Masking polypeptides, activatable cytokine constructs, and related compositions and methods

Abstract

Provided herein are activatable cytokine constructs comprising a novel masking moiety, a cytokine polypeptide, and a cleavable moiety between the masking moiety and the cytokine polypeptide. In some embodiments, the ACC is a monomer. In some embodiments, the ACC is a complex of two, three, four or more constructs. In some embodiments, the ACC is a dimer complex comprising a first monomeric construct comprising a first cytokine polypeptide, a first masking moiety, and a first dimerization domain, and a second monomeric construct comprising a second cytokine polypeptide, a second masking moiety, and a second dimerization domain, the second monomeric construct comprises a second cytokine polypeptide and / or an agonist of the first cytokine polypeptide and a second dimerization domain and optionally a second masking moiety, wherein the first masking moiety and / or the second masking moiety are the novel masking moiety.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 495,754, filed April 12, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] sequence list This application contains a sequence list that has been submitted electronically in XML format and is hereby incorporated in its entirety by reference. The XML copy was created on April 12, 2024, named "4862-146.xml", and is 819,200 bytes in size. Technical Field

[0003] This disclosure relates to the field of biotechnology, and more specifically to isolated peptides and cytokine constructs, including cytokine constructs. Background Technology

[0004] Cytokines are a family of naturally occurring small proteins and glycoproteins produced and secreted by most nucleated cells in response to viral infection and / or other antigenic stimulation. Interleukins are another subclass of cytokines. Interleukins regulate cell growth, differentiation, and motility. They are particularly important in stimulating immune responses such as inflammation. Interleukins have been used to treat cancer, autoimmune diseases, and other conditions. For example, interleukin-2 (IL2) has been indicated for the treatment of melanoma, graft-versus-host disease (GVHD), neuroblastoma, renal cell carcinoma (RCC), and is also considered for conditions including: acute coronary syndrome, acute myeloid syndrome, atopic dermatitis, autoimmune liver disease, basal cell carcinoma, bladder cancer, breast cancer, candidiasis, colorectal cancer, cutaneous T-cell lymphoma, endometrioma, HIV infection, ischemic heart disease, rheumatoid arthritis, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, systemic lupus erythematosus, tuberculosis, and other conditions. Interleukin-15 (IL-15) is known to promote the differentiation and expansion of T cells, B cells, and natural killer (NK) cells, thereby enhancing antitumor responses. IL-15 has been considered a promising candidate for anticancer therapy and has been tested in numerous clinical trials. Despite this prospect, IL-15 is known to exhibit undesirable pro-inflammatory effects and is associated with the pathogenesis of a variety of autoimmune diseases. The maximum tolerated dose of recombinant IL-15 has been reported to be 2 μg / kg. Conlon KC et al., “IL15 by Continuous Intravenous Infusion to Adult Patients with Solid Tumors in a Phase I Trial Induced Dramatic NK-Cell Subset Expansion.” Clin Cancer Res. 2019 Aug 15;25(16):4945-4954. It has also been reported that recombinant soluble IL-15 has a short half-life in vivo, which hinders its application as a treatment. Berraondo, P et al., “Cytokines in clinical cancer immunotherapy.” Br J Cancer 120, 6–15 (2019). Other interleukins such as IL-4, IL-6, IL-7, IL-9, IL-12, and IL-21 are also potential treatments for cancer and other conditions. However, interleukin therapy is often accompanied by unwanted side effects, including flu-like symptoms, nausea, vomiting, diarrhea, hypotension, and cardiac arrhythmias.

[0005] Therefore, there has always been a need for a cytokine therapy with fewer adverse side effects than existing cytokine therapies. Summary of the Invention

[0006] This disclosure provides isolated peptides and cytokine-activating constructs (ACC) comprising one or more novel masking portions.

[0007] In one aspect, this disclosure includes a cytokine masking portion (MM) comprising: (a) A first masking subunit, the first masking subunit being encoded by a first subsequence of the amino acid sequence of a first receptor polypeptide encoding a cytokine receptor, and (b) A second masking subunit, which is encoded by a second amino acid sequence selected from the group consisting of: (i) a second subsequence encoding an amino acid sequence of a cytokine receptor polypeptide, said cytokine receptor polypeptide being selected from the group consisting of an amino acid sequence encoding a first receptor polypeptide and an amino acid sequence encoding a second receptor polypeptide for a cytokine receptor, wherein the first and second subsequences are discontinuous within the amino acid sequence encoding the first receptor polypeptide; and (ii) The amino acid sequence encoding the blocking portion (BM).

[0008] In one aspect, this disclosure includes an activatable cytokine construct (ACC) comprising a masking portion (MM), a cleavable portion (CM), and a cytokine peptide (CP), wherein the MM comprises: (a) A first masking subunit, the first masking subunit being encoded by a first subsequence of the amino acid sequence of a first receptor polypeptide encoding the receptor for CP, and (b) A second masking subunit, which is encoded by a second amino acid sequence selected from the group consisting of: (i) a second subsequence encoding an amino acid sequence of a cytokine receptor polypeptide, said cytokine receptor polypeptide being selected from the group consisting of an amino acid sequence encoding a first receptor polypeptide and an amino acid sequence encoding a second receptor polypeptide for a receptor of CP, wherein the first and second subsequences are discontinuous within the amino acid sequence encoding the first receptor polypeptide; and (ii) The amino acid sequence encoding the blocking portion (BM).

[0009] In one aspect, this disclosure includes an isolated polypeptide comprising a polypeptide sequence comprising a tandem sequence of two or more receptor subsequences, wherein each of the receptor subsequences is derived from a receptor of a cytokine polypeptide (CP); and the two or more receptor subsequences: i) It is discontinuous in the sequence of the receptor; and ii) They are directly or indirectly connected to each other.

[0010] In one aspect, this disclosure includes an ACC comprising a masking portion (MM), a cleavable portion (CM), and a cytokine peptide (CP), wherein: a) The MM comprises a polypeptide sequence, the polypeptide sequence comprising a tandem sequence of two or more receptor subsequences; b) Each receptor from the CP in the receptor subsequence; and c) The two or more receptor subsequences: i) It is discontinuous in the sequence of the receptor; and ii) They are directly or indirectly connected to each other.

[0011] In one aspect, each of the two or more receptor subsequences comprises a continuous amino acid of the receptor, the continuous amino acid having at least one atom (C, O, N, or S) of any atom (C, O, N, or S) of the cytokine amino acid in the co-crystal structure of the receptor and the cytokine complex (cytokine-receptor complex co-crystal structure) within the range of 1.0 to 8.0 Å, 2.0 to 8.0 Å, or 2.0 to 7.0 Å. In one aspect, the isolated polypeptide further comprises a linker disposed between at least two of the two or more receptor subsequences. In one aspect, each of the receptor subsequences comprises 2, 3, 4, or more amino acids. In one aspect, at least one of the two or more receptor subsequences comprises a conserved substitution of at least one amino acid relative to the sequence of the receptor. In one aspect, the two or more receptor subsequences include a first receptor subsequence and a second receptor subsequence, wherein the second receptor subsequence is located at the C-terminus of the first receptor subsequence in the sequence of the receptor, wherein the linker comprises X amino acids, where X = n / y, where n = the distance between the C-terminus of the N-terminal peptide of the first receptor subsequence and the N-terminus of the C-terminal peptide of the second receptor subsequence in the co-crystal structure of the cytokine-receptor complex, in angstroms, and y is a number in the range of 1.5-3.5, or 2-3.5, or 2-3, or 2-2.5, and if X is not an integer, X is rounded to the next integer. In some aspects, the two or more receptor subsequences include a first receptor subsequence and a second receptor subsequence, wherein the second receptor subsequence is located at the C-terminus of the first receptor subsequence in the sequence of the receptor, wherein the linker comprises X amino acids, where X = n / 2.5, and if X is not an integer, X is rounded to the next integer.

[0012] As described in this paper, masking motifs containing tandem receptor subsequences have been found to exhibit excellent masking properties in interfering with the binding of cytokines to their receptors. Furthermore, ACCs containing these MMs exhibit very good restoration of cytokine activity upon activation.

[0013] In one aspect, this disclosure includes an isolated polypeptide comprising the amino acid sequence ALTTVX-linker-ASHYFE (SEQ ID NO: 508) (MM), wherein X is D, K, or R, and wherein the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid (as in SEQ ID NO: 515-518). In some aspects, the N-terminal alanine residue is substituted with lysine. In some aspects, the N-terminus, C-terminus, or both extend for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some respects, the isolated polypeptide comprises a sequence selected from the following: SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737). In some respects, the linker consists of 1 to 20 amino acids. In some aspects, the isolated polypeptide comprises an amino acid sequence selected from the following: ALTTVDGGGGSASHYFE (SEQ ID NO: 512), ALTTVDGGGGSASHYFER (SEQ ID NO: 236), ALTTVDGGGGSASHYFEK (SEQ ID NO: 237), ALTTVKGGGGSASHYFE (SEQ ID NO: 513), ALTTVKGGGGSASHYFER (SEQ ID NO: 238), ALTTVKGGGGSASHYFEK (SEQ ID NO: 239), ALTTVVRGGGGSASHYFE (SEQ ID NO: 514), ALTTVVRGGGGSASHYFER (SEQ ID NO: 240), or ALTTVVRGGGGSASHYFEK (SEQ ID NO: 241), or wherein the N-terminal alanine residue in each sequence is optionally absent or optionally substituted with any other amino acid. In some aspects, the N-terminal alanine residue is substituted with lysine. In some respects, the N-terminus, C-terminus, or both of the MM sequence in this paper are extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0014] According to this disclosure, the amino acid sequence is a masking part that inhibits the binding of cytokines to their receptors.

[0015] In one aspect, this disclosure includes isolated polypeptides further comprising cytokines. In some aspects, the isolated polypeptide is disposed in a complex comprising two or more polypeptides, and wherein said complex comprises cytokines. In one aspect, this disclosure includes a complex comprising a polypeptide containing cytokines compounded with the isolated polypeptide of this disclosure. In some aspects, the cytokines are disposed in polypeptides compounded with isolated polypeptides. In some aspects, the cytokines are cytokines that bind to IL2 / IL15 receptor β and / or IL2 / IL15 receptor γ. In some aspects, the cytokines are cytokines that bind to IL-15Rα. In some aspects, the cytokines are cytokines that bind to IL-2Rα.

[0016] In one aspect, this disclosure includes an activatable cytokine construct (ACC) comprising a cytokine polypeptide (CP), a cleavable portion (CM), and a separated polypeptide (MM) of this disclosure, wherein the MM is coupled to the CP via the CM and inhibits the binding of the CP to its receptor.

[0017] In one aspect, this disclosure includes an ACC comprising a first monomer construct and a second monomer construct, wherein the first monomer construct comprises a first cytokine polypeptide (CP1), a first cleavable portion (CM1), a first dimerizing domain (DD1) coupled to CP1 via CM1, and a first masking portion (MM1), and the second monomer construct comprises a second cytokine polypeptide (CP2), a second cleavable portion (CM2), a second dimerizing domain (DD2) coupled to CP2 via CM2, and a second masking portion (MM2), wherein DD1 and DD2 bind to each other to form a dimer of the first monomer construct and the second monomer construct, and MM1 and / or MM2 comprise the isolated polypeptide of this disclosure.

[0018] In one aspect, this disclosure includes an ACC comprising a first monomer construct and a second monomer construct, wherein the first monomer construct comprises a first cytokine polypeptide (CP1), a first dimerizing domain (DD1), and a first masking portion (MM1), and the second monomer construct comprises a second cytokine polypeptide (CP2), a first cleavable portion (CM1), a second dimerizing domain (DD2) coupled to CP2 via CM1, and a second masking portion (MM2), wherein MM1 and / or MM2 are isolated polypeptides of this disclosure, and DD1 and DD2 bind to each other to form a dimer of the first monomer construct and the second monomer construct.

[0019] In one aspect, this disclosure includes an ACC comprising a first monomer construct and a second monomer construct, wherein the first monomer construct comprises a first cytokine polypeptide (CP1), a first dimerizing domain (DD1), and a first masking moiety (MM1), and the second monomer construct comprises a second cytokine polypeptide (CP2), a second dimerizing domain (DD2), and a second masking moiety (MM2), wherein CP1 and / or CP2 comprise an amino acid sequence serving as a substrate for a protease, and DD1 and / or DD2 are coupled to CP1 and / or CP2 by an amino acid sequence, MM1 and / or MM2 are isolated polypeptides of this disclosure, and DD1 and DD2 bind to each other to form a dimer of the first monomer construct and the second monomer construct.

[0020] In one aspect, this disclosure includes an ACC comprising a first monomer construct and a second monomer construct, wherein the first monomer construct comprises the cytokine polypeptide (CP) of this disclosure, a first dimerizing domain (DD1), a first cleavable moiety (CM1), a second cleavable moiety (CM2), and an isolated polypeptide or masking moiety (MM), wherein the isolated polypeptide or MM is coupled to CP via CM1, and DD1 is coupled to CP via CM2, the second monomer construct comprising an agonist of CP, a third cleavable moiety (CM3), and a second dimerizing domain (DD2) coupled to the agonist via CM3, and DD1 and DD2 are bound to each other to form a dimer of the first monomer construct and the second monomer construct.

[0021] In one aspect, this disclosure includes polynucleotides encoding isolated polypeptides or monomeric constructs of this disclosure. This disclosure also includes vectors, host cells, compositions, manufacturing methods, and treatment methods according to the following disclosures. Attached Figure Description

[0022] Figures 1A to 1B This shows IL-15 and its receptor ( Figure 1A (left) or IL-2 ( Figure 1B The complex of (left) and IL-15 ( Figure 1A (right) or IL-2 ( Figure 1B (Right) An exemplary MM that binds to and interrupts the binding between interleukin and its receptor.

[0023] Figure 2This is a schematic diagram of an illustrative activatable cytokine construct comprising, from the N-terminus to the C-terminus: (1) a first monomer construct 110 having optional MM1119, optional CM3117, CP1115, CM1113, and DD1111; and (2) a second monomer construct 120 having optional MM2129, optional CM4127, CP2125, CM2123, and DD2121; and (3) one or more covalent or non-covalent bonds (← →) binding the first monomer construct 110 to the second monomer construct 120. The ACC may also include one or more of optional linkers 112, 114, 116, 118, 122, 124, 126, and 128 between the components. In one example, DD1111 and DD2121 are identical. In another example, DD1111 and DD2121 are different.

[0024] Figures 3A to 3E Additional examples of ACC are illustrated schematically. These include MM (e.g., β-peptide), CM (“substrate”), and CP (e.g., IL-15). Figure 3A )as well as Figure 3B An exemplary ACC containing DD1 and DD2 (Fc). An exemplary ACC having a cytokine agonist (e.g., a sushi domain) and an optional histidine tag (“His tag”). Figures 3C to 3E ).

[0025] Figure 4 An implementation scheme of an ACC representing the connection zone (LR) of an ACC is schematically shown.

[0026] Figure 5 A schematic diagram of the structure of an exemplary ACC ProC2970 is shown (top left), the tertiary structure of a monomeric construct containing interleukin, a cleavable portion and MM (top right), and the tertiary structure of a monomeric construct complexed with its receptor (the α, β and γ chains of the receptor shown) is shown (bottom).

[0027] Figure 6 The electrophoretic test results of uPA on the lysis of the exemplary ACC ProC2970 are shown.

[0028] Figure 7 The masking efficiency of the exemplary ACC ProC2970, tested by reporter gene assay, is shown and compared with that of the ProC1879.

[0029] Figure 8 The activity of exemplary ACC ProC2970 against PMBC proliferation is shown and compared with ProC1879.

[0030] Figures 9A to 9E The activation of ACC is shown. Figures 9A to 9B Electrophoretic images of ACC before and after uPA cleavage are shown. Figures 9C to 9E The study showed that the EC50 of ACC decreased after uPA-mediated activation in the HEK-Blue reporter gene assay.

[0031] Figure 10 The diagram shows the structure of an ACC without a Model (top row) and an exemplary ACC construct with a Model (bottom row). Detailed Implementation

[0032] Although aspects of the subject matter of this disclosure may be embodied in many forms, the following description is only intended to disclose some of these forms as specific examples of the subject matter covered by this disclosure. Therefore, the subject matter of this disclosure is not limited to the forms or aspects described.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including definitions, shall prevail.

[0034] Other features and advantages of the invention will be apparent from the following detailed description, accompanying drawings, and claims.

[0035] The term "a / an" refers to one or more grammatical objects of the article (i.e., at least one). By way of example, "a cell" encompasses one or more cells.

[0036] As used herein, the terms “about” and “approximately” when used to modify a numerical value or a quantity specified within a range indicate the numerical value and a reasonable deviation from a value known to those skilled in the art. For example, where appropriate, ±20%, ±10%, or ±5% may be within the intended meaning of the value.

[0037] Concentration, amount, and other numerical data may be expressed or presented in range format herein. It should be understood that this range format is used for convenience and brevity only, and therefore should be interpreted flexibly to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges covered within that range, as if each value and subrange were explicitly stated. For example, the numerical range “about 0.01 to 2.0” should be interpreted to include not only the explicitly stated values ​​of about 0.01 to about 2.0, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 0.5, 0.7, and 1.5, and subranges such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5, etc. Furthermore, this interpretation should apply regardless of the width of the range or the characteristics described. Additionally, it should be noted that unless otherwise specified, all percentages are by weight.

