Activatable cytokine constructs and methods of use

Abstract

Provided herein is a first monomer construct comprising an optional first peptide mask (PM1), an optional third cleavable portion (CM3), a first mature cytokine protein (CP1), a first cleavable portion (CM1) and a first dimerization domain (DD1), where CM1 is disposed between CP1 and DD1; an optional second peptide mask (PM2), an optional fourth cleavable portion (CM4), a second mature cytokine protein (CP2), a second cleavable portion (CM2) and a second dimerization domain (DD2). and a second monomeric construct comprising CM2 disposed between CP2 and DD2, wherein DD1 and DD2 bind to one another, thereby forming a dimer of the first and second monomeric constructs, and wherein the ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of the at least one activity of CP1 and / or CP2.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 253,893, filed October 8, 2021, and U.S. Provisional Application No. 63 / 328,525, filed April 7, 2022. The entire contents of the above-identified applications are hereby incorporated by reference in their entireties.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing entitled "CYTX086PCT.xml" (size: 907,197 bytes, and creation date: October 3, 2022) are incorporated by reference in their entirety into this specification.

[0003] Technical Field The present disclosure relates to the field of biotechnology, and more specifically to activatable cytokine constructs, including activatable cytokine constructs for use in immuno-oncology therapy. [Background technology]

[0004] Antibody-based therapeutics have been used to treat a variety of diseases with varying degrees of success, and in some cases, their therapeutic efficacy has been limited by toxicity due to widespread target expression. Furthermore, antibody-based therapeutics exhibit other limitations, such as rapid clearance from the circulation after administration. Combination therapies with antibody-based therapeutics have also been used, but are often limited by increased toxicity from each active drug.

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

[0006] Interferon therapy has many clinical benefits. For example, interferon is known to upregulate the immune system and to have antiviral and antiproliferative properties. Due to these biological properties, interferon has been used clinically as a therapeutic agent for the treatment of viral infections and malignancies. In addition, interferon is useful for recruiting the patient's innate immune system to identify and attack cancer cells. Thus, interferon therapy has been widely used in cancer and antiviral treatment, such as the treatment of hepatitis, Kaposi's sarcoma, hairy cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, and other disease states. However, systemic administration of interferon is associated with dose-dependent toxicities, particularly strong flu-like symptoms, neurological symptoms, liver toxicity, bone marrow suppression, and arrhythmias. In a study of melanoma patients, the combination of pembrolizumab with pegylated IFNa resulted in an ORR of 60.5%. This combination treatment was also associated with 49% G3 / G4 adverse events, requiring dose reduction of pegylated IFNa (Davar et al., J. Clin. Oncol., 2018). These undesirable side effects limit the dosage of interferon therapy and may lead to interruption or delay of interferon therapy.

[0007] Interleukins are another subclass of cytokines. Interleukins control cell growth, differentiation, and motility. Interleukins are particularly important in stimulating immune responses, such as inflammation. Interleukins have been used to treat cancer, autoimmune diseases, and other diseases. For example, interleukin-2 (IL2) is indicated for the treatment of melanoma, graft-versus-host disease (GVHD), neuroblastoma, renal cell carcinoma (RCC), and is also considered useful in conditions including acute coronary syndrome, acute myeloid syndrome, atopic dermatitis, autoimmune liver disease, basal cell carcinoma, bladder cancer, breast cancer, candidiasis, colon cancer, malignant cutaneous T-cell lymphoma, endometrioma, HIV invention, ischemic heart disease, rheumatoid arthritis, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, systemic lupus erythematosus, tuberculosis, and other diseases. Other interleukins, particularly IL-6, IL-7, IL-12 and IL-21, are potential treatments for cancer and other diseases. Interleukin therapy is often associated with undesirable side effects, including flu-like symptoms, nausea, vomiting, diarrhea, low blood pressure and cardiac arrhythmias, among others.

[0008] Under conditions of long-term stimulation, T cells upregulate and maintain the expression of the inhibitory receptor PD-1 to negatively regulate the quality and magnitude of T cell responses. PD-L1, the primary ligand of PD-1, is upregulated on many tumor cells and is associated with the inhibition of antitumor T cell immunity through the engagement of PD-1 on tumor-infiltrating T cells. Clinical trials have confirmed that antibody blockade of either PD-1 or PD-L1 can restore the activity of persistent tumor-specific immunity in patients across multiple tumor types (Herbst et al., 2014; Lipson et al., 2015). However, PD-1 / PD-L1 monotherapy has drawbacks, such as a significant number of non-responsive patients and / or patients who exhibit side effects related to relapse, tumor resistance, and autoimmune reactions.

[0009] Therefore, there is a great need and desire for improved specificity and selectivity of cytokine therapy to desired targets. Increasing the targeting of cytokine therapy to disease sites may reduce toxicity based on systemic mechanisms and lead to broader therapeutic utility. Combination therapy is also needed to improve the efficacy of therapies aimed at specifically and selectively inducing immune responses against various targets. Summary of the Invention

[0010] The present disclosure provides combinations, compositions, kits, and methods for treating a subject by administering to the subject a combination of an activatable cytokine construct (ACC) and a PD-1 / PD-L1 pathway inhibitor. In certain embodiments, the combination enhances efficacy in treatment. In certain embodiments, the combination reduces toxicity of one or both of the combination components when administered to a subject. In certain embodiments, the combination reduces or inhibits tumor growth, proliferation, and / or metastasis. In certain embodiments, the combination treats a subject suffering from cancer or an infectious disease. In certain embodiments, the combination enhances or increases therapeutic efficacy and / or therapeutic index compared to conventional cytokine therapy and / or conventional PD-1 / PD-L1 inhibitor therapy in the subject. In certain embodiments, the combination enhances or increases therapeutic efficacy and / or therapeutic index compared to conventional cytokine and PD-1 / PD-L1 inhibitor combination therapy in the subject. In certain embodiments, the combination enhances or increases therapeutic efficacy and / or therapeutic index compared to administration of ACC alone.

[0011] In one aspect, the ACC may contain (a) a first monomer comprising a first peptide mask (PM1), a first mature cytokine protein (CP1), a first and a third cleavable portion (CM1 and CM3), and a first dimerization domain (DD1), wherein CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1, and (b) a second monomer comprising a second mature cytokine protein (CP2), a second cleavable portion (CM2), and a second dimerization domain (DD2), wherein CM2 is disposed between CP2 and DD2, wherein CM1, CM2, and CM3 function as substrates for a protease, and DD1 and DD2 bind to each other, and characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2. The protease(s) that cleave CM1, CM2, and CM3 may be overexpressed in diseased tissue (e.g., tumor tissue) compared to healthy tissue. ACC may be activated by cleavage of CM1, CM2, and / or CM3 in diseased tissue (e.g., tumor microenvironment) to allow cytokines to exert their activity, while in healthy tissue, cytokine activity is reduced. Thus, the ACC provided herein can provide reduced toxicity, allow higher effective dosages of cytokines, and / or widen the therapeutic window of cytokines compared to conventional cytokine therapy.

[0012] Provided herein is an activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct, wherein (a) the first monomer construct comprises a first peptide mask (PM1), a first mature cytokine protein (CP1), first and third cleavable portions (CM1 and CM3), and a first dimerization domain (DD1), wherein CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1; and (b) the second monomer construct comprises a second mature cytokine protein (CP1), a first peptide mask (PM1), a first mature cytokine protein (CP1), a first and third cleavable portion (CM1 and CM3), and a first dimerization domain (DD1), wherein CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1. An activatable cytokine construct (ACC) is provided, comprising a protein (CP2), a second cleavable portion (CM2) and a second dimerization domain (DD2), wherein CM2 is positioned between CP2 and DD2, and DD1 and DD2 bind to each other, whereby the first monomer construct and the second monomer construct form a dimer, and wherein the activatable cytokine construct (ACC) has a reduced activity level of at least one of CP1 and / or CP2 compared to a control level of at least one of the activities of CP1 and / or CP2.

[0013] In some embodiments, the second monomer construct further comprises a second peptide mask (PM2) and a fourth cleavable portion (CM4), where CM4 is located between PM2 and CP2. In some embodiments, the first monomer construct comprises a first polypeptide comprising PM1, CM3, CP1, CM1 and DD1. In some embodiments, the second monomer construct comprises a second polypeptide comprising CP2, CM2 and DD2. In some embodiments, the second monomer construct comprises a second polypeptide comprising PM2, CM4, CP2, CM2 and DD2.

[0014] In some embodiments, PM1 comprises a sequence selected from the group consisting of SEQ ID NOs: 297, 298, 292, and 299-336, and CP1 is interferon; PM1 comprises a sequence selected from the group consisting of SEQ ID NOs: 297, 298, 292, and 299-332, and CP1 is interferon α; PM1 comprises a sequence selected from the group consisting of SEQ ID NOs: 299-328, and 330-332, and CP1 is interferon β; PM1 comprises a sequence selected from the group consisting of SEQ ID NOs: 299-328, and 333-336. and CP1 is interferon gamma; PM1 comprises a sequence selected from the group consisting of SEQ ID NOs: 337 to 341, and CP1 is IL-12; PM1 comprises a sequence selected from the group consisting of SEQ ID NOs: 342 to 349, 436 to 444, and 445, and CP1 is IL-15; PM1 comprises a sequence selected from the group consisting of SEQ ID NOs: 350 to 435, and 436 to 445, and CP1 is IL-2; or PM1 comprises a sequence selected from the group consisting of SEQ ID NOs: 445 and 446, and CP1 is IL-21. In some embodiments, PM2 comprises a sequence selected from the group consisting of SEQ ID NOs: 297, 298, 292, and 299-336, and CP2 is interferon; PM2 comprises a sequence selected from the group consisting of SEQ ID NOs: 297, 298, 292, and 299-364, and CP2 is interferon α; PM2 comprises a sequence selected from the group consisting of SEQ ID NOs: 299-328, and 330-332, and CP2 is interferon β; PM2 comprises a sequence selected from the group consisting of SEQ ID NOs: 299-328, and 333-336. PM2 comprises a sequence selected from the group consisting of SEQ ID NOs: 337-341, and CP2 is IL-12; PM2 comprises a sequence selected from the group consisting of SEQ ID NOs: 342-349, 436-444, and 445, and CP2 is IL-15; PM2 comprises a sequence selected from the group consisting of SEQ ID NOs: 350-435, 436-445, and CP2 is IL-2; or PM2 comprises a sequence selected from the group consisting of SEQ ID NOs: 445 and 446, and CP2 is IL-21.In some embodiments, PM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 297, 298, 292, and 299-446. In some embodiments, PM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 297, 298, 292, and 299-446.

[0015] In some embodiments, DD1 and DD2 are a pair selected from the group consisting of a pair of Fc domains; a sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα) and soluble IL-15; barnase and barstar; protein kinase A (PKA) and A-kinase anchor protein (AKAP); a mutant RNase I fragment-based adaptor / docking tag module; an epitope and a single domain antibody (sdAb); an epitope and a single chain variable region fragment (scFv); a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin and SNAP25; an antigen binding domain and an epitope.

[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 domains each comprise a sequence at least 80% identical to SEQ ID NO:3. In some embodiments, the human Fc domains each comprise a sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3. In some embodiments, the human Fc domain comprises SEQ ID NO:3. In some embodiments, DD1 and DD2 comprise SEQ ID NOs:287 and 288, respectively. In some embodiments, DD1 and DD2 are identical. In some embodiments, the human Fc domains contain mutations to eliminate glycosylation and / or to reduce Fc-gamma receptor binding. In some embodiments the human Fc domain comprises the mutation N297Q, N297A or N297G; in some embodiments the human Fc domain comprises a mutation at position 234 and / or 235, such as L235E, or L234A and L235A (in IgG1), or F234A and L235A (in IgG4); in some embodiments the human Fc domain is an IgG2 Fc domain comprising the mutations V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or a combination thereof (all according to EU numbering).

[0017] Additional examples of engineered human Fc domains are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids where at least one amino acid mutation results in reduced Fc function include, but are not limited to, mutations at amino acids 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330 and 331 of the heavy chain constant region (according to EU numbering). Examples of combinations of mutated amino acids are also known in the art, including, but are not limited to, combinations of mutations at amino acids 234, 235 and 331, such as L234F, L235E and P331S, or combinations of mutations at amino acids 318, 320 and 322, such as E318A, K320A and K322A.

[0018] Further examples of engineered Fc domains include F243L / R292P / Y300L / V305I / P396 IgG1; S239D / I332E IgG1; S239D / I332E / A330L IgG1; S298A / E333A / K334A; L234Y / L235Q / G236W / S239M / H268D / D270E / S298A IgG1 in one heavy chain, and D270E / K326D, A330M / K334E IgG1; G236A / S239D / I332E IgG1; K326W / E333S IgG1; S267E / H268F / S324T in the opposite heavy chain. These include IgG1; E345R / E430G / S440Y IgG1; N297A or N297Q or N297G IgG1; L235E IgG1; L234A / L235A IgG1; F234A / L235A IgG4; H268Q / V309L / A330S / P331S IgG2; V234A / G237A / P238S / H268A / V309L / A330S / P331S IgG2; M252Y / S254T / T256E IgG1; M428L / N434S IgG1; S267E / L328F IgG1; N325S / L328F IgG1, etc. In some embodiments, the engineered Fc domain comprises one or more substitutions selected from the group consisting of N297A IgG1, N297Q IgG1, and S228P IgG4.

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

[0020] In some embodiments, CP1 and CP2 are mature cytokines. In some embodiments, CP1 and CP2 each comprise a mature cytokine sequence and further comprise a signal peptide. A signal peptide is also referred to herein as a "signal sequence." In some embodiments, CP1 and / or CP2 are each independently selected from the group consisting of interferon, interleukin, GM-CSF, G-CSF, LIF, OSM, CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β1, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF, and MSP, and optionally CP1 and / or CP2 are each independently selected from the group consisting of IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN β, IFN In some embodiments selected from gamma, GM-CSF, TGF-beta, LIGHT, GITR-L, CD40L, CD27L, 4-1BB-L, OX40, and OX40L, CP1 and CP2 are the same. In some embodiments, CP1 and CP2 are different. In some embodiments, CP1 and / or CP2 are interferons. In some embodiments, CP1 and CP2 are both interferons. In some embodiments, CP1 and CP2 are different interferons. In some embodiments, CP1 and CP2 are the same interferon. In some embodiments, one of CP1 or CP2 is an interferon and the other 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, one of CP1 or CP2 is a monomeric interferon and the other CP1 or CP2 is a different cytokine. In some embodiments, CP1 and / or CP2 contain a mutant cytokine sequence.In some embodiments, CP1 and / or CP2 contain a universal cytokine sequence, hi some embodiments, CP1 and / or CP2 contain a truncated sequence that retains cytokine activity.

[0021] In some embodiments, the interferon(s) is human wild-type mature interferon. In some embodiments, the interferon(s) may be type I and type II interferons, including but not limited to interferon-α, interferon-β, interferon-γ, interferon-ω, and interferon-τ. In some embodiments, the interferon is interferon-α. In some embodiments, the interferon(s) is selected from the group consisting of interferon α-2a, interferon α-2b, and interferon α-n3. In some embodiments, the interferon(s) is interferon α-2b. In some embodiments, the interferon(s) is a mutant interferon. In some embodiments, the interferon(s) is a mutant interferon in which an endogenous protease cleavage site has been rendered dysfunctional by substitution, deletion, or insertion of one or more amino acids. In some embodiments, the interferon(s) is a universal cytokine molecule, e.g., having a hybrid sequence of different cytokine subtypes, or a chimeric cytokine sequence, or a humanized cytokine sequence. In some embodiments, the interferon(s) is a universal interferon molecule. In some embodiments, the interferon(s) is a universal interferon alpha, e.g., a hybrid of interferon alpha 1 and interferon alpha 2a. In some embodiments, CP1 and / or CP2 comprises a sequence that is at least 80% identical to SEQ ID NO:1. In some embodiments, CP1 and / or CP2 comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1. In some embodiments, CP1 and / or CP2 comprises a sequence of SEQ ID NO:1. In some embodiments, the interferon is interferon beta. In some embodiments, the interferon beta is selected from the group consisting of interferon beta-1a and interferon beta-1b. In some embodiments, CP1 and / or CP2 comprises an IFab domain.In some embodiments, CP1 and / or CP2 comprise an interleukin, hi some embodiments, the interleukin is selected from the group consisting of IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-12, IL-10, IL-20, IL-14, IL-16, and IL-17.

[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 for different proteases. In some embodiments, CM1 and CM2 comprise substrates for the same protease. In some embodiments, the protease(s) are selected from the group consisting of ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cruzipain, legumain, otubain-2, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, meprin, neprilysin, PSMA, BMP-1, matrix metalloproteinases (e.g., M MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, MMP-27), activated protein C, cathepsin A, cathepsin G, chymase, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, granulocyte selenite, erythrocyte selenite, erythrocyte septum ... The enzyme is selected from the group consisting of immunoglobulin B, guanidinobenzoatase, HtrA1, human neutrophil lyase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matripase, TMPRSS2, TMPRSS3 and TMPRSS4.In some embodiments, the protease(s) is selected from the group consisting of uPA, legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-2, MMP-9, MMP-12, MMP-13, and MMP-14.

[0023] Suitable cleavable moieties are disclosed in WO2010 / 081173, WO2015 / 048329, WO2015 / 116933, WO2016 / 118629 and WO2020 / 118109, the specifications of which are incorporated herein by reference in their entireties.

[0024] In some embodiments, CM1 and / or CM2 are selected from the group consisting of LSGRSDNH (SEQ ID NO:5), TGRGPSWV (SEQ ID NO:6), PLTGRSGG (SEQ ID NO:7), TARGPSFK (SEQ ID NO:8), NTLSGRSENHSG (SEQ ID NO:9), NTLSGRSGNHGS (SEQ ID NO:10), TSTSGRSANPRG (SEQ ID NO:11), TSGRSANP (SEQ ID NO:12), VHMPLGFLGP (SEQ ID NO:13), AVGLLAPP (SEQ ID NO:14), AQNLLGMV (SEQ ID NO:15), QNQALRMA (SEQ ID NO:16), LAAPLGLL( SEQ ID NO:17), STFPFGMF (SEQ ID NO:18), ISSGLLSS (SEQ ID NO:19), PAGLWLDP (SEQ ID NO:20), VAGRSMRP (SEQ ID NO:21), VVPEGRRS (SEQ ID NO:22), ILPRSPAF (SEQ ID NO:23), MVLGRSLL (SEQ ID NO:24), QGRAITFI (SEQ ID NO:25), SPRSIMLA (SEQ ID NO:26), SMLRSMPL (SEQ ID NO:27), ISSGLLLSGRSDNH (SEQ ID NO:28), AVGLLAPPGGLSGRSDNH (SEQ ID NO:29), ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 30), LSGRSGNH (SEQ ID NO: 31), SGRSANPRG (SEQ ID NO: 32), LSGRSDDH (SEQ ID NO: 33), LSGRSDIH (SEQ ID NO: 34), LSGRSDQH (SEQ ID NO: 35), LSGRSDTH (SEQ ID NO: 36), LSGRSDYH (SEQ ID NO: 37), LSGRSDNP (SEQ ID NO: 38), LSGRSANP (SEQ ID NO: 39), LSGRSANI (SEQ ID NO: 40), LSGRSDNI (SEQ ID NO: 41), MIAPVAYR (SEQ ID NO: 42), RPSPMWAY (SEQ ID NO: 43), WATPRPMR (SEQ ID NO: 44), FRLLDWQW (SEQ ID NO: 45), ISSGL (SEQ ID NO: 46), ISSGLLS (SEQ ID NO: 47), ISSGLL (SEQ ID NO: 48), ISSGLLSGRSAMPRG (SEQ ID NO: 49), AVGLLAPPTSGRSANPRG (SEQ ID NO: 50), AVGLLAPPSGRSANPRG (SEQ ID NO: 51), ISSGLLSGRSDDH (SEQ ID NO: 52), ISSGLLSGRSDIH (SEQ ID NO: 53), ISSGLLSGRSDQH (SEQ ID NO: 54), ISSGLLSGRSDTH (SEQ ID NO: 55), ISSGLLSGRSDYH (SEQ ID NO: 56),ISSGLLSGRSDNP (SEQ ID NO: 57), ISSGLLSGRSANP (SEQ ID NO: 58), ISSGLLSGRSANI (SEQ ID NO: 59), AVGLLAPPGGLSGRSDDH (SEQ ID NO: 60), AVGLLAPPGGLSGRSDIH (SEQ ID NO: 61), AVGLLAPPGGLSGRSDQH (SEQ ID NO: 62), AVGLLAPPGGLSGRSDTH (SEQ ID NO: 63), AVGLLAPPGGLSGRSDYH (SEQ ID NO: 64), AVGLLAPPGGLSGRSDNP (SEQ ID NO: 65) ), AVGLLAPPGGLSGRSANP (SEQ ID NO: 66), AVGLLAPPGGLSGRSANI (SEQ ID NO: 67), ISSGLLSGRSDNI (SEQ ID NO: 68), AVGLLAPPGGLSGRSDNI (SEQ ID NO: 69), GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 70), GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 71), LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 72), ISSGLSS (SEQ ID NO: 73), PVGYTSSL (SEQ ID NO: 74), DW LYWPGI (SEQ ID NO: 75), LKAAPRWA (SEQ ID NO: 76), GPSHLLVLT (SEQ ID NO: 77), LPGGLSPW (SEQ ID NO: 78), MGLFSEAG (SEQ ID NO: 79), SPLPLRVP (SEQ ID NO: 80), RMHLRSLG (SEQ ID NO: 81), LLAPSHRA (SEQ ID NO: 82), GPRSFGL (SEQ ID NO: 83), GPRSFG (SEQ ID NO: 84), SARGPSRW (SEQ ID NO: 85), GGWHTGRN (SEQ ID NO: 86), HTGRSGAL (SEQ ID NO: 87), AARGPAIH (SEQ ID NO: 88), 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 NO: 100). In some embodiments, the CM comprises a sequence selected from the group consisting of ISSGLLSGRSDNH (SEQ ID NO: 28), LSGRSDDH (SEQ ID NO: 33), ISSGLLSGRSDQH (SEQ ID NO: 54),In some embodiments, the protease(s) is produced by a tumor in the subject, e.g., the protease is produced in greater amounts in the tumor than in healthy tissue in the subject. In some embodiments, the subject has been diagnosed or identified as having cancer.

[0025] In some embodiments, in the first monomer construct, CP1 and CM1 are directly adjacent to each other. In some embodiments, in the first monomer construct, CM1 and DD1 are directly adjacent to each other. In some embodiments, in the second monomer construct, CP2 and CM2 are directly adjacent to each other. In some embodiments, in the second monomer construct, CM2 and DD2 are directly adjacent to each other. In some embodiments, the first monomer construct comprises CP1 directly adjacent to CM1 and CM1 directly adjacent to DD1, CM1 comprising 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, CM2 comprising 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, CM1 comprising a sequence of 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids or less in length. In some embodiments, the second monomer construct comprises CP2 which is directly adjacent to CM2 and CM2 which is directly adjacent to DD2, where CM2 comprises a sequence of no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids in length. In some embodiments, the first monomer construct and the second monomer construct are each configured such that the cytokine (CM1 and CM2, respectively) is directly adjacent to a cleavable portion (CM1 and CM2, respectively) that is no more than 10, 9, 8, 7, 6, 5, or 4 amino acids in length, which is directly adjacent to a dimerization domain (DD1 and DD2, respectively) that is an Fc region of human IgG, where the N-terminus of the Fc region is the first cysteine ​​residue (reading N-terminus to C-terminus) in the hinge region that participates in a disulfide bond with the second Fc domain (e.g., cysteine ​​226 of human IgG1 using EU numbering). In some embodiments, the dimerization domain is an IgG Fc region with the upper hinge residues deleted.For example, the Fc is a mutant lacking the N-terminal sequence EPKSCDKTHT (SEQ ID NO: 522), ERK, ELKTPLGDTTHT (SEQ ID NO: 523) or ESKYGPP (SEQ ID NO: 524).

[0026] In some embodiments, the first monomer construct comprises at least one linker. In some embodiments, the at least one linker is a linker L1 disposed between PM1 and CM3 and / or a linker L2 disposed between CM3 and CP1. In some embodiments, the second monomer construct comprises at least one linker. In some embodiments, the at least one linker is a linker L3 disposed between PM2 and CM4 and / or a linker L4 disposed between CM4 and CP2. In some embodiments, the first monomer construct comprises a linker L1 and the second monomer construct comprises a linker L3. In some embodiments, L1 and L3 are the same. In some embodiments, the first monomer construct comprises a linker L2 and the second monomer construct comprises a linker L4. In some embodiments, L2 and L4 are the same. In some embodiments, the first monomer construct comprises a linker between CP1 and CM1 and / or a linker between CM1 and DD1. In some embodiments, the second monomeric construct comprises a linker between CP2 and CM2 and / or a linker between CM2 and DD2. In some embodiments, each linker has an overall length of 1 amino acid to about 15 amino acids. In some embodiments, each linker has an overall length of at least 5 amino acids.

[0027] In some embodiments, the first monomeric construct comprises at least one linker, each linker being 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); GGGGSGGGGSGGGGGS (SEQ ID NO:213); GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:214); GGGGSGGGGS (SEQ ID NO:215); GGGGS (SEQ ID NO:216); GS; GGGGSGS (SEQ ID NO:217); GGGGSGGGGSGGGGSGS (SEQ ID NO:218); GGSLDPKGGGGS (SEQ ID NO:219); PKSCDKTHTCPPCPAPELLG (SEQ ID NO:220); SKYGPPCPPCPAPEFLG (SEQ ID NO:221); GKSSGSGSESKS (SEQ ID NO:222); GSTSGSGKSSEGKG (SEQ ID NO:223); 3);GSTSGSGKSSEGSGSTKG (SEQ ID NO:224);GSTSGSGKPGSGEGSTKG (SEQ ID NO:225);GSTSGSGKPGSSEGST (SEQ ID NO:226);(GS)n, (GGS)n, (GSGGS)n (SEQ ID NO:227), (GGGS)n (SEQ ID NO:228), (GGGGS)n (SEQ ID NO:216), where each n is an integer that is at least 1;GGSG (SEQ ID NO:229);GGSGG (SEQ ID NO:230);GSGSSG (SEQ ID NO:231;GSGGGG (SEQ ID NO:232);GGGSSG (SEQ ID NO:233);GSSSG (SEQ ID NO:234);GGGGSGGGGSGGGGGS (SEQ ID NO:213);GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:214); andGSTSGSSGKPGSSEGST (SEQ ID NO:226). In some embodiments, the linker comprises the sequence of GGGS (SEQ ID NO:2).

[0028] In some embodiments, the first monomeric construct comprises DD1 linked directly or indirectly to PM1, CM3, CP1, CM1, and the C-terminus of CM1 in the N-terminal to C-terminal direction. In some embodiments, the first polypeptide comprises DD1 linked directly or indirectly to PM1, CM3, CP1, CM1, and the N-terminus of CM1 in the C-terminal to N-terminal direction. In some embodiments, the second polypeptide comprises DD2 linked directly or indirectly to PM2, CM4, CP2, CM2, and the C-terminus of CM2 in the N-terminal to C-terminal direction. In some embodiments, the second polypeptide comprises DD2 linked directly or indirectly to PM2, CM4, CP2, CM2, and CM2 in the C-terminal to N-terminal direction.

[0029] In some embodiments, the first monomer construct comprises, in the N-terminal to C-terminal direction, CP1, an optional linker, CM1, an optional linker and DD1, where DD1 is an IgG Fc region, the N-terminus of the Fc region is the first cysteine ​​residue (reading N-terminal to C-terminal direction) in the hinge region that participates in a disulfide bond with the second Fc domain (e.g., cysteine ​​226 of human IgG1 or IgG4 using EU numbering), and where CM1 and the optional linker(s) located between CP1 and the N-terminal cysteine ​​of DD1 (the "linking region" or "LR") together have an overall length of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acids or less, preferably 10 amino acids or less, particularly preferably 7 amino acids or less. In some such embodiments, the first monomeric construct further comprises, in an N-terminal to C-terminal direction, PM1, an optional linker, CM3, and an optional linker attached to the N-terminus of CP1. In some embodiments, the second monomer construct comprises, in the N-terminal to C-terminal direction, CP2, an optional linker, CM2, an optional linker and DD2, where DD2 is an IgG Fc region, the N-terminus of the Fc region is the first cysteine ​​residue (reading N-terminal to C-terminal direction) in the hinge region that participates in a disulfide bond with the second Fc domain (e.g., cysteine ​​226 of human IgG1 or IgG4, using EU numbering), and where CM2 and the optional linker(s) ("linking region" or "LR") located between CP2 and the N-terminal cysteine ​​of DD2 together have an overall length of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acids or less, preferably 10 amino acids or less, particularly preferably 7 amino acids or less. In some such embodiments, the second monomer construct further comprises, in an N-terminal to C-terminal direction, PM2, the optional linker, CM4, and the optional linker attached to the N-terminus of CP2. In some aspects, there is no linker or spacer between the peptide mask and the cleavable moiety. In some aspects, there is no linker or spacer between the cytokine protein and the cleavable moiety.In some embodiments, there is no linker or spacer between the cleavable moiety and the dimerization domain.

[0030] In some embodiments, the ACC is a homodimer in which the first and second monomeric constructs are identical and comprise the amino acid sequence of SEQ ID NO: 290. In some embodiments, the first and second monomeric constructs each comprise an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 290. In some embodiments, the ACC is a homodimer in which the first and second monomeric constructs are identical and comprise the amino acid sequence of SEQ ID NO: 290 without the N-terminal spacer sequence (QSGQ). In some embodiments, the first monomer construct and the second monomer construct each comprise, from N-terminus to C-terminus, SEQ ID NO:292; an optional flexible linker of 0-10 amino acids; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:68, and SEQ ID NO:100; an optional flexible linker of 0-10 amino acids; SEQ ID NO:1; a second CM comprising 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 dimerization domain.

