ala Phe Asn Pro Met 1 5 <210> 90 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 90 Ser Ser Arg Gly Pro Ala Tyr Leu 1 5 <210> 91 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 91 Arg Gly Pro Ala Thr Pro Ile Met 1 5 <210> 92 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 92 Arg Gly Pro Ala 1 <210> 93 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 93 Gly Gly Gln Pro Ser Gly Met Trp Gly Trp 1 5 10 <210> 94 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 94 Phe Pro Arg Pro Leu Gly Ile Thr Gly Leu 1 5 10 <210> 95 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 95 Ser Pro Leu Thr Gly Arg Ser Gly 1 5 <210> 96 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 96 Ser Ala Gly Phe Ser Leu Pro Ala 1 5 <210> 97 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 97 Leu Ala Pro Leu Gly Leu Gln Arg Arg 1 5 <210> 98 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 98 Ser Gly Gly Pro Leu Gly Val Arg 1 5 <210> 99 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 99 Pro Leu Gly Leu 1 <210> 100 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 100 Ser Gly Arg Ser Asp Asn Ile 1 5 <210> 101 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 101 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln​​​​​​​​​​​​​​​​​​​​​​​​ 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 <210> 102 <211> 169 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 102 Cys Asp Leu Pro His Thr His Asn Leu Arg Asn Lys Arg Ala Phe Thr 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Leu Ser Pro Val Ser Cys Leu Lys Asp 20 25 30 Arg Lys Asp Phe Gly Phe Pro Leu Glu Lys Val Asp Gly Gln Gln Ile 35 40 45 Gln Lys Ala Gln Ala Ile Pro Val Leu His Glu Leu Thr Gln Gln Ile 50 55 60 Leu Ser Leu Phe Thr Ser Lys Glu Ser Ser Thr Ala Trp Asp Ala Ser 65 70 75 80 Leu Leu Asp Ser Phe Cys Asn Asp Leu Gln Gln Gln Leu Ser Gly Leu 85 90 95 Gln Ala Cys Leu Met Gln Gln Val Gly Val Gln Glu Ser Pro Leu Thr 100 105 110 Gln Glu Asp Ser Leu Leu Ala Val Arg Glu Tyr Phe His Arg Ile Thr 115 120 125 Val Tyr Leu Arg Glu Lys Lys His Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Val Trp Arg Ala Leu Ser Ser Ser Ala Asn Leu Leu Gly 145 150 155 160 Arg Leu Arg Glu Glu Arg Asn Glu Ser 165 <210> 103 <211> 167 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 103 Cys Asp Leu Pro His Thr Tyr Asn Leu Arg Asn Lys Arg Ala Leu Lys 1 5 10 15 Val Leu Ala Gln Met Arg Arg Leu Pro Phe Leu Ser Cys Leu Lys Asp 20 25 30 Arg Gln Asp Phe Gly Phe Pro Leu Glu Lys Val Asp Asn Gln Gln Ile 35 40 45 Gln Lys Ala Gln Ala Ile Pro Val Leu Arg Asp Leu Thr Gln Gln Thr 50 55 60 Leu Asn Leu Phe Thr Ser Lys Ala Ser Ser Ala Ala Trp Asn Ala Thr 65 70 75 80 Leu Leu Asp Ser Phe Cys Asn Asp Leu His Gln Gln Leu Asn Asp Leu 85 90 95 Gln Thr Cys Leu Met Gln Gln Val Gly Val Gln Glu Pro Pro Leu Thr 100 105 110 Gln Glu Asp Ala Leu Leu Ala Val Arg Lys Tyr Phe His Arg Ile Thr 115 120 125 Val Tyr Leu Arg Glu Lys Lys His Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Val Trp Arg Ala Leu Ser Ser Ser Val Asn Leu Leu Pro 145 150 155 160 Arg Leu Ser Glu Glu Lys Glu 165 <210> 104 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 104 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 <210> 105 <211> 495 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 105 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser 165 170 175 Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe 180 185 190 Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe 195 200 205 Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met 210 215 220 Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala 225 230 235 240 Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln 245 250 255 Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu 260 265 270 Thr Pro Leu Met Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe 275 280 285 Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala 290 295 300 Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr 305 310 315 320 Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Cys Asp Leu Pro Gln Thr 325 330 335 His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg 340 345 350 Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg Arg Asp Phe Gly Phe 355 360 365 Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro 370 375 380 Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys 385 390 395 400 Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr 405 410 415 Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly 420 425 430 Val Gly Val Thr Glu Thr Pro Leu Met Asn Glu Asp Ser Ile Leu Ala 435 440 445 Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys 450 455 460 Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser 465 470 475 480 Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu 485 490 495 <210> 106 <211> 166 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 106 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 <210> 107 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 107 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser 1 5 10 15 Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys 20 25 30 Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln 35 40 45 Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn 50 55 60 Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu 65 70 75 80 Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His 85 90 95 Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg 100 105 110 Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile 115 120 125 Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile 130 135 140 Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr 145 150 155 160 Gly Tyr Leu Arg Asn 165 <210> 108 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 108 Met Asn Asn Arg Trp Ile Leu His Ala Ala Phe Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Ile Asn Tyr Lys Gln Leu Gln Leu Gln Glu Arg 20 25 30 Thr Asn Ile Arg Lys Cys Gln Glu Leu Leu Glu Gln Leu Asn Gly Lys 35 40 45 Ile Asn Leu Thr Tyr Arg Ala Asp Phe Lys Ile Pro Met Glu Met Thr 50 55 60 Glu Lys Met Gln Lys Ser Tyr Thr Ala Phe Ala Ile Gln Glu Met Leu 65 70 75 80 Gln Asn Val Phe Leu Val Phe Arg Asn Asn Phe Ser Ser Thr Gly Trp 85 90 95 Asn