[0038] In understanding the scope of this disclosure, the terms “comprising” or “including” and their derivatives as used herein are intended to specify the presence of 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 foregoing also applies to words with similar meanings, such as the terms “comprising,” “having,” and their derivatives. The term “consisting of” and its derivatives as used herein are intended to specify the presence of 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 “substantially constitutes” as used herein is intended to specify the presence of stated features, elements, components, groups, integers and / or steps, as well as those features, elements, components, groups, integers and / or steps that do not substantially affect one or more basic and novel features of the features, elements, components, groups, integers and / or steps. It should be understood that reference to any of these transitional terms (i.e., "comprising," "consisting of," or "substantially composed of") provides direct support for substitution with any other transitional term not specifically used. For example, since this definition applies to any element disclosed throughout this disclosure, modifying the term from "comprising" to "substantially composed of" or "consisting of" would be directly supported. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0039] As used herein, for convenience, multiple compounds, elements, or steps may be presented in a common list. However, these lists should be interpreted as if each member of the list were individually identified as a separate and distinct member. Therefore, based solely on their presentation in a common group without any indication to the contrary, individual members of such a list should not be construed as de facto equivalents of any other member of the same list.

[0040] Furthermore, certain molecules, constructs, compositions, elements, portions, excipients, symptoms, diseases, properties, steps, etc., may be discussed in the context of a particular embodiment or aspect or in a separate paragraph or section of this disclosure. It should be understood that this is merely for convenience and brevity, and any such disclosure is equally applicable to any other embodiment or aspect seen anywhere in this disclosure and the claims, and is intended to be combined with any such other embodiment or aspect to form the present application and the invention claimed at the filing date. For example, a list of constructs, molecules, method steps, kits, or compositions described with respect to constructs, compositions, or methods is intended and does indeed provide direct support for embodiments relating to the constructs, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not restated in the context or section of that embodiment or aspect.

[0041] The terms "cleavable moiety" and "CM" are used interchangeably herein and refer to a polypeptide whose amino acid sequence contains a substrate of a sequence-specific protease. The cleavable moiety of ACC as used herein includes any protease substrate known in the art. Exemplary cleavable moies are described in more detail below.

[0042] The terms "masking portion" or "MM" are used interchangeably herein to refer to a peptide or protein that reduces or inhibits one or more activities of a cytokine polypeptide. In some embodiments, when located proximal to the cytokine polypeptide, the MM interferes with the binding of the cytokine polypeptide to its binding partner (e.g., its receptor). In some embodiments, the MM is an amino acid sequence of fewer than 50 amino acids, including any number or range of amino acids in the range of 1 to 50. In some embodiments, the MM is no more than 40 amino acids in length. In a preferred embodiment, the MM is no more than 20 amino acids in length. In some embodiments, the MM is no more than 19, 18, 17, 16, or 15 amino acids in length. In some aspects, the MM is at least 1, 2, 3, or 4 amino acids. In some aspects, the MM is 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids.

[0043] As used herein, the term "masking efficiency" refers to the activity (e.g., EC50) of unlyslaughtered ACC divided by the activity of a control interleukin, which may be a lysate of ACC or an interleukin used as a CP for ACC. ACCs with reduced levels of at least one interleukin have a masking efficiency greater than 10. In some embodiments, the ACCs described herein have a masking efficiency greater than 10, greater than 100, greater than 1000, or greater than 5000. In some embodiments, the ACC has a masking efficiency of about 10 to about 100, or about 10 to about 200, or about 50 to about 150, or about 50 to about 80, as measured by the ratio of the EC50 of unlyslaughtered ACC to the EC50 of ACC lysate in IL-2 / IL-15 responsive HEK293 cells.

[0044] As used herein, the term “blocking fraction” or “BM” refers to a subsequence that reduces or inhibits the interaction between a cytokine peptide and its binding partner but does not contain a receptor sequence or a cytokine receptor sequence.

[0045] As used herein, the term "subsequence" means that the portion does not include the full-length amino acid sequence of the cytokine receptor sequence, but rather all amino acids in a sequence having fewer amino acids than the cytokine receptor sequence. Therefore, as used herein, the subunit, monomer, construct, peptide, or amino acid sequence "encoded" by the subsequence does not include the full-length amino acid sequence of the cytokine receptor sequence, but rather all amino acids in a sequence having fewer amino acids than the cytokine receptor sequence.

[0046] As used in this article, in the context of acceptor subsequence, "continuous" means two or more adjacent amino acids in the subsequence that have the same order from the N-terminus to the C-terminus.

[0047] When used with respect to cytokine constructs, the term "activatable" means that a cytokine construct exhibits a first level of one or more activities, followed by exposure to conditions that cause cleavage of one or more cleavable portions, resulting in a second level of cytokine construct exhibiting said one or more activities, wherein said second activity level is greater than the first activity level. Non-limiting examples of activity include any exemplary activities of cytokines described herein or known in the art.

[0048] The term "mature cytokine polypeptide" herein refers to a cytokine polypeptide lacking a signaling sequence. A cytokine polypeptide (e.g., an interleukin polypeptide) can be a mature cytokine polypeptide or a cytokine polypeptide having a signaling peptide. Therefore, in some aspects, the ACC of this disclosure may include a mature cytokine polypeptide sequence. In some aspects, the ACC of this disclosure may include a mature cytokine polypeptide sequence and an additional signaling sequence. In some aspects, the ACC of this disclosure may include sequences disclosed herein, which may include or lack the signaling sequence described herein.

[0049] The terms “dimerizing domain” and “DD” are used interchangeably herein to refer to one member of a pair of dimerizing domains, wherein each member of the pair is capable of binding to the other member through one or more covalent or non-covalent interactions. The first DD and the second DD may be the same or different. Exemplary DDs applicable as DD1 and / or DD2 are described in more detail below.

[0050] As used herein, a polypeptide (such as a cytokine or an Fc domain) can be a wild-type polypeptide (e.g., a naturally occurring polypeptide) or a variant of a wild-type polypeptide. A variant can be a polypeptide modified by substitution, insertion, deletion, and / or addition of one or more amino acids of a wild-type polypeptide, provided that the variant retains the essential function or activity of the wild-type polypeptide. In some instances, the variant may have altered (e.g., increased or decreased) function or activity compared to the wild-type polypeptide. In some aspects, a variant may be a functional fragment of a wild-type polypeptide. The term "functional fragment" means that the sequence of a polypeptide (e.g., a cytokine) may include fewer amino acids than the full-length polypeptide sequence, but has sufficient polypeptide chain length to confer activity (e.g., cytokine activity).

[0051] In the context of two or more nucleic acid or amino acid sequences, the term "at least [certain] % identical" means that when comparing and aligning on a comparison window or specified nucleic acid or amino acid sequences to achieve maximum correspondence, the two or more sequences share a common nucleotide or amino acid residue within a given percentage (i.e., the sequences have at least 90% (%) identity). The percentage identity of nucleic acid or amino acid sequences can be measured using the BLAST sequence comparison algorithm with default parameters or by manual alignment and visual inspection (see, for example, blast.ncbi.nlm.nih.gov / Blast.cgi). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. For example, the % sequence identity of a given amino acid sequence A with or against a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a certain % sequence identity of a pair with or against a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y Where X is the number of amino acid residues marked as complete matches in the sequence alignment of programs A and B, and Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity percentage of A to B will not be equal to the amino acid sequence identity percentage of B to A.

[0052] As used herein, "isolated polypeptide" means a polypeptide of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, which, depending on its source or derived origin, is substantially free of components endogenously expressed in host cells such as mammalian cells, or, in the case of a cell-free expression system, is substantially free of cell-free expression reagents, and does not occur in nature. Using conventional isolation techniques (e.g., chromatography), isolated polypeptides may be substantially free of components endogenously expressed in host cells or substantially free of cell-free expression reagents. According to aspects of this disclosure, isolated polypeptides may be disposed in a complex comprising two or more polypeptides, including wherein the complex contains cytokines. Unless otherwise specified, "nucleic acid sequence encoding protein" includes all nucleotide sequences that are degenerate to each other and therefore encode the same amino acid sequence.

[0053] Unless otherwise specified, “nucleic acid sequence encoding protein” includes all nucleotide sequences that are degenerate to each other and therefore encode the same amino acid sequence.

[0054] When referring to the position of a first domain or sequence relative to a second domain or sequence in the primary amino acid sequence of a polypeptide, the term "N-terminus" 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, additional sequences and / or domains may exist between the first domain or sequence and the second domain or sequence.

[0055] When referring to the position of a first domain or sequence relative to a second domain or sequence in the primary amino acid sequence of a polypeptide, the term "C-terminus" 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, additional sequences and / or domains may exist between the first domain or sequence and the second domain or sequence.

[0056] The term "exogenous" means any material introduced from or derived from outside a cell, tissue, or organism that is not produced by or derived from the aforementioned cell, tissue, or organism into which it is introduced.

[0057] The terms “transduction,” “transfection,” or “transformation” refer to the process of introducing or transferring exogenous nucleic acids into cells. “Transduced,” “transfected,” or “transformed” cells (e.g., mammalian cells) are cells that have been transduced, transfected, or transformed with exogenous nucleic acids (e.g., vectors), including exogenous nucleic acids encoding any of the activatable cytokine constructs described herein.

[0058] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, or combinations thereof. Unless specifically limited, the term covers nucleic acids containing known analogs of a natural nucleotide that has similar binding properties to a reference nucleotide. Unless otherwise indicated, a particular nucleic acid sequence also implicitly covers complementary sequences as well as explicitly indicated sequences. In some embodiments of any nucleic acid described herein, the nucleic acid is DNA. In some embodiments of any nucleic acid described herein, the nucleic acid is RNA.

[0059] As used herein, the phrase “specific binding” means that ACC binds to its receptor or target and does not react with other peptides, or with a much lower affinity (e.g., about or greater than 10). -6 M) combination.

[0060] The term "treatment" refers to the improvement of at least one symptom of a condition. In some implementations, the condition being treated is cancer. In some implementations, the condition being treated is an autoimmune disease. In some implementations, the condition being treated is an inflammatory disease.

[0061] Peptides containing novel masking components This article provides isolated peptides containing amino acid sequences that can be used as masking portions in constructs of activating cytokines.

[0062] In one aspect, this disclosure includes a cytokine masking portion (MM) comprising: (a) A first masking subunit, the first masking subunit being encoded by a first subsequence of the amino acid sequence of a first receptor polypeptide encoding a cytokine receptor, and (b) A second masking subunit, which is encoded by a second amino acid sequence selected from the group consisting of: (i) a second subsequence encoding an amino acid sequence of a cytokine receptor polypeptide, said cytokine receptor polypeptide being selected from the group consisting of the amino acid sequence encoding the first receptor polypeptide and the amino acid sequence encoding the receptor for said cytokine, wherein the first subsequence and the second subsequence are discontinuous within the amino acid sequence encoding the first receptor polypeptide; and (ii) The amino acid sequence encoding the blocking portion (BM).

[0063] The cytokine MM described herein is a non-naturally occurring molecule. In some respects, the length of one or both of the first and second masking subunits does not exceed 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids. Cytokine MM can be expressed in the form of a polypeptide comprising a cytokine MM directly or indirectly coupled to a cleavable moiety (CM) directly or indirectly coupled to a cytokine polypeptide (CP).

[0064] In some aspects, this disclosure provides isolated polypeptides comprising a masking portion (MM) that interrupts the binding between an interleukin and its binding partner. For example, the MM may comprise a tandem peptide from a cytokine receptor, including amino acid residues at the receptor-cytokine interaction site. The tandem peptide of this disclosure comprises one or more receptor subsequences from a cytokine receptor sequence. The receptor subsequence is an amino acid sequence from a CP receptor. The receptor subsequences in the MM comprising the tandem peptide may be different from or identical to each other. Thus, in some instances, the receptor subsequences are not identical. In some instances, the receptor sequences may be identical.

[0065] When cytokine receptors have multiple subunits, MMs can contain receptor subsequences from different subunits.

[0066] As described in this paper, masking motifs containing tandem receptor subsequences have been found to exhibit excellent masking properties in interfering with the binding of cytokines to their receptors. Furthermore, ACCs containing these MMs exhibit very good restoration of cytokine activity upon activation.

[0067] The MMs described below with reference to specific amino acid sequences are examples of MMs that contain the tandem amino acid sequences of this disclosure.

[0068] In some aspects, each of the two or more receptor subsequences comprises a consecutive amino acid of the receptor, the consecutive amino acid having at least one atom (C, O, N, or S) of any atom (C, O, N, or S) of the cytokine amino acid in the co-crystal structure of the receptor and the cytokine complex (cytokine-receptor complex co-crystal structure) within the range of 1.0 to 8.0 Å, 2.0 to 8.0 Å, or 2.0 to 7.0 Å. In some aspects, the ACC also comprises a linker disposed between at least two of the two or more receptor subsequences. In some aspects, each of the receptor subsequences comprises 2, 3, 4, or more amino acids. In some aspects, at least one of the two or more subsequences comprises a conserved substitution of at least one amino acid relative to the sequence of the receptor.

[0069] Conservative amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine), and hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other amino acid families include: aliphatic hydroxy amino acids (e.g., serine and threonine), amide families (e.g., asparagine and glutamine), aliphatic families (e.g., alanine, valine, leucine, and isoleucine), and aromatic families (e.g., phenylalanine, tryptophan, and tyrosine).

[0070] In some aspects, the two or more receptor subsequences include a first receptor subsequence and a second receptor subsequence, wherein the second subsequence is located at the C-terminus of the first receptor subsequence in the sequence of the receptor, wherein the linker comprises X amino acids, where X = n / y, where n = the distance between the C-terminus of the N-terminal peptide of the first receptor subsequence and the N-terminus of the C-terminal peptide of the second receptor subsequence in the co-crystal structure of the cytokine-receptor complex, in angstroms, and y is a number in the range of 1.5-3.5, or 2-3.5, or 2-3, or 2-2.5, or y is 2.5, and if X is not an integer, X is rounded to the next integer. In some aspects, the receptor subsequences are different from each other. In some aspects, two or more of the receptor subsequences are different or the same.

[0071] In one aspect, this disclosure provides polypeptides (e.g., isolated polypeptides) comprising one or more masking portions (MMs).

[0072] In some respects, MM is generated by tandemly forming a peptide of the receptor protein that constitutes at least a portion of the cytokine-receptor interface. The receptor peptide (e.g., 3-20 Å long) that constitutes at least a portion of the cytokine-receptor interface is identified from the co-crystal structure of the receptor-cytokine complex as a consecutive amino acid in the receptor located at any atom (C, O, N, or S) of the cytokine amino acid within the range of 2.0-7.0 Å. Distances can also be calculated in the range of 1-20 Å based on the distance between the C-α atoms. Many receptor / cytokine crystal structures have been reported in the literature and can be used to identify the receptor peptide in this paper, such as the structure reported, for example, in Wang, Xinquan et al., “Structural biology of shared cytokine receptors.” Annual review of immunology 27 (2009): 29-60, Ring, A., Lin, JX., Feng, D. et al. Mechanistic and structural insight into the functional dichotomy between IL-2and IL-15. Nat Immunol 13, 1187–1195 (2012) (doi.org / 10.1038 / ni.2449) (pdb:4GS7, IL-15), Josephson, Kristopher, Naomi J. Logsdon, and Mark R. Walter. “Crystalstructure of the IL-10 / IL-10R1 complex reveals a shared receptor binding site.” Immunity 15.1 (2001): 35-46 (doi.org / 10.1016 / s1074-7613(01)00169-8) (pdb:1J7V, IL-10),Tsutsumi, N., Kimura, T., Arita, K. et al. Thestructural basis for receptor recognition of human interleukin-18. Nat Commun 5, 5340 (2014). (doi.org / 10.1038 / ncomms6340) (pdb:3WO4, IL-18),Mendoza, JL, Escalante, NK, Jude, KM et al. Structure of the IFNγ receptorcomplex guides design of biased agonists. Nature References 567, 56–60 (2019) (doi.org / 10.1038 / s41586-019-0988-7) (pdb:6E3K, IFNγ), etc., are incorporated herein by reference in their entirety. Suitable peptides can also be identified using homology modeling and molecular docking protocols using tools such as BIOVIA Discovery Studio (Dassault system), Schrodinger (Schrodinger, Inc.). These receptor peptides can be tandemly linked to linkers (e.g., flexible linkers containing Gly, Ser, Thr, Asn, Pro, such as those disclosed below). The length of the linker can be determined based on the distance between the individual peptides in the cocrystal. In some instances, if the distance between the C-terminus of an N-terminal peptide and the N-terminus of a C-terminal peptide is n Å, the length of the linker will be chosen to be equal to or greater than n / 2.5 (rounded to the next integer) (because the average C-α distance between amino acids is approximately 2.5 Å).

[0073] In some embodiments, MM comprises the sequence ALTTVX-linker-ASHYFE (SEQ ID NO: 508), where X is D, K, or R, and wherein the N-terminal alanine residue in each sequence is optionally absent or optionally substituted with any other amino acid. In some aspects, the N-terminal alanine residue is substituted with lysine. In some aspects, the N-terminus, C-terminus, or both are extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some respects, MM comprises sequences selected from the following: SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO:731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO:734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO:736) or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO:737).

[0074] The MM sequence is connected to one or more connector sequences, such as flexible connectors, connectors containing Gly, Ser, Thr, Asn, Pro (such as those disclosed below), connectors designed to impart a specific structure, etc.

[0075] In some embodiments, the linker consists of 1 to 22 amino acids. In one example, the linker consists of 1 amino acid. In another example, the linker consists of 2 amino acids. In another example, the linker consists of 3 amino acids. In another example, the linker consists of 4 amino acids. In another example, the linker consists of 5 amino acids. In another example, the linker consists of 6 amino acids. In another example, the linker consists of 7 amino acids. In another example, the linker consists of 8 amino acids. In another example, the linker consists of 9 amino acids. In another example, the linker consists of 10 amino acids. In another example, the linker consists of 11 amino acids. In another example, the linker consists of 12 amino acids. In another example, the linker consists of 13 amino acids. In another example, the linker consists of 14 amino acids. In another example, the linker consists of 15 amino acids. In another example, the linker consists of 16 amino acids. In another example, the linker consists of 17 amino acids. In another example, the linker consists of 18 amino acids. In another example, the linker consists of 19 amino acids. In another example, the linker consists of 20 amino acids. In another example, the linker consists of 21 amino acids. In another example, the linker consists of 22 amino acids. In some examples, the linker consists of 21–53 amino acids, for example, 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, 50, 51, 52, or 53 amino acids.