[0031] In some embodiments, the at least one CP1 and / or CP2 activity is the binding affinity of CP1 and / or CP2 to its cognate receptor, determined using surface plasmon resonance. For example, if CP1 or CP2 is an interferon, the cognate receptor may be the interferon-α / β receptor (IFNAR). In some embodiments, the at least one CP1 and / or CP2 activity is the proliferation level of lymphoma cells. In some embodiments, the at least one CP1 and / or CP2 activity is the activation level of the JAK / STAT / ISGF3 pathway in lymphoma cells. In some embodiments, the at least one activity is the production level of secreted alkaline phosphatase (SEAP) in lymphoma cells. In some embodiments, ACC is characterized by at least one of the CP1 and CP2 activities being at least two-fold lower compared to the control level. In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold, 3000-fold, or 4000-fold lower compared to the level of a control. In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 5000-fold lower compared to the level of a control. In some embodiments, the control level of at least one CP1 and / or CP2 activity is the CP1 and / or CP2 activity of the ACC after exposing the ACC to a protease(s). In some embodiments, the control level of at least one CP1 and / or CP2 activity is the corresponding CP1 and / or CP2 activity of the corresponding wild-type mature cytokine.

[0032] In some embodiments, the ACC is characterized by producing a cleavage product after exposure to a protease(s), the cleavage product comprising at least one activity of CP1 and / or CP2. In some embodiments, the at least one activity of CP1 and / or CP2 is an anti-proliferative activity. In some embodiments, the control level is the EC50 value of the wild-type mature cytokine, and the ratio of EC50 (cleavage product) to EC50 (wild-type control level) is less than about 10, or less than about 9, or less than about 8, or less than about 7, or less than about 6, or less than about 5, or less than about 4, or less than about 3, or less than about 2, or less than about 1.5, or equal to about 1. In some embodiments, the EC50 of the cleavage product is approximately the same as the EC50 of the wild-type mature cytokine, indicating that after cleavage, CP1 and / or CP2 activity is fully or nearly fully restored.

[0033] In some embodiments, the ACC comprises (a) a first monomer comprising a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerization domain (DD1), where the CM1 is disposed between the CP1 and the DD1, and (b) a second monomer comprising a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerization domain (DD2), where the CM2 is disposed between the CP2 and the DD2, where the CM1 and CM2 serve as substrates for a protease, and the DD1 and DD2 bind to each other, and the ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2. The protease(s) that cleave CM1 and CM2 may be overexpressed in diseased tissue (e.g., tumor tissue) compared to healthy tissue. In diseased tissues (e.g., the tumor microenvironment), ACC may become activated through cleavage of CM1 and / or CM2, allowing cytokines to exert their activity, whereas in healthy tissues, cytokine activity is reduced.

[0034] Provided herein is an activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct, characterized in that (a) the first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable portion (CM1) and a first dimerization domain (DD1), wherein CM1 is disposed between CP1 and DD1, and (b) the second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable portion (CM2) and a second dimerization domain (DD2), wherein CM2 is disposed between CP2 and DD2, and DD1 and DD2 bind to each other, whereby the first monomer construct and the second monomer construct form a dimer, and the activatable cytokine construct (ACC) has a reduced activity level of at least one of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2.

[0035] The present disclosure provides an activatable cytokine construct (ACC) comprising: (a) a first monomer comprising a first mature cytokine protein (CP1), a first dimerization domain (DD1); and (b) a second monomer comprising a second mature cytokine protein (CP2), a cleavable moiety (CM), and a second dimerization domain (DD2), wherein the CM is disposed between CP2 and DD2, wherein the CM functions as a substrate for a protease, and the DD1 and DD2 bind to each other, and the ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2.

[0036] The present disclosure provides an activatable cytokine construct (ACC) comprising: (a) a first monomer comprising a first mature cytokine protein (CP1), a cleavable moiety (CM), and a first dimerization domain (DD1), wherein the CM is disposed between the CP1 and the DD1; and (b) a second monomer comprising a second mature cytokine protein (CP2) and a second dimerization domain (DD2), wherein the CM functions as a substrate for a protease, and the DD1 and DD2 bind to each other, and the ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2.

[0037] The present disclosure provides an activatable cytokine construct (ACC) comprising: (a) a first monomer comprising a first mature cytokine protein (CP1) and a first dimerization domain (DD1); and (b) a second monomer comprising a second mature cytokine protein (CP2) and a second dimerization domain (DD2), wherein CP1, CP2, or both CP1 and CP2 comprise an amino acid sequence that functions as a substrate for a protease, and DD1 and DD2 bind to each other, and the ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2.

[0038] Thus, the ACC of the present disclosure does not require that CP1 and CP2 be connected to a peptide mask, such as an affinity masking moiety, and such a peptide mask is an optional feature of certain ACCs of the present disclosure.

[0039] In some embodiments, ACC is administered in combination with a PD-1 / PD-L1 pathway inhibitor. The present disclosure provides inhibitors that specifically bind to programmed cell death protein 1 (PD-1), also known as CD279, SLEB2, and / or hSLE1, and inhibitors that specifically bind to programmed death ligand 1, also known as cluster of differentiation 274 or B7 homolog 1 and / or B7-H1. Use of the term "PD-1" or "PD-L1" is intended to cover any variants thereof, such as, by way of non-limiting example, PD1 and / or PD-1, and PDL1 and / or PD-L1, all variants being used interchangeably herein.

[0040] In some embodiments, the ACC administered in combination with a PD-1 / PD-L1 pathway inhibitor is CP1 and / or CP2, each independently selected from the group consisting of interferon, interleukin, GM-CSF, G-CSF, LIF, OSM, CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β1, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF, and MSP, and optionally said CP1 and / or said CP2 are independently selected from the group consisting of IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN β, IFN gamma, GM-CSF, TGF-β, LIGHT, GITR-L, CD40L, CD27L, 4-1BB-L, OX40, and OX40L. In a preferred embodiment, CP1 and / or CP2 are each individually selected from interferons as described above. In a more preferred embodiment, the ACC administered in combination with a PD-1 / PD-L1 pathway inhibitor comprises CP1 and CP2, each of which is interferon alpha-2b.

[0041] In some embodiments, the PD-1 / PD-L1 pathway inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1. In some embodiments, the antibody or antigen-binding fragment thereof that binds to PD-1 or PD-L1 is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab') 2 The antibody or antigen-binding fragment thereof that binds to PD-1 or PD-L1 is a murine, other rodent, chimeric, humanized monoclonal antibody, or fully human monoclonal antibody. In some embodiments, the antibody comprises an isolated antibody or antigen-binding fragment thereof (AB) that specifically binds to a mammalian PD-1 or PD-L1, the AB having one or more characteristics selected from the group consisting of: (a) the AB inhibits binding of the mammalian PD-1 to the mammalian PDL1. In some embodiments, the PD-1 / PD-L1 pathway inhibitor is an activatable antibody.

[0042] In some embodiments, the antibody includes an isolated antibody or antigen-binding fragment thereof (AB) that specifically binds to mammalian PD-1 or PD-L1, wherein the AB has one or more characteristics selected from the group consisting of: (a) the AB inhibits the binding of mammalian PD-1 to mammalian PDL1 with an EC50 value of less than 5 nM; (b) the AB inhibits the binding of mammalian PD-1 to mammalian PDL2 with an EC50 value of less than 5 nM; and (c) the AB specifically binds human PD-1 and cynomolgus PD-1.

[0043] In some embodiments, the antibody is at a concentration of 0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.1 nM to 5 nM, 0.2 nM to 5 nM, 0.3 nM to 5 nM, 0.4 nM to 5 nM, 0.5 nM to 5 nM, 0.75 nM to 5 nM, 1 nM to 5 nM, 2 nM to 5 nM, 0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.1 nM to 2 nM, 0.2 nM to 2 nM, 0.3 nM to 2 nM, 0.4 nM~2nM, 0.5nM~2nM, 0.75nM~1nM, 1nM~2nM, 0.01nM~1nM, 0.05nM~1nM, 0.1nM~1nM, 0.2nM~1nM, 0.3nM~1 nM, 0.4nM~1nM, 0.5nM~1nM, 0.75nM~1nM, 0.01nM~0.75nM, 0.05nM~0.75nM, 0.1nM~0.75nM, 0.2nM~0.75 nM, 0.3nM~0.75nM, 0.4nM~0.75nM, 0.5nM~0.75nM, 0.01nM~0.5nM, 0.05nM~0.5nM, 0.1nM~0.5nM, 0.2nM ~0.5nM, 0.3nM~0.5nM, 0.4nM~0.5nM, 0.01nM~0.4nM, 0.05nM~0.4nM, 0.1nM~0.4nM, 0.2nM~0.4nM, 0.3n specifically binds to mammalian PD-1 or PD-L1 with a dissociation constant of M-0.4 nM, 0.01 nM-0.3 nM, 0.05 nM-0.3 nM, 0.1 nM-0.3 nM, 0.2 nM-0.3 nM, 0.01 nM-0.2 nM, 0.05 nM-0.2 nM, 0.1 nM-0.2 nM, 0.01 nM-0.1 nM, 0.05 nM-0.1 nM, or 0.01 nM-0.05 nM.

[0044] In some embodiments, the mammalian PD-1 / PD-L1 is selected from the group consisting of human PD-1 / PD-L1 and cynomolgus PD-1 / PD-L1. In some embodiments, the mammalian PD-1 / PD-L1 is mouse PD-1 / PD-L1. In some embodiments, the antibody specifically binds human PD-1 / PD-L1 or cynomolgus PD-1 / PD-L1 with a dissociation constant of 1 nM or less. In some embodiments, the mammalian PD-1 / PD-L1 is human PD-1 / PD-L1.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a mammalian PD-1 or PD-L1 with a dissociation constant of 0.01 nM or less, 0.05 nM or less, 0.1 nM or less, 0.2 nM or less, 0.3 nM or less, 0.4 nM or less, 0.5 nM or less, 0.75 nM or less, and 1 nM or less.

[0046] In some embodiments, the antibodies have one or more properties selected from the group consisting of: (a) AB specifically binds human PD-1 or PD-L1 and cynomolgus PD-1 or PD-L1; (b) AB inhibits the binding of human PDL1 and human PDL2 to human PD-1; (c) AB inhibits the binding of cynomolgus PDL1 and cynomolgus PDL2 to cynomolgus PD-1; (d) AB specifically binds mouse PD-1; and (e) AB inhibits the binding of mouse PDL1 and mouse PDL2 to mouse PD-1.

[0047] In some embodiments, the antibody is at a concentration of 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, 0.1 nM to 1 nM, 0.1 nM to 0.5 nM, 0.1 nM to 0.25 nM, 0.25 nM to 10 nM, 0.25 nM to 5 nM, 0.25 nM to 3 nM, 0.25 nM to 2 nM, 0.25 nM to 1 nM, 0.25 nM to 0.5 nM, 0.5 nM to Interferes with the ability of a natural ligand to bind to mammalian PDL1 with an EC50 of 10 nM, 0.5 nM-5 nM, 0.5 nM-3 nM, 0.5 nM-2 nM, 0.5 nM-1 nM, 1 nM-10 nM, 1 nM-5 nM, 1 nM-3 nM, 1 nM-2 nM, 2 nM-10 nM, 2 nM-5 nM, 2 nM-3 nM, 3 nM-10 nM, 3 nM-5 nM, or 5 nM-10 nM. In some embodiments, the natural ligand is mammalian PDL1 or mammalian PDL2. In some embodiments, the natural ligand is selected from the group consisting of human PDL1, human PDL2, cynomolgus PDL1, and cynomolgus PDL2. In some embodiments, the natural ligand is mouse PDL1 or mouse PDL2.

[0048] In some embodiments, the antibody interferes with the ability of a natural ligand to bind to mammalian PDL1 with an EC50 of 0.1 nM or less, 0.25 nM or less, 0.5 nM or less, 1 nM or less, 2 nM or less, 3 nM or less, 4 nM or less, 5 nM or less, or 10 nM or less.

[0049] In some embodiments, the anti-PD-1 antibody comprises a heavy chain comprising, or derived from, an amino acid sequence selected from the group consisting of SEQ ID NOs: 610-614 and 620-628, and a light chain comprising, or derived from, an amino acid sequence selected from the group consisting of SEQ ID NOs: 615-619 and 629-639.

[0050] In some embodiments, the anti-PD-1 antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 610-614 and 620-628, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 615-619 and 629-639.

[0051] In some embodiments, the anti-PD-1 antibody comprises (a) a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 487, and 642-645; (b) a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 488, and 646-650; (c) a variable heavy chain complementarity determining region 3 (VH CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 489, and 652-655; (d) a variable light chain complementarity determining region 1 (VL CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 656-663; (e) a variable light chain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 491, and 664-666; and (f) a variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 492, and 667-670.

[0052] In some embodiments, an anti-PD-1 antibody comprises a combination of a variable heavy chain complementarity determining region 1 (VHCDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VHCDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VHCDR3, also referred to herein as CDRH3) sequence, wherein the VHCDR1 sequence comprises GITFSNSG (SEQ ID NO:525), the VHCDR2 sequence comprises IWYDGSKR (SEQ ID NO:526), ​​and the VHCDR3 sequence comprises TNDDY (SEQ ID NO:527).

[0053] In some embodiments, the anti-PD-1 antibody comprises a combination of a variable light chain complementarity determining region 1 (VLCDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VLCDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VLCDR3, also referred to herein as CDRL3) sequence, wherein the VLCDR1 sequence comprises QSVSSY (SEQ ID NO:528), the VLCDR2 sequence comprises DAS, and the VLCDR3 sequence comprises QQSSNWPRT (SEQ ID NO:529).

[0054] In some embodiments, the anti-PD-1 antibody comprises a combination of a variable heavy chain complementarity determining region 1 (VHCDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VHCDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VHCDR3, also referred to herein as CDRH3) sequence, wherein the VHCDR1 sequence comprises GYTFTNYY (SEQ ID NO:530), the VHCDR2 sequence comprises INPSNGGT (SEQ ID NO:531), and the VHCDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO:532).

[0055] In some embodiments, the anti-PD-1 antibody comprises a combination of a variable light chain complementarity determining region 1 (VLCDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VLCDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VLCDR3, also referred to herein as CDRL3) sequence, wherein the VLCDR1 sequence comprises KGVSTSGYSY (SEQ ID NO:533), the VLCDR2 sequence comprises LAS, and the VLCDR3 sequence comprises QHSRDLPLT (SEQ ID NO:534).

[0056] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain that comprises, or is derived from, an amino acid sequence selected from the group consisting of SEQ ID NO: 673-694, and a light chain that comprises, or is derived from, an amino acid sequence selected from the group consisting of SEQ ID NO: 671 or SEQ ID NO: 672.

[0057] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:673-694, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:671 or SEQ ID NO:672.

[0058] In some embodiments, the anti-PD-L1 antibody comprises a combination of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 sequences, wherein at least one CDR sequence is selected from the group consisting of a VLCDR1 sequence comprising RASQSISSYLN (SEQ ID NO:535), a VLCDR2 sequence comprising AASSLQS (SEQ ID NO:536), a VLCDR3 sequence comprising DNGYPST (SEQ ID NO:537), a VHCDR1 sequence comprising SYAMS (SEQ ID NO:538), a VHCDR2 sequence comprising SSIWRNGIVTVYADS (SEQ ID NO:539), and a VHCDR3 sequence comprising WSAAFDY (SEQ ID NO:540).

[0059] In some embodiments, the PD-1 / PD-L1 pathway inhibitor is nivolumab, pembrolizumab, tislelizumab, spartalizumab, camrelizumab, cetrelimab, cemiplimab, valstilimab, dostallimab, prorugolimab, sasanlimab, zimberelimab, atezolizumab, avelumab, durvalumab, adebrelimab, lodapolimab, embafolimab, cosibelimab, budigalimab, ezalidom ... The compound is selected from the group consisting of benlimab, phynotonlimab, geptanolimab, lodapolimab, penprimab, pimivalimab, pucotenlimab, serplulimab, sintilimab, toripalimab, zelvalimab, iparomulimab, nofazinelimab, ruronilimab, galibrimab, manelimab, opcolimab, pakmilimab (CX-072), suduvulimab, sugemalimab, socazolimab, and tagitanlimab.

[0060] In some embodiments, the PD1 / PD-L1 pathway inhibitor comprises pakmiliumab (CX-072 (SEQ ID NO:485-HC, SEQ ID NO:496-LC); CX-075 (SEQ ID NO:485-HC, SEQ ID NO:497-LC), CX-171 (SEQ ID NO:504 or 505-HC, SEQ ID NO:506-LC), or CX-188 (SEQ ID NO:483-HC, SEQ ID NO:484-LC).

[0061] The present disclosure also provides an activatable antibody comprising an antibody or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1 coupled to a masking moiety (MM). The coupling of the MM reduces the ability of the antibody or antigen-binding fragment thereof to bind to PD-1 or PD-L1. In some embodiments, the MM is attached via a cleavable moiety (CM) that includes a sequence that serves as a substrate for a protease. The activatable anti-PD-1 or anti-PD-L1 antibody of the present disclosure is activated when the cleavable moiety is cleaved by a protease. For example, the protease is produced by a tumor that is in the vicinity of T cells expressing PD-1 or PD-L1. In some embodiments, the protease is produced by a tumor that is co-localized with T cells expressing PD-1 or PD-L1.

[0062] The activatable anti-PD-1 or anti-PD-L1 antibodies provided herein, also referred to herein as anti-PD-1 activatable antibodies or anti-PD-L1 activatable antibodies, or PD-1 activatable antibodies and anti-PD-L1 activatable antibodies, are stable in circulation, are activated at the intended site of treatment and / or diagnosis, but not in normal, e.g., healthy tissue or other tissue not targeted for treatment and / or diagnosis, and, upon activation, exert binding to PD-1 or PD-L1 that is at least comparable to the corresponding unmodified antibody.

[0063] The invention also provides methods of treating, preventing, and / or delaying the progression of, or alleviating a condition associated with aberrant expression and / or activity of PD-1 or PD-L1 in a subject using antibodies or activatable antibodies that bind to PD-1 or PD-L1, particularly activatable antibodies that bind to and neutralize or otherwise inhibit at least one biological activity of PD-1 or PD-L1, either alone or in combination with an activatable cytokine, such as an activatable interferon.

[0064] In some embodiments, the activatable anti-PD-1 or anti-PD-L1 antibody comprises an activatable antibody that specifically binds to mammalian PD-1 or PD-L1 in its activated state, the activatable antibody comprising an antibody or antigen-binding fragment thereof (AB) that specifically binds to mammalian PD-1 or anti-PD-L1, a masking moiety (MM) that inhibits binding of the AB to mammalian PD-1 or PD-L1 when the activatable antibody is in an uncleaved state, and a cleavable moiety (CM) coupled to the AB, where the CM is a polypeptide that functions as a substrate for a protease.

[0065] In some embodiments, the activatable anti-PD-1 or anti-PD-L1 antibody, in its activated state, comprises (a) an activatable antibody that specifically binds to a mammalian PD-1 or PD-L1, and (b) an antibody that specifically interferes with the binding of a natural ligand of PD-1 to the mammalian PD-1, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a mammalian PD-1 or anti-PD-L1, a masking moiety (MM) that inhibits binding of the AB to the mammalian PD-1 or PD-L1 when the activatable antibody is in an uncleaved state, and a cleavable moiety (CM) linked to the AB, where the CM is a polypeptide that functions as a substrate for a protease.

[0066] In some embodiments, the uncleaved activatable antibody may be present in a concentration range of 0.5 nM to 1 nM, 0.5 nM to 2 nM, 0.5 nM to 5 nM, 0.5 nM to 10 nM, 0.5 nM to 15 nM, 0.5 nM to 20 nM, 0.5 nM to 25 nM, 0.5 nM to 50 nM, 0.5 nM to 75 nM, 0.5 nM to 100 nM, 0.5 nM to 150 nM, 0.5 nM to 200 nM, 0.5 nM to 300 nM, 0.5nM~400nM, 1nM~2nM, 1nM~5nM, 1nM~10nM, 1nM~15nM, 1nM~20nM, 1nM~25nM , 1nM~50nM, 1nM~75nM, 1nM~100nM, 1nM~150nM, 1nM~200nM, 1nM~300nM, 1n M~400nM, 2nM~5nM, 2nM~10nM, 2nM~15nM, 2nM~20nM, 2nM~25nM, 2nM~50nM, 2nM~75nM, 2nM~100nM, 2nM~150nM, 2nM~200nM, 2nM~300nM, 2nM~400nM, 5nM ~10nM, 5nM~15nM, 5nM~20nM, 5nM~25nM, 5nM~50nM, 5nM~75nM, 5nM~100nM, 5nM~150nM, 5nM~200nM, 5nM~300nM, 5nM~400nM, 10nM~15nM, 10nM~20nM, 10 nM~25nM, 10nM~50nM, 10nM~75nM, 10nM~100nM, 10nM~150nM, 10nM~200nM, 10nM~300nM, 10nM~400nM, 15nM~20nM, 15nM~25nM, 15nM~50nM, 15nM~75nM, 15nM~100nM, 15nM~150nM, 15nM~200nM, 15nM~300nM, 15nM~400nM, 20nM~2 5nM, 20nM~50nM, 20nM~75nM, 20nM~100nM, 20nM~150nM, 20nM~200nM, 20nM~ 300nM, 20nM~400nM, 25nM~50nM, 25nM~75nM, 25nM~100nM, 25nM~150nM, 25 nM~200nM, 25nM~300nM, 25nM~400nM, 50nM~75nM, 50nM~100nM, 50nM~150nM , 50nM-200nM, 50nM-300nM, 50nM-400nM, 75nM-100nM, 75nM-150nM, 75nM-200nM, 75nM-300nM, 75nM-400nM, 100nM-150nM, 100nM-200nM, 100nM-300nM, 100nM-400nM, 150nM-200nM, 150nM-300nM, 150nM-400nM, 200nM-300nM, 200nM-400nM, or 300nM-400nM.

[0067] In some embodiments, an activatable antibody in an activated state has a concentration of 0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.1 nM to 5 nM, 0.2 nM to 5 nM, 0.3 nM to 5 nM, 0.4 nM to 5 nM, 0.5 nM to 5 nM, 0.75 nM to 5 nM, 1 nM to 5 nM, 2 nM to 5 nM, 0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.1 nM to 2 nM, 0.2 nM to 2 nM, 0.3 nM to 5 nM, M~2nM, 0.4nM~2nM, 0.5nM~2nM, 0.75nM~1nM, 1nM~2nM, 0.01nM~1nM, 0.05nM~1nM, 0.1nM~1nM, 0.2nM~1nM, 0.3nM~1nM, 0.4nM~1nM, 0.5nM~1nM, 0.75nM~1nM, 0.01nM~0.75nM, 0.05nM~0.75nM, 0.1nM~0.75nM, 0.2nM ~0.75nM, 0.3nM~0.75nM, 0.4nM~0.75nM, 0.5nM~0.75nM, 0.01nM~0.5nM, 0.05nM~0.5nM, 0.1nM~0.5nM, 0. 2nM~0.5nM, 0.3nM~0.5nM, 0.4nM~0.5nM, 0.01nM~0.4nM, 0.05nM~0.4nM, 0.1nM~0.4nM, 0.2nM~0.4nM, 0.3 and specifically binds to mammalian PD-1 or PD-L1 with a dissociation constant of 0.4 nM to 0.4 nM, 0.01 nM to 0.3 nM, 0.05 nM to 0.3 nM, 0.1 nM to 0.3 nM, 0.2 nM to 0.3 nM, 0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM, 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or 0.01 nM to 0.05 nM.

[0068] In some embodiments, the activatable antibody is at least 0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.1 nM to 5 nM, 0.2 nM to 5 nM, 0.3 nM to 5 nM, 0.4 nM to 5 nM, 0.5 nM to 5 nM, 0.75 nM to 5 nM, 1 nM to 5 nM, 2 nM to 5 nM, 0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.1 nM to 2 nM, 0.2 nM to 2 nM, 0.3 nM to 2 nM, 0 .4nM~2nM, 0.5nM~2nM, 0.75nM~1nM, 1nM~2nM, 0.01nM~1nM, 0.05nM~1nM, 0.1nM~1nM, 0.2nM~1nM, 0.3nM~ 1nM, 0.4nM~1nM, 0.5nM~1nM, 0.75nM~1nM, 0.01nM~0.75nM, 0.05nM~0.75nM, 0.1nM~0.75nM, 0.2nM~0.75n M, 0.3nM~0.75nM, 0.4nM~0.75nM, 0.5nM~0.75nM, 0.01nM~0.5nM, 0.05nM~0.5nM, 0.1nM~0.5nM, 0.2nM~0 .5nM, 0.3nM~0.5nM, 0.4nM~0.5nM, 0.01nM~0.4nM, 0.05nM~0.4nM, 0.1nM~0.4nM, 0.2nM~0.4nM, 0.3nM~0. and an AB that specifically binds to mammalian PD-1 or PD-L1 with a dissociation constant of 4 nM, 0.01 nM to 0.3 nM, 0.05 nM to 0.3 nM, 0.1 nM to 0.3 nM, 0.2 nM to 0.3 nM, 0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM, 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or 0.01 nM to 0.05 nM.

[0069] In some embodiments, the mammalian PD-1 or PD-L1 is selected from the group consisting of human PD-1 or human PD-L1 and cynomolgus PD-1 or cynomolgus PD-L1. In some embodiments, the AB specifically binds to human PD-1 or PD-L1 or cynomolgus PD-1 or PD-L1 with a dissociation constant of 1 nM or less. In some embodiments, the mammalian PD-1 or PD-L1 is human PD-1 or PD-L1. In some embodiments, the AB has one or more properties selected from the group consisting of: (a) the AB specifically binds to human PD-1 or PD-L1 and cynomolgus PD-1 or PD-L1; (b) the AB inhibits the binding of human PDL1 and human PDL2 to human PD-1; and (c) the AB inhibits the binding of cynomolgus PDL1 and cynomolgus PDL2 to cynomolgus PD-1.

[0070] In some embodiments, the mammalian PD-1 or PD-L1 is mouse PD-1 or PD-L1. In some embodiments, the activatable antibody comprises an AB that specifically binds to mouse PD-1 or PD-L1, or inhibits binding of mouse PDL1 and mouse PDL2 to mouse PD1.

[0071] In some embodiments, an uncleaved activatable antibody specifically binds to a mammalian PD-1 or PD-L1 with a dissociation constant of 0.5 nM or more, 1 nM or more, 2 nM or more, 3 nM or more, 4 nM or more, 5 nM or more, 10 nM or more, 15 nM or more, 20 nM or more, 25 nM or more, 50 nM or more, 75 nM or more, 100 nM or more, 150 nM or more, 200 nM or more, 300 nM or more, and / or 400 nM or more.

[0072] In some embodiments, the activatable antibody, when in an activated state, specifically binds to a mammalian PD-1 or PD-L1 with a dissociation constant of 0.01 nM or less, 0.05 nM or less, 0.1 nM or less, 0.2 nM or less, 0.3 nM or less, 0.4 nM or less, 0.5 nM or less, 0.75 nM or less, and 1 nM or less.

[0073] In some embodiments, the activatable antibody comprises an AB that specifically binds to a mammalian PD-1 or PD-L1 with a dissociation constant of 0.01 nM or less, 0.05 nM or less, 0.1 nM or less, 0.2 nM or less, 0.3 nM or less, 0.4 nM or less, 0.5 nM or less, 0.75 nM or less, and 1 nM or less.

[0074] In some embodiments, the activatable antibody is at a concentration of 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, 0.1 nM to 1 nM, 0.1 nM to 0.5 nM, 0.1 nM to 0.25 nM, 0.25 nM to 10 nM, 0.25 nM to 5 nM, 0.25 nM to 3 nM, 0.25 nM to 2 nM, 0.25 nM to 1 nM, 0.25nM~0.5nM, 0.5nM~10nM, 0.5nM~5nM, 0.5nM~3nM, 0.5nM~2nM, 0.5nM~1nM, 1nM~10nM, 1nM EC of ~5nM, 1nM~3nM, 1nM~2nM, 2nM~10nM, 2nM~5nM, 2nM~3nM, 3nM~10nM, 3nM~5nM, or 5nM~10nM 50 The present invention includes an AB that interferes with the ability of a natural ligand to bind to mammalian PDL1 having the structure

[0075] In some embodiments, the natural ligand is a mammalian PDL1 or a mammalian PDL2. In some embodiments, the natural ligand is selected from the group consisting of human PDL1, human PDL2, cynomolgus monkey PDL1, and cynomolgus monkey PDL2.

[0076] An activatable antibody binds to PD-1 or PD-L1 in its activated state and comprises (i) an antibody or antigen-binding fragment thereof (AB) that specifically binds to PD-1 or PD-L1, (ii) a masking moiety (MM) that inhibits the binding of the AB to PD-1 or PD-L1 when the activatable antibody is in an uncleaved state, and (c) a cleavable moiety (CM) coupled to the AB, where the CM is a polypeptide that functions as a substrate for a protease.

[0077] In some embodiments, the activatable PD-1 or PD-L1 antibody, when uncleaved, has the following structural configuration from N-terminus to C-terminus: MM-CM-AB, or AB-CM-MM.

[0078] In some embodiments, the activatable PD-1 or PD-L1 antibody comprises a linking peptide between the MM and CM.

[0079] In some embodiments, the activatable PD-1 or PD-L1 antibody comprises a CM as defined herein. In some embodiments, the activatable PD-1 or PD-L1 antibody comprises a linking peptide between the CM and the AB.

[0080] In some embodiments, an activatable PD-1 or PD-L1 antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the uncleaved activatable antibody has the following structural arrangement, from N-terminus to C-terminus: MM-LP1-CM-LP2-AB, or AB-LP2-CM-LP1-MM. In some embodiments, the two connecting peptides need not be identical to each other. In some embodiments, LP1 and LP2 are each a peptide about 1-20 amino acids in length.

[0081] In some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 227) and (GGGS) n (SEQ ID NO:228), where n is an integer of at least 1.