Glu Thr Ile Val Val Arg Leu Leu Asp Glu Leu His Gln Gln Thr 100 105 110 Val Phe Leu Lys Thr Val Leu Glu Glu Lys Gln Glu Glu Arg Leu Thr 115 120 125 Trp Glu Met Ser Ser Thr Ala Leu His Leu Lys Ser Tyr Tyr Trp Arg 130 135 140 Val Gln Arg Tyr Leu Lys Leu Met Lys Tyr Asn Ser Tyr Ala Trp Met 145 150 155 160 Val Val Arg Ala Glu Ile Phe Arg Asn Phe Leu Ile Ile Arg Arg Leu 165 170 175 Thr Arg Asn Phe Gln Asn 180 <210> 109 <211> 184 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 109 Met Ala Asn Arg Trp Thr Leu His Ile Ala Phe Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Ile Asp Tyr Lys Gln Leu Gln Phe Arg Gln Ser 20 25 30 Thr Ser Ile Arg Thr Cys Gln Lys Leu Leu Arg Gln Leu Asn Gly Arg 35 40 45 Leu Asn Leu Ser Tyr Arg Thr Asp Phe Lys Ile Pro Met Glu Val Met 50 55 60 His Pro Ser Gln Met Glu Lys Ser Tyr Thr Ala Phe Ala Ile Gln Val 65 70 75 80 Met Leu Gln Asn Val Phe Leu Val Phe Arg Ser Asn Phe Ser Ser Thr 85 90 95 Gly Trp Asn Glu Thr Ile Val Glu Ser Leu Leu Asp Glu Leu His Gln 100 105 110 Gln Thr Glu Leu Leu Glu Ile Ile Leu Lys Glu Lys Gln Glu Glu Arg 115 120 125 Leu Thr Trp Val Thr Ser Thr Thr Thr Leu Gly Leu Lys Ser Tyr Tyr 130 135 140 Trp Arg Val Gln Arg Tyr Leu Lys Asp Lys Lys Tyr Asn Ser Tyr Ala 145 150 155 160 Trp Met Val Val Arg Ala Glu Val Phe Arg Asn Phe Ser Ile Ile Leu 165 170 175 Arg Leu Asn Arg Asn Phe Gln Asn 180 <210> 110 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 110 Met Cys Asp Leu Pro Gln Asn His Gly Leu Leu Ser Arg Asn Thr Leu 1 5 10 15 Val Leu Leu His Gln Met Arg Arg Ile Ser Pro Phe Leu Cys Leu Lys 20 25 30 Asp Arg Arg Asp Phe Arg Phe Pro Gln Glu Met Val Lys Gly Ser Gln 35 40 45 Leu Gln Lys Ala His Val Met Ser Val Leu His Glu Met Leu Gln Gln 50 55 60 Ile Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala Ala Trp Asn Met 65 70 75 80 Thr Leu Leu Asp Gln Leu His Thr Gly Leu His Gln Gln Leu Gln His 85 90 95 Leu Glu Thr Cys Leu Leu Gln Val Val Gly Glu Gly Glu Ser Ala Gly 100 105 110 Ala Ile Ser Ser Pro Ala Leu Thr Leu Arg Arg Tyr Phe Gln Gly Ile 115 120 125 Arg Val Tyr Leu Lys Glu Lys Lys Tyr Ser Asp Cys Ala Trp Glu Val 130 135 140 Val Arg Met Glu Ile Met Lys Ser Leu Phe Leu Ser Thr Asn Met Gln 145 150 155 160 Glu Arg Leu Arg Ser Lys Asp Arg Asp Leu Gly Ser Ser 165 170 <210> 111 <211> 271 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 111 Met Ala Lys Val Pro Asp Met Phe Glu Asp Leu Lys Asn Cys Tyr Ser 1 5 10 15 Glu Asn Glu Glu Asp Ser Ser Ser Ile Asp His Leu Ser Leu Asn Gln 20 25 30 Lys Ser Phe Tyr His Val Ser Tyr Gly Pro Leu His Glu Gly Cys Met 35 40 45 Asp Gln Ser Val Ser Leu Ser Ile Ser Glu Thr Ser Lys Thr Ser Lys 50 55 60 Leu Thr Phe Lys Glu Ser Met Val Val Val Ala Thr Asn Gly Lys Val 65 70 75 80 Leu Lys Lys Arg Arg Leu Ser Leu Ser Gln Ser Ile Thr Asp Asp Asp 85 90 95 Leu Glu Ala Ile Ala Asn Asp Ser Glu Glu Glu Ile Ile Lys Pro Arg 100 105 110 Ser Ala Pro Phe Ser Phe Leu Ser Asn Val Lys Tyr Asn Phe Met Arg 115 120 125 Ile Ile Lys Tyr Glu Phe Ile Leu Asn Asp Ala Leu Asn Gln Ser Ile 130 135 140 Ile Arg Ala Asn Asp Gln Tyr Leu Thr Ala Ala Ala Leu His Asn Leu 145 150 155 160 Asp Glu Ala Val Lys Phe Asp Met Gly Ala Tyr Lys Ser Ser Lys Asp 165 170 175 Asp Ala Lys Ile Thr Val Ile Leu Arg Ile Ser Lys Thr Gln Leu Tyr 180 185 190 Val Thr Ala Gln Asp Glu Asp Gln Pro Val Leu Leu Lys Glu Met Pro 195 200 205 Glu Ile Pro Lys Thr Ile Thr Gly Ser Glu Thr Asn Leu Leu Phe Phe 210 215 220 Trp Glu Thr His Gly Thr Lys Asn Tyr Phe Thr Ser Val Ala His Pro 225 230 235 240 Asn Leu Phe Ile Ala Thr Lys Gln Asp Tyr Trp Val Cys Leu Ala Gly [[ID=1�]]245 250 255 Gly Pro Pro Ser Ile Thr Asp Phe Gln Ile Leu Glu Asn Gln Ala 260 265 270 <210> 112 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 112 Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser 1 5 10 15 Glu Asn Glu Asp Tyr Ser Ser Ala Ile Asp His Leu Ser Leu Asn Gln 20 25 30 Lys Ser Phe Tyr Asp Ala Ser Tyr Gly Ser Leu His Glu Thr Cys Thr 35 40 45 Asp Gln Phe Val Ser Leu Arg Thr Ser Glu Thr Ser Lys Met Ser Asn 50 55 60 Phe Thr Phe Lys Glu Ser Arg Val Thr Val Ser Ala Thr Ser Ser Asn 65 70 75 80 Gly Lys Ile Leu Lys Lys Arg Arg Leu Ser Phe Ser Glu Thr Phe Thr 85 90 95 Glu Asp Asp Leu Gln Ser Ile Thr His Asp Leu Glu Glu Thr Ile Gln 100 105 110 Pro Arg Ser Ala Pro Tyr Thr Tyr Gln Ser Asp Leu Arg Tyr Lys Leu 115 120 125 Met Lys Leu Val Arg Gln Lys Phe Val Met Asn Asp Ser Leu Asn Gln 130 135 140 Thr Ile Tyr Gln Asp Val Asp Lys His Tyr Leu Ser Thr Thr Trp Leu 145 150 155 160 Asn Asp Leu Gln Gln Glu Val Lys Phe Asp Met Tyr Ala Tyr Ser Ser 165 170 175 Gly Gly Asp Asp Ser Lys Tyr Pro Val Thr Leu Lys Ile Ser Asp Ser 180 185 190 Gln Leu Phe Val Ser Ala Gln Gly Glu Asp Gln Pro Val Leu Leu Lys 195 200 205 Glu Leu Pro Glu Thr Pro Lys Leu Ile Thr Gly Ser Glu Thr Asp Leu 210 215 220 Ile Phe Phe Trp Lys Ser Ile Asn Ser Lys Asn Tyr Phe Thr Ser Ala 225 230 235 240 Ala Tyr Pro Glu Leu Phe Ile Ala Thr Lys Glu Gln Ser Arg Val His 245 250 255 Leu Ala Arg Gly Leu Pro Ser Met Thr Asp Phe Gln Ile Ser 260 265 270 <210> 113 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 113 Met Ala Glu Val Pro Glu Leu Ala Ser Glu Met Met Ala Tyr Tyr Ser 1 5 10 15 Gly Asn Glu Asp Asp Leu Phe Phe Glu Ala Asp Gly Pro Lys Gln Met 20 25 30 Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile 35 40 45 Gln Leu Arg Ile Ser Asp His His Tyr Ser Lys Gly Phe Arg Gln Ala 50 55 60 Ala Ser Val Val Val Ala Met Asp Lys Leu Arg Lys Met Leu Val Pro 65 70 75 80 Cys Pro Gln Thr Phe Gln Glu Asn Asp Leu Ser Thr Phe Phe Pro Phe 85 90 95 Ile Phe Glu Glu Glu Pro Ile Phe Phe Asp Thr Trp Asp Asn Glu Ala 100 105 110 Tyr Val His Asp Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp 115 120 125 Ser Gln Gln Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala 130 135 140 Leu His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met 145 150 155 160 Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu 165 170 175 Gly Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp 180 185 190 Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys 195 200 205 Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn 210 215 220 Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr 225 230 235 240 Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly 245 250 255 Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser 260 265 <210> 114 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 114 Met Ala Thr Val Pro Glu Leu Asn Cys Glu Met Pro Pro Phe Asp Ser 1 5 10 15 Asp Glu Asn Asp Leu Phe Phe Glu Val Asp Gly Pro Gln Lys Met Lys 20 25 30 Gly Cys Phe Gln Thr Phe Asp Leu Gly Cys Pro Asp Glu Ser Ile Gln 35 40 45 Leu Gln Ile Ser Gln Gln His Ile Asn Lys Ser Phe Arg Gln Ala Val 50 55 60 Ser Leu Ile Val Ala Val Glu Lys Leu Trp Gln Leu Pro Val Ser Phe 65 70 75 80 Pro Trp Thr Phe Gln Asp Glu Asp Met Ser Thr Phe Phe Ser Phe Ile 85 90 95 Phe Glu Glu Glu Pro Ile Leu Cys Asp Ser Trp Asp Asp Asp Asp Asn 100 105 110 Leu Leu Val Cys Asp Val Pro Ile Arg Gln Leu His Tyr Arg Leu Arg 115 120 125 Asp Glu Gln Gln Lys Ser Leu Val Leu Ser Asp Pro Tyr Glu Leu Lys 130 135 140 Ala Leu His Leu Asn Gly Gln Asn Ile Asn Gln Gln Val Ile Phe Ser 145 150 155 160 Met Ser Phe Val Gln Gly Glu Pro Ser Asn Asp Lys Ile Pro Val Ala 165 170 175 Leu Gly Leu Lys Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp 180 185 190 Gly Thr Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Gln Tyr Pro 195 200 205 Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Val Lys 210 215 220 Ser Lys Val Glu Phe Glu Ser Ala Glu Phe Pro Asn Trp Tyr Ile Ser 225 230 235 240 Thr Ser Gln Ala Glu His Lys Pro Val Phe Leu Gly Asn Asn Ser Gly 245 250 255 Gln Asp Ile Ile Asp Phe Thr Met Glu Ser Val Ser Ser 260 265 <210> 115 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 115 Met Glu Ile Cys Arg Gly Leu Arg Ser His Leu Ile Thr Leu Leu Leu 1 5 10 15 Phe Leu Phe His Ser Glu Thr Ile Cys Arg Pro Ser Gly Arg Lys Ser 20 25 30 Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe[[ID=3D]] 35 40 45 Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn [[ID=3Y]]50 55 60 Val Asn Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala 65 70 75 80 Leu Phe Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser Cys Val Lys 85 90 95 Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Ala Val Asn Ile Thr Asp 100 105 110 Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser 115 120 125 Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp 130 135 140 Phe Leu Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn 145 150 155 160 Met Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp 165 170 175 Glu <210> 116 <211> 178 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 116 Met Glu Ile Cys Trp Gly Pro Tyr Ser His Leu Ile Ser Leu Leu Leu 1 5 10 15 Ile Leu Leu Phe His Ser Glu Ala Ala Cys Arg Pro Ser Gly Lys Arg 20 25 30 Pro Cys Lys Met Gln Ala Phe Arg Ile Trp Asp Thr Asn Gln Lys Thr 35 40 45 Phe Tyr Leu Arg Asn Asn Gln Leu Ile Ala Gly Tyr Leu Gln Gly Pro 50 55 60 Asn Ile Lys Leu Glu Glu Lys Ile Asp Met Val Pro Ile Asp Leu His 65 70 75 80 Ser Val Phe Leu Gly Ile His Gly Gly Lys Leu Cys Leu Ser Cys Ala 85 90 95 Lys Ser Gly Asp Asp Ile Lys Leu Gln Leu Glu Glu Val Asn Ile Thr 100 105 110 Asp Leu Ser Lys Asn Lys Glu Glu Asp Lys Arg Phe Thr Phe Ile Arg 115 120 125 Ser Glu Lys Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly 130 135 140 Trp Phe Leu Cys Thr Thr Leu Glu Ala Asp Arg Pro Val Ser Leu Thr 145 150 155 160<00,​​​​​​​​​​​​​​​​​​​​Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met 1 5 10 15 Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn 20 25 30 Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile 35 40 45 Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro 50 55 60 Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg 65 70 75 80 Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met 85 90 95 Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys 100 105 110 Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile 115 120 125 Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly 130 135 140 His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe 145 150 155 160 Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys 165 170 175 Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu 180 185 190 Asp <210> 118 <211> 192 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 118 Met Ala Ala Met Ser Glu Asp Ser Cys Val Asn Phe Lys Glu Met Met 1 5 10 15 Phe Ile Asp Asn Thr Leu Tyr Phe Ile Pro Glu Glu Asn Gly Asp Leu 20 25 30 Glu Ser Asp Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg 35 40 45 Asn Ile Asn Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe 50 55 60 Glu Asp Met Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg 65 70 75 80 Leu Ile Ile Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val 85 90 95 Thr Leu Ser Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn 100 105 110 Lys Ile Ile Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp 115 120 125 Ile Gln Ser Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn 130 135 140 Lys Met Glu Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys 145 150 155 160 Gln Lys Glu Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu 165 170 175 Asn Gly Asp Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser 180 185 190 <210> 119 <211> 150 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthesis <400> 119 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser 145 150 <210> 120 <211> 169 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 120 Met Tyr Ser Met Gln Leu Ala Ser Cys Val Thr Leu Thr Leu Val Leu 1 5 10 15 Leo Val Asn Ser Ala Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala 20 25 30 Glu Ala Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Leu 35 40 45 Glu Gln Leu Leu Met Asp Leu Gln Glu Leu Leu Ser Arg Met Glu Asn 50 55 60 Tyr Arg Asn Leu Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu 65 70 75 80 Pro Lys Gln Ala Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu 85 90 95 Leu Gly Pro Leu Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe 100 105 110 Gln Leu Glu Asp Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val 115 120 125 Val Lys Leu Lys Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp 130 135 140 Glu Ser Ala Thr Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys 145 150 155 160 Gln Ser Ile Ile Ser Thr Ser Pro Gln 165 <210> 121 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 121 Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu Ala 1 5 10 15 Cys