[0076] Examples of connectors include sequences such as SEQ ID NO: 2 and 210-235, 245 or 250. In one example, the connector is GGGGS (SEQ ID NO: 216).

[0077] In some embodiments, the MM comprises the sequence ALTTVD-connector-ASHYFE (SEQ ID NO: 509), ALTTVD-connector-ASHYFER (SEQ ID NO: 242), or ALTTVD-connector-ASHYFEK (SEQ ID NO: 243), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some embodiments, the MM comprises the sequence ALTTVK-connector-ASHYFE (SEQ ID NO: 510), ALTTVK-connector-ASHYFER (SEQ ID NO: 244), or ALTTVK-connector-ASHYFEK (SEQ ID NO: 246), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some embodiments, MM comprises the sequence ALTTVR-connector-ASHYFE (SEQ ID NO: 511), ALTTVR-connector-ASHYFER (SEQ ID NO: 247), or ALTTVR-connector-ASHYFEK (SEQ ID NO: 248), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some aspects, the N-terminal alanine residue is substituted with lysine. In some aspects, the N-terminus, C-terminus, or both extend by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some respects, MM comprises sequences selected from the following: SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736) or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0078] In some embodiments, the MM consists of the sequence ALTTVD-connector-ASHYFER (SEQ ID NO: 242) or ALTTVD-connector-ASHYFE(R / K) (SEQ ID NO: 502), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some embodiments, the MM consists of the sequence ALTTVK-connector-ASHYFE (SEQ ID NO: 510) or ALTTVK-connector-ASHYFE(R / K) (SEQ ID NO: 503), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some embodiments, the MM consists of the sequence ALTVR-connector-ASHYFE (SEQ ID NO: 511) or ALTVR-connector-ASHYFE(R / K) (SEQ ID NO: 504), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some aspects, the N-terminal alanine residue is substituted with lysine. In some aspects, the N-terminus, C-terminus, or both extend for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some aspects, MM comprises a sequence selected from the following: SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0079] In one example, the MM contains the sequence ALTTVDGGGGSASHYFE (SEQ ID NO: 512) or ALTTVDGGGGSASHYFE(R / K) (SEQ ID NO: 505), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In another example, the MM contains the sequence ALTTVKGGGGSASHYFE (SEQ ID NO: 513) or ALTTVKGGGGSASHYFE(R / K) (SEQ ID NO: 506), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In yet another example, the MM contains the sequence ALTVRGGGGSASHYFE (SEQ ID NO: 514) or ALTVRGGGGSASHYFE(R / K) (SEQ ID NO: 507), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some aspects, the N-terminal alanine residue is substituted with lysine. In some respects, the N-terminus, C-terminus, or both extend for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some respects, MM comprises a sequence selected from the following: SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0080] In one example, the MM consists of the sequence ALTTVDGGGGSASHYFE (SEQ ID NO: 512) or ALTTVDGGGGSASHYFE(R / K) (SEQ ID NO: 505), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In another example, the MM consists of the sequence ALTTVKGGGGSASHYFE (SEQ ID NO: 513) or ALTTVKGGGGSASHYFE(R / K) (SEQ ID NO: 506), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In yet another example, the MM consists of the sequence ALTVRGGGGSASHYFE (SEQ ID NO: 514) or ALTVRGGGGSASHYFE(R / K) (SEQ ID NO: 507), or wherein the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some aspects, the N-terminal alanine residue is substituted with lysine. In some respects, the N-terminus, C-terminus, or both extend for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some respects, MM comprises a sequence selected from the following: SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0081] The linker in a MM can contain any one or more amino acids and any combination of amino acid sequences. In some respects, the linker is a flexible linker. In other respects, the linker is designed to give the MM the desired two-dimensional and / or three-dimensional structure.

[0082] In some embodiments, the masking portion (MM) "masks" or reduces or otherwise inhibits the activity of the cytokine peptide. In some embodiments, the MM masks, reduces, or otherwise inhibits the binding of the cytokine peptide to its receptor. In some embodiments, the coupling or modification of the cytokine peptide with the MM inhibits the ability of the cytokine peptide to specifically bind to its receptor through inhibition known in the art (e.g., structural changes and receptor binding competition). In some embodiments, the coupling or modification of the cytokine peptide with the MM causes a structural change that reduces or inhibits the ability of the protein to specifically bind to its receptor. In some embodiments, the coupling or modification of the cytokine peptide with the MM spatially blocks, reduces, or inhibits the ability of the cytokine peptide to specifically bind to its receptor.

[0083] Cytokine activator construct In one aspect, this disclosure provides an activatable cytokine construct (ACC) comprising a cytokine polypeptide (CP), such as an interleukin polypeptide, a cleavable moiety (CM), and an MM conjugated to the CP via the CM as described herein.

[0084] In some instances, the ACC contains a cytokine polypeptide -CM-MM or MM-CM-cytokine polypeptide from its N-terminus to its C-terminus. As used herein and unless otherwise stated, each dash (-) between ACC components indicates a direct bond or a bond via, for example, one or more linkers.

[0085] In one aspect, this disclosure includes an ACC comprising a masking portion (MM), a cleavable portion (CM), and a cytokine peptide (CP), wherein: a) The MM comprises a polypeptide sequence, the polypeptide sequence comprising a tandem sequence of two or more receptor subsequences; b) Each receptor from the CP in the receptor subsequence; and c) The two or more receptor subsequences: i) It is discontinuous in the sequence of the receptor; and ii) They are directly or indirectly connected to each other.

[0086] In some respects, this disclosure provides various forms of activatable cytokine constructs (ACC) that integrate MM.

[0087] In one aspect, this disclosure includes an activatable cytokine construct (ACC) comprising a masking portion (MM), a cleavable portion (CM), and a cytokine peptide (CP), wherein the MM comprises: (a) A first masking subunit, the first masking subunit being encoded by a first subsequence of the amino acid sequence of a first receptor polypeptide encoding the receptor of said CP, and (b) A second masking subunit, which is encoded by a second amino acid sequence selected from the group consisting of: (i) a second subsequence encoding an amino acid sequence of a cytokine receptor polypeptide, said cytokine receptor polypeptide being selected from the group consisting of the amino acid sequence encoding the first receptor polypeptide and the amino acid sequence encoding the receptor for said CP, wherein the first subsequence and the second subsequence are discontinuous within the amino acid sequence encoding the first receptor polypeptide; and (ii) The amino acid sequence encoding the blocking portion (BM).

[0088] In some aspects, this disclosure provides an activatable cytokine construct (ACC) that exhibits a reduced level of activity of at least one of the corresponding cytokines, but produces a cytokine product with substantially restored activity upon exposure to activating conditions. In some embodiments, the ACC comprises a cytokine polypeptide (CP), a cleavable moiety (CM), and a masking moiety (MM) according to this disclosure. In some embodiments, the MM disrupts the interaction between the CP and its binding partner (e.g., its receptor). In some embodiments, the MM interacts with IL-15 (… Figure 1A ) or IL-2 ( Figure 1B It binds to and interrupts the binding between interleukin and its receptor.

[0089] The inventors unexpectedly discovered that, compared with corresponding ACCs that do not contain such MMs, ACCs containing the MMs disclosed herein have improved characteristics, such as higher masking efficiency.

[0090] The ACC disclosed herein can be selectively activated upon exposure to diseased tissue, but not activated in normal tissue. Cytokine activity is restored after activation of the ACC following lysis of the cleavable portion, indicating that the released masking portion does not appear to bind to cytokines after lysis, nor does it interfere with or compete with cytokines for binding to their targets. Therefore, the ACC has the potential to confer benefits to cytokine-based therapies while potentially exhibiting fewer toxicities associated with certain cytokine-based therapies and improved pharmacokinetics.

[0091] This document also provides related intermediates, compositions, kits, nucleic acids, and recombinant cells, as well as related methods, including methods for using any of the ACCs described herein and methods for generating and delivering any of the ACCs described herein.

[0092] In some implementations, ACC is characterized by a reduction in cytokine activity levels of at least 1000, 2000, 3000, 4000, 5000, or 6000 times compared to the corresponding recombinant wild-type cytokine. For example, ACC is characterized by an EC50 that is at least 1000, 2000, 3000, 4000, 5000, or 6000 times higher than that of recombinant wild-type IL-15, as measured in IL-2 / IL15-responsive HEK293 cells.

[0093] In some implementations, the ACC also includes an agonist of CP, such as a sushi domain. For example, the ACC may include MM, CM, CP (e.g., IL-15 or a mutant thereof) as described herein, and an agonist of CP (e.g., a sushi domain). In some instances, the agonist (e.g., the sushi domain) may be coupled to CP via a linker.

[0094] As used herein, the term "sushi domain" has its general meaning in the art and refers to a domain that begins at the first cysteine ​​residue (C1) following the signal peptide of IL-15Rα and ends at the fourth cysteine ​​residue (C4) following the signal peptide. The sushi domain, corresponding to a portion of the extracellular region of IL-15Rα, is essential for its binding to IL-15 (Wei et al., J. Immunol., Vol. 167(1), pp. 277-282, 2001, which is incorporated herein by reference in its entirety). In one instance, the sushi domain comprises the sequence of SEQ ID NO: 520. In another instance, the sushi domain comprises a functional fragment of the sequence of SEQ ID NO: 520. The sushi domain of IL-15Rα or a derivative thereof has at least 10%, for example at least 25%, and more preferably at least 50%, of the binding activity of the sushi domain of human IL-15Rα to human interleukin-15. The binding activity can be easily determined using the method disclosed by Wei et al. 2001.

[0095] In some embodiments, the sushi domain is covalently linked to an interleukin polypeptide, MM, DD1, or DD2. In some embodiments, the covalent bond is a non-α-carbon covalent bond, such as an isopeptide bond. In some embodiments, the isopeptide bond is located between a lysine residue and a glutamic acid or aspartic acid residue. In some embodiments, the non-α-carbon covalent bond is located between MM and a functional group substituted into the α-carbon in the cytokine. In some embodiments, the isopeptide bond is located between the γ-carboxamide group of glutamine and the ε-amino group of the lysine side chain. In some embodiments, the non-α-carbon covalent bond is an ester bond located between threonine and glutamine. In some embodiments, the non-α-carbon covalent bond is a thioester bond located between cysteine ​​and glutamine. In some embodiments, the non-α-carbon covalent bond is a thioether bond located between cysteine ​​and tyrosine. In some embodiments, the non-α-carbon covalent bond is formed by a crosslink between histidine and tyrosine (e.g., this type of histidine-tyrosine crosslink is known to exist in cytochromes). c (In oxidases). In some embodiments, the non-α-carbon covalent bond is a nitrogen-oxygen-sulfur (NOS) bond formed between lysine and cysteine. In some embodiments, the non-α-carbon covalent bond is a disulfide bond.

[0096] In the ACC, the MM can couple to the cytokine peptide via the CM and optionally one or more linkers, as described in more detail herein. In some embodiments, when the ACC is not activated, the MM prevents the cytokine peptide from binding to its receptor; however, when the ACC is activated (when the CM between the MM and the cytokine peptide is cleaved by a protease), the MM does not substantially or significantly interfere with the binding of the cytokine peptide to its receptor.

[0097] In the ACC, the MM can be directly or indirectly (e.g., through one or more linkers) coupled to cytokine peptides. Alternatively, the MM can be directly or indirectly coupled to components of the ACC that are not cytokine peptides. For example, the MM can be directly or indirectly coupled to different cytokine peptides. In another example, the MM can be directly or indirectly coupled to the DD. In either case, in the tertiary or quaternary structure of the activatable structure, the MM can be positioned to allow the MM to mask the cytokine peptide (e.g., close to the cytokine peptide to be masked).

[0098] In some implementations, the ACC also includes an agonist of CP, such as the sushi domain described below. For example, the ACC may include MM, CM, CP (e.g., IL-15 or a mutant thereof) and an agonist of CP (e.g., the sushi domain) as described herein. In some instances, the agonist (e.g., the sushi domain) is coupled to CP via a linker.

[0099] Interleukin polypeptide In some embodiments, CP is an interleukin polypeptide. Examples of interleukin polypeptides in the ACC described herein may include IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-10, IL-20, IL-21, IL-14, IL-15, IL-16, IL-17, and IL-21.

[0100] In some instances, CP is IL-15. For example, CP may contain SEQ ID NO: 348, 129, or 130, or a functional fragment thereof. In some instances, CP may contain a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 348, 129, or 130.

[0101] In some embodiments, the interleukin peptide is wild-type IL-15. In some embodiments, the interleukin peptide is wild-type human IL-15. In some embodiments, the interleukin peptide is mutant IL-15. In some embodiments, the interleukin peptide is mutant human IL-15. In some embodiments, the interleukin peptide is at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 348. In some embodiments, the interleukin peptide is at least 85% identical to IL-15 (SEQ ID NO: 348), wherein the amino acid at position 45 of the interleukin peptide is not leucine. In some instances, the interleukin peptide is at least 90% identical to SEQ ID NO: 348, wherein the amino acid at position 45 of the interleukin peptide is not leucine. In some instances, the interleukin peptide is at least 95% identical to SEQ ID NO: 348, wherein the amino acid at position 45 of the interleukin peptide is not leucine. In some instances, the interleukin polypeptide is at least 99% identical to SEQ ID NO: 348, wherein the amino acid at position 45 of the interleukin polypeptide is not leucine. The position of the mutation is relative to a reference sequence. Thus, for example, when the mutation is located at position 45, it is relative to the reference sequence. For example, when the reference sequence is: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:348), the amino acids at positions 45 and 52 are shown in bold.

[0102] In some instances, the amino acid at position 45 of SEQ ID NO: 348 in the interleukin polypeptide is aspartic acid. In some instances, the amino acid at position 45 of SEQ ID NO: 348 in the interleukin polypeptide is asparagine. In some instances, the amino acid at position 45 of SEQ ID NO: 348 in the interleukin polypeptide is threonine.

[0103] In some embodiments, the interleukin peptide is at least 85% identical to SEQ ID NO: 348, wherein the amino acid at position 52 of the interleukin peptide is not leucine. In some examples, the interleukin peptide is at least 90% identical to SEQ ID NO: 348, wherein the amino acid at position 52 of the interleukin peptide is not leucine. In some examples, the interleukin peptide is at least 95% identical to SEQ ID NO: 348, wherein the amino acid at position 52 of the interleukin peptide is not leucine. In some examples, the interleukin peptide is at least 99% identical to SEQ ID NO: 348, wherein the amino acid at position 52 of the interleukin peptide is not leucine.

[0104] In some instances, the amino acid at position 52 of SEQ ID NO: 348 in the interleukin polypeptide is aspartic acid. In some instances, the amino acid at position 52 of SEQ ID NO: 348 in the interleukin polypeptide is asparagine. In some instances, the amino acid at position 52 of SEQ ID NO: 348 in the interleukin polypeptide is threonine.

[0105] In some embodiments, the interleukin peptide is at least 85% identical to SEQ ID NO: 348, wherein the amino acid at position 45 of the interleukin peptide is not leucine, and the amino acid at position 52 is not leucine. In some examples, the interleukin peptide is at least 90% identical to SEQ ID NO: 348, wherein the amino acid at position 45 of the interleukin peptide is not leucine, and the amino acid at position 52 is not leucine. In some embodiments, the interleukin peptide is at least 95% identical to SEQ ID NO: 348, wherein the amino acid at position 45 of the interleukin peptide is not leucine, and the amino acid at position 52 is not leucine.

[0106] In some instances, the amino acids at positions 45 and 52 of the interleukin polypeptide corresponding to SEQ ID NO: 348 are aspartic acid. In some instances, the amino acids at positions 45 and 52 of the interleukin polypeptide corresponding to SEQ ID NO: 348 are asparagine. In some instances, the amino acids at positions 45 and 52 of the interleukin polypeptide corresponding to SEQ ID NO: 348 are threonine.

[0107] In some instances, the amino acids at positions 45 and 52 of the interleukin polypeptide corresponding to SEQ ID NO: 348 are aspartic acid, asparagine, or threonine.

[0108] In some embodiments, the interleukin peptide comprises any one of SEQ ID NO: 402-422. In one example, the interleukin peptide comprises SEQ ID NO: 402. In another example, the interleukin peptide comprises SEQ ID NO: 403. In another example, the interleukin peptide comprises SEQ ID NO: 404. In another example, the interleukin peptide comprises SEQ ID NO: 405. In another example, the interleukin peptide comprises SEQ ID NO: 406. In another example, the interleukin peptide comprises SEQ ID NO: 407. In another example, the interleukin peptide comprises SEQ ID NO: 408. In another example, the interleukin peptide comprises SEQ ID NO: 409. In another example, the interleukin peptide comprises SEQ ID NO: 410. In another example, the interleukin peptide comprises SEQ ID NO: 411. In another example, the interleukin peptide comprises SEQ ID NO: 412. In another example, the interleukin peptide comprises SEQ ID NO: 413. In another example, the interleukin peptide comprises SEQ ID NO: 414. In another example, the interleukin peptide comprises SEQ ID NO: 415. In another example, the interleukin peptide comprises SEQ ID NO: 416. In another example, the interleukin peptide comprises SEQ ID NO: 417. In another example, the interleukin peptide comprises SEQ ID NO: 418. In another example, the interleukin peptide comprises SEQ ID NO: 419. In another example, the interleukin peptide comprises SEQ ID NO: 420. In another example, the interleukin peptide comprises SEQ ID NO: 421. In another example, the interleukin peptide comprises SEQ ID NO: 422.