[0082] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 229), GGSGG (SEQ ID NO: 230), GSGSG (SEQ ID NO: 231), GSGGG (SEQ ID NO: 232), GGGSG (SEQ ID NO: 233), and GSSSG (SEQ ID NO: 234).

[0083] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:541), GSSGGSGGSGG (SEQ ID NO:210), GSSGGSGGSGGS (SEQ ID NO:542), GSSGGSGGSGGSGGGS (SEQ ID NO:588), GSSGGSGGSG (SEQ ID NO:543), GSSGGSGGSGS (SEQ ID NO:544), GGGSSGGS (SEQ ID NO:545), or GGGSSGG (SEQ ID NO:546).

[0084] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:2), GSSGT (SEQ ID NO:548) or GSSG (SEQ ID NO:549).

[0085] In some embodiments, the activatable antibody also comprises a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of a signal peptide. In some embodiments, the spacer is directly conjugated to the MM of the activatable antibody. In some embodiments, the spacer is directly conjugated to the MM of the activatable antibody from N-terminus to C-terminus in the structural arrangement spacer-MM-CM-AB.

[0086] In some embodiments, the activatable anti-PD-1 antibody comprises a heavy chain comprising, or derived from, an amino acid sequence selected from the group consisting of SEQ ID NOs: 610-614 and 620-628, and a light chain comprising, or derived from, an amino acid sequence selected from the group consisting of SEQ ID NOs: 615-619 and 629-639. In some aspects, the present disclosure includes the activatable anti-PD-1 antibodies disclosed in WO2017011580, which is incorporated herein by reference in its entirety.

[0087] In some embodiments, the activatable anti-PD-1 antibody is selected from the group consisting of AMSGCSWSAFCPYLA (SEQ ID NO: 550), DVNCAIWYSVCITVP (SEQ ID NO: 551), LVCPLYALSSGVCMG (SEQ ID NO: 552), SVNCRIWSAVCAGYE (SEQ ID NO: 553), MLVCSLQPTAMCERV (SEQ ID NO: 554), APRCYMFASYCKSQY (SEQ ID NO: 555), VGPCELTPKPVCNTY (SEQ ID NO: 556), ETCNQYERSSGLCFA (SEQ ID NO: 557), A PRTCYTYQCSSFYT (SEQ ID NO: 558), GLCSWYLSSSGLCVD (SEQ ID NO: 559), VPWCQLTPRVMCMWA (SEQ ID NO: 560), NWLDCQFYSECSVYG (SEQ ID NO: 561), SCPLYVMSSFGGCWD (SEQ ID NO: 562), MSHCWMFSSSCDGVK (SEQ ID NO: 563), VSYCTWLIEVICLRG (SEQ ID NO: 564), VLCAAYALSSGICGG (SEQ ID NO: 565), TTCNLYQQSSMFCNA (SEQ ID NO: 566), APRC YMFASYCKSQY (SEQ ID NO: 567), PCDQNPYFYPYVCHA (SEQ ID NO: 568), SVCPMYALSSMLCGA (SEQ ID NO: 569), LSVECYVFSRCSSLP (SEQ ID NO: 570), FYCTYLVSLTCHPQ (SEQ ID NO: 571), SMAGCQWSSFCVQRD (SEQ ID NO: 572), IYSCYMFASRCTSDK (SEQ ID NO: 573), SRCSVYEVSSGLCDW (SEQ ID NO: 574), GMCSAYAYSSKLCTI (SEQ ID NO: 575), MTTNTCN The masking portion (MM) includes an amino acid sequence selected from the group consisting of LLCQQFLT (SEQ ID NO: 576), FQPCLMFASSCFTSK (SEQ ID NO: 577), WNCHPAGVGPVFCEV (SEQ ID NO: 578), ALCSMYLASSGLCNK (SEQ ID NO: 579), NYLSCQFFQNCYETY (SEQ ID NO: 580), GWCLFSDMWLGLCSA (SEQ ID NO: 581), EFCARDWLPYQCSSF (SEQ ID NO: 582), and TSYCSIEHYPCNTHH (SEQ ID NO: 583).

[0088] In some embodiments, the activatable anti-PD-1 antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 610-614 and 620-628, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 615-619 and 629-639.

[0089] In some embodiments, the activatable anti-PD-1 antibody comprises (a) a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 487, and 642-645; (b) a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 488, and 646-650; (c) a variable heavy chain complementarity determining region 3 (VH CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 489, and 652-655; (d) a variable light chain complementarity determining region 1 (VL CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 656-663; (e) a variable light chain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 491, and 664-666; and (f) a variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 683-687.

[0090] In some embodiments, an activatable anti-PD-1 antibody comprises a combination of a variable heavy chain complementarity determining region 1 (VHCDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VHCDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VHCDR3, also referred to herein as CDRH3) sequence, wherein the VHCDR1 sequence comprises GITFSNSG (SEQ ID NO:525), the VHCDR2 sequence comprises IWYDGSKR (SEQ ID NO:526), ​​and the VHCDR3 sequence comprises TNDDY (SEQ ID NO:527).

[0091] In some embodiments, an activatable anti-PD-1 antibody comprises a combination of a variable light chain complementarity determining region 1 (VLCDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VLCDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VLCDR3, also referred to herein as CDRL3) sequence, wherein the VLCDR1 sequence comprises QSVSSY (SEQ ID NO:528), the VLCDR2 sequence comprises DAS, and the VLCDR3 sequence comprises QQSSNWPRT (SEQ ID NO:529).

[0092] In some embodiments, an activatable anti-PD-1 antibody comprises a combination of a variable heavy chain complementarity determining region 1 (VHCDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VHCDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VHCDR3, also referred to herein as CDRH3) sequence, wherein the VHCDR1 sequence comprises GYTFTNYY (SEQ ID NO:530), the VHCDR2 sequence comprises INPSNGGT (SEQ ID NO:531), and the VHCDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO:532).

[0093] In some embodiments, an activatable anti-PD-1 antibody comprises a combination of a variable light chain complementarity determining region 1 (VLCDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VLCDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VLCDR3, also referred to herein as CDRL3) sequence, wherein the VLCDR1 sequence comprises KGVSTSGYSY (SEQ ID NO:533), the VLCDR2 sequence comprises LAS, and the VLCDR3 sequence comprises QHSRDLPLT (SEQ ID NO:534).

[0094] In some embodiments, the activatable anti-PD-L1 antibody comprises a heavy chain that comprises, or is derived from, an amino acid sequence selected from the group consisting of SEQ ID NOs: 673-694, and a light chain that comprises, or is derived from, an amino acid sequence selected from the group consisting of SEQ ID NO: 671 or SEQ ID NO: 672. In some aspects, the disclosure includes the activatable anti-PD-L1 antibodies disclosed in WO2016 / 149201, which is incorporated herein by reference in its entirety.

[0095] In some embodiments, the activatable anti-PD-L1 antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:673-694, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:671 or SEQ ID NO:672.

[0096] In some embodiments, the activatable anti-PD-L1 antibody comprises a combination of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 sequences, wherein at least one CDR sequence is selected from the group consisting of a VLCDR1 sequence comprising RASQSISSYLN (SEQ ID NO:535), a VLCDR2 sequence comprising AASSLQS (SEQ ID NO:536), a VLCDR3 sequence comprising DNGYPST (SEQ ID NO:537), a VHCDR1 sequence comprising SYAMS (SEQ ID NO:538), a VHCDR2 sequence comprising SSIWRNGIVTVYADS (SEQ ID NO:539), and a VHCDR3 sequence comprising WSAAFDY (SEQ ID NO:540).

[0097] In some embodiments, the activatable anti-PD-L1 antibody is YCEVSELFVLPWCMG (SEQ ID NO:584), SCLMHPHYAHDYCYV (SEQ ID NO:585), LCEVLMLLQHPWCMG (SEQ ID NO:586), IACRHFMEQLPFCHH (SEQ ID NO:587), FGPRCGEASTCVPYE (SEQ ID NO:588), LYCDSWGAGCLTRP (SEQ ID NO:589), GIALCPSHFCQLPQT (SEQ ID NO:590), DGPRCFVSGECSPIG (SEQ ID NO:591), LCYKLDYDDRSYCHI (SEQ ID NO:592), PCHPHPYDARPYCNV (SEQ ID NO:593), PCYWHPFFAYRYCNT (SEQ ID NO:594), VCYYMDWLGRNWCSS (SEQ ID NO:595), LCDLFKLREFPYCMG( SEQ ID NO: 596), YLPCHFVPIGACNNK (SEQ ID NO: 597), FCHMGVVVPQCANY (SEQ ID NO: 598), ACHPHPYDARPYCNV (SEQ ID NO: 599), PCHPAPYDARPYCNV (SEQ ID NO: 600), PCHPHAYDARPYCNV (SEQ ID NO: 601), PCHPHPADARPYCNV (SEQ ID NO: 602), PCHPHPYAARPYCNV (SEQ ID NO: 603), PCHPHPYDAAPYCNV (SEQ ID NO: 604), PCHPHPYDARPACNV (SEQ ID NO: 605), PCHPHPYDARPYCAV (SEQ ID NO: 606), PCHAHPYDARPYCNV (SEQ ID NO: 607), and PCHPHPYDARAYCNV (SEQ ID NO: 608).

[0098] Provided herein is a composition comprising any one of the ACCs described herein.In some embodiments, the composition is a pharmaceutical composition.Also provided herein is a kit comprising at least one dose of any one of the compositions described herein.

[0099] Provided herein is a composition comprising any one of the ACCs described herein and a PD-1 or PD-L1 antibody. Provided herein is a composition comprising any one of the ACCs described herein and an activatable PD-1 or PD-L1 antibody. Also provided herein is a kit comprising at least one dose of any one of the ACCs described herein and at least one dose of a PD-1 or PD-L1 antibody, or a PD-1 activatable antibody or a PD-L1 activatable antibody.

[0100] Provided herein is a method of treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of any one of the ACCs described herein or any one of the compositions described herein, with or without a PD1 / PD-L1 inhibitor selected from a PD-1 antibody, an activatable PD-1 antibody, a PD-L1 antibody, or an activatable PD-L1 antibody. In some embodiments, the subject has been identified or diagnosed as having cancer. In some non-limiting embodiments, the cancer is Kaposi's sarcoma, hairy cell leukemia, chronic myelogenous leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, bladder cancer, breast cancer, colorectal cancer, malignant cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), colon cancer, kidney cancer, ovarian cancer, pancreatic cancer. In some non-limiting embodiments, the cancer is a carcinoma. In some non-limiting embodiments, the cancer is a sarcoma. In some non-limiting embodiments, the cancer is lymphoma. In some non-limiting embodiments, the lymphoma is Burkitt's lymphoma.

[0101] Provided herein is a nucleic acid encoding a polypeptide comprising CP1 and CM1 of any one of the ACCs described herein. In some embodiments, the polypeptide further comprises any one of DD1 described herein. In some embodiments, the polypeptide further comprises any one of PM1 and CM3 described herein. Also provided herein is a nucleic acid encoding a polypeptide comprising CP2 and CM2 of any one of the ACCs described herein. When the monomers are identical, the present disclosure provides a single nucleic acid encoding the monomers that dimerize to form the ACC. In some embodiments, the polypeptide further comprises any one of DD2 described herein. In some embodiments, the polypeptide further comprises any one of PM2 and CM4 described herein. In certain embodiments, the first monomeric construct and the second monomeric construct comprise the same CP, CM and DD components. In some of these embodiments, the first monomeric construct and the second monomeric construct are encoded by the same polypeptide (i.e., the same amino acid sequence). In many cases, when the first monomeric construct and the second monomeric construct comprise the same amino acid sequence, they are encoded by the same nucleic acid (i.e., the same nucleic acid sequence). In some of these embodiments, the first monomeric construct and the second monomeric construct are encoded by the same nucleic acid. Also provided herein is a vector comprising any one of the nucleic acids described herein. In some embodiments, the vector is an expression vector. Also provided herein is a cell comprising any one of the nucleic acids described herein or any one of the vectors described herein.

[0102] Provided herein is a pair of nucleic acids that together encode a polypeptide comprising CP1 and CM1 of a first monomeric construct and a polypeptide comprising CP2 and CM2 of a second monomeric construct of any one of the ACCs described herein. Also provided herein is a pair of nucleic acids that together encode a polypeptide comprising PM1, CM3, CP1 and CM1 of a first monomeric construct and a polypeptide comprising PM2, CM4, CP2 and CM2 of a second monomeric construct of any one of the ACCs described herein. Also provided herein is a pair of vectors that together comprise any one of the pairs of nucleic acids described herein. In some embodiments, the vector pair is an expression vector pair. Also provided herein is a cell that comprises any one of the pairs of nucleic acids described herein or any one of the pairs of vectors described herein. In other embodiments, the invention provides a vector that comprises a pair of vectors.

[0103] Provided herein is a method of producing ACC, comprising culturing any one of the cells described herein in a liquid medium under conditions sufficient to produce ACC, and recovering the ACC from the cells or liquid medium. In some embodiments, the method further comprises isolating the ACC recovered from the cells or liquid medium. In some embodiments, the method further comprises formulating the isolated ACC into a pharmaceutical composition. In some embodiments, the method further comprises formulating the isolated ACC and a PD1 / PD-L1 inhibitor selected from a PD-1 antibody, an activatable PD-1 antibody, a PD-L1 antibody, or an activatable PD-L1 antibody into a pharmaceutical composition.

[0104] Provided herein is an ACC made by any one of the methods described herein.Also provided herein is a composition comprising any one of the ACCs described herein, with or without a PD1 / PD-L1 inhibitor selected from a PD-1 antibody, an activatable PD-1 antibody, a PD-L1 antibody, or an activatable PD-L1 antibody.Also provided herein is a composition of any one of the compositions described herein, wherein the composition is a pharmaceutical composition.Also provided herein is a kit comprising at least one dose of any one of the compositions described herein.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. This specification describes methods and materials for use herein. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0106] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

[0107] The terms "a" and "an" refer to one or more (i.e., to at least one) of the grammatical object of the article. As an example, "a cell" includes one or more cells.

[0108] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, refer not only to the numerical value but also to reasonable deviations from that value known to those of skill in the art, e.g., ±20%, ±10%, or ±5%, where appropriate, within the intended meaning of the recited value.

[0109] Concentrations, amounts, and other numerical data may be expressed or presented in a range format herein. It should be understood that such range formats are used for convenience and brevity only, and thus should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limitations, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. As an illustration, a numerical range of "about 0.01 to 2.0" should be interpreted to include not only the explicitly recited values ​​of about 0.01 to about 2.0, but also the individual numerical values ​​and subranges within the stated range. Thus, within this numerical range, individual values, such as 0.5, 0.7, and 1.5, as well as subranges, such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5, are included. Moreover, such interpretation should be applied regardless of the breadth or characteristics of the range described. In addition, it should be noted that all percentages are by weight unless otherwise indicated.

[0110] In understanding the scope of the present disclosure, the terms "including" or "comprising" and their derivatives, as used herein, are intended to be open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The above also applies to words of similar meaning, such as the terms "including" and "having" and their derivatives. The term "consisting" and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The term "consisting essentially of," as used herein, is intended to specify the presence of the described features, elements, components, groups, integers, and / or steps, as well as the presence of features, elements, components, groups, integers, and / or steps that do not substantially affect the basic and novel characteristic(s). Reference to any one of these transitional phrases (i.e., "comprising," "consisting," or "consisting essentially") is understood to provide direct support for the replacement of any of the other transitional phrases not specifically used. For example, the modification of the term "comprising" to "consisting essentially of" or "consisting of" finds direct support for any element disclosed throughout this disclosure to be so defined. Based on this definition, any element disclosed or incorporated by reference herein may be included or excluded from the claimed invention.

[0111] As used herein, for convenience, a plurality of compounds, elements, or steps may be presented in general lists. However, these lists should be construed as if each member of the list were individually identified as a separate and unique member. Thus, the individual members of such lists should not be construed as being in effect equal to any other member of the same list solely based on presentation in a general group, unless otherwise indicated.

[0112] Furthermore, certain molecules, constructs, compositions, elements, moieties, excipients, diseases, conditions, properties, steps, etc. may be discussed in the context of a particular embodiment or aspect of this disclosure, or in a separate paragraph or section. This is merely for convenience and brevity, and it is understood that any such disclosure is equally applicable and intended to be combined with any other embodiment or aspect found anywhere in this disclosure and claims, all of which form the application and claimed invention as of the filing date. For example, a listing of a construct, molecule, method step, kit, or composition described with respect to a construct, composition, or method is intended to find direct support for the relevant embodiment of the construct, composition, formulation, and method described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.

[0113] Unless otherwise specified, a "nucleic acid sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and therefore encode the same amino acid sequence.

[0114] The term "located at the N-terminus," when referring to the position of a first domain or sequence in the primary amino acid sequence of a polypeptide relative to a second domain or sequence, means that the first domain or sequence is located closer to the N-terminus of the primary amino acid sequence of the polypeptide than the second domain or sequence. In some embodiments, there may be additional sequences and / or domains between the first and second domains or sequences.

[0115] The term "located at the C-terminus," when referring to the position of a first domain or sequence in the primary amino acid sequence of a polypeptide relative to a second domain or sequence, means that the first domain or sequence is located closer to the C-terminus of the primary amino acid sequence of the polypeptide than the second domain or sequence. In some embodiments, there may be additional sequences and / or domains between the first and second domains or sequences.

[0116] The term "exogenous" means any substance introduced or derived from outside a cell, tissue or organism and that is not produced by or derived from the same cell, tissue or organism into which it is introduced.

[0117] The terms "transduced," "transfected," or "transformed" refer to the process by which exogenous nucleic acid is introduced or transferred into a cell. A "transduced," "transfected," or "transformed" cell (e.g., a mammalian cell) is a cell that has been transduced, transfected, or transformed with an exogenous nucleic acid (e.g., a vector) that contains an exogenous nucleic acid encoding any of the activatable cytokine constructs described herein.

[0118] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or combinations thereof, in single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the referenced nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses complementary sequences in addition to the sequence explicitly indicated. In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.

[0119] Modifications to the nucleotide sequence can be introduced by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with acidic side chains (e.g., aspartate and glutamate), amino acids with basic side chains (e.g., lysine, arginine and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine and threonine), hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine). Other families of amino acids include the aliphatic hydroxyamino acids (e.g., serine and threonine), the amide family (e.g., asparagine and glutamine), the aliphatic family (e.g., alanine, valine, leucine and isoleucine), and the aromatic family (e.g., phenylalanine, tryptophan and tyrosine).

[0120] As used herein, the phrases "specifically bind" or "immunoreact with" refer to an activatable antigen-binding protein complex that reacts with one or more antigenic determinants of a desired target antigen and does not react with other polypeptides, or does so with very low affinity, e.g., about 10 -6 It means that it bonds with M or more.

[0121] The term "treatment" means improving at least one symptom of a disease. In some embodiments, the disease being treated is cancer, and is for the purpose of alleviating at least one symptom of the cancer. [Brief description of the drawings]