Ala Gly Asn Phe Val His Gly His Lys Cys Asp Ile Thr Leu Gln 20 25 30 Glu Ile Ile Lys Thr Leu Asn Ser Leu Thr Glu Gln Lys Thr Leu Cys 35 40 45 Thr Glu Leu Thr Val Thr Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr 50 55 60 Glu Lys Glu Thr Phe Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr 65 70 75 80 Ser His His Glu Lys Asp Thr Arg Cys Leu Gly Ala Thr Ala Gln Gln 85 90 95 Phe His Arg His Lys Gln Leu Ile Arg Phe Leu Lys Arg Leu Asp Arg 100 105 110 Asn Leu Trp Gly Leu Ala Gly Leu Asn Ser Cys Pro Val Lys Glu Ala 115 120 125 Asn Gln Ser Thr Leu Glu Asn Phe Leu Glu Arg Leu Lys Thr Ile Met 130 135 140 Arg Glu Lys Tyr Ser Lys Cys Ser Ser 145 150 <210> 122 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 122 Met Gly Leu Asn Pro Gln Leu Val Val Ile Leu Leu Phe Phe Leu Glu 1 5 10 15 Cys Thr Arg Ser His Ile His Gly Cys Asp Lys Asn His Leu Arg Glu 20 25 30 Ile Ile Gly Ile Leu Asn Glu Val Thr Gly Glu Gly Thr Pro Cys Thr 35 40 45 Glu Met Asp Val Pro Asn Val Leu Thr Ala Thr Lys Asn Thr Thr Glu 50 55 60 Ser Glu Leu Val Cys Arg Ala Ser Lys Val Leu Arg Ile Phe Tyr Leu 65 70 75 80 Lys His Gly Lys Thr Pro Cys Leu Lys Lys Asn Ser Ser Val Leu Met 85 90 95 Glu Leu Gln Arg Leu Phe Arg Ala Phe Arg Cys Leu Asp Ser Ser Ile 100 105 110 Ser Cys Thr Met Asn Glu Ser Lys Ser Thr Ser Leu Lys Asp Phe Leu 115 120 125 Glu Ser Leu Lys Ser Ile Met Gln Met Asp Tyr Ser 130 135 140 <210> 123 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 123 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys 20 25 30 Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu 35 40 45 Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe 50 55 60 Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe 65 70 75 80 Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser 85 90 95 Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr 100 105 110 Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala 115 120 125 Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu 130 135 140 Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu 145 150 155 160 Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu 165 170 175 His <210> 124 <211> 154 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 124 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Ile Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Thr Ser Ser Glu Cys His Ile Lys Asp Lys 20 25 30 Glu Gly Lys Ala Tyr Glu Ser Val Leu Met Ile Ser Ile Asp Glu Leu 35 40 45 Asp Lys Met Thr Gly Thr Asp Ser Asn Cys Pro Asn Asn Glu Pro Asn 50 55 60 Phe Phe Arg Lys His Val Cys Asp Asp Thr Lys Glu Ala Ala Phe Leu 65 70 75 80 Asn Arg Ala Ala Arg Lys Leu Lys Gln Phe Leu Lys Met Asn Ile Ser 85 90 95 Glu Glu Phe Asn Val His Leu Leu Thr Val Ser Gln Gly Thr Gln Thr [[ID=]14]100 105 110 Leu Val Asn Cys Thr Ser Lys Glu Glu Lys Asn Val Lys Glu Gln Lys 115 120 125 Lys Asn Asp Ala Cys Phe Leu Lys Arg Leu Leu Arg Glu Ile Lys Thr 130 135 140 Cys Trp Asn Lys Ile Leu Lys Gly Ser Ile 145 150 <210> 125 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 125 Met Leu Leu Ala Met Val Leu Thr Ser Ala Leu Leu Leu Cys Ser Val 1 5 10 15 Ala Gly Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile Asn Phe 20 25 30 Leu Ile Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys Ser 35 40 45 Ala Asn Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp Asn Cys 50 55 60 Thr Arg Pro Cys Phe Ser Glu Arg Leu Ser Gln Met Thr Asn Thr Thr 65 70 75 80 Met Gln Thr Arg Tyr Pro Leu Ile Phe Ser Arg Val Lys Lys Ser Val 85 90 95 Glu Val Leu Lys Asn Asn Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro 100 105 110 Cys Asn Gln Thr Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu 115 120 125 Leu Glu Ile Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile 130 135 140 <210> 126 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 126 Met Leu Val Thr Tyr Ile Leu Ala Ser Val Leu Leu Phe Ser Ser Val 1 5 10 15 Leu Gly Gln Arg Cys Ser Thr Thr Trp Gly Ile Arg Asp Thr Asn Tyr 20 25 30 Leu Ile Glu Asn Leu Lys Asp Asp Pro Pro Ser Lys Cys Ser Cys Ser 35 40 45 Gly Asn Val Thr Ser Cys Leu Cys Leu Ser Val Pro Thr Asp Asp Cys 50 55 60 Thr Thr Pro Cys Tyr Arg Glu Gly Leu Leu Gln Leu Thr Asn Ala Thr 65 70 75 80 Gln Lys Ser Arg Leu Leu Pro Val Phe His Arg Val Lys Arg Ile Val 85 90 95 Glu Val Leu Lys Asn Ile Thr Cys Pro Ser Phe Ser Cys Glu Lys Pro 100 105 110 Cys Asn Gln Thr Met Ala Gly Asn Thr Leu Ser Phe Leu Lys Ser Leu 115 120 125 Leu Gly Thr Phe Gln Lys Thr Glu Met Gln Arg Gln Lys Ser Arg Pro 130 135 140 <210> 127 <211> 146 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 127 Met His Pro Leu Leu Asn Pro Leu Leu Leu Ala Leu Gly Leu Met Ala 1 5 10 15 Leu Leu Leu Thr Thr Val Ile Ala Leu Thr Cys Leu Gly Gly Phe Ala 20 25 30 Ser Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Glu Leu Ile Glu 35 40 45 Glu Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly 50 55 60 Ser Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala 65 70 75 80 Leu Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr 85 90 95 Gln Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln 100 105 110 Phe Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe 115 120 125 Val Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg 130 135 140 Phe Asn 145 <210> 128 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 128 Met Ala Leu Trp Val Thr Ala Val Leu Ala Leu Ala Cys Leu Gly Gly 1 5 10 15 Leu Ala Ala Pro Gly Pro Val Pro Arg Ser Val Ser Leu Pro Leu Thr 20 25 30 Leu Lys Glu Leu Ile Glu Glu Leu Ser Asn Ile Thr Gln Asp Gln Thr 35 40 45 Pro Leu Cys Asn Gly Ser Met Val Trp Ser Val Asp Leu Ala Ala Gly 50 55 60 Gly Phe Cys Val Ala Leu Asp Ser Leu Thr Asn Ile Ser Asn Cys Asn 65 70 75 80 Ala Ile Tyr Arg Thr Gln Arg Ile Leu His Gly Leu Cys Asn Arg Lys 85 90 95 Ala Pro Thr Thr Val Ser Ser Leu Pro Asp Thr Lys Ile Glu Val Ala 100 105 110 His Phe Ile Thr Lys Leu Leu Ser Tyr Thr Lys Gln Leu Phe Arg His 115 120 125 Gly Pro Phe 130 <210> 129 <211> 162 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 129 Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Ser <210> 130 <211> 162 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 130 Met Lys Ile Leu Lys Pro Tyr Met Arg Asn Thr Ser Ile Ser Cys Tyr 1 5 10 15 Leu Cys