[0109] In some embodiments, the interleukin peptide is composed of any one of SEQ ID NO: 402-422. In one example, the interleukin peptide is composed of SEQ ID NO: 402. In another example, the interleukin peptide is composed of SEQ ID NO: 403. In another example, the interleukin peptide is composed of SEQ ID NO: 404. In another example, the interleukin peptide is composed of SEQ ID NO: 405. In another example, the interleukin peptide is composed of SEQ ID NO: 406. In another example, the interleukin peptide is composed of SEQ ID NO: 407. In another example, the interleukin peptide is composed of SEQ ID NO: 408. In another example, the interleukin peptide is composed of SEQ ID NO: 409. In another example, the interleukin peptide is composed of SEQ ID NO: 410. In another example, the interleukin peptide is composed of SEQ ID NO: 411. In another example, the interleukin polypeptide is composed of SEQ ID NO: 412. In another example, the interleukin polypeptide is composed of SEQ ID NO: 413. In another example, the interleukin polypeptide is composed of SEQ ID NO: 414. In another example, the interleukin polypeptide is composed of SEQ ID NO: 415. In another example, the interleukin polypeptide is composed of SEQ ID NO: 416. In another example, the interleukin polypeptide is composed of SEQ ID NO: 417. In another example, the interleukin polypeptide is composed of SEQ ID NO: 418. In another example, the interleukin polypeptide is composed of SEQ ID NO: 419. In another example, the interleukin polypeptide is composed of SEQ ID NO: 420. In another example, the interleukin polypeptide is composed of SEQ ID NO: 421. In another example, the interleukin polypeptide is composed of SEQ ID NO: 422.

[0110] In some instances, CP is IL-2 or a functional segment thereof. For example, CP may contain SEQ ID NO: 119 or 120, or a functional segment thereof. In some instances, CP may contain a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 119 or 120.

[0111] In some instances, CP is IL-4 or a functional segment thereof. For example, CP may contain SEQ ID NO: 121 or 122, or a functional segment thereof. In some instances, CP may contain a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 121 or 122.

[0112] In some instances, CP is IL-7 or a functional segment thereof. For example, CP may contain SEQ ID NO: 123 or 124, or a functional segment thereof. In some instances, CP may contain a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 123 or 124.

[0113] In some instances, CP is IL-9 or a functional fragment thereof. For example, CP may contain SEQ ID NO: 125 or 126, or a functional fragment thereof. In some instances, CP may contain a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 125 or 126.

[0114] In some instances, CP is IL-21 or a functional segment thereof. For example, CP may contain SEQ ID NO: 521 or 522, or a functional segment thereof. In some instances, CP may contain a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 521 or 522.

[0115] ACC with dimerization domain In some embodiments, the ACC described herein is a dimeric complex comprising a first monomeric construct and a second monomeric construct. Dimerization of the monomeric components is facilitated by a pair of dimerizing domains. In one aspect, each monomeric construct comprises a cytokine peptide, the MM described herein, and a dimerizing domain (DD).

[0116] In a specific implementation, the present invention provides an ACC, the ACC comprising a first monomeric construct and a second monomeric construct, wherein: The first monomer construct comprises a first cytokine polypeptide (CP1), a first cleavable moiety (CM1), a first dimerizing domain (DD1) coupled to CP1 via CM1, and a first masking moiety (MM1). The second monomer construct comprises a second cytokine peptide (CP2), a second cleavable moiety (CM2), a second dimerizing domain (DD2) coupled to CP2 via the CM2, and a second masking moiety (MM2). The DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct, and MM1 and / or MM2 include the MMs described herein.

[0117] In some embodiments, the ACC is characterized by having a reduced level of said at least one CP1 and / or CP2 activity compared to a control level of at least one CP1 and / or CP2 activity.

[0118] In some embodiments, the first monomeric construct includes a third cleavable portion (CM3), and MM1 is coupled to CP1 via CM3. In some embodiments, MM1 is coupled to CP1 via CM1. In some embodiments, the second monomeric construct includes a fourth cleavable portion (CM4), and MM2 is coupled to CP2 via CM4. In some embodiments, MM2 is coupled to CP2 via CM2.

[0119] In some embodiments, the ACC further comprises a third monomer containing a sushi domain containing the sequence of SEQ ID NO: 520. In some embodiments, the ACC further comprises a fourth monomer containing a sushi domain containing the sequence of SEQ ID NO: 520. In some embodiments, the third monomer further comprises a tag (e.g., a peptide tag, such as a His tag, myc tag, etc.). In some embodiments, the fourth monomer further comprises a tag (e.g., a peptide tag, such as a His tag, myc tag, etc.).

[0120] In some embodiments, ACC comprises a first monomer construct and a second monomer construct, wherein the first monomer construct comprises a first cytokine peptide (CP1), a first dimerizing domain (DD1), and a first masking moiety (MM1). The second monomer construct comprises a second cytokine peptide (CP2), a first cleavable moiety (CM1), a second dimerizing domain (DD2) coupled to CP2 via CM1, and a second masking moiety (MM2). MM1 and / or MM2 are the MMs described herein, and DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct.

[0121] In some embodiments, the first monomeric construct further comprises a second cleavable portion (CM2), and MM1 is coupled to CP1 via CM2. In some embodiments, MM2 is coupled to CP2 via CM1. In some embodiments, the second monomeric construct further comprises a third cleavable portion (CM3), wherein MM2 is coupled to CP2 via CM3.

[0122] In some implementations, ACC comprises a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a first cytokine peptide (CP1), a first dimerizing domain (DD1), and a first masking moiety (MM1). The second monomer construct comprises a second cytokine peptide (CP2), a second dimerization domain (DD2), and a second masking moiety (MM2). CP1 and / or CP2 contain amino acid sequences that serve as substrates for the protease, and DD1 and / or DD2 are coupled to CP1 or CP2 via amino acid sequences. MM1 and / or MM2 are the MMs described herein, and DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct.

[0123] In some embodiments, CP1 comprises an amino acid sequence serving as a substrate for the protease, and MM1 is coupled to CP1 via the amino acid sequence. In some embodiments, the first monomer construct further comprises a first cleavable moiety (CM1), and MM1 is coupled to CP1 via CM1. In some embodiments, CP2 comprises an amino acid sequence serving as a substrate for the protease, and MM2 is coupled to CP2 via the amino acid sequence. In some embodiments, the second monomer construct further comprises a second cleavable moiety (CM2), and MM2 is coupled to CP2 via CM2.

[0124] In some implementations, ACC comprises a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a cytokine peptide (CP), a first dimerizing domain (DD1), a first cleavable moiety (CM1), a second cleavable moiety (CM2), and the MM described herein, wherein the MM is coupled to the CP via CM1, and the DD1 is coupled to the CP via CM2. The second monomer construct comprises an agonist of CP, a third cleavable moiety (CM3), and a second dimerizing domain (DD2) coupled to the agonist via CM3. DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct.

[0125] In some embodiments, CP is IL-15, and the agonist is a sushi domain comprising the sequence of SEQ ID NO: 520. In some embodiments, ACC comprises a linker between the sushi domain and CM3. In some aspects, the linker may comprise 1-10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some aspects, the linker may comprise 1-3 amino acids, such as 1, 2, or 3 amino acids, or in some aspects, it may consist of 2 amino acids.

[0126] In the ACC, which is a dimer complex, the first monomer construct and the second monomer construct may also contain additional elements, such as, for example, one or more linkers. These additional elements are described in more detail below. The organization of the CP, CM, MM, and DD components in each of the first and second monomer constructs may be arranged in the same order in each monomer construct. The CP1, CM1, MM1, and DD1 components may be the same as or different from the corresponding CP2, CM2, MM2, and DD2 components in terms of, for example, the molecular weight, size, and amino acid sequence of the CP and CM components (and the DD component in embodiments where the DD component is a polypeptide). Therefore, the resulting dimer may have symmetrical or asymmetrical monomer construct components.

[0127] In some embodiments, the first monomer construct comprises CP1, CM1, and DD1 directly or indirectly (through a linker) attached to the C-terminus of CM1 from the N-terminus to the C-terminus of the CP and CM components. In other embodiments, the first monomer construct comprises CP1, CM1, and DD1 directly or indirectly (through a linker) attached to the N-terminus of CM1 from the C-terminus to the N-terminus of the CP and CM components. In some embodiments, the second monomer construct comprises CP2, CM2, and DD2 directly or indirectly (through a linker) attached to the C-terminus of CM2 from the N-terminus to the C-terminus of the CP and CM components. In other embodiments, the second monomer construct comprises CP2, CM2, and DD2 directly or indirectly (through a linker) attached to the N-terminus of CM2 from the C-terminus to the N-terminus of the CP and CM components. In some embodiments, the first monomer comprising a first mature cytokine polypeptide (CP1) and / or the second monomer comprising a second mature cytokine polypeptide (CP2) comprises one or more MMs. In some embodiments, the ACC further comprises CM between the MM and the CP.

[0128] In some embodiments, the activatable cytokine construct (ACC) includes a first monomer construct and a second monomer construct, wherein: (a) the first monomer construct comprises a first masking portion (MM1), a first mature cytokine polypeptide (CP1), a first cleavable portion and a third cleavable portion (CM1 and CM3), and a first dimerizing domain (DD1), wherein CM1 is located between CP1 and DD1, and CM3 is located between MM1 and CP1; and (b) the second monomer construct comprises a second mature cytokine polypeptide (CP2), a second cleavable portion (CM2), and a second dimerizing domain (DD2), wherein CM2 is located between CP2 and DD2; wherein DD1 and DD2 bind to each other, thereby forming a dimer of the first monomer construct and the second monomer construct; and wherein the ACC is characterized by having a reduced level of the activity of at least one CP1 and / or CP2 compared to a control level of the activity of at least one CP1 and / or CP2.

[0129] In some embodiments, the second monomer construct further comprises a second masking portion (MM2) and a fourth cleavable portion (CM4), wherein CM4 is located between MM2 and CP2. In some embodiments, the first monomer construct comprises a first polypeptide comprising MM1, 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 MM2, CM4, CP2, CM2, and DD2.

[0130] The ACC structure was found to highly effectively reduce the activity of mature cytokine peptide components in a manner that does not substantially impair cytokine activity after activation. The activity of CP in the ACC can be reduced by both the structure of the ACC (e.g., a dimer structure) and one or more masking moieties within the ACC. In some embodiments, the activation condition of the ACC described herein is exposure to one or more proteases that can dissociate CP from both DD and MM. For example, one or more proteases can cleave CM between CP and MM and CM between CP and DD. As demonstrated in the examples, activation of the ACC results in a substantial recovery of cytokine activity. The results indicate that, in the case of the ACC, the conformation of the cytokine components is not irreversibly altered.

[0131] In some embodiments, when a cytokine peptide is conjugated to a MM and in the presence of the cytokine peptide's natural binding partner (e.g., its receptor), when measured in a masking efficiency assay, the binding of the cytokine peptide not conjugated to the MM is absent or substantially absent, or the binding of the cytokine peptide to its binding partner does not exceed 0.001%, 0.01%, 0.1%, 1%, or 2%. 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50%, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer. For example, a masking efficiency assay may involve measuring, for example, the affinity of ACC for binding to the cell surface displaying the candidate masking motif using FACS. Another non-limiting exemplary assay includes evaluating the ability of the masking motif to inhibit the binding of ACC to its binding partner at treatment-related concentrations and times. For the second approach, an immunoadsorption assay has been developed to measure the time-dependent binding of the proprotein to its binding partner, as described in US20200308243, which is incorporated herein by reference in its entirety. In an embodiment, the masking efficiency assay may involve measuring the production level of secreted alkaline phosphatase (SEAP) in IL-2 / IL15-responsive HEK293 cells.

[0132] In some embodiments, the first monomer builder and the second monomer builder are oriented such that the components in each member of the dimer are organized in the same order from the N-terminus to the C-terminus of the CP and CM components. Figure 2This is a schematic diagram of an illustrative activatable cytokine construct comprising, from the N-terminus to the C-terminus: (1) a first monomer construct 110 having optional MM1119, optional CM3117, CP1115, CM1113, and DD1111; and (2) a second monomer construct 120 having optional MM2129, optional CM4127, CP2125, CM2123, and DD2121; and (3) one or more covalent or non-covalent bonds (← →) binding the first monomer construct 110 to the second monomer construct 120. The ACC may also include one or more of optional linkers 112, 114, 116, 118, 122, 124, 126, and 128 between the components. In one example, DD1111 and DD2121 are identical. In another example, DD1111 and DD2121 are different. In some instances, DD1111 and DD2121 are different polypeptides that bind to each other.

[0133] In terms of alternatives, one of the two parts described as CP1115 and CP2125 is a mutant cytokine polypeptide lacking cytokine activity. Alternatively, one of the two parts described as CP1115 and CP2125 is a polypeptide sequence lacking cytokine activity, such as a signaling portion and / or a fragmented sequence. In another alternative, the first part of the two parts described as CP1115 and CP2125 is a polypeptide sequence that binds with high affinity to the second part of the two parts described as CP1115 and CP2125, and reduces the cytokine activity of the second part compared to a control level of the second part.

[0134] Figures 3A to 3E Additional exemplary embodiments of the ACC are shown. Any description of the substrate attached to the masking portion in these figures is optional and exemplary of the invention, and the CM or CM-MM features are optional and non-limiting. Figure 3C Examples of the constructs include ProC2982 (SEQ ID NO: 525 and SEQ ID NO: 526 combined and dimerized with a second identical monomer construct complex), and Figure 3DExamples of constructs include ProC3571 (SEQ ID NO: 528 dimerized with SEQ ID NO: 527 using a mortar-and-pepper Fc dimer). In some aspects, the ACC may include a cytokine peptide, such as IL-15, or a biologically active fragment thereof, CM, and MM. In some aspects, the ACC may include a first monomer construct and a second monomer construct, each monomer construct including a cytokine peptide (e.g., IL-15) or a biologically active fragment thereof, CM, MM, and DD, wherein the first monomer construct and the second monomer construct are dimerized by DD. In some aspects, the ACC may include a first monomer construct including a cytokine peptide (e.g., IL-15) or a biologically active fragment thereof, CM, MM, and DD, and a second monomer construct including a sushi domain or a fragment thereof, CM, and DD, wherein the first monomer construct and the second monomer construct are dimerized by DD. In some aspects, an ACC may include a first monomeric construct and a second monomeric construct, the first monomeric construct including a cytokine peptide (e.g., IL-15) or a biologically active fragment thereof linked to DD1 via CM1, and the second monomeric construct including a MM linked to DD2, wherein the first monomeric construct and the second monomeric construct are dimerized via DD1 and DD2. In some aspects, the MM is linked to DD2 via CM2 on the second monomeric construct. In some aspects, the cytokine peptide is linked to CM1 via a sushi domain on the first monomeric construct. In some aspects, an ACC may include a cytokine peptide (e.g., IL-15) or a biologically active fragment thereof, CM, MM, and IL-15 is linked to a sushi domain or a fragment thereof. In some aspects, an ACC may include a first monomeric construct and a second monomeric construct, each monomeric construct including a cytokine peptide (e.g., IL-15) or a biologically active fragment thereof, CM, MM, and DD, wherein the first monomeric construct and the second monomeric construct are dimerized via DD, and wherein each IL-15 is bound to a sushi domain or a fragment thereof.

[0135] The activation conditions of ACC described in this article involve a protease exposed to at least one of the cleavable fractions (CM) in a cleavable ACC. As demonstrated in the examples, activation of ACC leads to a significant restoration of cytokine activity. The results indicate that, in the case of ACC, the conformation of the cytokine components is not irreversibly altered.

[0136] The mature cytokine peptides CP1 and CP2 may be the same or different. In some specific embodiments, CP1 and CP2 are the same. In other embodiments, CP1 and CP2 are different. ACC may contain additional amino acid residues at either or both of the N-terminus and / or C-terminus of CP1 and / or CP2.

[0137] Dimerization domain (DD) Each monomer construct of the ACC as a dimer complex can employ any of a variety of dimerizing domains (DDs). Suitable DDs include moieties of polymeric (e.g., synthetic polymers, peptides, polynucleotides, etc.) and small molecule (non-polymeric moieties with a molecular weight of less than about 1,000 Daltons and sometimes less than about 800 Daltons) types. DD pairs can be any pair of moieties known in the art to bind together.

[0138] For example, in some embodiments, DD1 and DD2 are a pair of members selected from the group consisting of: a sushi domain from the human IL-15 receptor α chain (IL15Rα) and soluble IL-15; barnase and barnstar; PKA and AKAP; adaptor / docking tag molecules based on mutated RNase I fragments; a pair of antigen-binding domains (e.g., a pair of single-domain antibodies); a soluble N-ethyl-maleimide sensitizing factor attachment protein receptor (SNARE) module based on the interaction of protein synaptic proteins, synaptic binding proteins, small synaptic vesicle proteins, and SNAP25; and single-domain antibodies. (sdAb) and corresponding epitopes; antigen-binding domains (e.g., single-chain antibodies (such as single-chain variable fragments (scFv)), single-domain antibodies, etc.) and corresponding epitopes; coiled-coil polypeptide structures (e.g., Fos-Jun coiled-coil structures, acid / base coiled-coil helices, Glu-Lys coiled-coil helices, leucine zipper structures), small molecule binding pairs, such as biotin and avidin or streptoavidin, amine / aldehyde, lectin / carbohydrate; a pair of polymers that can bind to each other, such as a pair of sulfur-containing polymers or thiol-containing polymers (e.g., a pair of Fc domains, a pair of thiolized human serum albumin polypeptides, etc.); and so on.