[0122] [Figure 1] FIG. 1 is a schematic diagram of an exemplary activatable cytokine construct. [Diagram 2] FIG. 1 is a schematic diagram of an exemplary activatable cytokine construct. [Diagram 3] FIG. 1 is a schematic diagram of an exemplary activatable cytokine construct. [Figure 4] FIG. 1 is a schematic diagram of an exemplary activatable cytokine construct. [Figure 5A] 1 shows the cleavage reaction of the peptide unmasked cytokine construct, IFNα-2b-hIgG4 Fc (with cleavable moiety 1204dL or cleavable moiety 1490) with a protease (uPA or MT-SP1), which generates monomeric mature IFNα-2b. [Figure 5B] 1 shows the cleavage reaction of the peptide unmasked cytokine construct, IFNα-2b-hIgG4 Fc (with cleavable moiety 1204dL or cleavable moiety 1490) with a protease (uPA or MT-SP1), which generates monomeric mature IFNα-2b. [Figure 6A] 6B shows the activation of a cytokine component (ProC440) by the proteases uPa and MMP14. ProC440 in FIG. 6B has the sequence of SEQ ID NO: 286. [Figure 6B] 6B shows the activation of a cytokine component (ProC440) by the proteases uPa and MMP14. ProC440 in FIG. 6B has the sequence of SEQ ID NO: 286. [Figure 6C] 6B shows the activation of a cytokine component (ProC440) by the proteases uPa and MMP14. ProC440 in FIG. 6B has the sequence of SEQ ID NO: 286. [Figure 7A] Figure 2 shows the activity of a cytokine construct (ProC440) tested in vitro using IFN-responsive HEK293 cells. [Figure 7B] Figure 2 shows the activity of a cytokine construct (ProC440) tested in vitro using Daudi cells. [Figure 8] The sequence of the masked cytokine construct, ProC732, is shown, with the optional signal sequence in italics, the masking peptide sequence in double underline, the sequence of the cleavable portion in bold, and the sequence of mature IFNα-2b in underline. [Figure 9] Shown is the sequence of a masked cytokine construct, ProC733, with no cleavable subsequence between the cytokine and dimerization domains, with the optional signal sequence in italics, the masking peptide sequence in double underline, the cleavable subsequence in bold, and the sequence of mature IFNα-2b underlined. [Figure 10A] Schematic diagrams of ProC440, ProC732 ​​and ProC733 are shown. [Figure 10B] Figure 2 shows the activity of cytokine constructs (ProC440, ProC732 ​​and ProC733) tested using IFN-responsive HEK293 cells. [Figure 11A]Schematic diagram of the structure of the cytokine construct ProC286 and its activity compared to that of Sylatron® (pegylated interferon α-2b) in the Daudi apoptosis assay. ProC286 and Sylatron® showed similar levels of activity, suggesting that ProC286 could be used as a surrogate for the Sylatron® control to assess the tolerability of IFNα-2b in hamster studies. [Figure 11B] 1 shows a schematic diagram of the structure of ProC291 and its activity in the Daudi apoptosis assay compared to that of Sylatron®. ProC291 showed significantly reduced activity compared to Sylatron® and ProC286. [Figure 12] Shown are the specific activities of an IFNa control (recombinant interferon alpha, a non-naturally occurring type I interferon); the active cytokine cleavage product of ProC440 (ProC440+uPA); Sylatron® ("PEG-IFNa2b"); and ProC440, as well as predicted toxic doses in an in vivo dose escalation study, e.g., dose escalation studies of 0.08, 0.4, 2, 10 and 15 mg / kg ("mpk"). [Figure 13A] 1 shows the weight loss profile of animals in response to different doses of cytokine constructs ProC286, ProC440 and ProC732 ​​or a control (human IgG4) in a different dose tolerability study conducted in golden Syrian hamsters. Data is shown for a dose of 2 mg / kg ("2mpk") for each construct tested. [Figure 13B] Figure 1 shows the weight loss profile of animals in response to different doses of cytokine constructs ProC286, ProC440 and ProC732 ​​or a control (human IgG4) in a different dose tolerability study conducted in golden Syrian hamsters. Data are shown for a dose of 10 mg / kg for each construct tested. [Figure 13C]Figure 1 shows the weight loss profile of animals in response to different doses of cytokine constructs ProC286, ProC440 and ProC732 ​​or a control (human IgG4) in a different dose tolerability study conducted in golden Syrian hamsters. Data are shown for a dose of 15 mg / kg for each construct tested. [Figure 13D] 1 shows IFNa2b-mediated toxicity in animals dosed with unmasked IFNa2b / Fc, which corresponds to an increase in ALP and an increase in the therapeutic index of IFNa2b single-masked and double-masked. [Figure 14] FIG. 1 shows the results of clinical chemistry analyses (alkaline phosphatase, alanine transaminase, and aspartate transaminase) in Golden Syrian hamsters in response to different doses (2 mpk, 10 mpk, and 15 mpk) of the cytokine constructs ProC286, ProC440, and ProC732 ​​or a control (human IgG4) in a tolerability study. [Figure 15] FIG. 1 shows the results of hematology analyses (reticulocyte count, neutrophil count and white blood cell (WBC) count) in Golden Syrian hamsters in response to different doses (2 mpk, 10 mpk and 15 mpk) of the cytokine constructs ProC286, ProC440 and ProC732 ​​or a control (human IgG4) in a tolerability study. [Figure 16] FIG. 1 shows the effect of the length of the linking region (LR) on the activity of unmasked IFNα-2b-Fc fusion proteins as determined from a Daudi apoptosis assay. [Figure 17] Schematic representation of the cytokine construct without the peptide mask, including delineation of the linking region (LR). [Figure 18A] Figure 1 shows the antitumor activity of masked activatable IFNaA / D (ProC1023) at 10, 50, and 200 μg. [Figure 18B] FIG. 1 shows the in vivo activation of masked IFNaA / D(ProC1023) versus uncleaved masked IFNaA / D(ProC1549). [Figure 18C]The anti-tumor activity of masked IFNaA / D (ProC1023) in combination with a PD-L1 monoclonal antibody (CX-171) is shown compared to masked IFNaA / D (ProC1023) alone and compared to a PD-L1 monoclonal antibody (CX-171) alone. [Figure 19] Shown is immune memory in response to MC38 tumor cell rechallenge in mice previously treated with activated IFNaA / D (200 micrograms of ProC1023) (Figure 19B, bottom) compared with MC38 tumor cell challenge in untreated control mice (Figure 19A, top). [Figure 20] The effect of combining Pro-IFN-a2b with PD-L1 monoclonal antibody on IFN-γ release in patient tissues is shown compared to masked IFN-a2b, unmasked IFN-a2b, Peg-IFN-a2b alone, PD-L1 monoclonal antibody alone, and control in patient PBMCs (left) and patient dissociated tumor cells (right). [Figure 21] FIG. 1 shows activation-dependent induction of a type I interferon signature by unmasked IFN-a2b. [Figure 22] Figure 1 shows the pharmacokinetics of doubly masked IFN-a2b (ProC732) and control molecules in hamsters. [Figure 23] Antitumor activity of masked activatable IFNaA / D at 20 μg and 200 μg compared to control. [Figure 24A] The activity of ProC1023 compared to ProC859 in an IFNa reporter assay in B16 mouse melanoma cells is shown. [Figure 24B] The activity of ProC1023 compared to ProC1549 in an IFNa reporter assay in B16 mouse melanoma cells is shown. [Figure 24C] The activity of ProC1023 compared to ProC1549 in an IFNa reporter assay in B16 mouse melanoma cells is shown. [Diagram 25]1 shows the activity of ProC1239 and ProC732 ​​tested in vitro using IFN-responsive HEK293 cells. [Figure 26] The activity of ProC732, ProC1550 and ProC1552 in vitro using IFN-responsive HEK293 cells is shown in the uncleaved state and after protease activation with uPa or MTSP1. [Figure 27] The activity of recombinant IFNa2b, monomeric IFNa2b / Fc, activated homodimeric IFNa2b / Fc, and homodimeric IFNa2b / Fc in IFN-responsive HEK293 cells in the uncleaved state and after protease activation is shown. [Figure 28] Antitumor activity of increasing doses of single masked IFNa2b / Fc (top) and pegylated interferon (bottom) is shown. [Figure 29] 1 shows the structure of ACC ProC859 universal interferon (top), the antiproliferative effect of ACC ProC859 in a B16 mouse melanoma cell assay, and the activity of ACC ProC859 in an IFN-responsive HEK293 assay. [Diagram 30] CD14, CD3, PD-L1, and IFNAR1 positive cells in PBMC populations and myeloid cells from healthy donors compared to patient PBMCs and dissociated tumors are shown. [Diagram 31] The combined effect of activating IFN-a2b and PD-L1 monoclonal antibody on IFN-γ release in patient tissues is shown compared to no treatment, dual masked IFN-a2b, Silatron alone, and PD-L1 monoclonal antibody alone or dual masked IFN-a2b alone. [Diagram 32] The activity of activated and non-activated single-masked IFNa2b, and activated and non-activated double-masked IFNa2b, tested in vitro in IFN-responsive HEK293 cells in the uncleaved state and after protease activation, is shown. [Figure 33A] The antitumor activity of double-masked activatable IFNaA / D at 10 μg, 50 μg, and 200 μg is shown compared to double-masked non-activatable IFNaA / D. [Figure 33B] The antitumor activity of the combination of double-masked IFNaA / D with PD-L1 monoclonal antibody is shown compared to double-masked IFNaA / D alone or PD-L1 monoclonal antibody alone. [Diagram 34] A shows the antitumor activity of ProIFNaA / D(ProC1023) at 10, 50, and 200 μg compared to the PBS control, and B shows the antitumor activity of ProIFNaA / D(ProC1023) at 200 μg compared to IFNaA / DNSUB(ProC1549). [Diagram 35] A shows the antitumor activity of ProIFNaA / D (ProC1023) at 10, 50, and 200 μg compared to 200 μg PD-L1 monoclonal antibody (CX-171).B shows the antitumor activity of IFNaA / DNSUB (ProC1549) at 50 μg and 200 μg compared to PBS control. [Diagram 36] Schematic representation of the cytokine construct, including delineation of the linking region (LR) and masked linking region (MLR). [Figure 37] The graph shows the time course of tumor volume and survival rate of mice transplanted with syngeneic CT26 and B16 tumor models. [Figure 38A] Figure 38 shows the binding of single-masked Pb-IFN-a2b molecules to human IFNAR2. Ligands were captured on chips coated with immobilized anti-human Fc (Figures 38A-B) or anti-histidine antibodies (Figures 38C-D). IFN-a2b (ProC1640) at concentrations ranging from 25 nM to 1.5625 μM was pumped over the ligand-captured chip, and multi-cycle kinetic sensorgrams were generated (Figures 38A and 38C). Masked Pb-IFN-a2b molecules (ProC440 (Figure 38D), ProC1976 (Figure 38B)) at concentrations ranging from 250 nM to 15.625 μM were pumped over the ligand-captured chip, and multi-cycle kinetic sensorgrams were generated. [Figure 38B]Figure 38 shows the binding of single-masked Pb-IFN-a2b molecules to human IFNAR2. Ligands were captured on chips coated with immobilized anti-human Fc (Figures 38A-B) or anti-histidine antibodies (Figures 38C-D). IFN-a2b (ProC1640) at concentrations ranging from 25 nM to 1.5625 μM was pumped over the ligand-captured chip, and multi-cycle kinetic sensorgrams were generated (Figures 38A and 38C). Masked Pb-IFN-a2b molecules (ProC440 (Figure 38D), ProC1976 (Figure 38B)) at concentrations ranging from 250 nM to 15.625 μM were pumped over the ligand-captured chip, and multi-cycle kinetic sensorgrams were generated. [Figure 38C] Figure 38 shows the binding of single-masked Pb-IFN-a2b molecules to human IFNAR2. Ligands were captured on chips coated with immobilized anti-human Fc (Figures 38A-B) or anti-histidine antibodies (Figures 38C-D). IFN-a2b (ProC1640) at concentrations ranging from 25 nM to 1.5625 μM was pumped over the ligand-captured chip, and multi-cycle kinetic sensorgrams were generated (Figures 38A and 38C). Masked Pb-IFN-a2b molecules (ProC440 (Figure 38D), ProC1976 (Figure 38B)) at concentrations ranging from 250 nM to 15.625 μM were pumped over the ligand-captured chip, and multi-cycle kinetic sensorgrams were generated. [Figure 38D]Figure 38 shows the binding of single-masked Pb-IFN-a2b molecules to human IFNAR2. Ligands were captured on chips coated with immobilized anti-human Fc (Figures 38A-B) or anti-histidine antibodies (Figures 38C-D). IFN-a2b (ProC1640) at concentrations ranging from 25 nM to 1.5625 μM was pumped over the ligand-captured chip, and multi-cycle kinetic sensorgrams were generated (Figures 38A and 38C). Masked Pb-IFN-a2b molecules (ProC440 (Figure 38D), ProC1976 (Figure 38B)) at concentrations ranging from 250 nM to 15.625 μM were pumped over the ligand-captured chip, and multi-cycle kinetic sensorgrams were generated. [Figure 39A] 1 shows the restoration of NSUB(ProC649) activity by MMP. [Figure 39B] 1 shows conditional activation of ProC732 ​​and ProC1299 by uPA. [Figure 39C] IFNa2b (SEQ ID NO:1) compared to IFNaAD and ProC1301 compared to ProC732 ​​are shown to be resistant to activation. [Figure 40A] Figure 2 shows the binding of activated Pb-IFN-a2b to interferon alpha receptor in vitro. Human IFNAR1, human IFNAR2, cynomolgus IFNAR1, or cynomolgus IFNAR2 proteins were captured on a chip coated with immobilized anti-human Fc. Concentrations of activated IFN-a2b (ProC1640) ranging from 25 nM to 1.5625 μM were pumped over the ligand-captured chip to generate multi-cycle kinetic sensorgrams. [Figure 40B]Figure 2 shows the binding of activated Pb-IFN-a2b to interferon alpha receptor in vitro. Human IFNAR1, human IFNAR2, cynomolgus IFNAR1, or cynomolgus IFNAR2 proteins were captured on a chip coated with immobilized anti-human Fc. Concentrations of activated IFN-a2b (ProC1640) ranging from 25 nM to 1.5625 μM were pumped over the ligand-captured chip to generate multi-cycle kinetic sensorgrams. [Figure 40C] Figure 2 shows the binding of activated Pb-IFN-a2b to interferon alpha receptor in vitro. Human IFNAR1, human IFNAR2, cynomolgus IFNAR1, or cynomolgus IFNAR2 proteins were captured on a chip coated with immobilized anti-human Fc. Concentrations of activated IFN-a2b (ProC1640) ranging from 25 nM to 1.5625 μM were pumped over the ligand-captured chip to generate multi-cycle kinetic sensorgrams. [Figure 40D] Figure 2 shows the binding of activated Pb-IFN-a2b to interferon alpha receptor in vitro. Human IFNAR1, human IFNAR2, cynomolgus IFNAR1, or cynomolgus IFNAR2 proteins were captured on a chip coated with immobilized anti-human Fc. Concentrations of activated IFN-a2b (ProC1640) ranging from 25 nM to 1.5625 μM were pumped over the ligand-captured chip to generate multi-cycle kinetic sensorgrams. [Figure 41A] Assay of ProC732 ​​activation by tumor tissue (Figure 41A) and the results are shown. Fluorescently labeled ProC732 ​​was incubated on tumor tissue sections at 37°C. The collected solution was subsequently analyzed through capillary electrophoresis (Figure 41C) and using the HEK blue IFNA reporter model (Figure 41B), which allows quantification of active molecules. Enzymatically inactive samples were used as control tissues. [Figure 41B]Assay of ProC732 ​​activation by tumor tissue (Figure 41A) and the results are shown. Fluorescently labeled ProC732 ​​was incubated on tumor tissue sections at 37°C. The collected solution was subsequently analyzed through capillary electrophoresis (Figure 41C) and using the HEK blue IFNA reporter model (Figure 41B), which allows quantification of active molecules. Enzymatically inactive samples were used as control tissues. [Figure 41C] Assay of ProC732 ​​activation by tumor tissue (Figure 41A) and the results are shown. Fluorescently labeled ProC732 ​​was incubated on tumor tissue sections at 37°C. The collected solution was subsequently analyzed through capillary electrophoresis (Figure 41C) and using the HEK blue IFNA reporter model (Figure 41B), which allows quantification of active molecules. Enzymatically inactive samples were used as control tissues. [Figure 42A] Figure 42A shows the change in bioactivity of ProC732 ​​(Figure 42A) and recombinant IFN-a2b (Figure 42B) molecules analyzed by HEK blue IFNA reporter model after incubation with tumor tissue. The fold change in bioactivity calculated compared to the 0 h value for 10 ng / mL of ProC732 ​​or for 1 ng / mL of recombinant IFN-a2b is shown. Figure 42C shows the bioactivity of ProC732 ​​and IFN-a2b proteins incubated for 24 h in the absence of tumor tissue. Each line connects individual samples (concentrations ranging from 100 to 0.01 ng / mL) analyzed before and after 24 h of incubation. [Figure 42B] Figure 42A shows the change in bioactivity of ProC732 ​​(Figure 42A) and recombinant IFN-a2b (Figure 42B) molecules analyzed by HEK blue IFNA reporter model after incubation with tumor tissue. The fold change in bioactivity calculated compared to the 0 h value for 10 ng / mL of ProC732 ​​or for 1 ng / mL of recombinant IFN-a2b is shown. Figure 42C shows the bioactivity of ProC732 ​​and IFN-a2b proteins incubated for 24 h in the absence of tumor tissue. Each line connects individual samples (concentrations ranging from 100 to 0.01 ng / mL) analyzed before and after 24 h of incubation. [Figure 42C] Figure 42A shows the change in bioactivity of ProC732 ​​(Figure 42A) and recombinant IFN-a2b (Figure 42B) molecules analyzed by HEK blue IFNA reporter model after incubation with tumor tissue. The fold change in bioactivity calculated compared to the 0 h value for 10 ng / mL of ProC732 ​​or for 1 ng / mL of recombinant IFN-a2b is shown. Figure 42C shows the bioactivity of ProC732 ​​and IFN-a2b proteins incubated for 24 h in the absence of tumor tissue. Each line connects individual samples (concentrations ranging from 100 to 0.01 ng / mL) analyzed before and after 24 h of incubation. [Diagram 43] Figure 1 shows the pharmacokinetics of masked IFN-a2b and control molecules in non-human primates. Cynomolgus monkeys (N=2 per group) were treated with a single subcutaneous dose of 0.03 mg / kg, 0.3 mg / kg, 3 mg / kg, or 15 mg / kg ProC732. Plasma samples were collected at the indicated time points and analyzed for total ProC732 ​​concentration. [Figure 44A] Figure 1 shows the pharmacokinetics of masked IFN-a2b and control molecules in non-human primates. Cynomolgus monkeys (N=2 per group) were treated with a single subcutaneous dose of 0.03 mg / kg, 0.3 mg / kg, 3 mg / kg or 15 mg / kg ProC732. Serum IP-10 concentrations measured by MSD V-plex assay are shown. [Figure 44B] Figure 1 shows the pharmacokinetics of masked IFN-a2b and control molecules in non-human primates. Cynomolgus monkeys (N=2 per group) were treated with a single, subcutaneous dose of 0.03 mg / kg, 0.3 mg / kg, 3 mg / kg, or 15 mg / kg ProC732. Circulating Pb-IFN-a2b and IP-10 concentrations are plotted against each other on days 1 and 7 post-dose. [Diagram 45]Figure 1 shows concentration-based gene expression profile changes induced by ProC732 ​​in non-human primates. Cynomolgus monkeys (N=2 per group) were treated with a single, subcutaneous dose of 0.03mg / kg, 0.3mg / kg, 3mg / kg, or 15mg / kg ProC732. PBMCs from treated animals were harvested and analyzed by bulk RNAseq. [Diagram 46] Cynomolgus monkeys (N=2 per group) were treated with a single subcutaneous dose of 0.03mg / kg, 0.3mg / kg, 3mg / kg, or 15mg / kg ProC732. PBMCs from treated animals were harvested and analyzed by bulk RNAseq. Genes were considered differentially expressed if the change in read count was >3. [Figure 47] This shows that ProC1023 preferentially activates immune cells in tumor tissue. After 6 days of treatment, tumors and tissues were harvested and analyzed by flow cytometry. Gating was performed on viable CD45+CD3+ cells. [Figure 48] Following multiple doses of ProC732, we demonstrate that ProC732 ​​is well tolerated. Male golden Syrian hamsters (N=5) were treated with three weekly doses of intraperitoneal injections of 15, 30, or 60 mg / kg Pb-IFN-a2b (ProC732) or 3.75, 7.5, or 15 mg / kg unmasked Fc-IFN-a2b (ProC286) fusion protein. Survival outcomes include animals confirmed dead or experiencing greater than 15% weight loss. [Figure 49] We show that masking ProC732 ​​attenuates cytokine / chemokine release in non-human primates. [Figure 50] We show that double-masked Pb-IFN-a2b (ProC732) inhibits tumor growth in immune-competent rodents in vivo. Male Syrian golden hamsters (N=16) were implanted subcutaneously with 10 ml of RPMI-1846 hamster melanoma cells and administered 5, 10, or 20 mg / kg Pb-IFN-a2b intraperitoneally twice weekly. [Figure 51]Antitumor activity of increasing doses of double-masked IFN a2b / Fc and pegylated interferon. Beige / SCID mice (n=8 per group) were implanted subcutaneously with 10 mln human lymphoma (Daudi) cells and treated when the mean tumor volume reached approximately 200 mm3. Pb-IFN-a2b (ProC732), unmasked Fc-IFN-a2b (ProC286), and Peg-IFN-a2b were orally administered at the indicated doses once weekly for 3 weeks. [Figure 52] Pharmacokinetics of double masked Pb-IFN-a2b(ProC732) in beige / SCID mice. Beige / SCID mice (n=15 per group) were treated with a single dose of Pb-IFN-a2b(ProC732) at the indicated doses. Plasma for PK studies was collected at 1, 2, 3, 6, 24, 48, 72, 120 hours, 7 and 14 days post-dose. Samples were analyzed by MSD assay. [Figure 53] We show that Pb-IFN-a2b is stable in non-human primates. Cynomolgus monkeys (N=4 per group, 2 males + 2 females) were treated with 7.5, 15, 30 or 60 mg / kg Pb-IFN-a2b(ProC732) intravenously three times per week. In a satellite experiment, a single group of monkeys (N=2, 1 per sex) was treated with 30 mg / kg Pb-IFN-a2b once per week for 3 weeks. Plasma concentrations of Pb-IFN-a2b(ProC732) and its activation products were measured by LC-MS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0123] Provided herein are activatable cytokine constructs (ACCs) that exhibit reduced activity levels of at least one of the corresponding cytokines, but produce a cytokine product with substantially restored activity after exposure to activating conditions. The activatable cytokine constructs of the present invention can be designed to be selectively activated upon exposure to diseased tissues and not activated in normal tissues. Further provided herein are combination therapies or uses of the ACCs described herein in combination with a PD1 / PD-L1 inhibitor selected from a PD-1 antibody, an activatable PD-1 antibody, a PD-L1 antibody, or an activatable PD-L1 antibody. As such, these compounds have the potential to provide the benefits of cytokine-based therapy with potentially less toxicity associated with certain cytokine-based therapies. Furthermore, this combination therapy may provide the benefits of cytokine-based therapy and anti-PD1 therapy and / or anti-PD-L1 therapy with potentially less toxicity associated with the respective monotherapy and respective combination therapy that does not include the use of the ACCs disclosed herein.

[0124] Also provided herein are related intermediates, compositions, kits, nucleic acids and recombinant cells, as well as related methods, including methods of using and making any of the activatable cytokines described herein.

[0125] The inventors have surprisingly found that ACC with the specific elements and structural orientations described herein appear to be potentially effective in improving the safety and therapeutic index of cytokines, particularly in cancer therapy. While cytokines are regulators of the innate and adaptive immune systems and have broad antitumor activity in preclinical models, clinical success has been limited by systemic toxicity and poor systemic exposure to target tissues. The inventors have surprisingly found that ACC with the specific elements and structural orientations described herein appear to reduce the systemic toxicity associated with cytokine therapy and improve targeting and exposure to target tissues. Thus, the present disclosure provides a method for reducing target-mediated drug loss (TMDD) of cytokine therapy by administering ACC with the specific elements and structural orientations described herein to a subject. Thus, the present invention solves the problem that a significant proportion of the administered cytokine dose is segregated by normal tissues. This segregation is problematic because the proportion of the dose available in the systemic circulation limits the dose that reaches target tissues, such as cancerous tissues, in conventional cytokine therapy. The cytokine constructs of the invention localize target binding to tumor tissue, thereby maintaining potency, reducing side effects, enabling new targeting opportunities, improving the therapeutic window of approved targets, creating a therapeutic window for undruggable targets, and providing multiple binding modes.

[0126] The present disclosure further provides a method of administering ACC in combination with a PD1 / PD-L1 inhibitor selected from a PD-1 antibody, an activatable PD-1 antibody, a PD-L1 antibody, or an activatable PD-L1 antibody. In some embodiments, the combination of ACC and a PD1 / PDL1 inhibitor may enhance or increase the therapeutic effect and / or therapeutic index compared to conventional cytokine therapy. In some embodiments, the combination of ACC and a PD1 / PDL1 inhibitor may enhance or increase the therapeutic effect and / or therapeutic index compared to conventional PD1 / PDL1 inhibitor therapy. In some embodiments, the combination of ACC and a PD1 / PDL1 inhibitor may enhance or increase the therapeutic effect and / or therapeutic index compared to conventional cytokine and PD1 / PDL1 inhibitor combination therapy. In yet other embodiments, the combination of ACC and a PD1 / PDL1 inhibitor may enhance or increase the therapeutic effect and / or therapeutic index compared to administering the ACC of the present disclosure alone.

[0127] The present disclosure allows for safe and effective systemic delivery, thereby avoiding the dose-dependent toxicity of conventional systemic cytokine therapy and the need for intratumoral injection. The present disclosure provides a means for providing localized antiviral, immunomodulatory, antiproliferative and proapoptotic activity. The inventors have surprisingly found that by dimerizing the first and second monomeric constructs, greater reductions in cytokine activity can be achieved, and have also surprisingly found that by adding a peptide mask to the other end of the activatable construct, cytokine activity can be substantially reduced with extremely high masking efficiency. See, for example, Figures 10A-10B.

[0128] The applicant's U.S. Provisional Application No. 63 / 008,542, filed April 10, 2020, and U.S. Provisional Application No. 63 / 161,889, filed March 16, 2021, describe certain activatable cytokine constructs without affinity peptide masks, and are hereby incorporated by reference in their entireties.

[0129] Activatable cytokine constructs and activatable antibodies The activatable cytokine construct (ACC) of the present invention is a dimeric complex comprising a first monomeric construct and a second monomeric construct. The dimerization of the monomeric constructs is promoted by a pair of dimerization domains. In one embodiment, each monomeric construct comprises a cytokine protein (CP), one or more cleavable moieties (CM), a dimerization domain (DD), and a peptide mask (PM). The inventors of the present invention have unexpectedly found that the ACC structure comprising the dimerization domain and the peptide mask has improved masking efficiency, minimizing or eliminating the off-target effects and undesirable activities and / or toxic side effects of the cytokine.

[0130] In one particular embodiment, the present invention provides an activatable cytokine construct (ACC) comprising a first monomeric construct and a second monomeric construct, (a) a first monomer construct comprising a first peptide mask (PM1), a first mature cytokine protein (CP1), first and third cleavable portions (CM1 and CM2), and a first dimerization domain (DD1); CM1 is placed between CP1 and DD1, CM2 is placed between PM1 and CP1, (b) a second monomeric construct comprising a second mature cytokine protein (CP2), a second cleavable portion (CM3), and a second dimerization domain (DD2); CM3 is placed between CP2 and DD2, DD1 and DD2 bind to each other, whereby the first monomeric component and the second monomeric component form a dimer; The activatable cytokine construct (ACC) is characterized by having a reduced activity level of at least one of CP1 and / or CP2 compared to a control level of at least one of the activities of CP1 and / or CP2. In some embodiments, the second monomer construct further comprises a second peptide mask (PM2) and a fourth cleavable moiety (CM4) located between PM2 and CP2. In some embodiments, the first monomer construct and the second monomer construct are identical and combine with each other to form a homodimer. In other embodiments, in each of the first monomer construct and the second monomer construct, at least one of the CP, CM, PM or DD components is not identical and the first monomer construct and the second monomer construct combine with each other to form a heterodimer.

[0131] In another specific embodiment, ACC is used in combination therapy with an isolated antibody or antigen-binding fragment (AB) that specifically binds to mammalian PD-1 or PD-L1.

[0132] In a more specific embodiment, ACC is used in combination therapy with an activatable anti-PD-1 or anti-PD-L1 antibody that specifically binds to mammalian PD-1 or PD-L1 in an activated state, the activatable antibody comprising an antibody or antigen-binding fragment thereof (AB) that specifically binds to mammalian PD-1 or anti-PD-L1, a masking moiety (MM) that inhibits binding of the AB to mammalian PD-1 or PD-L1 when the activatable antibody is in an uncleaved state, a cleavable moiety (CM) linked to the AB, where the CM is a polypeptide that functions as a substrate for a protease, and optionally a first connecting peptide (LP1) and / or a second connecting peptide (LP2).

[0133] The term "activatable," as used in reference to cytokine constructs and activatable anti-PD-1 or anti-PD-L1 antibodies, means a cytokine construct that exhibits a first level of one or more activities and, upon exposure to conditions that cause cleavage of one or more cleavable moieties, results in the production of a cytokine construct, or an anti-PD-1 or anti-PD-L1 antibody, that exhibits a second level of one or more activities, where the second level of activity is greater than the first level of activity. Non-limiting examples of activities include any of the exemplary activities of a cytokine, anti-PD-1, or anti-PD-L1, respectively, described herein or known in the art.

[0134] The term "mature cytokine protein" as used herein refers to a cytokine protein lacking a signal sequence. A signal sequence is also referred to herein as a "signal peptide". A cytokine protein (CP) may be a mature cytokine protein or a cytokine protein with a signal peptide. Thus, in some aspects, an ACC disclosed in the present invention may contain a mature cytokine protein sequence. In some aspects, an ACC disclosed in the present invention may contain a mature cytokine protein sequence and additionally a signal sequence. In some aspects, an ACC disclosed in the present invention may contain a sequence disclosed herein containing or lacking a signal sequence recited herein. In some embodiments, the signal sequence is selected from the group consisting of SEQ ID NO: 468, SEQ ID NO: 469 and SEQ ID NO: 470.

[0135] The terms "cleavable moiety" and "CM" are used interchangeably herein to refer to a peptide whose amino acid sequence includes a substrate for a sequence-specific protease. Cleavable moieties suitable for use as a CM include any of the protease substrates known in the art. Exemplary cleavable moieties are described in more detail below.

[0136] The terms "peptide mask" and "PM" are used interchangeably herein and refer to an amino acid sequence of less than 50 amino acids that reduces or inhibits one or more activities of a cytokine protein. A PM may bind to a cytokine to limit the cytokine from interacting with its receptor. In some embodiments, a PM is 40 amino acids or less in length. In preferred embodiments, a PM is 20 amino acids or less in length. In some embodiments, a PM is 19, 18, 17, 16, or 15 amino acids or less in length. In some aspects, a PM has at least 13 amino acids (including any number between 13 and 49). In some aspects, a PM has at least 14 amino acids (including any number between 14 and 49). In some aspects, a PM has at least 15 amino acids (including any number between 15 and 49). In certain aspects, the number of amino acids in a PM may be counted as amino acids that bind to a cytokine protein. For example, a PM excludes large polypeptides. For example, a PM is not a latency associated peptide. For example, a PM is not a cytokine. For example, the PM is not a cytokine receptor. For example, the PM is not a fragment of a cytokine receptor. In some embodiments, the PM does not have an amino acid sequence that is at least 85% identical to a cytokine receptor. For example, the PM is not albumin. For example, the PM excludes proteins or polypeptides with more than 50 amino acids. In some embodiments, the PM excludes proteins or polypeptides with more than 25 amino acids. In some embodiments, the PM excludes proteins or polypeptides with more than 20 amino acids. In some embodiments, the PM excludes proteins or polypeptides with more than 15 amino acids. In some embodiments, the PM does not include amino acids that form flexible N-terminal or C-terminal tail regions.

[0137] A "masking moiety" or "MM" within an activatable macromolecule (not yet activated) "masks" or weakens or otherwise inhibits the binding of the activatable macromolecule to its target and / or epitope. In some embodiments, coupling of an anti-PD-1 or anti-PD-L1 antibody to an MM or modification of an anti-PD-1 or anti-PD-L1 antibody with an MM can inhibit the ability of the anti-PD-1 or anti-PD-L1 antibody to specifically bind to its target and / or epitope by inhibition known in the art (such as, but not limited to, conformational changes and competition of the antigen-binding domain). In some embodiments, coupling of an anti-PD-1 or anti-PD-L1 antibody to an MM or modification of an anti-PD-1 or anti-PD-L1 antibody with an MM can result in a conformational change that weakens or inhibits the ability of the protein to specifically bind to its target or epitope. In some embodiments, coupling of an anti-PD-1 or anti-PD-L1 antibody to an MM or modification of an anti-PD-1 or anti-PD-L1 antibody with an MM sterically blocks, weakens, or inhibits the ability of the anti-PD-1 or anti-PD-L1 antibody to specifically bind to its target and epitope. In some embodiments, the MM can be a polypeptide between about 2 and 50 amino acids in length. For example, the MM can be a polypeptide between 2 and 40, 2 and 30, 2 and 20, 2 and 10, 5 and 15, 10 and 20, 15 and 25, 20 and 30, 25 and 35, 30 and 40, 35 and 45, 40 and 50 amino acids in length. For example, the MM can be a polypeptide that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some examples, the MM can be a polypeptide that is more than 50 amino acids in length, e.g., 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids.

[0138] The terms "dimerization domain" and "DD" are used interchangeably herein and refer to one member of a pair of dimerization domains, each member of the pair being capable of binding to the other via one or more covalent or non-covalent interactions. The first DD and the second DD may be the same or different. Exemplary DDs suitable for use as DD1 and / or DD2 are described in more detail herein below.

[0139] As used herein, the terms "linker," "linking peptide," and "LP" refer to a peptide whose amino acid sequence is not a substrate for a protease. Exemplary linkers and LPs are described in more detail below.

[0140] As used herein, the term "linking region" or "LR" refers to a stretch of amino acid residues between the C-terminus of the cytokine and the amino acid residues adjacent at the N-terminus to the proximal point of interaction between the dimerization domains (i.e., the linking region does not contain the C-terminal amino acid of the cytokine or the N-terminal amino acid of the DD that forms the proximal point of interaction with the DD of the corresponding second monomer). For example, if the DD is a pair of Fc domains, the linking region is a stretch of amino acid residues between the C-terminus of the cytokine and the first N-terminal cysteine ​​residue of the Fc that participates in a disulfide bond with the second Fc domain (e.g., cysteine ​​226 of the Fc domain of IgG1 or IgG4 according to EU numbering). If the dimerization domain is not a polypeptide, the linking region is a stretch of amino acid residues from the C-terminus of the cytokine to the last amino acid. For example, if the DD is a biotin-streptavidin pair, the linking region of the biotin-containing monomer is the stretch of amino acid residues between the C-terminus of the cytokine and the biotin molecule, and the linking region of the streptavidin-containing monomer is the stretch of amino acid residues between the C-terminus of the cytokine and the streptavidin molecule.

[0141] As used herein, the term "masked linkage region" or "MLR" refers to a stretch of amino acid residues between the PM and the CP. As shown in FIG. 36, the MLR spans from the N-terminus of the CP to the C-terminus of the PM. Thus, the MLR may contain a PM, a PM and a linker, or a PM and two linkers. In some embodiments, the MLR spans 15-22 amino acids. In some embodiments, the MLR spans 16-21 amino acids. In some embodiments, the MLR spans 17-20 amino acids. In some embodiments, the MLR spans 18-20 amino acids. In some embodiments, the MLR spans 15, 16, 17, 18, 18, 20, 21, or 22 amino acids.

[0142] As used herein, the term "masking efficiency" refers to the activity (e.g., EC50) of uncleaved ACC, activatable anti-PD-1 antibody, or activatable anti-PD-L1 antibody separated by the activity of a control cytokine, anti-PD-1 antibody, or anti-PD-L1 antibody, which may be a cleavage product of ACC, activatable anti-PD-1 antibody, or activatable anti-PD-L1 antibody, or a cytokine, anti-PD-1 antibody, or anti-PD-L1 antibody used as a CP for ACC, activatable anti-PD-1 antibody, or activatable anti-PD-L1 antibody. ACC with reduced levels of at least one CP1 and / or CP2 activity has a masking efficiency of greater than 10. In some embodiments, the ACC, activatable anti-PD-1 antibody, or activatable anti-PD-L1 antibody described herein have a masking efficiency of greater than 10, greater than 100, greater than 1000, or greater than 5000.

[0143] As used herein, the term "spacer" refers herein to an amino acid residue or peptide that is incorporated into the free end of a mature ACC, for example, between a signal peptide and the N-terminus of the mature ACC. In some embodiments, the spacer (or "header") may contain a glutamine (Q) residue. In some embodiments, residues within the spacer minimize aminopeptidase and / or exopeptidase activity to prevent cleavage of the N-terminal amino acid. Exemplary, non-limiting spacer amino acid sequences may include or consist of any of the following exemplary amino acid sequences: QGQSGS (SEQ ID NO:471); GQSGS (SEQ ID NO:472); QSGS (SEQ ID NO:473); SGS; GS; S; QGQSGQG (SEQ ID NO:474); GQSGQG (SEQ ID NO:475); QSGQG (SEQ ID NO:476); SGQG (SEQ ID NO:477); GQG; QG; G; QGQSGQ (SEQ ID NO:478); GQSGQ (SEQ ID NO:479); QSGQ (SEQ ID NO:480); QGQSG (SEQ ID NO:481); QGQS (SEQ ID NO:482); SGQ; GQ; and Q. In some embodiments, the spacer sequence may be omitted.

[0144] As used herein, a polypeptide such as a cytokine or Fc domain may be a wild-type polypeptide (e.g., a naturally occurring polypeptide) or a variant of a wild-type polypeptide. A variant may be a polypeptide that has been modified by substitution, insertion, deletion and / or addition of one or more amino acids of the wild-type polypeptide, provided that the variant retains the basic function or activity of the wild-type polypeptide. In some examples, a variant may have an altered (e.g., increased or decreased) function or activity compared to the wild-type polypeptide. In some embodiments, a variant may be a functional fragment of a wild-type polypeptide. The term "functional fragment" refers to a sequence of a polypeptide (e.g., a cytokine) that may contain fewer amino acids than the full-length polypeptide sequence, but contains sufficient length of the polypeptide chain to confer an activity (e.g., cytokine activity).

[0145] The first and second monomeric constructs may further include additional elements, such as, for example, one or more linkers. The additional elements are described in more detail below. The organization of the CP, CM, PM and DD components in each of the first and second monomeric constructs may be arranged in the same order in each monomeric construct. The CP1, CM1, PM1 and DD1 components may be the same or different in terms of, for example, the molecular weight, size, amino acid sequence, etc. of the CP, CM and PM components (and the DD components in embodiments where the DD components are polypeptides) compared to the corresponding CP2, CM2, PM2 and DD2. Thus, the resulting dimer may have symmetric or asymmetric monomeric construct components.

[0146] In some embodiments, the first monomer construct comprises DD1 linked directly or indirectly (through a linker) to the C-terminus of PM1, CM3, CP1, CM1, and CM1 from the N-terminus to the C-terminus of the CP and CM components. In other embodiments, the first monomer construct comprises DD1 linked directly or indirectly (through a linker) to the N-terminus of PM1, CM3, CP1, CM1, and CM1 from the C-terminus to the N-terminus of the CP and CM components. In some embodiments, the second monomer construct comprises DD2 linked directly or indirectly (through a linker) to the C-terminus of PM2, CM4, CP2, CM2, and CM2 from the N-terminus to the C-terminus of the CP and CM components. In some embodiments, the second monomer construct comprises DD2 linked directly or indirectly (through a linker) to the N-terminus of PM2, CM4, CP2, PM2, and CM2 from the C-terminus to the N-terminus of the CP and CM components. In one example, the first monomeric construct comprises a first polypeptide comprising PM1, CM3, CP1, CM1 and DD1. In one example, the second monomeric construct comprises a second polypeptide comprising CP2, CM2 and DD2. In another example, the second monomeric construct comprises a second polypeptide comprising PM2, CM4, CP2, CM2 and DD2.