Phe Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Val Ser Val Gly Leu Pro Lys Thr Glu Ala 35 40 45 Asn Trp Ile Asp Val Arg Tyr Asp Leu Glu Lys Ile Glu Ser Leu Ile 50 55 60 Gln Ser Ile His Ile Asp Thr Thr Leu Tyr Thr Asp Ser Asp Phe His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Asn Cys Phe Leu Leu Glu Leu Gln 85 90 95 Val Ile Leu His Glu Tyr Ser Asn Met Thr Leu Asn Glu Thr Val Arg 100 105 110 Asn Val Leu Tyr Leu Ala Asn Ser Thr Leu Ser Ser Asn Lys Asn Val 115 120 125 Ala Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Thr Phe 130 135 140 Thr Glu Phe Leu Gln Ser Phe Ile Arg Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Serum <210> 131 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 131 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15 Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 20 25 30 Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 35 40 45 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 50 55 60 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 65 70 75 80 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 85 90 95 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 100 105 110 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 115 120 125 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 130 135 140 Thr Thr Leu Ser Leu Ala Ile Phe 145 150 <210> 132 <211> 166 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 132 Met Val Leu Ala Ser Ser Thr Thr Ser Ile His Thr Met Leu Leu Leu 1 5 10 15 Leu Leu Met Leu Phe His Leu Gly Leu Gln Ala Ser Ile Ser Gly Arg 20 25 30 Asp Thr His Arg Leu Thr Arg Thr Leu Asn Cys Ser Ser Ile Val Lys 35 40 45 Glu Ile Ile Gly Lys Leu Pro Glu Pro Glu Leu Lys Thr Asp Asp Glu 50 55 60 Gly Pro Ser Leu Arg Asn Lys Ser Phe Arg Arg Val Asn Leu Ser Lys 65 70 75 80 Phe Val Glu Ser Gln Gly Glu Val Asp Pro Glu Asp Arg Tyr Val Ile 85 90 95 Lys Ser Asn Leu Gln Lys Leu Asn Cys Cys Leu Pro Thr Ser Ala Asn 100 105 110 Asp Ser Ala Leu Pro Gly Val Phe Ile Arg Asp Leu Asp Asp Phe Arg 115 120 125 Lys Lys Leu Arg Phe Tyr Met Val His Leu Asn Asp Leu Glu Thr Val 130 135 140 Leu Thr Ser Arg Pro Pro Gln Pro Ala Ser Gly Ser Val Ser Pro Asn 145 150 155 160 Arg Gly Thr Val Glu Cys 165 <210> 133 <211> 134 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 133 Met Arg Met Leu Leu His Leu Ser Leu Leu Ala Leu Gly Ala Ala Tyr 1 5 10 15 Val Tyr Ala Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu 20 25 30 Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu 35 40 45 Thr Leu Arg Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr 50 55 60 Glu Glu Ile Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln 65 70 75 80 Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys 85 90 95 Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg Val 100 105 110 Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn Thr 115 120 125 Glu Trp Ile Ile Glu Ser 130 <210> 134 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 134 Met Arg Arg Met Leu Leu His Leu Ser Val Leu Thr Leu Ser Cys Val 1 5 10 15 Trp Ala Thr Ala Met Glu Ile Pro Met Ser Thr Val Val Lys Glu Thr 20 25 30 Leu Thr Gln Leu Ser Ala His Arg Ala Leu Leu Thr Ser Asn Glu Thr 35 40 45 Met Arg Leu Pro Val Pro Thr His Lys Asn His Gln Leu Cys Ile Gly 50 55 60 Glu Ile Phe Gln Gly Leu Asp Ile Leu Lys Asn Gln Thr Val Arg Gly 65 70 75 80 Gly Thr Val Glu Met Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys Tyr 85 90 95 Ile Asp Arg Gln Lys Glu Lys Cys Gly Glu Glu Arg Arg Arg Thr Arg 100 105 110 Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Ser Thr Glu 115 120 125 Trp Ala Met Glu Gly 130 <210> 135 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 135 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135 140 <210> 136 <211> 141 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 136 Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu 1 5 10 15 Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His 20 2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<223> synthesis <400> 137 Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala Phe Ser Leu 1 5 10 15 Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro 20 25 30 Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr 35 40 45 Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile 50 55 60 Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser 65 70 75 80 Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95 Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110 Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125 Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135 140 Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn 145 150 155 160 Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170 175 Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His 180 185 190 Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala 195 200 205 Leu Arg Gln Met 210 <210> 138 <211> 211 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 138 Met Lys Phe Leu Ser Ala Arg Asp Phe His Pro Val Ala Phe Leu Gly 1 5 10 15 Leu Met Leu Val Thr Thr Thr Ala Phe Pro Thr Ser Gln Val Arg Arg 20 25 30 Gly Asp Phe Thr Glu Asp Thr Thr Pro Asn Arg Pro Val Tyr Thr Thr 35 40 45 Ser Gln Val Gly Gly Leu Ile Thr His Val Leu Trp Glu Ile Val Glu 50 55 60 Met Arg Lys Glu Leu Cys Asn Gly Asn Ser Asp Cys Met Asn Asn Asp 65 70 75 80 Asp Ala Leu Ala Glu Asn Asn Leu Lys Leu Pro Glu Ile Gln Arg Asn 85 90 95 Asp Gly Cys Tyr Gln Thr Gly Tyr Asn Gln Glu Ile Cys Leu Leu Lys 100 105 110 Ile Ser Ser Gly Leu Leu Glu Tyr His Ser Tyr Leu Glu Tyr Met Lys 115 120 125 Asn Asn Leu Lys Asp Asn Lys Lys Asp Lys Ala Arg Val Leu Gln Arg 130 135 140 Asp Thr Glu Thr Leu Ile His Ile Phe Asn Gln Glu Val Lys Asp Leu 145 150 155 160 His Lys Ile Val Leu Pro Thr Pro Ile Ser Asn Ala Leu Leu Thr Asp 165 170 175 Lys Leu Glu Ser Gln Lys Glu Trp Leu Arg Thr Lys Thr Ile Gln Phe 180 185 190 Ile Leu Lys Ser Leu Glu Glu Phe Leu Lys Val Thr Leu Arg Ser Thr 195 200 205 Arg Gln Thr 210 <210> 139 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 139 Met Asn Cys Val Cys Arg Leu Val Leu Val Val Leu Ser Leu Trp Pro 1 5 10 15 Asp Thr Ala Val Ala Pro Gly Pro Pro Pro Gly Pro Pro Arg Val Ser 20 25 30 Pro Asp Pro Arg Ala Glu Leu Asp Ser Thr Val Leu Leu Thr Arg Ser 35 40 45 Leu