[0139] In some embodiments, DD1 and DD2 are non-peptide polymers. These non-peptide polymers can be covalently bonded to each other. In some instances, the non-peptide polymers are sulfur-containing polymers, such as sulfur-containing polyethylene glycol. In such cases, DD1 and DD2 are covalently bonded to each other via one or more disulfide bonds.

[0140] When the DD1 and DD2 pair are members of a pair of epitopes and antigen-binding domains, the epitopes can be native or non-native. Exemplary non-native epitopes include, for example, non-native peptides, such as multi-His peptides (e.g., His tags, etc.).

[0141] In some specific implementations, DD1 and DD2 are a pair of Fc domains. As used herein, an "Fc domain" refers to a continuous amino acid sequence of a single heavy chain of an immunoglobulin. A pair of Fc domains associate together to form the Fc region of an immunoglobulin.

[0142] In some embodiments, the Fc domain pair is a human Fc domain pair (e.g., a wild-type human Fc domain pair). In some embodiments, the human Fc domain is a human IgG1 Fc domain (e.g., a wild-type human IgG1 Fc domain), a human IgG2 Fc domain (e.g., a wild-type human IgG2 Fc domain), a human IgG3 Fc domain (e.g., a wild-type human IgG3 Fc domain), or a human IgG4 Fc domain (e.g., a wild-type human IgG4 Fc domain). In some embodiments, the human Fc domain contains a sequence that is at least 80% identical to SEQ ID NO: 3 (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical).

[0143] In some embodiments, the Fc domains are contained in the club and mortar mutants. The club and mortar mutants may interact with each other to promote dimerization. In some embodiments, the club and mortar mutants may contain one or more amino acid modifications within the interface between the two Fc domains (e.g., in the CH3 domain). In one example, the modification comprises amino acid substitution T366W and optional amino acid substitution S354C in one antibody heavy chain, and amino acid substitutions T366S, L368A, Y407V, and optional Y349C (numbered according to the EU index of the Kabat numbering system) in another antibody heavy chain. Examples of club and mortar mutants include the Fc mutants of SEQ ID NO: 315 and 316, and those mutants described in U.S. Patent Nos. 5,731,168; 7,695,936; and 10,683,368, which are incorporated herein by reference in their entirety. In some implementations, the dimerized domain contains sequences that are at least 80% identical to SEQ ID NO: 315 and 316 (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical). In some embodiments, the human Fc domain contains the mutations N297Q, N297A, or N297G; in some embodiments, the human Fc domain contains mutations at positions 234 and / or 235, such as L235E, or L234A and L235A (in IgG1), or F234A and L235A (in IgG4); in some embodiments, the human Fc domain is an IgG2 Fc domain containing the mutations V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or combinations thereof (all according to EU designations). Other examples of engineered human Fc domains are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids whose mutations in at least one amino acid result in reduced Fc function include, but are not limited to, mutations in amino acids 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330, and 331 in the heavy chain constant region (all according to EU designations). Examples of combinations of mutated amino acids are also known in the art, such as, but not limited to, combinations of mutations in amino acids 234, 235, and 331, such as L234F, L235E, and P331S, or combinations of amino acids 318, 320, and 322, such as E318A, K320A, and K322A.

[0144] Other examples of engineered Fc domains include F243L / R292P / Y300L / V305I / P396 IgG1; S239D / I332E IgG1; S239D / I332E / A330L IgG1; S298A / E333A / K334A; in one heavy chain, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A IgG1, and in the opposite heavy chain, D270E / K326D, A330M / K334EIgG; G236A / S239D / I332E IgG1; K326W / E333S IgG1; S267E / H268F / S324T IgG1; E345R / E430G / S440Y IgG1; N297A or N297Q or N297G IgG1; L235E IgG1; L234A / L235A IgG1; F234A / L235A IgG4; H268Q / V309L / A330S / P331S IgG2; V234A / G237A / P238S / H268A / V309L / A330S / P331S IgG2; M252Y / S254T / T256E IgG1; M428L / N434S IgG1; S267E / L328F IgG1; N325S / L328F IgG1, etc. In some implementations, the engineered Fc domain includes one or more substitutions selected from the group consisting of N297A IgG1, N297Q IgG1 and S228P IgG4.

[0145] In some respects, the dimerizing domain is an IgG Fc region in which the upper hinge residue has been deleted. For example, the Fc is a variant in which the N-terminal sequence EPKSCDKTHT (SEQ ID NO: 387), ERK, ELKTPLGDTTHT (SEQ ID NO: 388), or ESKYGPP (SEQ ID NO: 389) has been deleted.

[0146] In some aspects, DD or DD1 and / or DD2 may also include a serum half-life extension (e.g., a peptide that binds to serum proteins, such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HSA))). Examples of half-life extensions include hexahat GST (glutathione S-transferase) glutathione affinity, calmodulin-binding peptide (CBP), Strep tag, cellulose-binding domain, maltose-binding protein, S-peptide tag, chitin-binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, and VSV epitope.

[0147] In some embodiments, DD1 and / or DD2 each comprise a total of about 5 amino acids to about 250 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 250 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 20 amino acids, about 20 amino acids to about 250 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 120 amino acids. Amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 40 amino acids, about 40 amino acids to about 250 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 250 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 250 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 250 amino acids.Approximately 100 amino acids to approximately 200 amino acids, approximately 100 amino acids to approximately 180 amino acids, approximately 100 amino acids to approximately 160 amino acids, approximately 100 amino acids to approximately 140 amino acids, approximately 100 amino acids to approximately 120 amino acids, approximately 120 amino acids to approximately 250 amino acids, approximately 120 amino acids to approximately 200 amino acids, approximately 120 amino acids to approximately 180 amino acids, approximately 120 amino acids to approximately 160 amino acids, approximately 120 amino acids to approximately 140 amino acids, approximately 140 amino acids to approximately 250 amino acids, approximately 140 amino acids to approximately 200 amino acids The following amino acids are listed: approximately 140 to approximately 180 amino acids; approximately 140 to approximately 160 amino acids; approximately 160 to approximately 250 amino acids; approximately 160 to approximately 200 amino acids; approximately 160 to approximately 180 amino acids; approximately 180 to approximately 250 amino acids; approximately 180 to approximately 200 amino acids; approximately 200 to approximately 250 amino acids; approximately 210 to approximately 220 amino acids; approximately 215 to approximately 225 amino acids; approximately 215 to approximately 220 amino acids; approximately 217 to approximately 200 amino acids; or approximately 218 to approximately 200 amino acids. In some embodiments, DD1 and DD2 are each an Fc domain comprising a portion of a hinge region (the portion comprising two cysteine ​​residues), a CH2 domain, and a CH3 domain. In some embodiments, DD1 and DD2 are each the Fc domain of the first cysteine ​​residue read in the N-to-C direction in the hinge region (e.g., cysteine ​​226 of human IgG1 or IgG4, using EU numbering).

[0148] In some embodiments, the first monomer and / or the second monomer may each comprise a total of 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, or about 150 amino acids. 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 250 Approximately 250 amino acids to approximately 750 amino acids, approximately 250 amino acids to approximately 700 amino acids, approximately 250 amino acids to approximately 650 amino acids, approximately 250 amino acids to approximately 600 amino acids, approximately 250 amino acids to approximately 550 amino acids, approximately 250 amino acids to approximately 500 amino acids, approximately 250 amino acids to approximately 450 amino acids, approximately 250 amino acids to approximately 400 amino acids, approximately 250 amino acids to approximately 350 amino acids, approximately 250 amino acids to approximately 300 amino acids, approximately 300 amino acids to approximately 800 amino acids, approximately 300 amino acids to approximately 750 amino acids, approximately 300 amino acids to approximately 700 amino acids, approximately 300 amino acids to approximately 650 amino acids, approximately 3 00 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.Approximately 350 amino acids to approximately 400 amino acids, approximately 400 amino acids to approximately 800 amino acids, approximately 400 amino acids to approximately 750 amino acids, approximately 400 amino acids to approximately 700 amino acids, approximately 400 amino acids to approximately 650 amino acids, approximately 400 amino acids to approximately 600 amino acids, approximately 400 amino acids to approximately 550 amino acids, approximately 400 amino acids to approximately 500 amino acids, approximately 400 amino acids to approximately 450 amino acids, approximately 450 amino acids to approximately 800 amino acids, approximately 450 amino acids to approximately 750 amino acids, approximately 450 amino acids to approximately 700 amino acids, approximately 450 amino acids to approximately 650 amino acids, approximately 450 amino acids to approximately 600 amino acids, approximately 450 amino acids to approximately 550 amino acids, approximately 450 amino acids to approximately 500 amino acids, approximately 500 amino acids to approximately 800 amino acids, approximately 500 amino acids to approximately 750 amino acids, approximately 500 amino acids to Approximately 700 amino acids, approximately 500 amino acids to approximately 650 amino acids, approximately 500 amino acids to approximately 600 amino acids, approximately 500 amino acids to approximately 550 amino acids, approximately 550 amino acids to approximately 800 amino acids, approximately 550 amino acids to approximately 750 amino acids, approximately 550 amino acids to approximately 700 amino acids, approximately 550 amino acids to approximately 650 amino acids, approximately 550 amino acids to approximately 600 amino acids, approximately 600 amino acids to approximately 800 amino acids, approximately 600 amino acids to approximately 750 amino acids, approximately 600 amino acids to approximately 700 amino acids, approximately 600 amino acids to approximately 650 amino acids, approximately 650 amino acids to approximately 800 amino acids, approximately 650 amino acids to approximately 750 amino acids, approximately 650 amino acids to approximately 700 amino acids, approximately 700 amino acids to approximately 800 amino acids, approximately 700 amino acids to approximately 750 amino acids, or approximately 750 amino acids to approximately 800 amino acids.

[0149] Decomposable component (CM) In some embodiments, the ACC includes one or more CMs. The CM may be located between two components of the ACC, for example, between a cytokine peptide and a MM, between a cytokine peptide and a DD, and / or between a cytokine peptide and another component of the ACC. In some embodiments, the MM is coupled to the cytokine peptide via the CM, i.e., the CM is located between the interleukin and the MM.

[0150] In some embodiments, the CM is located directly or indirectly (e.g., through a connector) between the MM and the cytokine peptide. In some embodiments, the CM is located directly or indirectly (e.g., through a connector) between the cytokine peptide and the DD.

[0151] In some embodiments, the CM described herein may contain substrates of proteases that have been reported in one or more cancers. See, for example, La Roca et al., British J. Cancer 90(7):1414-1421, 2004. Substrates suitable for use in the CM components herein include those more commonly found in cancerous cells and tissues. Thus, in some embodiments, the CM contains substrates of proteases more commonly found in cancer-associated diseased tissues. In some embodiments, the cancer is selected from the group consisting of gastric cancer, breast cancer, osteosarcoma, and esophageal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is HER2-positive cancer. In some implementations, the cancer is Kaposi's sarcoma, hairy cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, breast cancer, ovarian cancer, bladder cancer, BCG-resistant non-muscle-invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer, or testicular cancer, etc. In some of the above implementations, the CM component contains substrates of proteases more commonly found in tumor tissue. For example, the protease may be produced by the subject's tumor.

[0152] The CM used in this ACC includes any protease substrate known in the art. In some instances, the CM may contain substrates of serine proteases (e.g., u-type plasminogen activator (uPA, also known as urokinase)) or proteolytic enzymes (also referred to herein as MT-SP1 or MTSP1). In some instances, the CM may contain substrates of matrix metalloproteinases (MMPs). In some instances, the CM may contain substrates of cysteine ​​proteases (CPs) (e.g., podin).

[0153] In some embodiments, the CM may comprise a substrate of the following: integrin and metalloproteinases (ADAM) or integrin and metalloproteinases (ADAMTS) having a thrombin-sensitive motif (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5), aspartic proteases (e.g., BACE, renin), aspartic cathepsins (e.g., cathepsin D, cathepsin E), apoptotic proteases (e.g., apoptotic proteases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14), cysteine. Acidic cathepsins (e.g., cathepsin A, cathepsin B, cathepsin C, cathepsin G, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P), cysteine ​​proteases (e.g., Cruzipain, podocyte protein, Otubain-2), chymotrypsin, DESC1, DPP-4, FAP, elastase, FVIIa, FIXA, Fxa, FXIa, FXIIa, granzyme B, guanidinylbenzoate, hepsin, HtrA1, human neutrophil elastase, KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14), metalloproteinases (e.g., meprin, enkephalin, PSMA, BMP-1), lactoferrin, marapsin, interstitial proteinase-2, MT-SP1 / interstitial proteinase, NS3 / 4A, PACE4, plasmin, PSA, MMP) (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27), TMPRSS2, TMPRSS3, TMPRSS4, tPA, thrombin, trypsin, and uPA.

[0154] In some embodiments, the protease substrate in the CM may contain a polypeptide sequence that is substantially different from (e.g., no more than 90%, 80%, 70%, 60%, or 50% identical) to any polypeptide sequence naturally cleaved by the same protease.

[0155] In some embodiments, CM comprises or consists of a sequence of LSGRSDNH (SEQ ID NO: 552) or PLGLAG (SEQ ID NO: 615). In some embodiments, CM comprises or consists of a sequence covered by a common sequence of any of the following disclosed in WO2015048329 (which is incorporated herein by reference in its entirety): SEQ ID NOs: 317-327, 329-335, 340-347, 352-363, 371-378, 394-401, 410-419, 425-433, 436-449, 453-456, 458-469, 473, 475-482, 485-495 and SEQ ID NOs: 1-162, 268-306 disclosed in WO2015116933 (which is incorporated herein by reference in its entirety).

[0156] In some implementations, CM includes SEQ ID NOs disclosed in WO2015048329: 14-52, 126-154, 159, 315-316, 328, 336-339, 348-351, 364-370, 379-393, 402-409, 420-424, 434-435, 450-452, 457, 470-472, 474, 483, 484; SEQ ID NOs disclosed in WO2015116933: 163-267, 307-384, 402-445, 665-683; and SEQ IDs disclosed in WO2016118629 (which are incorporated herein by reference in their entirety). Sequences of or composed of any of the sequences disclosed in SEQ ID NO: 1-16, 50-56, 60-63, 20, 70-76, 78-115, 120-128, 130-132, 135-140, 141, 152, 21-23, 17-19, 25-43 disclosed in WO2020118109 (which is incorporated herein by reference in its entirety) are also present. In some instances, the CM of the cysteine ​​protease contains or is composed of the sequence of AAN, SAN, or GPTN (SEQ ID NO: 152). Examples of CM also include WO 2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236 679. WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO20 The CMs described in WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974 (which are incorporated herein by reference in their entirety) are also included in this document.

[0157] In some implementations, a CM comprises or consists of any of the CM sequences in Table 1 below, or is covered by a common sequence of said sequences.

[0158] Table 1. CM sequences In some embodiments, the CM comprises a combination of, C-terminal truncated variants of, or N-terminal truncated variants of, the exemplary sequences discussed above. The truncated variants of the amino acid sequences mentioned above applicable to the CM are any truncated variants that retain the recognition site of the corresponding protease. These variants include C-terminal and / or N-terminal truncated variants that contain at least three consecutive amino acids of the aforementioned amino acid sequence retaining the protease recognition site, or at least four, five, six, or seven amino acids of the aforementioned amino acid sequence. In some embodiments, the truncated variants of the aforementioned amino acid sequences correspond to any of the above amino acid sequences, but are truncated at the C-terminus and / or N-terminus by 1 to about 10 amino acids, 1 to about 9 amino acids, 1 to about 8 amino acids, 1 to about 7 amino acids, 1 to about 6 amino acids, 1 to about 5 amino acids, 1 to about 4 amino acids, or 1 to about 3 amino acids, and: (1) have at least three amino acid residues; and (2) retain the amino acid sequence of the protease recognition site. In some of the foregoing embodiments, the truncated CM is an N-terminal truncated CM. In some implementations, the truncated CM is a CM with its C-end truncated. In some implementations, the truncated C is a CM with both its C-end and N-end truncated.

[0159] In some embodiments, the CM may contain a total of 3 to 25 amino acids. In some embodiments, the CM may contain a total of 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25 amino acids.

[0160] In some embodiments, CM is administered via at least one protease at a concentration of approximately 0.001-1500 × 10⁻⁶. 4 M -1 S -1Or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250 or 1500×10 4 M -1 S -1 The rate of fragmentation is specific. This rate can be determined by substrate fragmentation kinetics (k... cat / K m ) to measure, as disclosed in WO2016118629.

[0161] In some embodiments of any cytokine-activating construct described herein, CM comprises a total of about 3 amino acids to about 25 amino acids. In some embodiments, CM comprises 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.

[0162] In some embodiments, the ACC comprises multiple CMs, each CM containing a substrate of a different protease. In some embodiments, CM1 and CM2 in the dimer construct contain substrates of different proteases. In some embodiments, CM1 and CM2 in the dimer construct contain substrates of the same protease.

[0163] The first and second monomer builder of ACC, or ACC as a dimer complex, may contain one or more additional components, including one or more connectors, etc. In some embodiments, the first monomer may include a connector disposed between CP and CM. In some embodiments, CP and CM are directly adjacent to each other.

[0164] In some embodiments, in the ACC as a dimer complex, the first monomer may include a linker disposed between CP1 and CM1. In some embodiments, CP1 and CM1 are directly adjacent to each other in the first monomer. In some embodiments, the first monomer includes a linker disposed between CM1 and DD1. In some embodiments, the linker has a total length of 1 amino acid to about 15 amino acids. In some embodiments, CM1 and DD1 are directly adjacent to each other in the first monomer. In some embodiments, CM and any linker disposed between CP1 and DD1 have a total length of 3 to 15 amino acids, or 3 to 10 amino acids, or a combination of 3 to 7 amino acids.