[0147] In some embodiments, the CP and DD components are linked by a linker that is not cleavable by a protease. For example, the CP and DD components may be linked by a non-cleavable substrate sequence (NSUB). In some embodiments, one of the first and second monomeric constructs includes an NSUB between the CP and DD, and the other includes a CM between the CP and DD. In some aspects, the linker may contain glycine and serine residues, but may be an amino acid substrate sequence that is not susceptible to protease cleavage. Examples of non-cleavable linker sequences include those described in U.S. Pat. No. 10,611,845 B2, which is incorporated herein by reference in its entirety. In such cases, the CP and / or DD may have a cleavage site for the protease.

[0148] An example of an ACC of the present disclosure may be represented by the following formula (in the form monomer1 / monomer2, from N-terminus to C-terminus in each monomer): PM1-CM3-CP1-CM1-DD1 / PM2-CM4-CP2-CM2-DD2 PM1-CM3-CP1-CM1-DD1 / CP2-CM2-DD2 DD1-CM1-CP1-CM3-PM1 / DD2-CM2-CP2-CM4-PM2 DD1-CM1-CP1-CM3-PM1 / DD2-CM2-CP2 ACC may include one or more linkers between its components. For example, ACC may include one or more linkers between PM and CP and / or between CP and DD. Thus, as used herein and unless otherwise specified, each dash (-) between ACC components indicates a direct linkage or a linkage via one or more linkers.

[0149] In some embodiments, when ACC has an orientation of N-PM-CM1-CP-CM2-DD-C, the total span of amino acids from the N-terminus of ACC to the N-terminal amino acid of the cytokine is 17-71 amino acids in length. In some embodiments, when ACC has an orientation of N-DD-CM1-CP-CM2-PM-C, the total span of amino acids from the C-terminus of ACC to the C-terminal amino acid of the cytokine is 17-71 amino acids in length.

[0150] In certain embodiments, the first and second monomer constructs are oriented such that the components of each member of the dimer are arranged in the same order from the N-terminus to the C-terminus of the CP and CM components. A schematic diagram of an exemplary ACC is provided in FIG. 1. Referring to FIG. 1, the ACC comprises, from N-terminus to C-terminus, (1) a first monomer construct 110 having PM1 119, CM3 117, CP1 115, CM1 113, and DD1 111; (2) a second monomer construct 120, optionally having PM2 129, CM4 127, CP2 125, CM2 123, and DD2 121; and (3) one or more covalent or non-covalent bonds (←→) that link the first monomer construct 110 to the second monomer construct 120. The ACC may further comprise one or more optional linkers 112, 114, 116, 118, 122, 124, 126 and 128 between its components. In one example, DD1 111 and DD2 121 are the same. In another example, DD1 111 and DD2 121 are different.

[0151] A schematic diagram of a further exemplary ACC, in which the components of the ACC are arranged in the opposite orientation, is provided in Figure 2. Referring to Figure 2, the ACC comprises, from the N-terminus to the C-terminus of the CP and CM components, (1) a first monomeric construct 210 having DD1 211, CM1 213, CP1 215, CM3 217 and PM1 219; (2) a second monomeric construct 220 having DD2 221, CM2 223, CP2 225, and optionally CM4 227 and PM2 229; and (3) one or more covalent or non-covalent bonds (←→) linking the first monomeric construct 210 to the second monomeric construct 220. The ACC may further comprise one or more optional linkers 212, 214, 216, 218, 222, 224, 226 and 228 between the components.

[0152] The ACC may further comprise one or more optional linkers 212, 214, 216, 218, 222, 224, 226, and 228 between its components. In one example, DD1 211 and DD2 221 are the same. In another example, DD1 211 and DD2 221 are different.

[0153] A schematic diagram of another exemplary ACC is provided in Figure 3. Referring to Figure 3, the ACC comprises, from N-terminus to C-terminus, (1) a first monomeric construct 310 having PM1 319, CM3 317, CP1 315, CM1 313 and DD1 311; (2) a second monomeric construct 320 having CP2 325, CM2 323 and DD2 321, and optionally PM2 329 and CM4 327. DD1 311 and DD2 321 are binding partners, such as a ligand / receptor pair or an antigen / antigen-binding peptide pair, such that DD1 and DD2 are covalently or non-covalently linked together. The ACC may further comprise one or more optional linkers 312, 314, 316, 318, 322, 324, 326 and 328 between the components. In one example, DD1 311 and DD2 321 are the same. In another example, DD1 311 and DD2 321 are different. In another example, DD1 311 and DD2 321 are different.

[0154] In an alternative embodiment, either of the two moieties designated as CP1 315 and CP2 325 is a truncated cytokine protein lacking cytokine activity. For example, CP1 or CP2 may be a truncated interferon alpha 2b having the first 151 amino acids of wild-type interferon alpha 2b. In an alternative embodiment, either of the two moieties designated as CP1 315 and CP2 325 is a mutant cytokine protein lacking cytokine activity. For example, CP1 or CP2 may be a truncated interferon alpha 2b having a mutation of L130P (e.g., SEQ ID NO: 298). In an alternative embodiment, either of the two moieties designated as CP1 315 and CP2 325 is a polypeptide sequence lacking cytokine activity, such as a signal portion and / or a stub sequence. In an alternative embodiment, a first of the two moieties designated CP1 315 and CP2 325 is a polypeptide sequence that binds with high affinity to a second of the two moieties designated CP1 315 and CP2 325 and reduces the cytokine activity of the second moiety compared to a control level of the second moiety.

[0155] A schematic diagram of another exemplary ACC, with components organized in the opposite orientation, is provided in Figure 4. Referring to Figure 4, the ACC comprises, from the N-terminus to the C-terminus of the CP and CM components, (1) a first monomeric construct 410 having DD1 411, CM1 413, CP1 415, CM3 417, and PM1 419; and (2) a second monomeric construct 420 having DD2 421, CM2 423, CP2 425, and optionally CM4 427 and PM2 429. DD1 411 and DD2 421 are binding partners, such as a ligand / receptor pair or an antigen / antigen-binding peptide pair, such that DD1 and DD2 are bound together by covalent or non-covalent bonds. The ACC may further comprise one or more optional linkers 412, 414, 416, 418, 422, 424, 426, and 428 between its components. In one example, DD1 411 and DD2 421 are the same. In another example, DD1 411 and DD2 421 are different.

[0156] In certain aspects of the disclosure, PM1 and PM2 as shown in the figure may be absent in ACC used in combination with an anti-PD1 antibody or an anti-PD-L1 antibody.

[0157] It has been found that the ACC structure is extremely effective in reducing the activity of mature cytokine protein components such that the cytokine activity is not substantially impaired after activation. The activity of the CP in the ACC may be reduced by both the structure of the ACC (e.g., dimer structure) and the peptide mask(s) in the ACC. In some embodiments, the activation condition of the ACC described herein is exposure to one or more proteases that can cleave the CP from both the DD and the PM. For example, the one or more proteases may cleave the CM between the CP and the PM, and the CM between the CP and the DD. As shown in the examples, activation of the ACC resulted in a substantial recovery of the cytokine activity. These results suggest that within the context of the ACC, the conformation of the cytokine components was not irreversibly altered. Importantly, the inventors have found that the structure of the present invention utilizes both a dimerization domain and one or more peptide masks with specific binding affinity to the cytokine protein, which appears to achieve a substantial masking effect beyond the use of a peptide mask alone or a dimerization domain alone.

[0158] ACC may use any of a variety of mature cytokine proteins, cleavable portions, peptide masks, and dimerization domains as CP1, CP2, CM1, CM2, CM3, CM4, PM1, PM2, DD1, and DD2, respectively. For example, any of a variety of mature cytokine proteins known in the art, or any of their sequences and / or truncated variants, may be suitable for use as one or both of the CP1 and CP2 components of ACC. The mature cytokine proteins, CP1 and CP2, may be the same or different. In certain embodiments, CP1 and CP2 are the same. In other embodiments, CP1 and CP2 are different. ACC may include additional amino acid residues at one or both of the N-terminus and / or C-terminus of CP1 and / or CP2.

[0159] In some embodiments, CP1 and / or CP2 are each independently selected from the group consisting of interferon (e.g., interferon alpha, interferon beta, interferon gamma, interferon tau, and interferon omega), interleukin (e.g., IL-1 alpha, IL-1 beta, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, IL-21, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-69, IL-70, IL-71, IL-72, IL-73 , IL-14, IL-16, IL-17, CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD27, CD70, CD153, CD178, GITRL, LIGHT, OX40L, OX40, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β2, TGF-β3, EPOo, TPO, Flt-3L, SCF, M-CSF and MSP, and the like, and sequences and / or truncated variants thereof. In particular, the ACC used in the combination may contain IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN-β, IFN-γ, GM-CSF, TGF-β, LIGHT, GITR-L, CD40L, CD27L, 4-1BB-L, OX40, OX40L. For example, sequences of such proteins include those in Table 23, and additional examples of sequences can be obtained from ncbi.nlm.nih.gov / protein. Suitable truncated variants for use in the ACC of the present invention include any cytokine truncated at the N-terminus or C-terminus that retains cytokine activity.Exemplary truncated variants for use in the present invention include any of the truncated cytokine polypeptides known in the art (see, e.g., Slutzki et al., J. Mol. Biol. 360:1019-1030, 2006 and US 2009 / 0025106), as well as cytokine polypeptides truncated at the N- and / or C-terminus by 1 to about 40 amino acids, 1 to about 35 amino acids, 1 to about 30 amino acids, 1 to about 25 amino acids, 1 to about 20 amino acids, 1 to about 15 amino acids, 1 to about 10 amino acids, 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids that retain cytokine activity. In some of the foregoing embodiments, the truncated CP is a CP truncated at the N-terminus. In other embodiments, the truncated CP is a CP truncated at the C-terminus. In certain embodiments, the truncated CP is a CP truncated at both the C-terminus and the N-terminus.

[0160] In some embodiments, CP1 and / or CP2 are each independently selected from SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:12, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, No. 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, 190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, and SEQ ID NO:209.The amino acid sequence of the peptide sequence of the present invention includes an amino acid sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical. The percentage of sequence identity refers to the level of amino acid sequence identity between two or more peptide sequences when aligned using a sequence alignment program, such as the BLAST suite of programs publicly available on the Internet at the NCBI website. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990. In some embodiments, the ACC contains an interferon alpha 2b mutant, such as an interferon alpha 2b molecule having a mutation at position L130, such as an L130P mutation (e.g., SEQ ID NO: 298), as CP1 or CP2, compared to SEQ ID NO: 1. In some embodiments, the ACC contains an interferon alpha 2b variant having a mutation at the position of I24, F64, I60, I63, F64, W76, I116, L117, F123, or L128, or a combination thereof. For example, the interferon alpha 2b variant may contain a mutation of I116 to T, N, or R; L128 to N, H, or R; I24 to P or Q; L117H; or L128T, or a combination thereof. In some embodiments, the interferon alpha 2b variant may contain a mutation of I24Q, I60T, F64A, W76H, I116R, and L128N, or a subset thereof. In some embodiments, the ACC contains a truncated interferon alpha 2b molecule lacking cytokine activity as either CP1 or CP2. For example, truncated interferon α2b may consist of 151 or less amino acids of interferon α2b, such as 1-151, 1-150, 1-149, 1-148, ... 1-10, 1-9, 1-8, 1-7, 1-6, or 2-151, 3-151, 4-151, 5-150, 6-149, 7-148, 8-147 amino acids from the N-terminus to the C-terminus of the wild-type interferon α2b sequence, or any intervening sequence of these amino acids or variants.

[0161] In certain embodiments, CP1 and / or CP2 comprise an interferon. Interferons suitable for use as CP1 and / or CP2 in the compositions of the invention include, for example, interferon alpha, interferon beta, interferon gamma, interferon omega, and interferon tau. In some embodiments, when the interferon is interferon alpha, it may be interferon alpha-2a, interferon alpha-2b, or interferon alpha-n3. Further examples of interferon alpha include interferon alpha-1, interferon alpha-4, interferon alpha-5, interferon alpha-6, interferon alpha-7, interferon alpha-8, interferon alpha-10, interferon alpha-13, interferon alpha-14, interferon alpha-16, interferon alpha-17, and interferon alpha-21. In some embodiments, the interferon is recombinant or purified interferon alpha. In certain embodiments, when the interferon is interferon beta, it is selected from the group consisting of interferon beta-1a and interferon beta-1b. In some embodiments, CP1 and / or CP2 comprise an IFab domain, which is a conserved protein domain found in interferon alpha or interferon beta. The IFab domain is responsible for the cytokine release and antiviral functions of interferons. Exemplary IFab sequences are provided in SEQ ID NOs: 449-458. In one example, CP1 and CP2 are different interferons. In another example, CP1 and CP2 are the same interferon.

[0162] In some embodiments, CP1 and / or CP2 exhibit interferon activity and contain an amino acid sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical, or 100% identical to an interferon alpha 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 certain embodiments, the interferon alpha reference sequence is SEQ ID NO:1 (human interferon alpha-2b). In some embodiments, CP1 and / or CP2 comprise a 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 certain 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-described embodiments, CP1 and CP2 comprise identical amino acid sequences.

[0163] In other embodiments, CP1 and / or CP2 exhibit interferon activity and contain an amino acid sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical, or 100% identical to an interferon beta 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 certain embodiments, the interferon beta reference sequence is a human interferon beta 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 a mature beta 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-mentioned embodiments, CP1 and CP2 comprise identical amino acid sequences.

[0164] In some embodiments, CP1 and / or CP2 exhibit interferon activity and contain an amino acid sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical, or 100% identical to an interferon omega reference sequence corresponding to SEQ ID NO: 110 (human interferon omega). In certain embodiments, CP1 and / or CP2 comprise a mature human omega interferon having the amino acid sequence of SEQ ID NO: 110. In some of the above-described embodiments, CP1 and CP2 comprise identical amino acid sequences.

[0165] In some embodiments, CP1 and / or CP2 exhibit interleukin activity and are selected from the group consisting of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:12, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:143 , SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160. In some embodiments, CP1 and / or CP2 comprise a mature interleukin 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: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-described embodiments, CP1 and CP2 comprise the same amino acid sequence.

[0166] In some embodiments, CP1 and / or CP2 exhibit interleukin activity and are 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-144), SEQ ID NO:145 (human IL-146), SEQ ID NO:147 (human IL-148), SEQ ID NO:149 (human IL-15), SEQ ID NO:150 (human IL-150), SEQ ID NO:151 (human IL-151), SEQ ID NO:152 (human IL-152), SEQ ID NO:153 (human IL-153), SEQ ID NO:154 (human IL-154), SEQ ID NO:155 (human IL-155), SEQ ID NO:156 (human IL-156), SEQ ID NO:157 (human IL-157), SEQ ID NO:158 (human IL-158), SEQ ID NO:159 (human IL-160), SEQ ID NO:161 (human IL-161), SEQ ID NO:162 (human IL- The present invention relates to an interleukin reference sequence comprising an amino acid sequence that is at least 80% identical, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an interleukin reference sequence selected from the group consisting of: 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 certain of these embodiments, CP1 and / or CP2 are 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 ( In some of the above-described embodiments, CP1 and CP2 comprise an amino acid sequence from the group consisting of: SEQ ID NO: 133 (human IL-5), SEQ ID NO: 137 (human IL-6), SEQ ID NO: 139 (human IL-11), SEQ ID NO: 143 (human IL-12α), SEQ ID NO: 144 (human IL-12β), SEQ ID NO: 151 (human IL-10), SEQ ID NO: 153 (human IL-20); SEQ ID NO: 155 (human IL-14), SEQ ID NO: 157 (human IL-16) and SEQ ID NO: 159 (human IL-17). In some of the above-described embodiments, CP1 and CP2 comprise the same amino acid sequence.

[0167] The number of amino acids in the sequence of the cytokine protein used may vary depending on the particular cytokine protein used. In some embodiments, CP1 and / or CP2 may be a total of about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 25 ... 0 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 20 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 250 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 60 amino acids acid, about 20 amino acids to about 40 amino acids, about 40 amino acids to about 700 amino acids, about 40 amino acids to about 650 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 350 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 250 amino acids,About 40 amino acids to about 200 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids , about 60 amino acids to about 350 amino acids, about 60 amino acids to about 300 amino acids, about 60 amino acids to about 250 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 700 amino acids, about 80 amino acids to about 650 amino acids, about 80 amino acids to about 600 amino acids, about 80 amino acids to about 550 amino acids, about 80 amino acids to about 500 amino acids amino acids, about 80 amino acids to about 450 amino acids, about 80 amino acids to about 400 amino acids, about 80 amino acids to about 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 55 0 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids,About 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 amino acids, about 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 about 200 to about 550 amino acids, about 200 to about 500 amino acids, about 200 to about 450 amino acids, about 200 to about 400 amino acids, about 200 to about 350 amino acids, about 200 to about 300 amino acids, about 200 to about 250 amino acids, about 250 to about 700 amino acids, about 250 to about 650 amino acids, about 250 to about 600 amino acids, about 250 to about 550 amino acids, about 250 to about About 500 amino acids, about 250 amino acids to about 450 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 350 amino acids, about 250 amino acids to about 300 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids , about 300 amino acids to about 350 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids,About 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 700 amino acids, about 50 In some embodiments, CP1 and / or CP2 are mature wild-type human cytokine proteins.

[0168] Each monomeric component of ACC may use any of a variety of dimerization domains. Suitable DDs include both polymeric (e.g., synthetic polymers, polypeptides, polynucleotides, etc.) and small molecule (non-polymeric moieties with molecular weights of less than about 1 kilodalton, sometimes less than about 800 daltons) moieties. A pair of DDs may be any pair of moieties known in the art to bind to each other.

[0169] For example, in some embodiments, DD1 and DD2 are selected from the group consisting of sushi domains from the alpha chain of the human IL-15 receptor (IL15Rα) and soluble IL-15; barnase and barstar; protein kinase A (PKA) and A kinase anchor protein (AKAP); mutant RNase I fragment-based adaptor / docking tag molecules; a pair of antigen binding domains (e.g., a pair of single domain antibodies); soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs); modules based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin and SNAP25; single domain antibodies (sdAbs) and corresponding epitopes; A pair of members selected from the group consisting of an antigen-binding domain (e.g., a single chain antibody such as a single chain variable fragment (scFv), a single domain antibody, etc.) and the corresponding epitope; a coiled-coil polypeptide structure (e.g., a Fos-Jun coiled-coil structure, an acid / base coiled-coil helix, a Glu-Lys coiled-coil helix, a leucine zipper structure), a pair of small molecules conjugated such as biotin and avidin or streptavidin, an amine / aldehyde, a lectin / carbohydrate; a pair of polymers capable of binding to each other, for example, a pair of sulfur- or thiol-containing polymers (e.g., a pair of Fc domains, a pair of thiolated human serum albumin polypeptides, etc.); and the like.

[0170] In some embodiments, DD1 and DD2 are non-polypeptide polymers. The non-polypeptide polymers may be covalently bonded to each other. In some examples, the non-polypeptide polymer may be a sulfur-containing polymer, such as a sulfur-containing polyethylene glycol. In such cases, DD1 and DD2 may be covalently bonded to each other via one or more disulfide bonds.

[0171] When a pair of DD1 and DD2 is a member of a pair of epitope and antigen-binding domain, the epitope may be a natural or non-natural epitope. Exemplary non-natural epitopes include non-natural peptides such as poly-His peptides (e.g., His tags).

[0172] In certain embodiments, DD1 and DD2 are a pair of Fc domains. As used herein, "Fc domain" refers to the adjacent amino acid sequence of a single heavy chain of an immunoglobulin. A pair of Fc domains bind together to form the Fc region of an immunoglobulin.

[0173] In some embodiments, the pair of Fc domains is a pair of human Fc domains (e.g., a pair of wild-type human Fc domains). In some embodiments, the human Fc domain is a human IgG1 Fc domain (e.g., a wild-type human IgG1 Fc domain), a human IgG2 Fc domain (e.g., a wild-type human IgG2 Fc domain), a human IgG3 Fc domain (e.g., a wild-type human IgG3 Fc domain), or a human IgG4 Fc domain (e.g., a wild-type human IgG4 Fc domain). In some embodiments, the human Fc domain comprises a sequence at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NO:3.

[0174] In some embodiments, a pair of Fc domains includes knob and hole variants of the Fc domain. The knob and hole variants may interact with each other to promote dimerization. In some embodiments, the knob and hole variants may include one or more amino acid modifications in the interface between the two Fc domains (e.g., in the CH3 domain). In one example, the modifications include amino acid substitutions T366W and optionally S354C in one of the antibody heavy chains, and amino acid substitutions T366S, L368A, Y407V, and optionally Y349C in the other antibody heavy chain (numbered according to the EU index of the Kabat numbering system). Examples of knob and hole variants include the Fc variants of SEQ ID NOs: 287 and 288, as well as those described in U.S. Patent Nos. 5,731,168, 7,695,936, and 10,683,368, which are incorporated herein by reference in their entireties. In some embodiments, the dimerization domain comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NOs: 287 and 288, respectively.

[0175] In some embodiments, DD1 and / or DD2 may further comprise a serum half-life extending moiety (e.g., a polypeptide that binds to a serum protein such as an immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA)). Examples of half-life extending moieties include hexa-hat GST (glutathione S-transferase) glutathione affinity, calmodulin binding peptide (CBP), strep tag, cellulose binding domain, maltose binding protein, S-peptide tag, chitin binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitope, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, and VSV epitope.

[0176] In some embodiments, DD1 and / or DD2 each contain a total of about 5 amino acids to about 250 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 20 amino acids, , about 5 amino acids to about 10 amino acids, about 10 amino acids to about 250 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 20 amino acids, about 0 amino acids to about 250 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 40 amino acids, about 40 amino acids to about 250 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 250 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 140 amino acids,About 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 250 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids The present invention relates to a method for preparing a nucleic acid comprising the steps of: a) preparing a nucleic acid sequence comprising: a) a nucleic acid sequence comprising: a nucleic acid sequence comprising: a nucleic acid sequence comprising: a nucleic acid sequence comprising: a nucleic acid sequence comprising: a nucleic acid sequence comprising: a nucleic acid sequence comprising: a nucleic acid sequence comprising: a nucleic acid sequence comprising: In some embodiments, DD1 and DD2 are Fc domains that include a portion of the hinge region containing two cysteine ​​residues, a CH2 domain, and a CH3 domain. In some embodiments, DD1 and DD2 are Fc domains that are N-terminal to the first cysteine ​​residue (reading from N-terminus to C-terminus) in the hinge region that participates in a disulfide bond with a second Fc domain (e.g., cysteine ​​226 in human IgG1 or IgG4 using EU numbering).

[0177] In some aspects, located directly or indirectly (e.g., via a linker) between the CP and DD components and / or between the CP and PM components is a cleavable moiety (CM) that comprises a substrate for a protease. In some embodiments, the CM is each independently selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, Caspase 10, Caspase 14, Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin G, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Chymase, Cruzipain, DESC1, DPP-4, FAP, Legumain, Otubain-2, Elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoate hepsin, HtrA1, human neutrophil elastase, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, lactoferrin, marapsin, matriptase-2, meprin, MT-SP1 / matriptase, neprilysin, NS3 / 4A, PACE4, plasmin, PSMA, PSA, BMP-1, 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, tryptase and uPA, and any combination of two or more thereof.

[0178] In some embodiments of any of the ACCs described herein, the protease that cleaves any of the CMs described herein is selected from the group consisting of ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, caspase 15, caspase 16, caspase 17, caspase 18, caspase 19, caspase 20, caspase 21, caspase 22, caspase 23, caspase 24, caspase 25, caspase 26, caspase 27, caspase 28, caspase 29, caspase 30, caspase 31, caspase 32, caspase 33, caspase 34, caspase 35, caspase 36, caspase 37, caspase 38, caspase 39, caspase 40, caspase 41, caspase 42, caspase 43, caspase 44, caspase 45, caspase 46, caspase 47, caspase 48, caspase 49 ...9, Caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cruzipain, legumain, otubain-2, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, meprin, neprilysin, PSMA, BMP -1, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, MMP-27, 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, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3 and TMPRSS4, and any combination of two or more thereof.

[0179] In some embodiments of any of the ACC described herein, the protease is selected from the group of uPA, legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-2, MMP-9, MMP-12, MMP-13, and MMP-14.

[0180] Elevated levels of proteases with known substrates have been reported in many cancers. See, e.g., La Roca et al., British J.Cancer 90(7):1414-1421, 2004. Suitable substrates for use in the CM components used herein include substrates that are more prevalently found in cancer cells and tissues. Thus, in certain embodiments, each CM independently comprises a substrate for a protease that is more prevalently found in diseased tissues associated with the cancer. In some embodiments, the cancer is selected from the group consisting of gastric cancer, breast cancer, osteosarcoma, and esophageal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the cancer is Kaposi's sarcoma, hairy cell leukemia, chronic myelogenous leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, malignant cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, breast cancer, ovarian cancer, bladder cancer, BCG-resistant non-muscle invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer, or testicular cancer, etc. In some of the above-described embodiments, the CM component comprises a substrate for a protease(s) that is more prevalent in the tumor tissue.

[0181] In some embodiments, the CMs each independently contain a sequence selected from the group consisting of SEQ ID NO:5 to SEQ ID NO:100, and C-terminal and N-terminal truncated variants thereof.

[0182] In some embodiments, the CM contains a sequence selected from the group consisting of ISSGLLSGRSDNH (SEQ ID NO: 28), LSGRSDDH (SEQ ID NO: 33), ISSGLLSGRSDQH (SEQ ID NO: 54), and ISSGLLSGRSDNI (SEQ ID NO: 68).

[0183] In certain embodiments, CM1 and / or CM1 contain a sequence selected from the group consisting of AQNLLGMY (SEQ ID NO:237), LSGRSDNHGGAVGLLAPP (SEQ ID NO:238), VHMPLGFLGPGGLSGRSDNH (SEQ ID NO:239), LSGRSDNHGGVHMPLGFLGP (SEQ ID NO:240), LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO:241), ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO:242), LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO:243), QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO:244), LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO:245), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO:246), ISSGLLSGRSGNH (SEQ ID NO:247), and C-terminal and N-terminal truncated variants thereof. Further examples of CMs include those described in U.S. Patent Application Publication Nos. US20160289324, US20190284283, Publication Nos. WO2010 / 081173, WO2015 / 048329, WO2015 / 116933, WO2016 / 118629, and WO2020 / 118109, which are incorporated by reference in their entireties.

[0184] The truncated variants of the above-mentioned amino acid sequences suitable for use in CMs are any truncated variants that retain the recognition site of the corresponding protease. These include C-terminal and / or N-terminal truncated variants that contain at least 3 consecutive amino acids of the above-mentioned amino acid sequences, or at least 4, or at least 5, or at least 6, or at least 7 amino acids of the aforementioned amino acid sequences that retain the recognition site of the protease. In certain embodiments, the truncated variants of the above-mentioned amino acid sequences are amino acid sequences that correspond to any of the above, but are truncated at the C-terminus and / or N-terminus by 1 to about 10 amino acids, 1 to about 9 amino acids, 1 to about 8 amino acids, 1 to about 7 amino acids, 1 to about 6 amino acids, 1 to about 5 amino acids, 1 to about 4 amino acids, or 1 to about 3 amino acids, (1) having at least 3 amino acid residues; (2) retaining the recognition site of the protease. In some of the above embodiments, the truncated CM is an N-terminally truncated CM. In some embodiments, the truncated CM is a CM truncated at the C-terminus. In some embodiments, the truncated C is a CM truncated at the C-terminus and the N-terminus.

[0185] In some embodiments of any of the ACC or activatable antibodies described herein, the CM may comprise a total of about 3 amino acids to about 25 amino acids. In some embodiments, the CM may 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.

[0186] In some embodiments, the ACC, activatable anti-PD1, or activatable anti-PD-L1 may include multiple CMs that include substrates for different proteases. In some embodiments, the ACC, activatable anti-PD1, or activatable anti-PD-L1 may include multiple CMs that are substrates for the same protease. In one example, the CM(s) between each CP and PM may be substrates for the same protease as each other, and the CM(s) between each CP and DD may be substrates for the same protease as each other, but different proteases from the CM(s) between the CP and PM. In another example, the CM(s) between the CP and PM and the CM(s) between the CP and DD may include substrates for the same protease. In another example, the CM(s) between the CP and PM may include substrates for different proteases. In another example, the CM(s) between the CP and PM may include substrates for the same protease. In another example, the CM(s) between the CP and DD may include substrates for different proteases. In another example, the CM(s) between the CP and DD may comprise a substrate for the same protease. In one example, the CM(s) between each activatable anti-PD1 or activatable anti-PD-L1 and MM may be a substrate for the same protease as each other. In another example, the CM(s) between activatable anti-PD1 or activatable anti-PD-L1 and MM may comprise a substrate for a different protease. In another example, the CM(s) between activatable anti-PD1 or activatable anti-PD-L1 and MM may comprise a substrate for the same protease.