Leu Ala Asp Thr Arg Gln Leu Ala Ala Gln Leu Arg Asp Lys Phe 50 55 60 Pro Ala Asp Gly Asp His Asn Leu Asp Ser Leu Pro Thr Leu Ala Met 65 70 75 80 Ser Ala Gly Ala Leu Gly Ala Leu Gln Leu Pro Gly Val Leu Thr Arg 85 90 95 Leu Arg Ala Asp Leu Leu Ser Tyr Leu Arg His Val Gln Trp Leu Arg 100 105 110 Arg Ala Gly Gly Ser Ser Leu Lys Thr Leu Glu Pro Glu Leu Gly Thr 115 120 125 Leu Gln Ala Arg Leu Asp Arg Leu Leu Arg Arg Leu Gln Leu Leu Met 130 135 140 Ser Arg Leu Ala Leu Pro Gln Pro Pro Pro Asp Pro Pro Ala Pro Pro 145 150 155 160 Leu Ala Pro Pro Ser Ser Ala Trp Gly Gly Ile Arg Ala Ala His Ala 165 170 175 Ile Leu Gly Gly Leu His Leu Thr Leu Asp Trp Ala Val Arg Gly Leu 180 185 190 Leu Leu Leu Lys Thr Arg Leu 195 <210> 140 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 140 Met Asn Cys Val Cys Arg Leu Val Leu Val Val Leu Ser Leu Trp Pro 1 5 10 15 Asp Arg Val Val Ala Pro Gly Pro Pro Ala Gly Ser Pro Arg Val Ser 20 25 30 Ser Asp Pro Arg Ala Asp Leu Asp Ser Ala Val Leu Leu Thr Arg Ser 35 40 45 Leu Leu Ala Asp Thr Arg Gln Leu Ala Ala Gln Met Arg Asp Lys Phe 50 55 60 Pro Ala Asp Gly Asp His Ser Leu Asp Ser Leu Pro Thr Leu Ala Met 65 70 75 80 Ser Ala Gly Thr Leu Gly Ser Leu Gln Leu Pro Gly Val Leu Thr Arg 85 90 95 Leu Arg Val Asp Leu Met Ser Tyr Leu Arg His Val Gln Trp Leu Arg 100 105 110 Arg Ala Gly Gly Pro Ser Leu Lys Thr Leu Glu Pro Glu Leu Gly Ala 115 120 125 Leu Gln Ala Arg Leu Glu Arg Leu Leu Arg Arg Leu Gln Leu Leu Met 130 135 140 Ser Arg Leu Ala Leu Pro Gln Ala Ala Pro Asp Gln Pro Val Ile Pro 145 150 155 160 Leu Gly Pro Pro Ala Ser Ala Trp Gly Ser Ile Arg Ala Ala His Ala 165 170 175 Ile Leu Gly Gly Leu His Leu Thr Leu Asp Trp Ala Val Arg Gly Leu 180 185 190 Leu Leu Leu Lys Thr Arg Leu 195 <210> 141 <211> 207 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 141 Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro 20 25 30 Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 35 40 45 Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys 50 55 60 Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 65 70 75 80 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 85 90 95 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 100 105 110 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 115 120 125 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 130 135 140 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 145 150 155 160 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 165 170 175 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 180 185 190 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 195 200 205 <210> 142 <211> 208 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 142 Met Ala Gln Leu Ser Ala Gln Arg Arg Met Lys Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp Gln Ser Ala Leu Trp Ser Gly Arg Glu Ala Val Pro 20 25 30 Leu Val Thr Val Ser Ala Leu Pro Pro Ser Leu Pro Leu Pro Arg Ser 35 40 45 Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Ala Ser Gly 50 55 60 Ser Val Leu Leu Glu Gln Leu Cys Ala Thr Tyr Lys Leu Cys His Pro 65 70 75 80 Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Lys Ala Ser 85 90 95 Leu Ser Gly Cys Ser Ser Gln Ala Leu Gln Gln Thr Gln Cys Leu Ser 100 105 110 Gln Leu His Ser Gly Leu Cys Leu Tyr Gln Gly Leu Leu Gln Ala Leu 115 120 125 Ser Gly Ile Ser Pro Ala Leu Ala Pro Thr Leu Asp Leu Leu Gln Leu 130 135 140 Asp Val Ala Asn Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Asn Leu 145 150 155 160 Gly Val Ala Pro Thr Val Gln Pro Thr Gln Ser Ala Met Pro Ala Phe 165 170 175 Thr Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Ala Ile Ser Tyr 180 185 190 Leu Gln Gly Phe Leu Glu Thr Ala Arg Leu Ala Leu His His Leu Ala 195 200 205 <210> 143 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 143 Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val Leu Leu 1 5 10 15 Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro 20 25 30 Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val 35 40 45 Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys 50 55 60 Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser 65 70 75 80 Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys 85 90 95 Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala 100 105 110 Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr 115 120 125 Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys 130 135 140 Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu 145 150 155 160 Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr 165 170 175 Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys 180 185 190 Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr 195 200 205 Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 210 215 <210> 144 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 144 Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65 70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175 Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile 195 200 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220 Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300 Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305 310 315 320 Glu Trp Ala Ser Val Pro Cys Ser 325 <210> 145 <211> 335 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> synthesis <400> 145 Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu 1 5 10 15 Val Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val 20 25 30 Val Glu Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln 50 55 60 Arg His Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Lys 65 70 75 80 Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr 85 90 95 Leu Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp 100 105 110 Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys 115 120 125 Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln 130 135 140 Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro 145 150 155 160 Asp Ser Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys 165 170 175 Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln 180 185 190 Glu Asp Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu 195 200 205 Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser 210 215 220 Phe Phe Ile Arg Asp Ile Ile Lys Pro ...