[0165] In some embodiments, the second monomer includes a linker disposed between CP2 and CM2. In some embodiments, CP2 and CM2 are directly adjacent to each other in the second monomer. In some embodiments, the second monomer includes a linker disposed between CM2 and DD2. In some embodiments, the linker has a total length of 1 amino acid to about 15 amino acids. In some embodiments, the linker includes the sequence G; GG; or GGGS (SEQ ID NO: 2). In some embodiments, CM2 (e.g., any of the cleavable moieties described herein) and DD2 (e.g., any of the DDs described herein) are directly adjacent to each other in the second monomer. In some embodiments, CM and any linker disposed between CP2 and DD2 have a total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0166] Cytokine masking fraction (MM) and blocking fraction (BM) In some implementations, the cytokine masking portion (MM) includes a connector disposed between a first masking subunit and a second masking subunit.

[0167] In some embodiments, the second masking subunit is encoded by a second subsequence of an amino acid sequence encoding a receptor polypeptide selected from the group consisting of the amino acid sequence encoding the first receptor polypeptide and the amino acid sequence of a second receptor polypeptide encoding the receptor for the cytokine, wherein the first subsequence and the second subsequence are discontinuous within the amino acid sequence encoding the first receptor polypeptide.

[0168] In some implementations, the receptor polypeptide of the cytokine receptor is the extracellular domain of the receptor. The extracellular domains of cytokine receptors have been identified in the literature, such as Wang, Xinquan et al., “Structuralbiology of shared cytokine receptors.” Annual review of immunology27 (2009):29-60, which is incorporated herein by reference in its entirety.

[0169] In some respects, such peptides with binding affinity for cytokines include the sequences disclosed in Table 2.

[0170] Table 2. Examples of masking peptides (MMs) associated with appropriate cytokines In some embodiments, the second masking subunit is encoded by the second amino acid sequence encoding the blocking portion (BM). In some embodiments, the BM is a polypeptide that binds to a cytokine peptide, but not a cytokine receptor peptide or a subsequence of a cytokine receptor peptide. Therefore, the BM specifically and / or selectively binds to the cytokine peptide instead of a tumor antigen or tissue antigen. In some instances, the BM can be an antibody or antibody fragment (e.g., Fab fragment, F(ab')2 fragment, scFv, scAb, variable domain of a camel-type nanobody (VHH), dAb, single-domain heavy chain antibody, and single-domain light chain antibody), a non-immunoglobulin mimicking antibody binding and / or structures (e.g., anti-coagulant, avidin, avidiosome molecules, avidiosome, avidin, alphabet, avimer, DARPin, fynomer, kunitz domain peptide, monomeric antibody), and a binding domain based on other engineered scaffolds (such as SpA, GroEL, fibronectin, lipid carrier proteins, and CTLA4 scaffolds) that binds to the cytokine peptide, thereby disrupting the binding of the cytokine peptide to its target. In some instances, BM can be a peptide that binds to cytokine peptides and interrupts the binding of cytokine peptides to their targets.

[0171] This document provides methods for screening candidate peptides to obtain BM peptides that selectively and / or specifically bind cytokine polypeptides, and may include protein display methods and methods for screening candidate peptides to identify BM, such as those described in US20200308243A1, WO2009025846A2, and WO2010081173, which are incorporated herein by reference in their entirety.

[0172] In some respects, BM includes the masked portion sequences disclosed in WO2022 / 197764, WO2010096838A2, WO2020 / 069398 A1 and WO2019 / 246392, which are incorporated herein by reference in their entirety.

[0173] In some respects, BM contains scFvs that have binding affinity for cytokines.

[0174] In some respects, BM is a spatial mask that inhibits the binding of cytokines to their binding partners through spatial steric hindrance.

[0175] In some implementations, the first subsequence and the second subsequence are the same. In other implementations, the first subsequence and the second subsequence are different.

[0176] In some embodiments, the first receptor polypeptide and the second receptor polypeptide are the same. In other embodiments, the first receptor polypeptide and the second receptor polypeptide are different.

[0177] In some implementations, the cytokine MM is operatively linked to the cytokine. In some aspects, the cytokine MM is linked to the cytokine directly or via a connector and placed in a functional relationship such that the cytokine MM can bind to the cytokine and inhibit the interaction between the cytokine peptide and its binding partner in the subject's body.

[0178] Additional cover (MM) In some embodiments, the ACC described herein may include one or more additional MMs besides the MMs described above. The following disclosures and embodiments discussing additional MMs also apply to BMs according to any embodiment described herein. In some embodiments, the additional MM interacts with the cytokine peptide, thereby reducing or inhibiting the interaction between the cytokine peptide and its binding partner. In some embodiments, the additional MM comprises at least some or all of the amino acid sequence of a naturally occurring binding partner of the cytokine peptide. For example, the additional MM may be a fragment of a naturally occurring binding partner. The fragment may retain no more than 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, or 20% homology of the nucleic acid or amino acid sequence to the naturally occurring binding partner. As used herein, the term "naturally occurring" when applied to an object means the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from natural sources and is not intentionally modified by humans in a laboratory or otherwise is considered naturally occurring.

[0179] In some embodiments, the additional MM comprises a non-naturally occurring amino acid sequence or does not contain the amino acid sequence of a naturally occurring binding partner. In some embodiments, the MM is not a natural binding partner of the cytokine peptide. The additional MM may be a modified binding partner of the cytokine peptide containing amino acid variations that reduce the binding affinity and / or co-occurrence with the cytokine peptide. In some embodiments, the additional MM contains little or no nucleic acid or amino acid homology to the natural binding partner of the cytokine peptide. In other embodiments, the additional MM contains no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the natural binding partner of the cytokine peptide.

[0180] In some implementations, the additional MM does not specifically bind to the cytokine peptide, but rather interferes with the binding of the cytokine peptide to its binding partner through non-specific interactions such as steric hindrance (“spatial masking”). For example, the additional MM may be located in the ACC, such that the tertiary or quaternary structure of the ACC allows the additional MM to mask the cytokine peptide through charge-based interactions, thereby keeping the additional MM in place to interfere with the binding partner’s access to the cytokine peptide.

[0181] In some embodiments, the dissociation constant of the additional MM to the cytokine peptide is no greater than the dissociation constant of the cytokine peptide to its binding pair. In some embodiments, the additional MM, in its cleaved state, does not interfere with or compete for the binding of the cytokine peptide to its binding pair.

[0182] The structural properties of MM can be selected based on factors such as the minimum amino acid sequence required to interfere with the binding of the protein to the binding partner, the target binding partner protein-protein binding pair, the size of the cytokine peptide, and the presence or absence of the linker.

[0183] In some embodiments, the additional MM is unique for the coupled cytokine peptide. Examples of additional MMs include MMs (e.g., affinity masks) that are specifically screened to bind to the binding domain of the cytokine peptide or a fragment thereof. Methods for screening MMs to obtain MMs that are unique for the cytokine peptide and those that specifically and / or selectively bind to the binding domain of the binding partner are provided herein and may include protein display methods.

[0184] In some implementations, the additional MM is a polypeptide with a length of about 2 to 50 amino acids. For example, the additional MM can be a polypeptide with a length of 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, or 40 to 50 amino acids. For example, the additional MM can be a polypeptide with a length of 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 some instances, the additional MM can be a polypeptide with a length exceeding 50 amino acids, such as 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids.

[0185] In some embodiments, when measured in an in vitro immunoadsorption assay as described, for example, as in US20200308243A1, in the inactive state of the ACC containing the cytokine peptide and interfering MM, in the presence of the binding partner of the cytokine peptide, the cytokine peptide is not bound to its binding partner or is substantially not bound, or the binding to the corresponding antibody without interfering MM is no more than 0.001%, 0.01%, 0.1%, or 1%. 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50%, lasting for at least 0.1, 0.5, 1, 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0186] Under MM interference, the binding affinity of cytokine peptides to their binding partners can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, and 50,000,000 times lower than that when MM interference is absent. Alternatively, the binding affinity of cytokine peptides to their binding partners can be 5-10, 10-100, 10-1,000, 10-10,000, and 10-100 times lower than that when MM interference is absent. ,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-10,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-10,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times.

[0187] The dissociation constant of the masked cytokine peptide by the metamorphic membrane (MM) may be greater than that of the cytokine peptide with its binding partner. The dissociation constant of the MM for the masked cytokine peptide 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 that of the cytokine peptide with its binding partner. Conversely, the binding affinity of the MM for the masked cytokine peptide may be lower than that of the cytokine peptide with its binding partner. The binding affinity of MM to cytokine peptides can 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 that of cytokine peptides to their binding partners.

[0188] In some embodiments, the additional MM contains genetically encoded amino acids or genetically non-coding amino acids. Examples of genetically non-coding amino acids include, but are not limited to, D-amino acids, β-amino acids, and γ-amino acids. In specific embodiments, the MM contains no more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% genetically non-coding amino acids.

[0189] In some embodiments, once released from the ACC and in a free state, the additional MM possesses biological activity or therapeutic properties, such as binding capacity. For example, the free peptide may bind to the same or different binding partners. In some embodiments, the free MM exerts a therapeutic effect, thereby providing a secondary function to the compositions disclosed herein. In some embodiments, once uncoupled from the TB and in a free state, the MM may advantageously not exhibit biological activity. For example, in some embodiments, the free MM does not elicit an immune response in a subject.

[0190] Suitable additional MMs can be identified and / or further optimized from candidate ACCs with variable MMs through screening procedures. For example, cytokine peptides and CMs can be selected to provide the desired enzyme / target combination, and the amino acid sequence of the additional MM can be identified through screening procedures described below to identify MMs that provide a transformational phenotype. For example, random peptide libraries (e.g., random peptide libraries containing peptides of 2 to 40 or more amino acids) can be used in the screening methods disclosed herein to identify suitable MMs.

[0191] This document provides methods for screening candidate peptides to obtain MMs specific to cytokine peptides, and may include protein display methods and methods for screening candidate peptides to identify masking portions (MMs), blocking portions (BMs), or additional MMs, such as those described in US20200308243A1, WO2009025846A2, and WO2010081173, which are incorporated herein by reference in their entirety.

[0192] Examples of additional MM and BM peptides include polypeptides that bind to IL-15 and / or IL-2, such as any one of SEQ ID NO: 358-374.

[0193] connector In some embodiments of any ACC described herein, one or more adapters (e.g., flexible adapters) are introduced into the activatable cytokine construct to provide flexibility at one or more junctions between domains, between portions, between portions and domains, or at any other junction where the adapter would be beneficial. In some embodiments, when the ACC is provided as a conformationally constrained construct, the insertion of flexible adapters facilitates the formation and maintenance of structures in the uncleaved activatable cytokine construct. Any adapter described herein can provide the desired flexibility to facilitate inhibition of binding to a binding partner (e.g., a cytokine receptor) or to facilitate the cleavage of CM by a protease. In some embodiments, a wholly or partially flexible adapter is included in the ACC, such that the adapter may include a flexible adapter and one or more portions that impart less flexibility to provide the desired ACC. Some adapters may include cysteine ​​residues that can form disulfide bonds and reduce the flexibility of the construct. In some embodiments, reducing the length of the adapter or linker region reduces the activity of the mature cytokine peptide in the ACC. In most cases, the linker length is determined by counting the number of amino acids in the N-to-C direction from the N-terminus of the linker adjacent to the C-terminus of the previous component to the C-terminus of the linker adjacent to the N-terminus of the subsequent component (i.e., the linker length does not include the C-terminus of the previous component or the N-terminus of the subsequent component). In embodiments where the linker is used at the N-terminus of the DD containing the Fc domain, the linker length is determined by counting the number of amino acids from the N-terminus of the linker adjacent to the C-terminus of the previous component to the C-terminus of the linker adjacent to the first cysteine ​​residue of the Fc hinge region (i.e., the linker length does not include the C-terminus of the previous component or the first cysteine ​​residue of the Fc hinge region).

[0194] In some embodiments, the ACC of this disclosure includes the CP and an amino acid segment between the proximal interaction sites between the dimerizing domains (see [link]). Figure 4(Examples in the text). The segment of amino acids may be referred to as the linker region (LR). As used herein, the term "linker region" or "LR" refers to a segment of amino acid residues between the nearest amino acid residues of the C-terminus and N-terminus of a cytokine that interact with the dimerizing domains adjacent to it (i.e., the linker region does not include the C-terminal amino acid of the cytokine or the N-terminal amino acid of the DD, which forms the nearest point of interaction with the DD of the corresponding second monomer). For example, when the DD is a pair of Fc domains, the linker region is a segment of amino acid residues between the C-terminus of the cytokine and the first N-terminal cysteine ​​residue involved in the disulfide bond of the Fc (e.g., cysteine ​​226 of the Fc domain of IgG1 or IgG4, according to EU designation). When the dimerizing domain is not a peptide, the linker region is a segment of amino acid residues from the C-terminus of the cytokine to the last amino acid. For example, when DD is a biotin-streptavidin pair, the linker region of the biotin-containing monomer is an amino acid residue chain between the C-terminus of the cytokine and the biotin molecule, and the linker region of the streptavidin-containing monomer is an amino acid residue chain between the C-terminus of the cytokine and the streptavidin molecule. In some aspects, the linker region may contain no more than 24, 18, 14, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids, for example, 5 to 14, 7 to 12, 7 to 11, or 8 to 11 amino acids.

[0195] In some embodiments, the additional amino acid sequence is located 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 of receptors present on cells in target tissues) and serum half-life extension moieties (e.g., peptides that bind serum proteins, such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (I))).

[0196] In some embodiments of any activatable cytokine construct described herein, the linker may comprise a total of about 1 amino acid to about 25 amino acids (e.g., about 1 amino acid to about 24 amino acids, about 1 amino acid to about 22 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 18 amino acids, about 1 amino acid to about 16 amino acids, about 1 amino acid to about 15 amino acids, about 1 amino acid to about 14 amino acids, about 1 amino acid to about 12 amino acids, about 1 amino acid to about 10 amino acids, about 1 amino acid to about 8 amino acids, about 1 amino acid to about 6 amino acids, about 1 amino acid to about 5 amino acids, about 1 amino acid to about 4 amino acids, about 1 amino acid...). About 3 amino acids, about 1 amino acid to about 2 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 24 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 15 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 2 amino acids to about 3 amino acids, about 4 amino acids to Approximately 25 amino acids, approximately 4 amino acids to approximately 24 amino acids, approximately 4 amino acids to approximately 22 amino acids, approximately 4 amino acids to approximately 20 amino acids, approximately 4 amino acids to approximately 18 amino acids, approximately 4 amino acids to approximately 16 amino acids, approximately 4 amino acids to approximately 15 amino acids, approximately 4 amino acids to approximately 14 amino acids, approximately 4 amino acids to approximately 12 amino acids, approximately 4 amino acids to approximately 10 amino acids, approximately 4 amino acids to approximately 8 amino acids, approximately 4 amino acids to approximately 6 amino acids, approximately 4 amino acids to approximately 5 amino acids, approximately 5 amino acids to approximately 25 amino acids, approximately 5 amino acids to approximately 24 amino acids, approximately 5 amino acids to approximately 22 amino acids, approximately 5 amino acids to approximately 20 amino acids, approximately 5 amino acids The amino acids are approximately 18 amino acids, approximately 5 amino acids are approximately 16 amino acids, approximately 5 amino acids are approximately 15 amino acids, approximately 5 amino acids are approximately 14 amino acids, approximately 5 amino acids are approximately 12 amino acids, approximately 5 amino acids are approximately 10 amino acids, approximately 5 amino acids are approximately 8 amino acids, approximately 5 amino acids are approximately 6 amino acids, approximately 6 amino acids are approximately 25 amino acids, approximately 6 amino acids are approximately 24 amino acids, approximately 6 amino acids are approximately 22 amino acids, approximately 6 amino acids are approximately 20 amino acids, approximately 6 amino acids are approximately 18 amino acids, approximately 6 amino acids are approximately 16 amino acids, approximately 6 amino acids are approximately 15 amino acids, approximately 6 amino acids are approximately 14 amino acids, approximately 6 amino acids are approximately 12 amino acids.About 6 amino acids to about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 24 amino acids, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 18 amino acids, about 8 amino acids to about 16 amino acids, about 8 amino acids to about 15 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 amino acids to about 22 amino groups. Acid, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 18 amino acids, about 10 amino acids to about 16 amino acids, about 10 amino acids to about 15 amino acids, about 10 amino acids to about 14 amino acids, about 10 amino acids to about 12 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 15 amino acids, about 12 amino acids to about 14 amino acids Approximately 14 amino acids to approximately 25 amino acids, approximately 14 amino acids to approximately 24 amino acids, approximately 14 amino acids to approximately 22 amino acids, approximately 14 amino acids to approximately 20 amino acids, approximately 14 amino acids to approximately 18 amino acids, approximately 14 amino acids to approximately 16 amino acids, approximately 14 amino acids to approximately 15 amino acids, approximately 15 amino acids to approximately 25 amino acids, approximately 15 amino acids to approximately 24 amino acids, approximately 15 amino acids to approximately 22 amino acids, approximately 15 amino acids to approximately 20 amino acids, approximately 15 amino acids to approximately 18 amino acids, approximately 15 amino acids to approximately 16 amino acids, approximately 16 amino acids to approximately 25 amino acids. Approximately 16 amino acids to approximately 24 amino acids, approximately 16 amino acids to approximately 22 amino acids, approximately 16 amino acids to approximately 20 amino acids, approximately 16 amino acids to approximately 18 amino acids, approximately 18 amino acids to approximately 25 amino acids, approximately 18 amino acids to approximately 24 amino acids, approximately 18 amino acids to approximately 22 amino acids, approximately 18 amino acids to approximately 20 amino acids, approximately 20 amino acids to approximately 25 amino acids, approximately 20 amino acids to approximately 24 amino acids, approximately 20 amino acids to approximately 22 amino acids, approximately 22 amino acids to approximately 25 amino acids, approximately 22 amino acids to approximately 24 amino acids, or approximately 24 amino acids to approximately 25 amino acids.