[0187] The first monomer construct and the second monomer construct may include one or more additional components, such as one or more linkers. In some embodiments, the first monomer can include a linker disposed between CP1 and CM1. In some embodiments, in the first monomer, CP1 and CM1 are directly adjacent to each other. In some embodiments, the first monomer includes a linker disposed between CM1 and DD1. In some embodiments, in the first monomer, CM1 and DD1 are directly adjacent to each other. In some embodiments, the first monomer can include a linker disposed between CP1 and CM3. In some embodiments, in the first monomer, CP1 and CM3 are directly adjacent to each other. In some embodiments, the first monomer can include a linker disposed between CP1 and PM1. In some embodiments, in the first monomer, CP1 and PM1 are directly adjacent to each other. In some embodiments, the linker has an overall length of 1 amino acid to about 15 amino acids. In some embodiments, the CM and any linker disposed between CP1 and DD1 have a combined total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0188] In some embodiments, the second monomer comprises a linker disposed between CP2 and CM2. In some embodiments, in the second monomer, CP2 and CM2 are directly adjacent to each other. In some embodiments, the second monomer comprises a linker disposed between CM2 and DD2. In some embodiments, in the second monomer, CM2 (e.g., any of the cleavable moieties described herein) and DD2 (e.g., any of the DDs described herein) are directly adjacent to each other. In some embodiments, the second monomer can contain a linker disposed between CP2 and CM4. In some embodiments, in the second monomer, CP2 and CM4 are directly adjacent to each other. In some embodiments, the second monomer can contain a linker disposed between CP2 and PM2. In some embodiments, in the second monomer, CP2 and PM2 are directly adjacent to each other. In some embodiments, the linker has an overall length of between 1 amino acid and about 15 amino acids. In some embodiments, the linker comprises a sequence of GGGS (SEQ ID NO:2). In some embodiments, the CM and any linker located between CP2 and DD2 have a combined total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0189] In some embodiments, the first monomer and / or the second monomer may have a total of about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, About 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about About 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids , about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids,About 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 800 amino acids, about about 250 to about 750 amino acids, about 250 to about 700 amino acids, about 250 to about 650 amino acids, about 250 to about 600 amino acids, about 250 to about 550 amino acids, about 250 to about 500 amino acids, about 250 to about 450 amino acids, about 250 to about 400 amino acids, about 250 to about 350 amino acids, about 250 to about 300 amino acids, about 300 to about 800 amino acids, about 300 to about About 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 750 amino acids, about 350 amino acids to about 700 amino acids , about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids,About 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 The amino acid sequence may contain about 550 to about 800 amino acids, about 550 to about 750 amino acids, about 550 to about 700 amino acids, about 550 to about 650 amino acids, about 550 to about 600 amino acids, about 600 to about 800 amino acids, about 600 to about 750 amino acids, about 600 to about 700 amino acids, about 600 to about 650 amino acids, about 650 to about 800 amino acids, about 650 to about 750 amino acids, about 650 to about 700 amino acids, about 700 to about 800 amino acids, about 700 to about 750 amino acids, or about 750 to about 800 amino acids.

[0190] In some embodiments of any of the ACCs described herein, one or more linkers (e.g., flexible linkers) can be introduced into the activatable cytokine construct to provide flexibility at one or more of the junctions between domains, between moieties, between moieties and domains, or at any other junctions where a linker would be beneficial. In some embodiments, when the ACC is provided as a conformationally constrained construct, a flexible linker can be inserted to facilitate the formation and maintenance of structure in the uncleaved activatable cytokine construct. Any of the linkers described herein can provide the desired flexibility to facilitate inhibition of target (e.g., cytokine receptor) binding or facilitate cleavage of the CM by proteases. In some embodiments, the linker contained in the ACC is fully or partially flexible, such that the linker can contain, in addition to the flexible linker, one or more moieties that impart a less flexible structure to provide the desired ACC. Some linkers may contain cysteine ​​residues, which may form disulfide bonds to reduce the flexibility of the construct. It has been found that reducing the length of the linker or linking region reduces the activity of the mature cytokine protein in the ACC (see, for example, FIG. 16, which shows data for ACC without peptide affinity mask). In most cases, the length of the linker is determined by counting the number of amino acids in the N-terminal to C-terminal direction from the N-terminus of the linker adjacent to the C-terminal amino acid of the previous component to the C-terminus of the linker adjacent to the N-terminal amino acid of the next component (i.e., where the length of the linker does not contain either the C-terminal amino acid of the previous component or the N-terminal amino acid of the next component). In embodiments where the linker is used at the N-terminus of a DD containing an Fc domain, the length of the linker is determined by counting the number of amino acids from the N-terminus of the linker adjacent to the C-terminal amino acid of the previous component to the C-terminus of the linker adjacent to the first cysteine ​​of the Fc hinge region that participates in a disulfide bond with the second Fc domain (i.e., where the length of the linker does not contain the C-terminal amino acid of the previous component or the first cysteine ​​of the Fc hinge region).

[0191] As is evident from the present disclosure and FIG. 17, the ACC of the present disclosure contains a stretch of amino acids between the CP and the proximal point of interaction between the dimerization domains. That stretch of amino acids may be referred to as the linking region (LR). As used herein, the term "linking region" or "LR" refers to a stretch of amino acid residues between the C-terminus of the cytokine and the amino acid residues adjacent at the N-terminus to the proximal point of interaction between the dimerization domains (i.e., the linking region does not contain the C-terminal amino acid of the cytokine or the N-terminal amino acid of the DD that forms the proximal point of interaction with the DD of the corresponding second monomer). For example, when the DD is a pair of Fc domains, the linking region is a stretch of amino acid residues between the C-terminus of the cytokine and the first N-terminal cysteine ​​residue of the Fc that participates in a disulfide bond with the second Fc domain (e.g., cysteine ​​226 of the Fc domain of IgG1 or IgG4 according to EU numbering). If the dimerization domain is not a peptide, the linking region is a stretch of amino acid residues from the C-terminus of the cytokine to the last amino acid, for example, if DD is a biotin-streptavidin pair, the linking region of the biotin-containing monomer is the stretch of amino acid residues between the C-terminus of the cytokine and the biotin molecule, and the linking region of the streptavidin-containing monomer is the stretch of amino acid residues between the C-terminus of the cytokine and the streptavidin molecule.

[0192] In some embodiments, additional amino acid sequences may be placed at the N-terminus or C-terminus of any domain of any ACC. Examples include, but are not limited to, targeting moieties (e.g., ligands for receptors on cells present in target tissues) and serum half-life extending moieties (e.g., polypeptides that bind to serum proteins such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HSA))).

[0193] In some embodiments of any of the activatable cytokine constructs described herein, the linker comprises a total of about 1 amino acid to about 25 amino acids (e.g., about 1 amino acid to about 24 amino acids, about 1 amino acid to about 22 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 18 amino acids, about 1 amino acid to about 16 amino acids, about 1 amino acid to about 15 amino acids, about 1 amino acid to about 14 amino acids, about 1 amino acid to about 12 amino acids, about 1 amino acid to about 10 amino acids, about 1 1 to about 8 amino acids, about 1 to about 6 amino acids, about 1 to about 5 amino acids, about 1 to about 4 amino acids, about 1 to about 3 amino acids, about 1 to about 2 amino acids, about 2 to about 25 amino acids, about 2 to about 24 amino acids, about 2 to about 22 amino acids, about 2 to about 20 amino acids, about 2 to about 18 amino acids, about 2 to about 16 amino acids, about 2 to about 15 amino acids, about 2 to about About 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 2 amino acids to about 3 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 4 amino acids to about 5 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 24 amino acids, about 5 amino acids to about 22 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 18 amino acids, about 5 amino acids to about 16 amino acids,about 5 amino acids to about 15 amino acids, about 5 amino acids to about 14 amino acids, about 5 amino acids to about 12 amino acids, about 5 amino acids to about 10 amino acids, about 5 amino acids to about 8 amino acids, about 5 amino acids to about 6 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 15 amino acids, about 6 1 to about 14 amino acids, about 6 to about 12 amino acids, about 6 to about 10 amino acids, about 6 to about 8 amino acids, about 8 to about 25 amino acids, about 8 to about 24 amino acids, about 8 to about 22 amino acids, about 8 to about 20 amino acids, about 8 to about 18 amino acids, about 8 to about 16 amino acids, about 8 to about 15 amino acids, about 8 to about 14 amino acids, about 8 to about 12 amino acids, about 8 From about 10 amino acids, from about 10 amino acids to about 25 amino acids, from about 10 amino acids to about 24 amino acids, from about 10 amino acids to about 22 amino acids, from about 10 amino acids to about 20 amino acids, from about 10 amino acids to about 18 amino acids, from about 10 amino acids to about 16 amino acids, from about 10 amino acids to about 15 amino acids, from about 10 amino acids to about 14 amino acids, from about 10 amino acids to about 12 amino acids, from about 12 amino acids to about 25 amino acids, from about 12 amino acids to about 24 amino acids, from about 12 amino acids to about 2 2 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 15 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 14 amino acids to about 15 amino acids,About 15 amino acids to about 25 amino acids, about 15 amino acids to about 24 amino acids, about 15 amino acids to about 22 amino acids, about 15 amino acids to about 20 amino acids, about 15 amino acids to about 18 amino acids, about 15 amino acids to about 16 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acids to about 25 amino acids, about 22 amino acids to about 24 amino acids, or about 24 amino acids to about 25 amino acids.

[0194] In some embodiments of any of the ACCs described herein, the linker contains a total of about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, or about 25 amino acids.

[0195] Surprisingly, applicants have found that ACC that does not contain any linker between CP and DD shows the most significant reduction in cytokine activity with respect to wild-type mature cytokine, compared to ACC that contains linker or additional sequence in the linking region.See, for example, FIG. 16 (showing data of ACC without peptide affinity mask). Furthermore, the configuration without linker between CP and DD still allows effective cleavage of CM placed between CP and DD.See, for example, FIG. 7A, FIG. 7B, FIG. 10A and FIG. 10B. Thus, in some embodiments, ACC does not contain any linker between CP and DD, and CM between CP and DD contains 10, 9, 8, 7, 6, 5, 4 or 3 amino acids or less. In some embodiments, the total number of amino acids in the LR includes no more than 25 amino acids, e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids, or any range or specific number of amino acids selected from the range encompassed by 3-10 amino acids, or 5-15 amino acids, or 7-12 amino acids, or 3-25 amino acids.

[0196] In some embodiments of any of the ACCs described herein, the linker can be rich in glycine (Gly or G) residues. In some embodiments, the linker can be rich in serine (Ser or S) residues. In some embodiments, the linker can be rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs). In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences). In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences). In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences).

[0197] In some embodiments of any of the ACCs described herein, the linker is 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), GGGGSGGGGSGGGGGS (SEQ ID NO:213), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:214), GGGGSGGGGS (SEQ ID NO:215), GGGGS (SEQ ID NO:216), GS, GGGGSGS (SEQ ID NO:217), GGGGS It contains any one or a combination of one or more of GGGGSGGGGSGS (SEQ ID NO: 218), GGSLDPKGGGGS (SEQ ID NO: 219), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 220), SKYGPPCPPCPAPEFLG (SEQ ID NO: 221), GKSSGSGSESKS (SEQ ID NO: 222), GSTSGSGKSSEGKG (SEQ ID NO: 223), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 224), and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 225).

[0198] Non-limiting examples of linkers can contain a sequence that is at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to GGGS (SEQ ID NO:2), GSSGGSGGSGG (SEQ ID NO:210), GGGGSGGGGSGGGGS (SEQ ID NO:213), GGGGSGS (SEQ ID NO:217), GGGGSGGGGSGGGGSGS (SEQ ID NO:218), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:214), GGSLDPKGGGGS (SEQ ID NO:215), and GSTSGSGKPGSSEGST (SEQ ID NO:226).

[0199] In some embodiments, the linker contains a sequence selected from the group of GGSLDPKGGGGS (SEQ ID NO:219), GGGGSGGGGSGGGGSGS (SEQ ID NO:218), GGGGSGS (SEQ ID NO:217), GS, (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO:227) and (GGGS)n (SEQ ID NO:228), GGSG (SEQ ID NO:229), GGSGG (SEQ ID NO:230), GSGSG (SEQ ID NO:231), GSGGG (SEQ ID NO:232), GGGSG (SEQ ID NO:233), GSSSG (SEQ ID NO:234), GGGGSGGGGSGGGGGS (SEQ ID NO:213), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:214), GSTSGSGKPGSSEGST (SEQ ID NO:226), (GGGGS)n (SEQ ID NO:216), where n is an integer of at least 1. In some embodiments, the linker contains a sequence selected from the group consisting of GGSLDPKGGGGS (SEQ ID NO:219), GGGGSGGGGSGGGGSGS (SEQ ID NO:218), GGGGSGS (SEQ ID NO:217), and GS. In some embodiments of any of the ACCs described herein, the linker contains a sequence selected from the group of GGGSGGGGSGGGGGS (SEQ ID NO:213), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:214), and GSTSGSGKPGSSEGST (SEQ ID NO:226). In some embodiments of any of the activatable cytokine constructs described herein, the linker contains a sequence selected from the group of GGGSGGGGSGGGGGS (SEQ ID NO:213) or GGGGS (SEQ ID NO:216). In some embodiments, the linker comprises the sequence of GGGS (SEQ ID NO:2). Additional examples of linkers include those listed in Table 23.

[0200] In some embodiments, the ACC can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., linker sequences that are the same or different from any of the exemplary linker sequences described herein or known in the art). In some embodiments, the linkers include sulfo-SIAB, SMPB, and sulfo-SMPB, which linkers react with primary amine sulfhydryls.

[0201] In some embodiments of any of the ACC described herein, the ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2. In some embodiments, the control level can be the activity level of recombinant CP1 and / or CP2 (e.g., commercially available recombinant CP1 and / or CP2, recombinant wild-type CP1 and / or CP2, etc.). In some embodiments, the control level can be the activity level of a truncated (activated) form of ACC. In certain embodiments, the control level can be the activity level of pegylated CP1 and / or CP2.

[0202] In some embodiments, the at least one activity is the binding affinity of CP1 and / or CP2 to its cognate receptor, as determined using surface plasmon resonance (e.g., performed in phosphate buffered saline at 25 degrees Celsius). In certain embodiments, the at least one activity is the level of proliferation of lymphoma cells. In other embodiments, the at least one activity is the level of activation of the JAK / STAT / ISGF3 pathway in lymphoma cells. In some embodiments, the at least one activity is the level of production of SEAP in lymphoma cells. In further embodiments, the at least one activity of CP1 and / or CP2 is the level of cytokine-stimulated gene induction, for example, using RNAseq analysis (see, e.g., Zimmerer et al., Clin. Cancer Res. 14(18):5900-5906, 2008; Hilkens et al., J. Immunol. 171:5255-5263, 2003).

[0203] In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 2-fold lower compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 5-fold lower compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 10-fold lower compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 20-fold lower compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, ACC is characterized by an activity of at least one of CP1 and / or CP2 that is at least 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 5000-fold or 5,000-fold lower compared to a control level of at least one activity of CP1 and / or CP2. In some embodiments, the ACC has an activity of at least one of CP1 and / or CP2 that is at least 1-20 fold lower, 200-2000 fold lower, 300-2000 fold lower, 400-2000 fold lower, 500-2000 fold lower, 1000-2000 fold lower, 1500-2000 fold lower, 100-1500 fold lower, 200-1500 fold lower, 300-1500 fold lower, 400-1500 fold lower, 500-1500 fold lower, 600-1500 fold lower, 700-1500 fold lower, 800-1500 fold lower, 900-1500 fold lower, 100-1500 fold lower, 1500-1500 fold lower, 100-1500 fold lower, 200-1500 fold lower, 300-1500 fold lower, 400-1500 fold lower, 500-1500 fold lower, 1 ... 0 times lower, 1000 to 1500 times lower, 100 to 1000 times lower, 200 to 1000 times lower, 300 to 1000 times lower, 400 to 1000 times lower, 500 to 1000 times lower, 1000 to 5000 times lower, 2000 to 5000 times lower, 3000 to 5000 times lower, 4000 to 5000 times lower, 1000 to 4000 times lower, 2000 to 4000 times lower, 3000 to 4000 times lower, 1000 to 3000 times lower, 2000 to 3000 times lower, or 1000 to 2000 times lower.

[0204] In some embodiments, the control level of at least one activity of CP1 and / or CP2 is the activity of CP1 and / or CP2 released from ACC following cleavage of the CM by a protease(s) (the "cleavage product"). In some embodiments, the control level of at least one activity of CP1 and / or CP2 is the activity of the corresponding wild-type mature cytokine (e.g., a recombinant wild-type mature cytokine).

[0205] In some embodiments, incubation of ACC with a protease produces activated cytokine product(s), and the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are greater than the CP1 and / or CP2 activity(s) of untreated ACC. In some embodiments, the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are at least 1-fold greater than the CP1 and / or CP2 activity(s) of ACC. In some embodiments, the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are at least 2-fold greater than the CP1 and / or CP2 activity(s) of ACC. In some embodiments, the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are at least 5-fold greater than the CP1 and / or CP2 activity(s) of ACC. In some embodiments, the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are at least 10-fold greater than the CP1 and / or CP2 activity(s) of ACC. In some embodiments, the activity of one or more of CP1 and / or CP2 of the activated cytokine product(s) is at least 20-fold greater than the activity of one or more of CP1 and / or CP2 of ACC.In some embodiments, the activity of one or more of CP1 and / or CP2 of the activated cytokine product(s) is at least 1-20 fold greater, 200-2000 fold greater, 300-2000 fold greater, 400-2000 fold greater, 500-2000 fold greater, 1000-2000 fold greater, 1500-2000 fold greater, 100-1500 fold greater, 200-1500 fold greater, 300-1500 fold greater, 400-1500 fold greater, 500-1500 fold greater, 600-1500 fold greater, 700-1500 fold greater, 800-1500 fold greater, 900-1500 fold greater, 100-1500 fold greater, 1500-1500 fold greater, 100-1500 fold greater, 200-1500 fold greater, 300-1500 fold greater, 400-1500 fold greater, 500-1500 fold greater, 100-1500 fold greater, 15 ...100-1500 fold greater, 100-1500 fold greater, 1500-1500 fold greater, 100-1500 fold greater, 100-1500 fold greater, 1500-1500 500 times larger, 1000-1500 times larger, 100-1000 times larger, 200-1000 times larger, 300-1000 times larger, 400-1000 times larger, 500-1000 times larger, 1000-5000 times larger, 2000-5000 times larger, 3000-5000 times larger, 4000-5000 times larger, 1000-4000 times larger, 2000-4000 times larger, 3000-4000 times larger, 1000-3000 times larger, 2000-3000 times larger, or 1000-2000 times larger.

[0206] In some embodiments, ACC can contain a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 290 or 291. In some embodiments, ACC can be encoded by a nucleic acid that contains a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100%) identical to a nucleic acid encoding SEQ ID NO: 290 or 291. In some aspects, ACC may contain such sequences but may not include the signal sequences of those sequences. The signal sequence is not particularly limited. Some non-limiting examples of signal sequences include, for example, the corresponding residues and nucleotides of SEQ ID NO: 470 and other sequences, or those substituted with a signal sequence from another species or cell line. Other examples of signal sequences include MRAWIFFLLCLAGRALA (SEQ ID NO:468) and MALTFALLVALLVLSCKSSCSVG (SEQ ID NO:469).

[0207] Various exemplary embodiments of these ACC and activatable antibodies are described below, which may be used in any combination, without limitation, in the methods provided herein. Exemplary embodiments of ACC and activatable antibodies and methods of making ACC and activatable antibodies are described below.

[0208] In some embodiments, the CM is selected for use with a particular protease. The protease may be a protease produced by tumor cells (e.g., tumor cells may express a greater amount of a protease than healthy tissue). In some embodiments, the CM is a substrate for at least one protease selected from the group of ADAM17, BMP-1, cysteine ​​proteases such as cathepsins, HtrA1, legumain, matriptase (MT-SP1), matrix metalloproteases (MMPs), neutrophil elastase, TMPRSS such as TMPRSS3 or TMPRSS4, thrombin, and u-type plasminogen activator (uPA, also known as urokinase).

[0209] In some embodiments, the CM is a substrate for at least one matrix metalloprotease (MMP). Examples of MMPs include MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27. In some embodiments, the CM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, the CM is a substrate for MMP7. In some embodiments, the CM is a substrate for MMP9. In some embodiments, the CM is a substrate for MMP14. In some embodiments, the CM is a substrate for two or more MMPs. In some embodiments, the CM is a substrate for at least MMP9 and MMP14. In some embodiments, the CM contains two or more substrates of the same MMP. In some embodiments, the CM contains at least two or more MMP9 substrates. In some embodiments, the CM contains at least two or more MMP14 substrates.

[0210] In some embodiments, the CM is a substrate for MMPs and contains the sequences ISSGLLSS (SEQ ID NO: 19); QNQALRMA (SEQ ID NO: 16); AQNLLGMV (SEQ ID NO: 15); STFPFGMF (SEQ ID NO: 18); PVGYTSSL (SEQ ID NO: 74); DWLYWPGI (SEQ ID NO: 75); MIAPVAYR (SEQ ID NO: 42); RPSPMWAY (SEQ ID NO: 43); WATPRPMR (SEQ ID NO: 44); FRLLDWQW (SEQ ID NO: 45); LKAAPRWA (SEQ ID NO: 76); GPSHLVLT (SEQ ID NO: 77); LPGGLSPW (SEQ ID NO: 78); MGLFSEAG (SEQ ID NO: 79); SPLPLRVP (SEQ ID NO: 80); RMHLRSLG (SEQ ID NO: 81); LAAPLGLL (SEQ ID NO: 17); AVGLLAPP (SEQ ID NO: 14); LLAPSHRA (SEQ ID NO: 82); PAGLWLDP (SEQ ID NO: 20); and / or ISSGLSS (SEQ ID NO: 73).

[0211] In some embodiments, the CM is a substrate for thrombin. In some embodiments, the CM is a substrate for thrombin and contains the sequence GPRSFGL (SEQ ID NO: 83) or GPRSFG (SEQ ID NO: 84).

[0212] In some embodiments, the CM contains an amino acid sequence selected from the group of NTLSGRSENHSG (SEQ ID NO: 9); NTLSGRSGNHGS (SEQ ID NO: 10); TSTSGRSAMPRG (SEQ ID NO: 11); TSGRSAMP (SEQ ID NO: 12); VAGRSMRP (SEQ ID NO: 21); VVPEGRRS (SEQ ID NO: 22); ILPRSPAF (SEQ ID NO: 23); MVLGRSLL (SEQ ID NO: 24); QGRAITFI (SEQ ID NO: 25); SPRSIMLA (SEQ ID NO: 26); and SMLRSMPL (SEQ ID NO: 27).

[0213] In some embodiments, the CM is a substrate for neutrophil elastase. In some embodiments, the CM is a substrate for a serine protease. In some embodiments, the CM is a substrate for uPA. In some embodiments, the CM is a substrate for legumain. In some embodiments, the CM is a substrate for matriptase. In some embodiments, the CM is a substrate for a cysteine ​​protease. In some embodiments, the CM is a substrate for a cysteine ​​protease, such as a cathepsin.

[0214] In some embodiments, the CM is ISSGLLSGRSDNH (SEQ ID NO: 28); ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 30); AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 275); TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 276); VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 277); TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 278); AVGLLAPPGGLSGRSDNH (SEQ ID NO: 29); LSGRSDNHGGAVGLLAPP (SEQ ID NO: 70); VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 266); LS Contains the sequence of GRSDNHGGVHMPLGFLGP (SEQ ID NO: 267); LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 268); LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 279); ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 269); LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 270); QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 271); LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 272); QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 273) and / or ISSGLLSGRSGNH (SEQ ID NO: 274).

[0215] In some embodiments, the CM comprises a sequence selected from the group consisting of SEQ ID NO:5 through SEQ ID NO:100. In some embodiments, the CM comprises a sequence selected from the group of ISSGLLSGRSDNH (SEQ ID NO:28), LSGRSDDH (SEQ ID NO:33), ISSGLLSGRSDQH (SEQ ID NO:54), SGRSDNI (SEQ ID NO:100), and ISSGLLSGRSDNI (SEQ ID NO:68). Any one or combination of the CMs disclosed herein may be used in conjunction with any of the ACCs and activatable antibodies of the present disclosure.

[0216] In some embodiments, the ACC comprises a first monomer comprising CP1 selected from SEQ ID NOs: 1 and 101-209, CM1 selected from SEQ ID NOs: 5-100 and 237-281, PM1 selected from SEQ ID NOs: 297, 298, 292 and 299-446, CM3 selected from SEQ ID NOs: 5-100 and 237-281, and DD1, and the first monomer is dimerized with a second monomer comprising CP2 selected from SEQ ID NOs: 1 and 101-209, CM2 selected from SEQ ID NOs: 5-100 and 237-281, PM2 selected from SEQ ID NOs: 297, 298, 292 and 299-446, CM3 selected from SEQ ID NOs: 5-100 and 237-281, and DD2. In some embodiments, ACC may contain a linker between CP1 and CM1, between CP1 and PM1, between CP1 and CM3, between PM1 and CM3, and / or between CM1 and DD1 selected from SEQ ID NOs: 2 and 210-263, and between CP2 and CM2, between CP2 and PM2, between CP2 and CM4, between PM2 and CM4, and / or between CM2 and DD2 selected from SEQ ID NOs: 2 and 210-2236. In some embodiments, PM1 is selected for use with CP1 according to Table 24, and PM2 is selected for use with CP2 according to Table 24.

[0217] In some embodiments, the ACC contains DD1 and / or DD2 having an amino acid sequence at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the ACC contains DD1 having an amino acid sequence at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NO: 287 or SEQ ID NO: 288. In some embodiments, the ACC contains a DD2 having an amino acid sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NO:287 or SEQ ID NO:288.

[0218] One or both monomers of the ACC herein may include one or more peptide masks (PM), which can prevent the CP from binding to a binding partner (e.g., a receptor). In some embodiments, when the ACC is not activated, the PM in the ACC prevents the CP from binding to a target, but when the ACC is activated, the PM does not substantially or significantly prevent the CP from binding to a binding partner. In some embodiments, the PM is linked to the CP by a CM and, optionally, one or more linkers described herein.

[0219] In some embodiments, PM may interact with CP, thereby reducing or inhibiting the interaction between CP and binding partner.In some embodiments, PM may not specifically bind to CP, but rather prevents CP from binding to binding partner through non-specific interaction, such as steric hindrance.For example, PM may be placed in uncleaved ACC, such that the tertiary or quaternary structure of ACC allows PM to mask CP through charge-based interaction, thereby holding PM in place and preventing binding partner from approaching CP.

[0220] The structural characteristics of the PM can be selected according to factors such as the minimum amino acid sequence required to prevent the protein from binding to the target, the target protein-protein binding pair of interest, the size of the cytokine, the presence or absence of a linker, and the like.

[0221] PMs can be identified and / or further optimized from a library of candidate ACCs with various PMs through a screening procedure. For example, CPs and CMs can be selected to provide a desired enzyme / target combination, and the amino acid sequence of the PM can be identified through a screening procedure described below to identify PMs that provide a switchable phenotype. For example, a random peptide library (e.g., a peptide library containing about 2 to about 40 or more amino acids) can be used in the screening methods disclosed herein to identify suitable PMs. In certain embodiments, PMs with a particular binding affinity for a CP can be identified through a screening procedure that includes providing a library of peptide scaffolds consisting of candidate PMs, each scaffold being composed of a transmembrane protein and a candidate PM. The library can then be contacted with all or a portion of a protein, such as a full-length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing the protein (capable of binding to a binding partner of interest) to identify one or more candidate PMs having detectably bound proteins. Screening may be performed by one or more rounds of magnetic activated sorting (MACS) or fluorescence activated sorting (FACS) and determining the binding affinity of the PM to the CP and subsequent masking efficiency, e.g., as described in US20200308243A1, the specification of which is incorporated herein by reference in its entirety.

[0222] In some embodiments, the PM is unique to the linked CP. Examples of PMs include PMs that are specifically screened for binding to the binding domain of a cytokine or a protein fragment (e.g., affinity peptide mask). Methods for screening PMs to obtain PMs that are unique to a cytokine and that specifically and / or selectively bind to the binding domain of a binding partner / target are provided herein and can include protein display methods. Table 7 discloses exemplary PMs suitable for use with various exemplary CPs.

[0223] In some embodiments, the CP is linked to a PM and, in the presence of a natural binding partner for the CP, is capable of binding to the PM for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 ... , 10, 11, 12 months or more, there is no or substantially no binding of the CP to its binding partner, or the binding of the CP to its binding partner is less than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% compared to binding of the CP not linked to the PM.

[0224] PMs contemplated by the present disclosure may range from 1 to 50 amino acids (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, or 40 amino acids, or no more than 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids). In some examples, PMs may be 8 to 15 amino acids in length.

[0225] The PM may contain genetically encoded or non-genetically encoded amino acids. Examples of non-genetically encoded amino acids include, but are not limited to, D-amino acids, β-amino acids, and γ-amino acids. In certain embodiments, the PM contains no more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% non-genetically encoded amino acids.

[0226] The binding affinity of the cytokine for its target or binding partner when linked to the PM is at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold, 2,500-fold, 5,000-fold, 10,000-fold, 50,000-fold, 100,000-fold, 500,000-fold, 1,000,000-fold, 5,000,000-fold, 10,000,000-fold, 50,000,000-fold or more less than the binding affinity of the cytokine for its binding partner when not linked to the PM, or is 5-10-fold, 10-100-fold, 10-100-fold, 10-100-fold, or more less than the binding affinity of the cytokine for its binding partner when not linked to the PM. ,000x, 10~10,000x, 10~100,000x, 10~1,000,000x, 10~10,000,000x, 100~1,000x, 100 ~10,000x, 100~100,000x, 100~1,000,000x, 100~10,000,000x, 1,000~10,000x, 1,000 It could be ~100,000 times, 1,000-1,000,000 times, 1000-10,000,000 times, 10,000-100,000 times, 10,000-1,000,000 times, 10,000-10,000,000 times, 100,000-1,000,000 times, or 100,000-10,000,000 times lower.

[0227] When a cytokine is linked to a PM and in the presence of a binding partner, specific binding of the cytokine to the binding partner is reduced or inhibited compared to specific binding of a cytokine not linked to a PM to its binding partner. The ability of the cytokine to bind to a binding partner when linked to a PM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more as measured in an in vivo or masking efficiency assay, e.g., as described in Example 1, in an in vitro immunosorbent assay, e.g., as described in US20200308243A1.

[0228] K in response to PM cytokines D is the K of a cytokine for its binding partner D PM's K for cytokines is usually larger than that of D is the K of a cytokine for its binding partner D The binding affinity of the PM to the cytokine may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the binding affinity of the cytokine to its binding partner. The binding affinity of the PM to the cytokine may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times less than the binding affinity of the cytokine to its binding partner.