Claims

1. An activatable cytokine construct comprising a first monomer construct and a second monomer construct, wherein: (a) the first monomer construct comprises a first mature cytokine protein, a first cleavable moiety, and a first dimerization domain; (b) the second monomer construct comprises a second mature cytokine protein, a second cleavable moiety, and a second dimerization domain; (c) the first monomer construct is a polypeptide comprising, in an N-terminal to C-terminal direction, the first mature cytokine protein, the first cleavable moiety, and the first dimerization domain, and the first monomer construct is characterized in that the first mature cytokine protein and the first dimerization domain are connected by a linking region of no more than 18 amino acids, such that the linking region of no more than 18 amino acids comprises the first cleavable moiety; wherein the first mature cytokine protein and the second mature cytokine protein are each a mature interferon alpha; wherein the first cleavable moiety and the second cleavable moiety are each a substrate for a protease and cleave upon exposure to the protease; (d) further wherein: (i) the second monomer construct is identical to the first monomer construct, (ii) the first monomer construct and the second monomer construct are covalently bound to one another by at least one disulfide bond, (iii) the first dimerization domain and the second dimerization domain are a pair of human IgG Fc domains; (e) the first dimerization domain and the second dimerization domain are covalently bound to one another by at least one disulfide bond, thereby forming a dimer of the first monomer construct and the second monomer construct; and (f) the activatable cytokine construct is characterized as having a level of at least one first mature cytokine protein and second mature cytokine protein activity that is reduced compared to a corresponding control level of the at least one first mature cytokine protein and second mature cytokine protein activity.

2. The activatable cytokine construct of claim 1, wherein the first mature cytokine protein is a mature human interferon alpha.

3. The activatable cytokine construct of claim 1, wherein the first mature cytokine protein is a mature interferon alpha-2b.

4. The activatable cytokine construct of any one of claims 1 to 3, wherein the first mature cytokine protein comprises the sequence of SEQ ID NO:

1.

5. The activatable cytokine construct of any one of claims 1 to 3, wherein the first cleavable moiety and the second cleavable moiety each comprise no more than 7 amino acids.

6. The activatable cytokine construct of any one of claims 1 to 3, wherein the first cleavable moiety and the second cleavable moiety are each independently a substrate for urokinase (uPa) and / or a matrix metalloproteinase (MMP). ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 7. The activatable cytokine construct of any one of claims 1 to 3, wherein the first cleavable moiety and the second cleavable moiety each comprise a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:68, and SEQ ID NO:

100.

8. The activatable cytokine construct of claim 1, wherein the first dimerization domain and the second dimerization domain are a pair of human IgG4 Fc domains.

9. The activatable cytokine construct of claim 8, wherein the first dimerization domain and the second dimerization domain are a pair of human IgG4 Fc domains truncated at the N-terminus to cysteine 226, numbered as EU numbering.

10. The activatable cytokine construct of claim 8, wherein the human IgG4 Fc domain comprises a S228P mutation, numbered as EU numbering.

11. The activatable cytokine construct of any one of claims 8 to 10, wherein the amino acid sequence of the first dimerization domain and the second dimerization domain are each SEQ ID NO:

3.

12. The activatable cytokine construct of any one of claims 1 to 3, wherein the first monomer construct and the second monomer construct are covalently bound to one another by at least two disulfide bonds.

13. The activatable cytokine construct of any one of claims 1 to 3, wherein the first monomer construct and the second monomer construct are covalently bound to one another by at least three disulfide bonds.

14. The activatable cytokine construct of any one of claims 1 to 3, wherein the first monomer construct and the second monomer construct are covalently bound to one another by at least four disulfide bonds.

15. The activatable cytokine construct of claim 1, wherein the first monomer construct and the second monomer construct each comprise the sequence of SEQ ID NO:

313.

16. An activatable cytokine construct comprising a first monomer construct and a second monomer construct, wherein: (a) the first monomer construct comprises, in an N-terminal to C-terminal direction, a first mature cytokine protein, a first cleavable moiety, and a first dimerization domain, wherein the first mature cytokine protein and the first cleavable moiety are directly adjacent to one another, and the first cleavable moiety and the first dimerization domain are directly adjacent to one another; the first monomer construct is characterized in that the first mature cytokine protein and the first dimerization domain are connected by a linking region of no more than 18 amino acids, such that the linking region of no more than 18 amino acids comprises the first cleavable moiety; (b) the second monomer construct comprises, in an N-terminal to C-terminal direction, a second mature cytokine protein, a second cleavable moiety, and a second dimerization domain, wherein the second mature cytokine protein and the second cleavable moiety are directly adjacent to one another, and the second cleavable moiety and the second dimerization domain are directly adjacent to one another. ​ ​ (c) wherein the first and second mature cytokine proteins are each a mature interferon alpha, and wherein the amino acid sequences of the first and second cleavable moieties are each SEQ ID NO: 100; (d) further wherein: (i) the second monomeric construct is identical to the first monomeric construct; and (ii) the first and second dimerization domains are a pair of human IgGl or IgG4 Fc domains; (e) the first and second dimerization domains are covalently bound to one another by at least one disulfide bond, thereby forming a dimer of the first and second monomeric constructs; and (f) the activatable cytokine construct is characterized by a level of interferon alpha activity that is reduced compared to the interferon alpha activity of peginterferon alpha-2b.