[0197] In some embodiments of any ACC described herein, the linker comprises a total of about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, or about 25 amino acids.

[0198] In some embodiments, the ACC does not contain any linker between CP and DD. Such ACCs may exhibit the most significant reduction in cytokine activity relative to wild-type mature cytokines. Furthermore, the configuration without a linker between CP and DD still allows for efficient cleavage of the CM located between CP and DD. Therefore, in some embodiments, the ACC does not contain any linker between CP and DD, and the CM between CP and DD contains no more than 10, 9, 8, 7, 6, 5, 4, or 3 amino acids. In some embodiments, the total number of amino acids in the LR contains no more than 25 amino acids, for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids, or 3 to 10 amino acids, or 5 to 15 amino acids, or 7 to 12 amino acids, or any range or specified number of amino acids selected from the range covered by 3 to 25 amino acids.

[0199] In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in both glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs). In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS; SEQ ID NO: 228) 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; SEQ ID NO: 216) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences). In some embodiments, the connector has one or more Gly-Gly-Ser-Gly (GGSG; SEQ ID NO: 229) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more GGSG sequences).

[0200] In any of the ACC implementations described herein, the connector comprises any one or a combination of the following: G, GG, GSSGGSGGSGG (SEQ ID NO: 210), GGGS (SEQ ID NO: 2), GGGSGGGS (SEQ ID NO: 211), GGGSGGGSGGGS (SEQ ID NO: 212), GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 214), GGGGSGGGGS (SEQ ID NO: 215), GGGGS (SEQ ID NO: 216), GS, GGGGSGS (SEQ ID NO: 217), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGSLDPKGGGGS (SEQ ID NO: 219), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 219). 220), SKYGPPCPPCPAPEFLG (SEQ ID NO: 221), GKSGSGSESKS (SEQ ID NO: 222), GSTSGSGKSSEGKG (SEQ ID NO: 223), GSTGSSGKSSEGSGSTKG (SEQ ID NO: 224), and GSTGSSGKPGSGEGSTKG (SEQ ID NO: 225).

[0201] Non-limiting examples of connectors may include those with GGGS (SEQ ID NO: 2), GSSGGSGGSGG (SEQ ID NO: 210), GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGS (SEQ ID NO: 217), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 235), GGSLDPKGGGGS (SEQ ID NO: 219), and GSTGSGSGKPGSSEGST (SEQ ID NO: 219). 226) 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) sequences.

[0202] In some embodiments, the connector includes sequences selected from the group consisting of: GGSLDPKGGGGS (SEQ ID NO: 219), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGGGSGS (SEQ ID NO: 217), GS, (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 227) and (GGGS)n (SEQ ID NO: 228), GGSG (SEQ ID NO: 229), GGSGG (SEQ ID NO: 230), GGSSG (SEQ ID NO: 231), GGSGG (SEQ ID NO: 232), GGGSG (SEQ ID NO: 233), GSSSG (SEQ ID NO: 234), GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 214), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 215), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 217), GGGGSGGGGSGGGSGGGSGGGS (SEQ ID NO: 218 ...GGGS (SEQ ID NO: 218), GGGGSGGGGSGGGSGGGSGGGSGGGS (SEQ ID NO 235), GSTGSGSGKPGSSEGST (SEQ ID NO: 226), (GGGGS)n (SEQ ID NO: 216), where n is an integer at least 1. In some embodiments, the connector includes sequences selected from the group consisting of: GGSLDPKGGGGS (SEQ ID NO: 219), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGGGSGS (SEQ ID NO: 217), and GS. In some embodiments of any ACC described herein, the connector includes sequences selected from the group consisting of: GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 235), and GSTGSGSGKPGSSEGST (SEQ ID NO: 226). In some embodiments of any of the cytokine-activating constructs described herein, the linker comprises a sequence selected from the group consisting of GGGGSGGGGSGGGGS (SEQ ID NO: 213) or GGGGS (SEQ ID NO: 216). In some embodiments, the linker comprises the sequence of GGGS (SEQ ID NO: 2). In some embodiments, the linker comprises a sequence of a single glycine residue (G) or two glycine residues (GG).

[0203] In some embodiments, the ACC may include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences as described herein or known in the art). In some embodiments, the linkers comprise sulfon-SIAB, SMPB, and sulfon-SMPB, wherein the linker reacts with a primary amine hydrogen sulfide group.

[0204] In some aspects, spacers are employed in the peptides or constructs disclosed herein. As used herein, the terms "spacer" or "head" refer to an amino acid residue or amino acid sequence incorporated at the free terminus of a mature ACC, for example, between the signal peptide and the N-terminus of the mature ACC. In some aspects, the spacer comprises one or more glutamine (Q) residues. In some aspects, the residues in the spacer minimize the activity of aminopeptidases and / or exopeptidases to prevent cleavage of the N-terminal amino acid. The illustrative and non-limiting spacer amino acid sequences may comprise or consist of any of the following exemplary amino acid sequences: QGQSGS (SEQ ID NO: 375); GQSGS (SEQ ID NO: 376); QSGS (SEQ ID NO: 377); SGS; GS; S; QGQSGQG (SEQ ID NO: 378); GQSGQG (SEQ ID NO: 379); QSGQG (SEQ ID NO: 380); SGQG (SEQ ID NO: 381); GQG; QG; G; QGQSGQ (SEQ ID NO: 382); GQSGQ (SEQ ID NO: 383); QSGQ (SEQ ID NO: 384); QGQSG (SEQ ID NO: 385); QGQS (SEQ ID NO: 386); SGQ; GQ; and Q. In some embodiments, the spacer sequence is omitted.

[0205] In some embodiments of any ACC described herein, the ACC is characterized by a reduced activity of CP or CP1 and / or CP2 if the ACC is a dimer complex, compared to a control level of at least one activity of CP1 and / or CP2. In some embodiments, the control level is the activity level of recombinant CP or CP1 and / or CP2 (e.g., commercially available recombinant CP or CP1 and / or CP2, recombinant wild-type CP or CP1 and / or CP2, etc.). In some embodiments, the control level is the activity level of the cleaved (activated) form of ACC. In some embodiments, the control level is the activity level of PEGylated CP or PEGylated CP1 and / or CP2.

[0206] In some embodiments, at least one activity is the binding affinity (Kb) of CP or CP1 and / or CP2 to its homologous receptor. D ), such as as determined using surface plasmon resonance (e.g., in phosphate-buffered saline at 25°C). In some embodiments, at least one activity is the level of lymphoma cell proliferation. In other embodiments, at least one activity is the level of JAK / STAT / ISGF3 pathway activation in lymphoma cells. In some embodiments, at least one activity is the level of SEAP production in lymphoma cells. In some embodiments, at least one activity is the level of SEAP production in a cell-based assay using HEK cells. In another embodiment, at least one activity of CP or CP1 and / or CP2 is the level of gene induction by cytokine stimulation as determined using, for example, RNAseq methods (see, for example, Zimmerer et al.). Clin. Cancer Res. 14(18):5900-5906, 2008; Hilkens et al., J. Immunol. 171:5255-5263, 2003).

[0207] In some embodiments, ACC is characterized by a reduction of at least 2-fold in the activity of at least one CP or CP1 and / or CP2 compared to a control level of at least one CP or CP1 and / or CP2 activity. In some embodiments, ACC is characterized by a reduction of at least 5-fold in the activity of at least one CP or CP1 and / or CP2 compared to a control level of at least one CP or CP1 and / or CP2 activity. In some embodiments, ACC is characterized by a reduction of at least 10-fold in the activity of at least one CP or CP1 and / or CP2 compared to a control level of at least one CP or CP2 activity. In some embodiments, ACC is characterized by a reduction of at least 20-fold in the activity of at least one CP or CP1 and / or CP2 activity compared to a control level of at least one CP or CP2 activity. In some embodiments, ACC is characterized in that, compared with a control level of at least one activity of CP or CP1 and / or CP2, the activity of at least one CP or CP1 and / or CP2 is reduced by at least 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold. In some embodiments, ACC is characterized in that, compared with a control level of at least one activity of CP or CP1 and / or CP2, the activity of at least one CP or CP1 and / or CP2 is reduced by at least 1-fold to 20-fold, 200-fold to 500-fold, 300-fold to 500-fold, 400-fold to 500-fold, 500-fold to 600-fold, 600-fold to 700-fold, 150-fold to 1000-fold, 100-fold to 1500-fold, 200-fold to 1500-fold, 300-fold to 1500-fold, 400-fold to 1500-fold, or more. 500 to 1500 times, reduced by 1000 to 1500 times, reduced by 100 to 1000 times, reduced by 200 to 1000 times, reduced by 300 to 1000 times, reduced by 400 to 1000 times, reduced by 500 to 1000 times, reduced by 100 to 500 times, reduced by 20 to 50 times, reduced by 30 to 50 times, reduced by 40 to 50 times, reduced by 100 to 400 times, reduced by 200 to 400 times or reduced by 300 to 400 times, reduced by 100 to 300 times, reduced by 200 to 300 times or reduced by 100 to 200 times.

[0208] In some embodiments, the ACC is characterized by producing lysis products upon exposure to the protease, wherein the lysis products contain at least one activity of CP1 and / or CP2. In some embodiments, at least one activity of CP1 and / or CP2 is antiproliferative activity. In some embodiments, the control level is the EC50 value of wild-type mature cytokines, and wherein the ratio of EC50 (lysis products) to EC50 (wild-type control level) is less than about 10, or less than about 9, or less than about 8, or less than about 7, or less than about 6, or less than about 5, or less than about 4, or less than about 3, or less than about 2, or less than about 1.5, or equal to about 1. In some embodiments, the EC50 of the lysis products is substantially the same as the EC50 of wild-type mature cytokines, thereby demonstrating that the activities of CP1 and / or CP2 are fully or almost fully restored after lysis. In some embodiments, the ratio of the EC50 of the lysate to the EC50 of the wild-type control is about 1 to about 10, or about 2 to about 8, or about 3 to about 7, or about 4 to about 6, demonstrating good recovery of cytokine activity after protease activation. In some embodiments, the ACC is characterized by having a lysate after protease activation, wherein the ratio of the EC50 of the lysate to the EC50 of recombinant IL-15 is 1 to about 10, or about 2 to about 8, or about 3 to about 7, or about 4 to about 6, or about 5 to about 7, or about 6, as measured in IL-2 / IL-15 responsive HEK293 cells.

[0209] In some embodiments, the control level for at least one activity of CP or CP1 and / or CP2 is the activity of CP or CP1 and / or CP2 released from the ACC after the protease cleaves CM or CM1 and CM2 (“cleavage products”). In some embodiments, the control level for at least one activity of CP or CP1 and / or CP2 is the activity of the corresponding wild-type mature cytokine (e.g., recombinant wild-type mature cytokine).

[0210] In some embodiments, ACC is incubated with a protease to produce one or more activated cytokine products, wherein the CP or CP1 and / or CP2 activities of the one or more activated cytokine products are greater than the CP or CP1 and / or CP2 activities of the intact ACC. In some embodiments, the CP or CP1 and / or CP2 activities of the one or more activated cytokine products are at least 1 times greater than the CP or CP1 and / or CP2 activities of the ACC. In some embodiments, the CP or CP1 and / or CP2 activities of the one or more activated cytokine products are at least 2 times greater than the CP or CP1 and / or CP2 activities of the ACC. In some embodiments, the CP or CP1 and / or CP2 activities of the one or more activated cytokine products are at least 5 times greater than the CP or CP1 and / or CP2 activities of the ACC. In some embodiments, the CP or CP1 and / or CP2 activities of the one or more activated cytokine products are at least 10 times greater than the CP or CP1 and / or CP2 activities of the ACC. In some embodiments, the activity of one or more activated cytokine products CP or CP1 and / or CP2 is at least 20 times greater than the activity of ACC CP or CP1 and / or CP2. In some embodiments, the activity of one or more activated cytokine products of CP or CP1 and / or CP2 is at least 1 to 20 times, 2 to 20 times, 3 to 20 times, 4 to 20 times, 5 to 20 times, 10 to 20 times, 15 to 20 times, 1 to 15 times, 2 to 15 times, 3 to 15 times, 4 to 15 times, 5 to 15 times, 10 to 15 times, 1 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 5 to 10 times, 1 to 5 times, 2 to 5 times, 3 to 5 times, 4 to 5 times, 1 to 4 times, 2 to 4 times, 3 to 4 times, 1 to 3 times, 2 to 3 times, or 1 to 2 times that of ACC.

[0211] In some embodiments, the ACC may include 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 any one of SEQ ID NO: 423. In some embodiments, the ACC may include 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 any one of SEQ ID NO: 424. In some embodiments, the ACC may include 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%) of the sequence identical to SEQ ID NO: 425. In some embodiments, the ACC may include 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%) of the sequence identical to SEQ ID NO: 426. In some embodiments, the ACC may include 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%) of the sequence identical to SEQ ID NO: 427. In some embodiments, the ACC may include 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%) of the sequence identical to SEQ ID NO: 428. In some embodiments, the ACC may include 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%) of the sequence identical to SEQ ID NO: 430.In some implementations, ACC may include 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%) of the sequence that is identical to SEQ ID NO: 431.

[0212] In some respects, ACC may include such sequences, but signal sequences may or may not have these sequences. The signal sequences are not specifically limited. Some non-limiting examples of signal sequences include, for example, MRAWIFFLLCLAGRALA (SEQ ID NO: 343), MALTFALLVALLVLSCKSSCSVG (SEQ ID NO: 344), and METDTLLLWVLLLWVPGSTG (SEQ ID NO: 345).

[0213] Various exemplary aspects of these activatable cytokine constructs are described below, and can be used in any combination in the methods provided herein without limitation. Exemplary aspects of the activatable cytokine constructs and methods for preparing them are described below.

[0214] In some aspects, ACC includes CP1 selected from SEQ ID NO: 402-422, CM1 and DD1 selected from SEQ ID No: 5-118, 131-209, 251-314, 432-499, 530-599 and 603-719, which dimerize with CP2 selected from SEQ ID NO: 402-422, CM2 and DD2 selected from SEQ ID No: 5-118, 131-209, 251-314, 432-499, 530-599 and 603-719. In some aspects, the ACC may include a linker selected from SEQ ID Nos: 2 and 210-235, 245, or 250 between CP1 and CM1 and / or between CM1 and DD1, and a linker selected from SEQ ID Nos: 2 and 210-235, 245, or 250 between CP2 and CM2 and / or between CM2 and DD2. In some embodiments, the ACC includes DD1 and / or DD2 having an amino acid sequence 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: 3 or SEQ ID NO: 4. In some embodiments, the ACC comprises DD1 having an amino acid sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 315 or SEQ ID NO: 316. In some embodiments, the ACC comprises DD2 having an amino acid sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 315 or SEQ ID NO: 316.

[0215] Conjugation This disclosure also provides methods and materials for including additional elements in any isolated peptides and ACCs described herein, said additional elements including, for example, targeting portions, agents (e.g., therapeutic agents, anti-hypertrophic agents), toxins, or fragments thereof for facilitating delivery to target cells or tissues.

[0216] In some embodiments, the ACC is conjugated to a cytotoxic agent, including but not limited to toxins (e.g., bacterial, fungal, plant, or animal-derived enzyme-active toxins or fragments thereof) or radioisotopes. In some embodiments of any ACC described herein, an activatable cytokine construct is conjugated to a cytotoxic agent, including but not limited to toxins (e.g., bacterial, fungal, plant, or animal-derived enzyme-active toxins or fragments thereof) or radioisotopes.

[0217] Non-limiting exemplary cytotoxic agents that can be conjugated to any ACC described herein include: dolastatin and its derivatives (e.g., auristatin E, AFP, monomethylauribatin D (MMAD), monomethylauribatin F (MMAF), monomethylauribatin E (MMAE), normethylauribatin E (DMAE), auristatin F, normethylauribatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone), and maytansinoids. (e.g., DM-1, DM-4), maytansine derivatives, duocarmycin, alpha-amanitin, turbostatin, phenstatin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocomstatin, pyrrolobenzimidazole (PBI), cibrostatin 6, doxaliform, cemadotin analogue (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracycline, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecin, 10,11-difluoromethylenedioxycamptothecin, comprbetastatin, debromoaplysiatoxin, KahaMide-F, discodermolide, and ecteinascidin.

[0218] Non-limiting exemplary enzymatic toxins that can be conjugated to any ACC described herein include: diphtheria toxin, and toxins derived from Pseudomonas aeruginosa. Pseudomonas aeruginosaThe following are exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, and tung oil ( Aleuriies fordii Protein, caryophyllin (dianfhin) protein, American pokeweed ( Phytoiaca Americana Proteins such as PAPI, PAPII, and PAP-8, momordica charantia inhibitors, curcin, crotirs, sapaonaria officinalis inhibitors, gelonin, mitogeliin, restrictocin, phenomycin, neomycin, and trichothecene.

[0219] Non-limiting exemplary anti-hypertrophic agents that can be conjugated to any ACC described herein include: adriamycin, cerubidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarbazine, and cytarabine.

[0220] Non-limiting exemplary antiviral agents that can be conjugated to any ACC described herein include acyclovir, vira A, and symmetrel.

[0221] Non-limiting exemplary antifungal agents that can be conjugated to any ACC described herein include nystatin.

[0222] Non-limiting exemplary conjugable assays that can be conjugated to any ACC described herein include: fluorescein and its derivatives, and fluorescein isothiocyanate (FITC).

[0223] Non-limiting exemplary antimicrobial agents that can be conjugated to any cytokine-activating construct described herein include: aminoglycoside, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin.