[0229] In some embodiments, the PM comprises at least a partial or complete amino acid sequence of a naturally occurring binding partner of the CP (e.g., a receptor for the CP). The PM may be a fragment of the naturally occurring binding partner. The fragment may retain no more than 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25% or 20% nucleic acid or amino acid sequence homology with the naturally occurring binding partner.

[0230] In some embodiments, the PM comprises an amino acid sequence that is not naturally occurring or does not contain the amino acid sequence of a naturally occurring binding partner or target protein. In certain embodiments, the PM is not a natural binding partner of the CP. The PM may be a modified binding partner of the CP that contains amino acid changes such that the binding affinity and / or binding activity to the CP is at least slightly reduced. In some embodiments, the PM contains no or substantially no nucleic acid or amino acid homology with the natural binding partner of the CP. In other embodiments, the PM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to the natural binding partner of the CP.

[0231] In some embodiments, the PM comprises an amino acid sequence that is at least 80% identical (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% identical) to a sequence selected from SEQ ID NOs: 297, 298, 292, and 299-446. An exemplary PM for use with a CP that is an interferon, preferably IFN-α, can contain the consensus sequence: TDVDYYREWXXXXXXXX (SEQ ID NO: 329), where X is any amino acid.

[0232] In some embodiments, the ACC may comprise a pair of PM1 and CP1, or a pair of PM2 and CP2, as listed in Table 7, which contains exemplary PMs for use with specific exemplary cytokines. In some examples, PM1 comprises a sequence selected from SEQ ID NOs: 297, 298, 292, and 299-336, and CP1 is an interferon; PM1 comprises a sequence selected from SEQ ID NOs: 297, 298, 292, and 299-332, and CP1 is interferon alpha; PM1 comprises a sequence selected from SEQ ID NOs: 299-328, and 330-332, and CP1 is interferon beta; PM1 comprises a sequence selected from SEQ ID NOs: 299-328, and 333-336, and CP1 is interferon beta. PM1 comprises a sequence selected from SEQ ID NOs: 337-341, and CP1 is IL-12; PM1 comprises a sequence selected from SEQ ID NOs: 342-349, 436-444, 478, and CP1 is IL-15; PM1 comprises a sequence selected from SEQ ID NOs: 350-435, 436-445, and CP1 is IL-2; or PM1 comprises a sequence selected from SEQ ID NOs: 445 and 446, and CP1 is IL-21. In some examples, PM2 comprises a sequence selected from SEQ ID NOs: 297, 298, 292, and 299-336, and CP2 is interferon; PM2 comprises a sequence selected from SEQ ID NOs: 297, 298, 292, and 299-332, and CP2 is interferon α; PM2 comprises a sequence selected from SEQ ID NOs: 299-328, and 330-332, and CP2 is interferon β; PM2 comprises a sequence selected from SEQ ID NOs: 299-328, and 333-336, and CP2 is interferon β. PM2 comprises a sequence selected from SEQ ID NOs: 337-341, and CP2 is IL-12; PM2 comprises a sequence selected from SEQ ID NOs: 342-349, 436-444, 478, and CP2 is IL-15; PM2 comprises a sequence selected from SEQ ID NOs: 350-435, 436-445, and CP2 is IL-2; or PM2 comprises a sequence selected from SEQ ID NOs: 445 and 446, and CP2 is IL-21.

[0233] In some embodiments, the PM may include an inactive cytokine. For example, the inactive cytokine may interact with the CP component of ACC to prevent interaction between the CP and a binding partner of the CP. In one example, the inactive cytokine may include IFNα-2b (SEQ ID NOs: 297 and 298) having a mutation, such as the L130P mutation. In another example, the inactive cytokine may be a truncated version of a wild-type cytokine, such as IFNα-2b having 1-150 amino acids.

[0234] In some embodiments, once the PM is detached from the cytokine and in a free state, it may have biological activity or therapeutic effect, such as binding ability. For example, the free peptide may bind to the same or different binding partner. In certain embodiments, the free PM (detached PM) may exert a therapeutic effect and provide a secondary function to the compositions disclosed herein. In some embodiments, once the PM is detached from the cytokine and in a free state, it may advantageously not exhibit biological activity. For example, in some embodiments, the free PM does not induce an immune response in a subject.

[0235] Conjugation to Reagents The present disclosure also provides methods and materials for including additional elements, such as targeting moieties, drugs (e.g., therapeutic drugs, antitumor drugs), toxins, or fragments thereof, in any of the ACC and antibodies described herein to facilitate delivery to cells or tissues of interest. Any of the following disclosures regarding conjugation of drugs to ACC should be construed as applying to and supporting the conjugation of drugs to the antibodies of the present disclosure as well.

[0236] In some embodiments of any of the ACCs described herein, the ACC can be conjugated to a cytotoxic agent, such as, but not limited to, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope. In some embodiments of any of the ACCs described herein, the activatable cytokine construct can be conjugated to a cytotoxic agent, such as, but not limited to, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope.

[0237] Non-limiting exemplary cytotoxic agents that can be conjugated to any of the ACCs described herein include dolastatins and their derivatives (e.g., auristatin E, AFP, monomethylauristatin D (MMAD), monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), desmethylauristatin E (DMAE), auristatin F, desmethylauristatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolones), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycins, alpha-amanitin, turbostatin, fenstatin, and the like). camptothecin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocompstatin, pyrrolobenzimidazole (PBI), sibulostatin 6, doxaliform, cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) mutant, Pseudomonas toxin A (ZZ-PE38) mutant, ZJ-101, anthracyclines, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecins, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaplysiatoxin, KahaMide-F, discodermolide, and ecteinascidin.

[0238] Non-limiting, exemplary enzymatically active toxins that can be conjugated to any of the ACCs described herein include diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuriies fordii proteins, dianfhin proteins, Phytoiaca Americana proteins (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotirs, sapaonaria officinalis inhibitor, geoinin, mitogeliin, restrictocin, phenomycin, neomycin, and the trichothecenes.

[0239] Non-limiting, exemplary antineoplastic agents that can be conjugated to any of the ACCs described herein include adriamycin, cerbidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarbazine, and cytarabine.

[0240] Non-limiting, exemplary antiviral drugs that can be conjugated to any of the ACCs described herein include acyclovir, virA, and symmetrel.

[0241] Non-limiting, exemplary antifungal agents that can be conjugated to any of the ACC described herein include nystatin.

[0242] Non-limiting, exemplary conjugable detection reagents that can be conjugated to any of the ACCs described herein include fluorescein and its derivative, fluorescein isothiocyanate (FITC).

[0243] 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.

[0244] Non-limiting, exemplary 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-α-L-arabinopyranoside) (OSW-1) that can be conjugated to any of the activatable cytokine constructs described herein include s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepines, calicheamicins, podophyiitoxins, taxanes, and vinca alkaloids.

[0245] Non-limiting, exemplary radiopharmaceuticals that can be conjugated to any of the activatable cytokine constructs described herein include: 123 I, 89 Zr, 125 I, 131 I, 99 mTc, 201 T1, 62 Cu, 18 F, 68 Ga, 13 N, 15 O. 38 K, 82 Rb, 111 In, 133 Xe, 11 C, and 99 One example is mTc (technetium).

[0246] Non-limiting exemplary heavy metals that can be conjugated to any of the ACCs described herein include barium, gold and platinum.

[0247] Non-limiting exemplary anti-mycoplasma agents that can be conjugated to any of the ACC described herein include tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0248] Those skilled in the art will appreciate that a wide variety of possible moieties can be conjugated to any of the activatable cytokine constructs described herein. Conjugation can include any chemical reaction that results in binding two molecules, so long as the ACC and other moieties retain their respective activities. Conjugation can include many chemical reaction mechanisms, such as covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In some embodiments, the preferred bond is a covalent bond. Covalent binding can be achieved either by direct condensation of existing side chains or by incorporation of an external bridging molecule. Many bivalent or multivalent binding agents are useful in conjugating any of the activatable cytokine constructs described herein. For example, conjugates can contain organic compounds, such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzene, and hexamethylenediamine. In some embodiments, the activatable cytokine construct can contain or otherwise introduce one or more non-natural amino acid residues to provide suitable sites for conjugation.

[0249] In some embodiments of any of the ACCs described herein, the drug and / or conjugate is attached to the antigen-binding domain by a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Since many cancers naturally release high levels of glutathione, a reducing agent, glutathione present in the cancer tissue microenvironment can reduce the disulfide bond, followed by release of the drug and / or conjugate at the delivery site.

[0250] In some embodiments of any of the ACCs described herein, when the conjugate binds to the target in the presence of complement within the target site (e.g., diseased tissue (e.g., cancer tissue)), the amide or ester bond attaching the conjugate and / or agent to the linker is cleaved, resulting in release of the conjugate and / or agent in an activated state. These conjugates and / or agents, when administered to a subject, will achieve delivery and release of the conjugate and / or agent at the target site (e.g., diseased tissue (e.g., cancer tissue)). These conjugates and / or agents are particularly effective for in vivo delivery of any of the conjugates and / or agents described herein.

[0251] In some embodiments, the linker is not cleavable by the enzymes of the complement system. For example, the conjugate and / or agent is released without complement activation, which ultimately lyses the target cell. In such embodiments, the conjugate and / or agent is to be delivered to the target cell (e.g., a hormone, enzyme, corticosteroid, neurotransmitter, or gene). Furthermore, the linker is made mildly susceptible to cleavage by serum proteases, and the conjugate and / or agent is slowly released at the target site.

[0252] In some embodiments of any of the ACC described herein, the conjugate and / or agent are designed such that the conjugate and / or agent is delivered to a target site (e.g., diseased tissue (e.g., cancerous tissue)) but the conjugate and / or agent is not released.

[0253] In some embodiments of any of the ACCs described herein, the conjugate and / or agent is attached to the antigen-binding domain either directly or via a non-cleavable linker. Exemplary non-cleavable linkers include amino acids (e.g., D-amino acids), peptides, or other organic compounds that can be modified by the methods described herein to contain a functional group that is subsequently available for attachment to the antigen-binding domain.

[0254] In some embodiments of any of the ACCs described herein, the ACC contains at least one point of conjugation of an agent. In some embodiments, all available points of conjugation are available for conjugation to an agent. In some embodiments, the one or more points of conjugation include, but are not limited to, sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain sulfide bonds but not intrachain disulfide bonds, and / or sulfur atoms of cysteine ​​or other amino acid residues that contain sulfur atoms. In such cases, the residues may be naturally occurring in the protein construct structure or may be incorporated into the protein construct using methods such as, but not limited to, site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.

[0255] The present disclosure also provides methods and materials for preparing ACC for conjugation. In some embodiments of any of the ACCs described herein, the ACC is modified to contain one or more interchain disulfide bonds. For example, the disulfide bonds in ACC can be reduced after exposure to a reducing agent, such as, but not limited to, TCEP, DTT or β-mercaptoethanol. In some cases, the reduction of disulfide bonds is only partial reduction. As used herein, the term "partial reduction" refers to the situation in which ACC is contacted with a reducing agent and some of all available sites of the conjugate are reduced (e.g., not all disulfide bonds are reduced). In some embodiments, an activatable cytokine construct is partially reduced if, after contact with a reducing agent, all available sites on the conjugate are reduced by less than 99% (e.g., less than 98%, less than 97%, less than 96%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%). In some embodiments, an ACC having reduction in one or more interchain disulfide bonds is conjugated to an agent that is reactive with free thiols.

[0256] The present disclosure also provides methods and materials for conjugating a therapeutic agent to a specific location on the ACC. In some embodiments of any of the ACCs described herein, the ACC is modified so that a therapeutic agent can be conjugated to the ACC at a specific location on the ACC. For example, the ACC may be partially reduced to facilitate conjugation to the ACC. In such cases, partial reduction of the ACC exists such that the conjugation site of the ACC is not reduced. In some embodiments, the conjugation site(s) on the ACC are selected to facilitate conjugation of the agent at a specific location on the protein construct. Upon treatment with a reducing agent, various factors may affect the "reduction level" of the ACC. For example, to achieve partial reduction of the ACC using the methods and materials described herein, optimization of, but not limited to, the ratio of reducing agent to ACC, the length of incubation, the incubation temperature, and / or the pH of the reduction reaction solution may be required. Any suitable combination of factors (e.g., the ratio of reducing agent to ACC, the length and temperature of incubation with the reducing agent, and / or the pH of the reducing agent) can be used to achieve partial reduction of ACC (e.g., total reduction of available conjugation sites, or reduction of specific conjugation sites).

[0257] An effective ratio of reducing agent to ACC can be any ratio that at least partially reduces ACC in a manner that allows for conjugation to a drug (e.g., overall reduction of available conjugation sites, or reduction of specific conjugation sites). In some embodiments, the ratio of reducing agent to ACC will be in the range of about 20:1 to 1:1, about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or about 1:1 to 1:1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0258] Effective incubation times and temperatures for treating ACC with a reducing agent can be any times and temperatures that at least partially reduce the ACC (e.g., total reduction of available conjugation sites, or reduction of specific conjugation sites) in a manner that allows for conjugation of the agent to the ACC. In some embodiments, incubation times and temperatures for treating ACC will range from about 1 hour at 37° C. to about 12 hours at 37° C. (or any subrange therein).

[0259] The effective pH of the reduction reaction for treating ACC with a reducing agent can be any pH that at least partially reduces ACC (e.g., total reduction of available conjugation sites, or reduction of specific conjugation sites) in a manner that allows for conjugation of ACC to an agent.

[0260] When the partially reduced ACC contacts with a thiol-containing drug, the drug can be conjugated to the interchain thiol in the ACC. The drug can be modified to contain a thiol using a thiol-containing reagent (e.g., cysteine ​​or N-acetylcysteine). For example, the ACC can be partially reduced after incubation with a reducing agent (e.g., TEPC) at a desired ratio of reducing agent to ACC for about 1 hour at about 37°C. The effective ratio of reducing agent to ACC can be any ratio that partially reduces at least two interchain disulfide bonds located in the ACC in a manner that allows conjugation of a thiol-containing drug (e.g., reduction of all available conjugation sites or reduction of specific conjugation sites).

[0261] In some embodiments of any of the ACCs described herein, the ACC is reduced by a reducing agent in a manner that avoids the reduction of any intrachain disulfide bonds. In some embodiments of any of the ACCs described herein, the ACC is reduced by a reducing agent in a manner that avoids the reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0262] In some embodiments of any of the ACCs described herein, the ACC can also contain a drug conjugated to the ACC. In some embodiments, the conjugated drug is a therapeutic drug.

[0263] In some embodiments, the agent (e.g., the agent conjugated to the activatable cytokine construct) is a detectable moiety, such as a label or other marker. For example, the agent is or contains a radiolabeled amino acid, one or more biotinyl moieties that can be detected by labeled avidin (e.g., streptavidin that contains a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety is attached by a spacer molecule.

[0264] In some embodiments, an agent (eg, a cytotoxic agent conjugated to an activatable cytokine construct) is linked to the ACC using a carbohydrate moiety, a sulfhydryl group, an amino group, or a carboxylate group.

[0265] In some embodiments of any of the ACCs described herein conjugated to an agent, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to the ACC via a linker and / or CM (also referred to as a cleavable sequence). In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to a cysteine ​​or lysine in the ACC. In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to another residue of the ACC, such as a residue disclosed herein. In some embodiments, the linker is a thiol-containing linker. Some non-limiting examples of linkers and / or CMs are shown in Table 1.

[0266] [Table 1] Those skilled in the art will appreciate that a wide variety of possible moieties can be linked to the ACC of the present disclosure. (See, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference.) In general, effective conjugation of a drug (e.g., a cytotoxic drug) to an ACC can be achieved by any chemical reaction that conjugates the drug to the ACC while also allowing the drug and ACC to retain functionality.

[0267] In some embodiments of any of the ACC conjugated to an agent, the agent can be conjugated to the ACC using a variety of bifunctional protein coupling agents, including, but not limited to, N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, radionucleotides can be conjugated to ACC using carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agents. (See, e.g., WO94 / 11026).

[0268] Suitable linkers and CMs are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives linked to ACC via oligopeptide linkers. In some embodiments, suitable linkers include (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co., Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimide) conjugated to EDC: Pierce Chem. Co., Cat. #2165-G). Chem. Co., Cat. #24510). Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

[0269] The above linkers and CMs contain components with different attributes, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. In addition, the linker SMPT contains a sterically hindered disulfide bond, which can form highly stable conjugates. Disulfide bonds are generally less stable than other bonds, as disulfide bonds are cleaved in vitro, resulting in less usable conjugates. Sulfo-NHS can particularly increase the stability of carbodiimide coupling. When carboimide coupling (e.g., EDC) is used in conjunction with sulfo-NHS, it forms esters that are more resistant to hydrolysis than the carboimide coupling reaction alone.

[0270] In some embodiments of any of the ACC, agents can be conjugated to the ACC using modified amino acid sequences encompassed by the amino acid sequence of the ACC. Protein constructs can be designed to control the placement and / or dosage of conjugated agents (e.g., cytotoxic agents) by inserting conjugable amino acids at specific positions within the amino acid sequence of the ACC. For example, the ACC can be modified to contain cysteine ​​amino acid residues at positions on the first, second, third and / or fourth monomers that provide reactive thiol groups, do not negatively affect protein folding and / or assembly and do not alter antigen binding properties. In some embodiments, the ACC can be modified to contain one or more non-natural amino acid residues within the amino acid sequence of the ACC to provide suitable sites for conjugation. In some embodiments, the ACC can be modified to contain enzymatically activatable peptide sequences within the amino acid sequence of the ACC.

[0271] nucleic acid Provided herein is a nucleic acid containing a sequence encoding a first monomeric construct (or a protein portion of the first monomeric construct) (e.g., any of the first monomeric constructs described herein) and a second monomeric construct (or a protein portion of the second monomeric construct) (e.g., any of the second monomeric constructs described herein) of any of the ACCs described herein. In some embodiments, a pair of nucleic acids together encodes the first monomeric construct (or the protein portion of the first monomeric construct) and the second monomeric construct (or the protein portion of the second monomeric construct). In some embodiments, the nucleic acid sequence encoding the first monomeric construct (or the protein portion of the first monomeric construct) is at least 70% identical (e.g., at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the nucleic acid sequence encoding the second monomeric construct (or the protein portion of the second monomeric construct).

[0272] In some embodiments, the nucleic acid encoding the protein portion of the first monomeric construct encodes a polypeptide comprising PM1, CP1, CM1 and CM3 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, the nucleic acid encoding the protein portion of the second monomer encodes a polypeptide comprising CP2, CM2, PM2 and CM4 portions. In some embodiments, a pair of nucleic acids together encodes the protein portion of the first monomeric construct and the protein portion of the second monomeric construct, which are subsequently conjugated to the DD1 and DD2 portions, respectively (in a subsequent conjugation step).

[0273] In some embodiments, the nucleic acid encoding the first monomeric construct encodes a polypeptide comprising a DD1 portion. In some embodiments, the nucleic acid encoding the second monomeric construct encodes a polypeptide comprising a DD2 portion.

[0274] vector Provided herein are vectors and vector sets that contain any of the nucleic acids described herein.Those skilled in the art will be able to select a suitable vector or vector set (e.g., expression vector) to generate any of the ACCs described herein, and use the vector or vector set to express any of the ACCs described herein.For example, in selecting a vector or vector set, the cell must be considered, since the vector(s) may need to be integrated into and / or replicate in the chromosome of the cell.Exemplary vectors that can be used to generate ACC are also described below.

[0275] As used herein, the term "vector" refers to a polynucleotide that can induce expression of a recombinant protein (e.g., a first or second monomer) in a cell (e.g., any of the cells described herein). A "vector" can deliver nucleic acids and their fragments into a host cell and contains control sequences (e.g., promoters, enhancers, poly(A) signals). An exogenous polynucleotide may be inserted into an expression vector to be expressed. The term "vector" also encompasses artificial chromosomes, plasmids, retroviruses and baculovirus vectors.

[0276] Methods for making vectors containing any one of the nucleic acids described herein and suitable for transforming a cell (e.g., a mammalian cell) are well known in the art. See, for example, Sambrook et al., Eds. "Molecular Cloning: A Laboratory Manual," 2 nd Ed., Cold Spring Harbor Press, 1989 and Ausubel et., Eds. "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0277] Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vectors), adeno-associated viral (AAV) vectors, lentiviral vectors, and retroviral vectors), as well as any Gateway® vectors. A vector can, for example, contain sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. One of skill in the art would be able to select suitable vectors and mammalian cells for making any of the ACCs described herein.

[0278] In some embodiments of any of the ACC described herein, the ACC may be made biosynthetically using recombinant DNA techniques and expression in eukaryotic or prokaryotic species.

[0279] In some embodiments, the vector contains a nucleic acid encoding a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the vector is an expression vector.

[0280] In some embodiments, the pair of vectors contains a pair of nucleic acids that together encode a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the pair of vectors is a pair of expression vectors.

[0281] cell Also provided herein is a host cell containing any of the vectors or sets of vectors described herein, which contain any of the nucleic acids described herein.

[0282] Any ACC and antibody described herein can be produced by any cell (e.g., a mammalian cell). In some embodiments, the host cell is a mammalian cell (e.g., a human cell), a rodent cell (e.g., a mouse cell, a rat cell, a hamster cell, or a guinea pig cell), or a non-human primate cell.

[0283] Methods for introducing nucleic acids and vectors (e.g., any of the vectors or sets of vectors described herein) into cells are known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids into cells include lipofection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transfection (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0284] In some embodiments, the introducing step includes introducing into the cell a vector (e.g., any of the vectors or sets of vectors described herein) containing nucleic acid encoding the monomers that make up any of the ACC and antibodies described herein.

[0285] In some embodiments of any of the methods described herein, the cell can be a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell that has a distinct, membrane-bound nucleus. Such cells can include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammalian, avian, plant, or insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells).

[0286] In some embodiments, the cell contains a nucleic acid encoding a first monomer and a second monomer of any one of the ACC and antibodies described herein. In some embodiments, the cell contains a pair of nucleic acids that together encode a first monomer and a second monomer of any of the ACC and antibodies described herein.

[0287] Methods for producing activatable cytokine constructs Provided herein is a method of producing any of the ACC described herein, the method comprising: (a) culturing any of the recombinant host cells described herein in a liquid medium under conditions sufficient to produce the ACC; and (b) recovering the ACC from the host cells and / or the liquid medium.

[0288] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor cell proliferation, cell differentiation, and cell growth. For example, cells can be cultured by contacting cells (e.g., any of the cells described herein) with a cell culture medium that contains the necessary growth factors and supplements sufficient to support cell viability and growth.

[0289] In some embodiments of any of the methods described herein, the method further comprises isolating the recovered ACC. Non-limiting examples of isolation methods include ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand-affinity chromatography, ion exchange chromatography (e.g., anion or cation), and hydrophobic interaction chromatography.

[0290] In some embodiments, the cells are capable of producing a protein portion of a first monomeric construct containing CP1, CM1, PM2 and CM3, and a protein portion of a second monomeric construct containing CP2 and CM2, and optionally PM2 and CM4, which protein portions are subsequently conjugated to the DD1 and DD2 portions, respectively.

[0291] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that allow for the formation of disulfide bonds between the dimerization domains, forming and maintaining the dimerization of ACC.

[0292] In some embodiments of any of the methods described herein, the method further comprises preparing the isolated ACC into a pharmaceutical composition.Various formulations are known in the art and described herein.Any of the isolated ACC and / or antibodies described herein can be prepared for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0293] Also provided herein is an ACC produced by any of the methods described herein.Also provided is a composition (e.g., a pharmaceutical composition) containing any of the ACC produced by any of the methods described herein.Also provided herein is a kit containing at least one dose of any of the compositions (e.g., a pharmaceutical composition) described herein.

[0294] Treatment method Provided herein are methods for treating a disease (e.g., cancer (e.g., any of the cancers described herein) or an infectious disease) in a subject, comprising administering to the subject a therapeutically effective amount of any of the ACC and antibodies described herein.

[0295] As used herein, the term "subject" refers to any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, a rat, a hamster, or a guinea pig), a non-human primate (e.g., a simian (e.g., a monkey (e.g., a baboon, a marmoset), or an ape (e.g., a chimpanzee, a gorilla, an orangutan, or a gibbon)), or a human. In some embodiments, the subject is a human.

[0296] In some embodiments, the subject has been previously identified or diagnosed as having a disease (e.g., cancer (e.g., any of the cancers described herein)).

[0297] As used herein, the term "treating" includes reducing the severity, frequency, or number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., cancer (e.g., any of the cancers described herein)) in a subject (e.g., any of the subjects described herein). In some embodiments where the disease is cancer, treating results in a reduction in cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduction in the risk of cancer recurrence in a subject with cancer.

[0298] In some embodiments, the disclosed methods and uses include administering ACC and a PD-1 / PD-L1 pathway inhibitor simultaneously or sequentially, e.g., sequentially in any order. In some embodiments, the disclosed methods and uses include administering ACC and a PD-1 / PD-L1 pathway inhibitor separately. In some aspects, therapeutic or sub-therapeutic doses of each agent are administered. In some aspects, the disclosed methods and uses include administering ACC and a PD-1 / PD-L1 pathway inhibitor sequentially or simultaneously, such that an additive or synergistic therapeutic effect is achieved in the subject. As used herein, the term "combination" broadly includes simultaneous or sequential administration, and also includes administering the active agents separately or in the same composition or container. Specifically, the ACC used in combination may include IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFNβ, IFNγ, GM-CSF, TGF-β, LIGHT, GITR-L, CD40L, CD27L, 4-1BB-L, OX40, and OX40L.

[0299] In some embodiments, the methods and uses of the present disclosure include any route of administration, such as intravenous, infusion, intratumoral, subcutaneous, intraperitoneal, intradermal, oral (e.g., inhalation), intranasal, transdermal (e.g., topical), transmucosal, and / or intramuscular.

[0300] In some embodiments of any of the methods described herein, the disease is cancer.Also provided herein is a method for treating a subject in need of treatment (e.g., any of the exemplary subjects described herein or known in the art), comprising administering to the subject a therapeutically effective amount of any of the ACCs described herein or any of the compositions described herein (e.g., pharmaceutical compositions).

[0301] In some embodiments of these methods, the subject has been identified or diagnosed with cancer. Non-limiting examples of cancer include solid tumors, hematological tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B cell neoplasms, multiple myeloma, lymphomas (e.g., B cell lymphoma, B cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, malignant cutaneous T cell lymphoma), leukemias (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloma, myeloma, leukemia ... The cancers include myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), Kaposi's sarcoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colon cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), squamous cell head and neck cancer, uterine cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is Burkitt's lymphoma. In some aspects, the subject has been identified or diagnosed as having a familial cancer syndrome, such as Li-Fraumeni syndrome, familial breast and ovarian cancer (BRCA1 or BRCA2 mutation) syndrome, and the like. The disclosed methods are also useful for treating non-solid cancers. Exemplary solid tumors include malignant tumors (e.g., sarcomas, adenocarcinomas, and carcinomas) of various organ systems, such as the lung, breast, lymphatic system, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., renal, urothelial, or testicular tumors), pharynx, prostate, and ovary. Exemplary adenocarcinomas include colorectal carcinoma, renal cell carcinoma, liver carcinoma, non-small cell carcinoma of the lung, and carcinoma of the small intestine.