17. An activatable cytokine construct comprising a first monomeric construct and a second monomeric construct, wherein: (a) the first monomeric construct comprises a first mature cytokine protein, a first cleavable moiety, and a first dimerization domain, wherein the first cleavable moiety is positioned between the first mature cytokine protein and the first dimerization domain, and the first monomeric construct is characterized in that the first mature cytokine protein and the first dimerization domain are connected by a linking region of no more than 18 amino acids, such that the linking region of no more than 18 amino acids comprises the first cleavable moiety; and (b) the second monomeric construct comprises a second mature cytokine protein, a second cleavable moiety, and a second dimerization domain, wherein the second cleavable moiety is positioned between the second mature cytokine protein and the second dimerization domain, and the second monomeric construct is characterized in that the second mature cytokine protein and the second dimerization domain are connected by a linking region of no more than 18 amino acids, such that the linking region of no more than 18 amino acids comprises the second cleavable moiety; wherein the first and second dimerization domains are bound to one another, thereby forming a dimer of the first and second monomeric constructs; wherein the activatable cytokine construct is characterized by a level of the first or second mature cytokine protein activity that is reduced compared to a control level of the first or second mature cytokine protein activity, and wherein the first and second monomeric constructs have the following structure, wherein in each construct, the first and second mature cytokine proteins are positioned N-terminal to the first and second dimerization domains, respectively; wherein the first and second mature cytokine proteins are each a mature interferon alpha; wherein the first and second dimerization domains are a pair of human IgG Fc domains; wherein the first and second cleavable moieties each function as a substrate for a protease and are cleaved upon exposure to the protease. (a) the first monomeric construct comprises a first mature cytokine protein and a first dimerization domain, and 18. An activatable cytokine construct comprising a first monomeric construct and a second monomeric construct, wherein: wherein the first and second dimerization domains are covalently bound to one another by at least one disulfide bond, thereby forming a dimer of the first and second monomeric constructs; and wherein the activatable cytokine construct is characterized by a level of interferon alpha activity that is reduced compared to the interferon alpha activity of peginterferon alpha-2b. (b) the second monomer construct comprises a second mature cytokine protein, a cleavable moiety, and a second dimerization domain, wherein the cleavable moiety is positioned between the second mature cytokine protein and the second dimerization domain, wherein the cleavable moiety serves as a substrate for a protease, and the second monomer construct is characterized in that the second mature cytokine protein and the second dimerization domain are connected by a linking region of no more than 18 amino acids, such that the linking region of no more than 18 amino acids comprises the cleavable moiety; wherein the first dimerization domain and the second dimerization domain bind to each other, thereby forming a dimer of the first monomer construct and the second monomer construct; wherein the activatable cytokine construct is characterized by a level of the first mature cytokine protein or the second mature cytokine protein activity that is reduced compared to a control level of the first mature cytokine protein or the second mature cytokine protein activity, and wherein the first monomer construct and the second monomer construct have the following structure, wherein in each construct, the first mature cytokine protein and the second mature cytokine protein are positioned N-terminal to the first dimerization domain and the second dimerization domain, respectively; wherein the first mature cytokine protein and the second mature cytokine protein are each a mature interferon alpha; wherein the first dimerization domain and the second dimerization domain are a pair of human IgG Fc domains; wherein the cleavable moiety serves as a substrate for a protease and is cleaved upon exposure to the protease.

19. An activatable cytokine construct comprising a first monomer construct and a second monomer construct, wherein: (a) the first monomer construct comprises a first mature cytokine protein, a cleavable moiety, and a first dimerization domain, wherein the cleavable moiety is positioned between the first mature cytokine protein and the first dimerization domain, and the first monomer construct is characterized in that the first mature cytokine protein and the first dimerization domain are connected by a linking region of no more than 18 amino acids, such that the linking region of no more than 18 amino acids comprises the cleavable moiety; and (b) the second monomer construct comprises a second mature cytokine protein and a second dimerization domain, wherein the first dimerization domain and the second dimerization domain bind to each other, thereby forming a dimer of the first monomer construct and the second monomer construct; wherein the activatable cytokine construct is characterized by a level of the first mature cytokine protein or the second mature cytokine protein activity that is reduced compared to a control level of the first mature cytokine protein or the second mature cytokine protein activity, and wherein the first monomer construct and the second monomer construct have the following structure, wherein in each construct, the first mature cytokine protein and the second mature cytokine protein are positioned N-terminal to the first dimerization domain and the second dimerization domain, respectively; wherein the first mature cytokine protein and the second mature cytokine protein are each a mature interferon alpha; wherein the first dimerization domain and the second dimerization domain are a pair of human IgG Fc domains; wherein the cleavable moiety serves as a substrate for a protease and is cleaved upon exposure to the protease.

20. The activatable cytokine construct of any one of claims 17-19, wherein the human Fc domain is a human IgGl Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain.

21. The activatable cytokine construct of claim 20, wherein the human Fc domain is a human IgG4 Fc domain.

22. The activatable cytokine construct of any one of claims 16-19, wherein the human Fc domain has an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 315, or SEQ ID NO:

316.

23. The activatable cytokine construct of any one of claims 16-19, wherein the first mature cytokine protein and / or the second mature cytokine protein comprises the sequence of SEQ ID NO:

1.

24. A composition comprising the activatable cytokine construct of any one of claims 1 to 23.

25. The composition of claim 24, wherein the composition is a pharmaceutical composition.

26. A container comprising at least one dose of the composition of claim 24 or 25.

27. A vial, syringe, or kit comprising at least one dose of the composition of claim 24 or 25.

28. An injection pen comprising at least one dose of the composition of claim 24 or 25.

29. Use of the activatable cytokine construct of any one of claims 1 to 23 or the composition of claim 24 or 25 in the manufacture of a medicament for treating Burkitt’s lymphoma in a subject in need thereof.

30. Use of the activatable cytokine construct of any one of claims 1 to 23 or the composition of claim 24 or 25 in the manufacture of a medicament for treating melanoma in a subject in need thereof.

31. A nucleic acid encoding a polypeptide comprising the first monomer of the activatable cytokine construct of any one of claims 1 to 17 and 19 to 23.

32. A vector comprising the nucleic acid of claim 31.

33. The vector of claim 32, wherein the vector is an expression vector.

34. A mammalian cell comprising the nucleic acid of claim 31.

35. The mammalian cell of claim 34, wherein the mammalian cell is a HEK293 cell or a CHO cell.

36. A mammalian cell comprising the vector of claim 32 or 33.

37. The mammalian cell of claim 36, wherein the mammalian cell is a HEK293 cell or a CHO cell.

38. A method of manufacturing an activatable cytokine construct, the method comprising: a. expressing the activatable cytokine construct in the mammalian cell of any one of claims 34-37; and b. purifying the expressed activatable cytokine construct. ​

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