[0302] Exemplary cancers described by the National Cancer Institute include: adult acute lymphocytic leukemia; childhood acute lymphocytic leukemia; adult acute myeloid leukemia; adrenal cortical carcinoma; childhood adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma (childhood, cerebellum); astrocytoma (cerebrum, child); extrahepatic bile duct cancer; bladder cancer; pediatric bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma; pediatric brain stem glioma; brain tumor (adult); brain tumor, pediatric brain stem glioma; brain tumor, astrocytoma (childhood, cerebellum); brain tumor, astrocytoma (cerebrum) / pediatric malignant glioma; brain tumor, pediatric ependymoma ;Brain tumors, medulloblastoma (pediatric);Brain tumors, supratentorial primitive neuroectodermal tumor (pediatric);Brain tumors, visual pathway and hypothalamic glioma (pediatric);Brain tumors, (pediatric, other);Breast cancer;Breast cancer and pregnancy;Pediatric breast cancer;Male breast cancer;Pediatric bronchial adenoma / carcinoid;Pediatric carcinoid tumors;Gastrointestinal carcinoid tumors;Adrenal cortical carcinoma;Pancreatic islet cell carcinoma;Cancer of unknown primary;Primary central nervous system lymphoma;Cerebellar astrocytoma (pediatric);Cerebral astrocytoma / malignant glioma (pediatric);Cervical cancer;Pediatric cancer;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Chronic myeloproliferative disorder;Clear cell sarcoma of tendon sheath;Colon cancer;Pediatric colon Rectal cancer;Malignant cutaneous T-cell lymphoma;Uterine cancer;Ependymoma (childhood);Epithelial ovarian cancer;Esophageal cancer;Childhood esophageal cancer;Ewing's sarcoma family of tumors;Childhood extracranial germ cell tumors;Extragonadal germ cell tumors;Extrahepatic bile duct cancer;Eye cancer, intraocular melanoma;Eye cancer, retinoblastoma;Gallbladder cancer;Gastric (stomach) cancer;Childhood gastric (stomach) cancer;Gastrointestinal carcinoid tumors;Childhood extracranial germ cell tumors;Extragonadal germ cell tumors;Ovarian germ cell tumors;Gestational trophoblastic tumors;Childhood brain stem gliomas;Visual pathway and hypothalamic gliomas (childhood);Hairy cell leukemia;Head and neck cancer;Adult primary hepatocellular (liver) cancer;Childhood primary hepatocellular (liver) cancer cystic (liver) cancer;adult Hodgkin's lymphoma;childhood Hodgkin's lymphoma;Hodgkin's lymphoma during pregnancy;hypopharyngeal cancer;hypothalamic and visual pathway glioma (childhood);intraocular melanoma;islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;kidney cancer;laryngeal cancer;childhood laryngeal cancer;adult acute lymphoblastic leukemia;childhood acute lymphoblastic leukemia;adult acute myeloid leukemia;childhood acute myeloid leukemia;chronic lymphocytic leukemia;chronic myeloid leukemia;hairy cell leukemia;lip and oral cavity cancer;adult primary liver cancer;childhood primary liver cancer;non-small cell lung cancer;small cell lung cancer;adult acute lymphocytic leukemia;childhood acute lymphocytic leukemia;chronic lymphocytic leukemia;AIDS-related lymphoma;primary central nervous system lymphoma;malignant cutaneous T-cell lymphoma;adult Hodgkin lymphoma;childhood Hodgkin lymphoma;Hodgkin lymphoma during pregnancy;adult non-Hodgkin lymphoma;childhood non-Hodgkin lymphoma;non-Hodgkin lymphoma during pregnancy;primary central nervous system lymphoma;Waldenstrom's macroglobulinemia;male breast cancer;malignant mesothelioma (adult);malignant mesothelioma (childhood);malignant thymoma;medulloblastoma (childhood);melanoma;intraocular melanoma;Merkel cell carcinoma;malignant mesothelioma;metastatic squamous cell carcinoma of the neck of unknown primary;multiple endocrine glands (childhood);multiple Myeloma / plasma cell neoplasms;mycosis fungoides;myelodysplastic syndrome;chronic myeloid leukemia;childhood acute myeloid leukemia;multiple myeloma;myeloproliferative disorders (chronic);nasal cavity and paranasal sinus cancer;nasopharyngeal cancer;pediatric nasopharyngeal cancer;neuroblastoma;adult non-Hodgkin's lymphoma;childhood non-Hodgkin's lymphoma;non-Hodgkin's lymphoma during pregnancy;non-small cell lung cancer;oral cavity cancer (childhood);cancer of the oral cavity and lip;oropharyngeal cancer;osteosarcoma / malignant fibrous histiocytoma of bone;childhood ovarian cancer;epithelial ovarian cancer;ovarian germ cell tumors;low-grade malignant tumors of the ovary;pancreatic cancer;childhood pancreatic cancer;pancreatic cancer, islet cell;sinus and nasal cancer;parathyroid cancer;penile cancer;brown Cell tumors;Pineal and supratentorial primitive neuroectodermal tumors (childhood);Pituitary tumors;Plasma cell neoplasms / multiple myeloma;Pleuropulmonary blastoma;Pregnancy and breast cancer;Pregnancy and Hodgkin's lymphoma;Pregnancy and non-Hodgkin's lymphoma;Primary central nervous system lymphoma;Adult primary liver cancer;Primary liver cancer in children;Prostate cancer;Rectal cancer;Renal cell (kidney) cancer;Pediatric renal cell carcinoma;Renal pelvis and ureter, transitional cell carcinoma;Retinoblastoma;Rhabdomyosarcoma (childhood);Salivary gland cancer;Pediatric salivary gland cancer;Sarcoma, Ewing's sarcoma family of tumors;Kaposi's sarcoma;Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone;Sarcoma, childhood rhabdomyosarcoma;Adult soft tissue sarcoma; pediatric soft tissue sarcoma;Sézary syndrome;skin cancer;skin cancer (childhood);skin cancer (melanoma);skin cancer, Merkel cell;small cell lung cancer;small intestine cancer;adult soft tissue sarcoma;pediatric soft tissue sarcoma;metastatic cervical squamous cell carcinoma of unknown primary;gastric (stomach) cancer;pediatric gastric (stomach) cancer;supratentorial primitive neuroectodermal tumor (childhood);malignant cutaneous T-cell lymphoma;testicular cancer;pediatric thymoma;malignant thymoma;thyroid cancer;pediatric thyroid cancer;renal pelvis and ureter, transitional cell carcinoma;gestational trophoblastic tumor;cancer of unknown primary (childhood);uncommon cancers of childhood;ureter and renal pelvis, transitional cell carcinoma;urethral cancer;uterine sarcoma;vaginal cancer;visual pathway and hypothalamic glioma (childhood);vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0303] Further exemplary cancers include diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).

[0304] Metastasis of the above cancers can also be treated or prevented according to the methods described herein.

[0305] In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of cancer in a subject (e.g., compared to the number, severity, or frequency of one or more symptoms of cancer in a subject before treatment).

[0306] In some embodiments of any of the methods described herein, the disease is an infectious disease. The ACC and antibodies of the present disclosure can also be used to prevent or treat infectious diseases and infectious diseases. The ACC and antibodies can be used to stimulate immune responses against pathogens, toxins, and self-antigens. The ACC and antibodies can be used to stimulate immune responses against pathogenic viruses, including, but not limited to, HIV, hepatitis (A, B, or C) viruses, herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus. The ACC and antibodies can also be used to stimulate an immune response to infections caused by bacteria, fungi, parasites, or other pathogens.

[0307] In some embodiments of any of the methods described herein, the method further comprises administering to the subject an additional therapeutic agent (e.g., one or more of the therapeutic agents listed in Table 2).

[0308] [Table 2] JPEG2024538706000004.jpg226159 JPEG2024538706000005.jpg228159 JPEG2024538706000006.jpg109159

[0309] Compositions / kits Also provided herein are compositions (e.g., pharmaceutical compositions) comprising any of the ACC and / or antibodies described herein and one or more (e.g., 1, 2, 3, 4 or 5) pharma- ceutically acceptable carriers (e.g., any of the pharma- ceutically acceptable carriers described herein), diluents or excipients.

[0310] In some embodiments, compositions (e.g., pharmaceutical compositions) containing any of the ACC and / or antibodies described herein can be placed into sterile vials or pre-filled syringes.

[0311] In some embodiments, compositions (e.g., pharmaceutical compositions) containing any of the ACC and / or antibodies described herein can be prepared for different routes of administration (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, or intratumoral).

[0312] In some embodiments, any of the pharmaceutical compositions described herein can contain one or more buffering agents (e.g., neutral buffered saline, phosphate buffered saline (PBS)), 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 a pharma- ceutically acceptable carrier (e.g., bacteriostatic water, PBS, or saline).

[0313] As used herein, the term "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Examples of such carriers include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and about 5% human serum albumin.

[0314] In some embodiments of any of the pharmaceutical compositions described herein, any of the ACC and / or antibodies described herein are prepared with carriers that protect against rapid elimination from the body, such as slow-release and controlled-release formulations, such as implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such pharmaceutical compositions and formulations are clear to those skilled in the art.

[0315] Also provided herein is a kit containing any of the ACC and / or antibodies described herein, any of the compositions containing any of the ACC and / or antibodies described herein, or any of the pharmaceutical compositions containing any of the ACC and / or antibodies described herein. Also provided is a kit containing, in addition to the ACC and / or antibodies described herein, one or more second therapeutic agent(s) selected from Table 2. The second therapeutic agent(s) may be provided in a dosage form separate from the ACC and / or antibody. Alternatively, the second therapeutic agent(s) may be formulated together with the ACC and / or antibody.

[0316] Any of the kits described herein can include instructions for using any of the compositions (e.g., pharmaceutical compositions) and / or any of the ACC and / or antibodies described herein. In some embodiments, the kit can include instructions for performing any of the methods described herein. In some embodiments, the kit can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kit can provide a syringe for administering any of the pharmaceutical compositions described herein.

[0317] Anti-PD1 sequence In some embodiments, the anti-PD-1, which in some aspects may be configured as an activatable antibody and in other aspects may not be configured as an activatable antibody, comprises the sequence shown below. m136-M13-MHC723 mIgG1 / K MHC723HC.1 variable heavy chain region amino acid sequence: EVKLVESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPAKRLEWVAYISNSGGNAHYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCTREDYGTSPFVYWGQGTLVTVSA (sequence number 610). MHC723LC.3 variable light chain region amino acid sequence: DIVLTQSPASLAVSLGQRTTISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAVYFCQQSKDVPWTFGGGTKLEIR (sequence number 615). MHC725HC.2 variable heavy chain region amino acid sequence: EVQLQQSGPELVKPGDSVKMSCKASGYTFTDYYMDWVKQSHGKSLEWIGYIYPKNGGSSYNQKFKGKATLTVDKSSSTAYMELHSLTSEDSAVYYCARKVVATDYWGQGTTLTVSS (sequence number 611). MHC725LC.2 variable light chain region amino acid sequence: DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIFWASIRESGVPDRFTGSGSGTDFTLTISSVKAEDRAVYYCQQCDSYPWTFGGGTKLEIK (sequence number 616). MHC728HC.4 variable heavy chain region amino acid sequence: EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYAMSWVRQTPAKRLEWVAYISNGGGDTHYPDSLKGRFTVSRDNAKNTLYLQMSSLKSEDTAMYYCARENYGTSPFVYWGQGTLVTVSA (sequence number 612). MHC728LC.2 variable light chain region amino acid sequence: DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKDVPWTFGGGTKLEIK (sequence number 617). MHC729HC.1 variable heavy chain region amino acid sequence: EVQLVESGGGLVKSGGSLKLSCAHSGFSFSSYDMSWVRQTPAKRLEWVATISGGGRYTYYPDSVKGRFTISRDNAKNTLYLQMSGLRSEDTAMYYCASNYYGFDYWGQGTTLTVSS (sequence number 613). MHC729LC.3 variable light chain region amino acid sequence: DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPWTFGGGTKLEIK (sequence number 618). MHC724HC.3 variable heavy chain region amino acid sequence: KVMLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGGGRDIYYADTVKGRFTISRDNAKNTLYLQMSSLRSEDTALYFCARLYLGFDYWGQGTTLTVSS (sequence number 614). MHC724LC.1 variable light chain region amino acid sequence: DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATSLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYSIPWTFGGGTKLEIK (sequence number 619). PD-1 A Hv variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNAHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSPFVYWGQGTLVTVSS (sequence number 620). PD-1 Ab Hv variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSYISNSGGNAHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEDYGTSPFVYWGQGTLVTVSS (sequence number 621). PD-1 Ae Hv variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDYGTSPFVYWGQGTLVTVSS (sequence number 622). PD-1 Af Hv variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNTHYADSLKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAREDYGTSPFVYWGQGTLVTVSS (sequence number 623). PD-1 Ba Hv variable heavy chain region amino acid sequence: QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYYMDWVRQAPGQGLEWIGYIYPKNGGSSYAQKFQGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARKVVATDYWGQGTLLTVSS (sequence number 624). PD-1 Bb Hv variable heavy chain region amino acid sequence: QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYYMDWVRQAPGQGLEWIGYIYPKNGGSSYAQKFQGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARKVVATDYWGQGTLLTVSS (sequence number 625). PD-1 C Hv variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVAYISNGGGDTHYADSLKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARENYGTSPFVYWGQGTLVTVSS (sequence number 626). PD-1 Ca Hv variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVAYISNQGGDTHYADSLKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARENYGTSPFVYWGQGTLVTVSS (sequence number 627). PD-1 D Hv variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAHSGFSFSSYDMSWVRQAPGKGLEWVATISGGGRYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASNYYGFDYWGQGTLLTVSS (sequence number 628). PD-11.0 Lv variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (sequence number 629). PD-11.1 Lv variable light chain region amino acid sequence: DIQLTQSPSSLSVSVGDRATITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (sequence number 630). Lv variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDQYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (sequence number 631). PD-11.4 Lv variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDSYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (sequence number 632). PD-11.5 Lv variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (sequence number 633). PD-11.6 Lv variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASDQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (sequence number 634). PD-11.7 Lv variable light chain region amino acid sequence: DIQLTQSPSSLSVSVGDRATITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (sequence number 635). PD-11.9 Lv variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKVEIK (sequence number 636). PD-11.10 Lv variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPYTFGQGTKLEIK (sequence number 637). PD-12 Lv variable light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTMTCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIFWASIRESGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSDSYPWTFGQGTKLEIK (sequence number 638). PD-14 Lv variable light chain region amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQYSSYPWTFGQGTKLEIK (sequence number 639). Kappa constant region amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 640). hIgG4 S228P amino acid sequence: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 641).

[0318] In some embodiments, the anti-PD1 CDR sequences comprise the sequences listed in the table below. [Table 3] [Table 4] In some embodiments, the PD-1 pathway inhibitor is an antibody comprising one or more sequences in Tables 7-9 of WO2017011580A2. In some embodiments, the PD1 pathway inhibitor comprises an activatable PD-1 antibody comprising: (i) an antibody or antigen-binding fragment thereof (AB) comprising one or more sequences in Tables 7-9 of WO2017011580A2, (ii) a masking moiety (MM) that inhibits binding of AB to PD-1 when the activatable antibody is in an uncleaved state, and (c) a cleavable moiety (CM) coupled to AB, where the CM is a polypeptide that functions as a substrate for a protease, and optionally a first connecting peptide and / or a second connecting peptide.

[0319] Any of the above polypeptides can be combined with human immunoglobulin constant regions to provide fully human IgG, including IgG1, IgG2, IgG4, or with mutant constant regions to provide human IgG with altered function, such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P. The above polypeptides are not limited by specific combinations and include any mask sequence that matches any substrate sequence that matches any VL sequence that matches any VH sequence. In addition to the substrate sequence, any CM disclosed herein can be used.

[0320] Anti-PD-L1 sequence In some embodiments, the anti-PD-L1, which in some aspects may be configured as an activatable antibody, and in other aspects may not be configured as an activatable antibody, comprises the sequences shown below. Variable light chain region amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYPSTFGQGTKVEIKR (SEQ ID NO: 671). Variable light chain region amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYPSTFGGGTKVEIKR (SEQ ID NO: 672). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGQSRPGFDYWGQGTLVTVSS (SEQ ID NO: 673). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGQSWPGFDYWGQGTLVTVSS (SEQ ID NO: 674). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGQSFPGFDYWGQGTLVTVSS (SEQ ID NO: 675). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSS (SEQ ID NO: 676). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAGYDYWGQGTLVTVSS (SEQ ID NO: 677). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSTGGATAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSKGFDYWGQGTLVTVSS (SEQ ID NO: 678). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWKQGIVTVYDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTV (SEQ ID NO: 679). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS (SEQ ID NO: 680). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDIWKQGMVTVYDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS (SEQ ID NO: 681). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRQGLATAYDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS (SEQ ID NO: 682). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSEIVATGILTSYDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS (SEQ ID NO: 683). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGRQGLITVYDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS (SEQ ID NO: 684). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWYQGLVTVYDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS (SEQ ID NO: 685). EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDIWKQGFATADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS (SEQ ID NO: 686). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWKQGIVTVYDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS (SEQ ID NO: 687). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRQGLATAYDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSAGFDYWGQGTLVTVSS (SEQ ID NO: 688). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSS (SEQ ID NO: 689). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAGYDYWGQGTLVTVSS (SEQ ID NO: 690). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSKGFDYWGQGTLVTVSS (SEQ ID NO: 691). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWYQGLVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVTVSS (SEQ ID NO: 692). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWYQGLVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAGYDYWGQGTLVTVSS (SEQ ID NO: 693). Variable heavy chain region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWYQGLVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSKGFDYWGQGTLVTVSS (SEQ ID NO: 694).

[0321] In some embodiments, the anti-PD-L1 CDR sequences comprise the sequences listed in the table below. [Table 5] [Table 6] Any of the above polypeptides can be combined with human immunoglobulin constant regions to provide fully human IgG, including IgG1, IgG2, IgG4, or with mutant constant regions to provide human IgG with altered function, such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P. The above polypeptides are not limited by specific combinations and include any mask sequence that matches any substrate sequence that matches any VL sequence that matches any VH sequence. In addition to the substrate sequence, any CM disclosed herein can be used.

[0322] As a non-limiting example, the spacer sequence and mask can be combined with a substrate and combined with a human kappa constant domain to obtain SEQ ID NO: 496. Or the mask can be combined with a substrate and combined with a human kappa constant domain to obtain SEQ ID NO: 728. Furthermore, the VH domain can be combined with a human immunoglobulin heavy chain constant domain to obtain human IgG1 (SEQ ID NO: 729), mutated human IgG4 S228P (SEQ ID NO: 485), mutated human IgG1 N297A (SEQ ID NO: 730), or mutated human IgG1 N297Q (SEQ ID NO: 731). Co-expression results in an activatable antibody.

[0323] Light chain sequence with spacer: [QGQSGS][GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYPSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 496). Light chain sequence without spacer: GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYPSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 728). EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 729). EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 485). EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 730). EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 731). In some embodiments, the PD-L1 pathway inhibitor is an antibody comprising one or more sequences in Tables 15-17 of WO2016149201A2. In some embodiments, the PD-L1 pathway inhibitor comprises an activatable PD-L1 antibody comprising (i) an antibody or antigen-binding fragment thereof (AB) comprising one or more sequences in Tables 15-17 of WO2016149201A2, (ii) a masking moiety (MM) that inhibits binding of AB to PD-L1 when the activatable antibody is in an uncleaved state, and (c) a cleavable moiety (CM) coupled to AB, where the CM is a polypeptide that functions as a substrate for a protease, and optionally a first connecting peptide and / or a second connecting peptide.

[0324] In some embodiments, the PD1 / PD-L1 pathway inhibitor is an antibody comprising one or more sequences in Table 7 below. In some embodiments, the PD1 / PD-L1 pathway inhibitor comprises an activatable PD-1 antibody or activatable PD-L1 antibody comprising: (i) an antibody or antigen-binding fragment thereof (AB) comprising one or more sequences in Table 7 below; (ii) a masking moiety (MM) that inhibits binding of AB to PD-1 or PD-L1 when the activatable antibody is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to AB, where the CM is a polypeptide that functions as a substrate for a protease, and optionally a first connecting peptide and / or a second connecting peptide.

[0325] [Table 7] JPEG2024538706000012.jpg231159 JPEG2024538706000013.jpg230159 JPEG2024538706000014.jpg229159 JPEG2024538706000015.jpg235159JPEG2024538706000016.jpg233159 JPEG2024538706000017.jpg233159 JPEG2024538706000018.jpg232159 JPEG2024538706000019.jpg233159 JPEG2024538706000020.jpg229159 JPEG2024538706000021.jpg227159 JPEG2024538706000022.jpg227159 JPEG2024538706000023.jpg230159 JPEG2024538706000024.jpg224159 JPEG2024538706000025.jpg221159 JPEG2024538706000026.jpg22315...

Claims

1. A composition for use in a method of treating a subject in need thereof, comprising an activatable cytokine component (ACC) and / or a PD-1 / PD-L1 pathway inhibitor; the method includes administering to the subject the activatable cytokine composition (ACC) and a PD-1 / PD-L1 pathway inhibitor; The ACC comprises a first monomeric component and a second monomeric component; (a) the first monomer construct comprises a first peptide mask (PM1), a first mature cytokine protein (CP1), first and third cleavable portions (CM1 and CM3), and a first dimerization domain (DD1), wherein the CM1 is disposed between the CP1 and the DD1, and the CM3 is disposed between the PM1 and the CP1; (b) the second monomer construct comprises a second peptide mask (PM2), a second mature cytokine protein (CP2), second and fourth cleavable portions (CM2 and CM4), and a second dimerization domain (DD2), wherein the CM2 is disposed between the CP2 and the DD2, and the CM4 is disposed between the PM2 and the CP2; the DD1 and the DD2 bind to each other, thereby forming a dimer of the first monomeric component and the second monomeric component; the first monomer and the second monomer are the same; composition. (a) said PM1 and said PM2 comprise a sequence at least 95% identical to SEQ ID NO:292; (b) said CP1 and / or said CP2 comprise a sequence at least 80% identical to SEQ ID NO:1 or SEQ ID NO:448; (c) the first monomer construct is characterized in that the CP1 and the DD1 are linked by a linking region of 18 or less amino acids, and the linking region of 18 or less amino acids contains the CM3; or (d) the second monomer construct is characterized in that the CP2 and the DD2 are connected by a linking region of 18 or less amino acids, and the linking region of 18 or less amino acids contains the CM2; The composition of claim 1.

3. The CM1, the CM2, the CM3, and / or the CM4 are selected from the group consisting of LSGRSDNH (SEQ ID NO: 5), TGRGPSWV (SEQ ID NO: 6), PLTGRSGG (SEQ ID NO: 7), TARGPSFK (SEQ ID NO: 8), NTLSGRSENHSG (SEQ ID NO: 9), NTLSGRSGNHGS (SEQ ID NO: 10), TSTSGRSANPRG (SEQ ID NO: 11), TSGRSANP (SEQ ID NO: 12), VHMPLGFLGP (SEQ ID NO: 13), AVGLLAPP (SEQ ID NO: 14), AQNLLGMV (SEQ ID NO: 15), QNQALRMA (SEQ ID NO: 16), LAAPLG LL (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), ISSGLLSSGGSGGSLSGRSD NH (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), LSGRSAN1 (SEQ ID NO: 40), LSGRSDNI (SEQ ID NO: 41), MIAPVAYR (SEQ ID NO: 42), RPSPMWAY (SEQ ID NO: 43), WATPRPMR (SEQ ID NO: 44) , FRLLDWQW (SEQ ID NO: 45), ISSGL (SEQ ID NO: 46), ISSGLLS (SEQ ID NO: 47), ISSGLL (SEQ ID NO: 48), ISSGLLSGRSANPRG (SEQ ID NO: 49), AVGLLAPPTSGRSANPRG (SEQ ID NO: 50), AVGLLAPPPSGRSANPRG (SEQ ID NO: 51), ISSGLLSGRSDDH (SEQ ID NO: 52), ISSGLLSGRSDIH (SEQ ID NO: 53), ISSGLLSGRSDQH (SEQ ID NO: 54), ISSGLLSGRSDTH (SEQ ID NO: 55), ISSGLLSGRSDYH (SEQ ID NO: 56),ISSGLLSGRSDNP (SEQ ID NO: 57), ISSGLLSGRSANP (SEQ ID NO: 58), ISSGLLSGRSANI (SEQ ID NO: 59), AVGLLAPPGGLSGRSDDH (SEQ ID NO: 60), AVGLLAPPGGLSGRSDIH (SEQ ID NO: 61), AVGLLAPPGGLSGRSDQH (SEQ ID NO: 62), AVGLLAPPGGLSGRSDTH (SEQ ID NO: 63), AVGLLAPPGGLSGRSDYH (SEQ ID NO: 64), AVGLLAPPGGLSGRSDNP (SEQ ID NO: 65), A VGLLAPPGGLSGRSANP (SEQ ID NO: 66), AVGLLAPPGGLSGRSANI (SEQ ID NO: 67), ISSGLLSGRSDNI (SEQ ID NO: 68), AVGLLAPPGGLSGRSDNI (SEQ ID NO: 69), GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 70), GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 71), LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 72), ISSGLSS (SEQ ID NO: 73), PVGYTSSL (SEQ ID NO: 74), DWLYWPGI (SEQ ID NO: 75), LKAAPRWA (SEQ ID NO: 76), GPSHLVLT (SEQ ID NO: 77), LPGGLSPW (SEQ ID NO: 78), MGLFSEAG (SEQ ID NO: 79), SPLPLRVP (SEQ ID NO: 80), RMHLRSLG (SEQ ID NO: 81), LLAPSHRA (SEQ ID NO: 82), GPRSFGL (SEQ ID NO: 83), GPRSFG (SEQ ID NO: 84), SARGPSRW (SEQ ID NO: 85), GGWHTGRN (SEQ ID NO: 86), HTGRSGAL (SEQ ID NO: 87), AARGPAIH (SEQ ID NO: 88), RG 2. The composition of claim 1, comprising a sequence selected from the group consisting of PAFNPM (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 NO: 100).

4. The composition described in claim 1, wherein CM1, CM2, CM3 and / or CM4 comprise an amino acid sequence selected from SEQ ID NOs: 41, 68 and 100. (a) the CM1, the CM2, and the CM3 contain a substrate for the same protease; (b) said CP1 and said CM1 are directly adjacent to each other in said first monomeric component; (c) the CM1 and the DD1 are directly adjacent to each other in the first monomeric component; (d) said CP2 and said CM2 are directly adjacent to each other in said second monomeric component; and / or (e) said CM2 and said DD2 are directly adjacent to each other in said second monomeric component; The composition of claim 1.

6. The composition of claim 1, (a) (i) the first monomeric component comprises, from N-terminal to C-terminal, the PM1, the CM3, the CP1, the CM1, and the DD1; or (ii) the first polypeptide comprises, from C-terminus to N-terminus, the PM1, the CM3, the CP1, the CM1, and the DD1; and / or (b) (i) the second polypeptide comprises, from N-terminal to C-terminal, the CP2, the CM2, and the DD2; or (ii) the second polypeptide comprises, from C-terminus to N-terminus, the CP2, the CM2, and the DD2; composition.

7. The composition of claim 1, (a) the first monomer construct comprises, from the N-terminus to the C-terminus, the PM1, the CM3, the CP1, the CM1, and the DD1; (i) said PM1 comprises a sequence at least 85% identical to SEQ ID NO: 292; (ii) the CM1 and the DD1 are directly adjacent to each other; (iii) the CM1 comprises a sequence at least 85% identical to SEQ ID NO: 41; (iv) the CP1 comprises a sequence at least 85% identical to SEQ ID NO:1; (b) the DD1 and the DD2 are a pair of human IgG4 Fc domains; (c) the DD1 and the DD2 are covalently bonded to each other via at least one disulfide bond, thereby forming a homodimer of the first monomeric construct and the second monomeric construct; and (d) the ACC is characterized by a reduced level of interferon activity compared to the corresponding wild-type interferon or the corresponding pegylated interferon; composition.

8. The DD1 and DD2 are an Fc domain of human IgG1, an Fc domain of human IgG2, an Fc domain of human IgG3, or an Fc domain of human IgG4; or said DD1 and said DD2 comprise SEQ ID NO:3; The composition of claim 1.

9. the first monomeric construct and the second monomeric construct each comprise a sequence at least 95% identical to SEQ ID NO: 290; or each of the first monomeric construct and the second monomeric construct comprises the sequence of SEQ ID NO: 290; The composition of claim 1.

10. The PD1 / PD-L1 pathway inhibitors include nivolumab, pembrolizumab, tislelizumab, spartalizumab, camrelizumab, cetrelimab, balstilimab, dostallimab, prorugolimab, sasanlimab, zimvelerimab, atezolizumab, avelumab, durvalumab, adebulerimab, rodapolimab, embafolimab, cosibelimab, budigalimab, and ezabenli 10. The composition of claim 1, comprising mab, phytonlimab, geptanolimab, rodapolimab, penprimimab, pimivalimab, pucotenlimab, serplulimab, sintilimab, toripalimab, zelvalimab, ipalomulimab, nofazinelimab, ruronilimab, galiflimab, manelimab, opucolimab, suduvulimab, sugemalimab, socazolimab, or tagitanlimab.

11. The composition of claim 1, wherein the PD1 / PD-L1 pathway inhibitor comprises pembrolizumab.

12. The composition described in claim 1, wherein the ACC comprises a linking region comprising 7 to 12 amino acids.

13. The composition of claim 1, wherein the first monomeric component and the second monomeric component are identical.

14. The composition described in claim 1, wherein the first monomer construct and the second monomer construct each comprise, from the N-terminus to the C-terminus, SEQ ID NO: 292; an optional linker of 0 to 10 amino acids; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 68 and 100; an optional linker of 0 to 10 amino acids; SEQ ID NO: 1; a second CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 68 and 100; and a dimerization domain.

15. The method of claim 1, wherein the first monomer construct and the second monomer construct comprise the amino acid sequence of SEQ ID NO: 290; the PD1 / PD-L1 pathway inhibitor is pembrolizumab; The composition of claim 1.

16. The composition of claim 1 , wherein the subject has been identified or diagnosed with cancer.

17. The composition of claim 1, wherein the subject has melanoma, renal cell carcinoma, or head and neck cancer.

18. The method of claim 17, wherein the first monomer construct and the second monomer construct are identical and comprise the amino acid sequence of SEQ ID NO: 290; the PD1 / PD-L1 pathway inhibitor is pembrolizumab; The composition of claim 1.

19. A composition for use in a method of treating a subject in need thereof, comprising an activatable cytokine component (ACC) and / or a PD-1 / PD-L1 pathway inhibitor; the method includes administering to the subject the activatable cytokine composition (ACC) and a PD-1 / PD-L1 pathway inhibitor; The ACC comprises a first monomeric component and a second monomeric component; (a) the first monomeric construct is a polypeptide comprising a first peptide mask (PM1), a first mature cytokine protein (CP1), first and third cleavable portions (CM1 and CM3), and a first dimerization domain (DD1); (b) the second monomeric construct is a polypeptide comprising a second peptide mask (PM2), a second mature cytokine protein (CP2), second and fourth cleavable portions (CM2 and CM4), and a second dimerization domain (DD2); (c) the first monomeric component comprises, from the N-terminus to the C-terminus, the PM1, the CM3, the CP1, the CM1, and the DD1; (d) the second monomeric component comprises, from the N-terminus to the C-terminus, the PM2, the CM4, the CP2, the CM2, and the DD2; (e) each of said CP1 and said CP2 is interferon-α, and said ACC is characterized by at least a 1000-fold reduction in interferon-α activity compared to wild-type interferon-α or the corresponding pegylated interferon; (f) the DD1 and the DD2 are covalently bonded to each other via a disulfide bond; (g) each of the CP1 and the CP2 is interferon-α-2b, and the ACC exhibits lower toxicity in vivo compared to wild-type interferon α-2b or pegylated interferon α-2b; (h) the first monomeric construct comprises a linking region comprising 12 or fewer amino acids between the C-terminus of the CP1 and the amino acid residue adjacent, N-terminally, to the proximal point of interaction between the DD1 and the DD2; (i) the second monomer construct comprises a linking region comprising 12 or fewer amino acids between the C-terminus of the CP2 and the amino acid residue adjacent, N-terminally, to the proximal point of interaction between the DD1 and the DD2; (j) said PM1 and said PM2 are not receptors for said CP1 and said CP2, respectively; (k) each of the PM1 and PM2 is not a fragment of the CP1 and CP2 receptors; composition.

20. The composition of any one of claims 1 to 19, wherein the ACC and the PD-1 / PD-L1 pathway inhibitor are administered sequentially.