[0224] 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 activatable cytokine construct described herein includes: an s-nitrobenzyloxycarbonyl derivative of O6-benzylguanine, a topoisomerase inhibitor, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepine dimer (PBD), functionalized pyrrolobenzodiazepine, calcicheamicin, podophyllotoxin, taxane, and vinca alkaloids.

[0225] Non-limiting exemplary radiopharmaceuticals that can be conjugated to any cytokine-activating construct described herein include: 123 I, 89 Zr、 125 I, 131 I, 99 mTc, 201 T1, 62 Cu、 18 F, 68 Ga、 13 N、 15 O、 38 K, 82 Rb、 111 In、 133 Xe, 11 C and 99 mTc (Technetium).

[0226] Non-limiting exemplary heavy metals that may be incorporated into any ACC described herein include barium, gold, and platinum.

[0227] Non-limiting exemplary antimycoplasma agents that can be conjugated to any ACC described herein include: tylosine, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0228] Those skilled in the art will recognize that a variety of possible moieties can be conjugated to any of the activatable cytokine constructs described herein. Conjugation may include any chemical reaction that binds the two molecules, as long as the ACC and other moieties retain their respective activities. Conjugation may include a number of chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordination binding, and complexation. In some embodiments, covalent binding is preferred. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linkers can be used to conjugate any of the activatable cytokine constructs described herein. For example, conjugation may include organic compounds such as thioesters, carbodiimides, succinimides, glutaraldehyde, diazobenzene, and hexamethylenediamine. In some embodiments, the activatable cytokine construct may include or otherwise introduce one or more non-natural amino acid residues to provide a suitable conjugation site.

[0229] In some embodiments of any of the ACCs described herein, the agent and / or conjugate is attached to the antigen-binding domain via a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Because many cancers naturally release high levels of the reducing agent glutathione, glutathione present in the cancerous tissue microenvironment can reduce the disulfide bond and subsequently release the agent and / or conjugate at the delivery site.

[0230] In some embodiments of any of the ACCs described herein, when the conjugate binds to its target in the presence of complement at the target site (e.g., diseased tissue (e.g., cancerous tissue)), the amide or ester bond attaching the conjugate and / or agent to the linker is cleaved, resulting in the release of the conjugate and / or agent in its active form. When administered to a subject, these conjugates and / or agents achieve delivery and release of the conjugate and / or agent at the target site (e.g., diseased tissue (e.g., cancerous tissue)). These conjugates and / or agents are particularly effective for in vivo delivery of any of the conjugates and / or agents described herein.

[0231] In some embodiments, the linker cannot be cleaved by enzymes of the complement system. For example, the conjugate and / or agent is released without complement activation, as complement activation would ultimately lyse the target cell. In such embodiments, the conjugate and / or agent will be delivered to the target cell (e.g., hormone, enzyme, corticosteroid, neurotransmitter, or gene). Furthermore, the linker is only slightly susceptible to cleavage by serum proteases, and the conjugate and / or agent is released slowly at the target site.

[0232] In some embodiments of any of the ACC described herein, the conjugates and / or agents are designed such that the conjugates and / or agents are delivered to the target site (e.g., diseased tissue (e.g., cancerous tissue)), but the conjugates and / or agents are not released.

[0233] In some embodiments of any ACC described herein, the conjugate and / or agent is attached to the ACC directly or via a non-cleavable linker. Exemplary non-cleavable linkers include amino acids (e.g., D-amino acids), peptides, or other organic compounds that may be modified to include functional groups that can subsequently be used to attach to the ACC via the methods described herein.

[0234] In some embodiments of any ACC described herein, the ACC includes at least one conjugation site for an agent. In some embodiments, all possible conjugation sites may be used for conjugation with an agent. In some embodiments, one or more conjugation sites include, but are not limited to, sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain disulfide bonds but not in intrachain disulfide bonds, and / or sulfur atoms of cysteine ​​or other amino acid residues containing sulfur atoms. In such cases, the residues may be naturally present in the protein construct structure or incorporated into the protein construct using methods including, but not limited to, site-directed mutagenesis, chemical transformation, or mis-incorporation of non-natural amino acids.

[0235] This disclosure also provides methods and materials for preparing ACCs for conjugation. In some embodiments of any ACC described herein, the ACC is modified to include one or more interchain disulfide bonds. For example, the disulfide bonds in the ACC may 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 the disulfide bonds is only partial. As used herein, the term partial reduction refers to a situation where the ACC is contacted with a reducing agent, and a portion of all possible conjugation sites are reduced (e.g., not all disulfide bonds are reduced). In some embodiments, the cytokine-activating construct is partially reduced after contact with a reducing agent if less than 99% (e.g., less than 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 5%) of all possible conjugation sites are reduced. In some implementations, a reduced ACC having one or more interchain disulfide bonds is conjugated to a drug that is reactive with free thiols.

[0236] This disclosure also provides methods and materials for conjugating therapeutic agents to specific sites on ACC. In some embodiments of any ACC described herein, the ACC is modified to allow the therapeutic agent to conjugate to the ACC at specific sites on the ACC. For example, the ACC may be partially reduced in a manner that facilitates conjugation to the ACC. In such cases, partial reduction of the ACC occurs in a manner that the conjugation site in the ACC is not reduced. In some embodiments, one or more conjugation sites on the ACC are selected to facilitate conjugation of the agent at specific sites on the protein construct. Various factors can affect the “reduction level” of the ACC after treatment with a reducing agent. Without limitation, for example, the ratio of reducing agent to ACC, the duration of incubation, the incubation temperature, and / or the pH of the reduction reaction solution may need to be optimized to achieve partial reduction of the ACC using the methods and materials described herein. Any suitable combination of factors (e.g., the ratio of reducing agent to ACC, the duration and temperature of incubation with the reducing agent, and / or the pH of the reducing agent) can be used to achieve partial reduction of the ACC (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0237] The effective ratio of reducing agent to ACC can be any ratio that at least partially reduces ACC in a manner that allows for conjugation with the agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites). In some implementations, 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.

[0238] Effective incubation time and temperature for treating ACC with a reducing agent can be any time and temperature that allows the agent to at least partially reduce ACC in a manner that allows the ACC to bind to itself (e.g., general reduction of possible binding sites or reduction at specific binding sites). In some embodiments, the incubation time and temperature for treating ACC will be in the range of about 1 hour at 37°C to about 12 hours at 37°C (or any subrange thereof).

[0239] The effective pH for the reduction reaction of ACC with a reducing agent can be any pH that allows ACC to be at least partially reduced in a manner that allows the agent to bind to the ACC (e.g., general reduction of possible binding sites or reduction at specific binding sites).

[0240] When partially reduced ACC is contacted with a thiol-containing agent, the agent can conjugate the interchain thiol in the ACC. The agent can be modified in a manner that includes thiols, using a thiol-containing reagent (e.g., cysteine ​​or N-acetylcysteine). For example, ACC can be partially reduced after incubation at about 37°C with a reducing agent (e.g., TCEP) at a desired ratio of reducing agent to ACC for about 1 hour. An effective ratio of reducing agent to ACC can be any ratio that allows for the partial reduction of at least two interchain disulfide bonds in the ACC in a manner that allows for conjugation of the thiol-containing agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0241] In some embodiments of any ACC described herein, the ACC is reduced by a reducing agent in a manner that avoids the reduction of any intra-chain disulfide bonds. In some embodiments of any ACC described herein, the ACC is reduced by a reducing agent in a manner that avoids the reduction of any intra-chain disulfide bonds and reduces at least one inter-chain disulfide bond.

[0242] In some embodiments of any ACC described herein, the ACC may also include an agent conjugated to the ACC. In some embodiments, the conjugated agent is a therapeutic agent.

[0243] In some embodiments, the agent (e.g., an agent conjugated to an activatable cytokine construct) is a detectable portion, such as a label or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotinylated moieties detectable by labeled avidin (e.g., streptavidin containing a fluorescent label or enzyme activity detectable by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzyme labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable portion is attached via a spacer molecule.

[0244] In some implementations, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is attached to the ACC using a carbohydrate moiety, a hydrogen sulfide group, an amino group, or a carboxylic acid ester group.

[0245] In some embodiments of any ACC described herein, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to the ACC via a linker and / or a CM (also 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 residue in the ACC. In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to another residue of the ACC, such as those disclosed herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a non-cleavable linker. Table 3 provides some non-limiting examples of cleavable portions and linkers.

[0246] Table 3. Those skilled in the art will recognize that a variety of possible components may be coupled to the ACC of this disclosure. (See, for example, “Conjugate Vaccines,” Contributions to Microbiology and Immunology, JMCruse and RE Lewis, Jr (eds.), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference). Generally, effective conjugation of an agent (e.g., a cytotoxic agent) to an ACC can be achieved by any chemical reaction that binds the agent to the ACC while also allowing the agent and the ACC to retain their functionality.

[0247] In some embodiments of any ACC conjugated to the agent, a variety of bifunctional protein conjugates can be used to conjugate the agent to the ACC, including but not limited to 3-(2-pyridyldithio)propionate N-succinimide ester (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of imine esters (e.g., dimethyl diimide adipate hydrochloride), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazidium compounds (e.g., bis(p-azidobenzoyl)hexamethylenediamine), diazinon derivatives (e.g., bis(p-diazinonbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and difluorinated compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). In some embodiments, a carbon-14 labeled 1-isothiocyanophenylmethyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agent can be used to conjugate radioactive nucleotides to ACC. (See, for example, WO94 / 11026).

[0248] Suitable linkers and CMs are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (m-maleimide benzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazine derivatives coupled to ACC via oligopeptide linkers. In some embodiments, suitable connectors include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimide-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene) (Pierce Chem. Co., catalog (21558G); (iii) SPDP (6-[3-(2-pyridyldithio)propamido]hexanoate succinimide) (Pierce Chem. Co., catalog 21651G); (iv) sulfonyl-LC-SPDP (6-[3-(2-pyridyldithio)propamido]hexanoate sulfosuccinimide) (Pierce Chem. Co., catalog 2165-G); and (v) sulfonyl-NHS (N-hydroxysulfonyl-succinimide: Pierce Chem.) conjugated to EDC. Co., catalog number 24510). Additional connectors include, but are not limited to, SMCC, sulfonated-SMCC, SPDB, or sulfonated-SPDB.

[0249] The CMs and connectors described above contain components with different properties, thus resulting in conjugates with different physicochemical properties. For example, sulfonated NHS esters of alkyl carboxylic acids are more stable than sulfonated NHS esters of aryl carboxylic acids. Connectors containing NHS esters are less soluble than sulfonated NHS esters. Furthermore, the SMPT connector contains sterically hindered disulfide bonds and can form conjugates with increased stability. Generally, disulfide bonds are less stable than other bonds because they are cleaved in vitro, resulting in fewer usable conjugates. In particular, sulfonated NHS can enhance the stability of carbodiimide couplings. Compared to carbodiimide coupling reactions alone, carbodiimide couplings (such as EDC) form esters that are more resistant to hydrolysis when used in combination with sulfonated NHS.

[0250] In some embodiments of any ACC, the agent may be conjugated to the ACC using a modified amino acid sequence included in the amino acid sequence of the ACC. By inserting amino acids at specific positions within the amino acid sequence of the ACC to enable conjugation, the protein construct can be designed to achieve controlled placement and / or dosing of the conjugated agent (e.g., a cytotoxic agent). For example, the ACC may be modified to include cysteine ​​amino acid residues at positions on the first, second, third, and / or fourth monomers that provide reactive thiol groups and do not negatively affect protein folding and / or assembly or alter target properties. In some embodiments, the ACC may be modified to include one or more non-natural amino acid residues within its amino acid sequence to provide suitable conjugation sites. In some embodiments, the ACC may be modified to include an enzymatically activated peptide sequence within its amino acid sequence.

[0251] Nucleic acid This document provides nucleic acids comprising sequences encoding isolated polypeptides or ACC, or, if the ACC is a dimeric complex, nucleic acids comprising sequences encoding a first monomeric construct (or a protein portion of the first monomeric construct) (e.g., any first monomeric construct described herein) and a second monomeric construct (or a protein portion of the second monomeric construct) (e.g., any second monomeric construct described herein). In some embodiments, a pair of nucleic acids together encode the first monomeric construct (or the protein portion of the first monomeric construct) and the second monomeric construct (or the protein portion of the second monomeric construct). In some implementations, the nucleic acid sequence encoding the first monomer construct (or the protein portion of the first monomer construct) is at least 70% identical to the nucleic acid sequence encoding the second monomer construct (or the protein portion of the second monomer construct) (e.g., at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical).

[0252] In some embodiments, the nucleic acid encoding the protein portion of the first monomer construct encodes a polypeptide comprising CP1 and CM1 portions. In some embodiments, the nucleic acid encoding the protein portion of the second monomer encodes a polypeptide comprising CP2 and CM2 portions. In some embodiments, a pair of nucleic acids together encode the protein portions of the first and second monomer constructs, wherein the protein portions are then conjugated to DD1 and DD2 portions, respectively (in a subsequent conjugation step).

[0253] In some embodiments, the nucleic acid encoding the first monomer construct encodes a polypeptide containing the DD1 moiety. In some embodiments, the nucleic acid encoding the second monomer construct encodes a polypeptide containing the DD2 moiety.

[0254] This disclosure includes polynucleotides encoding proteins or portions thereof as described herein, and uses of such polynucleotides for protein production and / or for therapeutic purposes. Such polynucleotides may include DNA and RNA molecules (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.) encoding proteins as defined herein. This disclosure includes compositions comprising such polynucleotides. In some aspects, such compositions may be used therapeutically or prophylactically.

[0255] Modifications can be introduced into nucleotide sequences using standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis.

[0256] carrier This document provides vectors and vector sets that include any of the nucleic acids described herein. Those skilled in the art will be able to select a suitable vector or vector set (e.g., an expression vector) for preparing any ACC described herein, and to express any ACC described herein using said vector or vector set. For example, when selecting a vector or vector set, the cell must be considered, as the one or more vectors may need to be able to integrate into the chromosome of the cell and / or replicate in the cell. Exemplary vectors that can be used to generate ACCs are also described below.

[0257] As used herein, the term "vector" refers to a polynucleotide capable of inducing the expression of a recombinant protein (e.g., a first or second monomer) in a cell (e.g., any cell described herein). A "vector" is capable of delivering nucleic acids and fragments thereof into a host cell and includes regulatory sequences (e.g., promoters, enhancers, poly(A)) signals). Exogenous polynucleotides may be inserted into expression vectors for expression. The term "vector" also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0258] Methods for constructing suitable vectors, including any of the nucleic acids described herein, and adapted for transformation of cells (e.g., mammalian cells), are well known in the art. See, for example, Sambrook et al., eds., *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Press, 1989, and Ausubel et al., eds., *Current Protocols in Molecular Biology*, Current Protocols, 1993.

[0259] Non-limiting examples of vectors include plasmids, transposons, granules, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector) and any Gateway® vector. Vectors may, for example, include cis-acting elements sufficient to achieve expression; other elements for expression may be provided by host mammalian cells or in an in vitro expression system. Skilled practitioners will be able to select suitable vectors and mammalian cells for the preparation of any ACC described herein.

[0260] In some implementations of any of the ACCs described herein, the ACC is prepared biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0261] In some embodiments, the vector comprises a first and second monomer of nucleic acid encoding any of the ACCs described herein. In some embodiments, the vector is an expression vector.

[0262] In some embodiments, a vector pair together comprises a pair of nucleic acids encoding a first and a second monomer of any ACC described herein. In some embodiments, the vector pair is an expression vector pair.

[0263] cell This document also provides host cells comprising any vector or vector group described herein, wherein any vector or vector group comprises any nucleic acid described herein.

[0264] Methods for introducing nucleic acids and vectors (e.g., any vector or group 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: liposome transfection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenovirus transduction, lentivirus transduction), nanoparticle transfection, and electroporation.

[0265] In some implementations, the introduction step includes introducing into the cell a vector (e.g., any vector or vector group described herein) comprising a nucleic acid encoding a monomer that constitutes any of the ACCs described herein.

[0266] In some embodiments of any of the methods described herein, the ACC can be generated by any cell, including prokaryotic cells (e.g., bacterial cells) or eukaryotic cells. As used herein, the term "eukaryotic cell" refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian, insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiaeIn some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammalian, bird, plant, or insect cell. Non-limiting examples of mammalian cells include rodent cells (e.g., mouse cells, rat cells, hamster cells, such as Chinese hamster ovary (CHO) cells), or non-human primate cells, or human cells, such as human embryonic kidney cells (e.g., HEK293 cells).

[0267] In some embodiments, the cell contains nucleic acids encoding a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the cell contains a nucleic acid pair encoding a first monomer and a second monomer together of any of the ACCs described herein. In some aspects, the nucleic acids encoding the first monomer and the second monomer are integrated into the genomic DNA of the host cell.

[0268] Methods for generating activatable cytokine constructs This document provides a method for generating any of the ACCs described herein, the method comprising: (a) culturing any of the recombinant host cells described herein in a liquid culture medium under conditions sufficient to generate the ACC; and (b) recovering the ACC from the host cells and / or the liquid culture medium.

[0269] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions favorable to cell proliferation, cell differentiation, and cell growth. For example, cells (such as any cells described herein) can be cultured by contacting a cell culture medium containing sufficient essential growth factors and supplements to support cell viability and growth.

[0270] In some embodiments of any of the methods described herein, the method further includes the separation and recovery of ACC. Non-limiting examples of separation methods include: ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand affinity chromatography, ion exchange chromatography (e.g., anion or cation) and hydrophobic interaction chromatography.

[0271] In some embodiments, this disclosure includes a method of inducing cells to produce: a first monomeric construct comprising protein moieties of CP1, CM1, MM2 and CM3, and a second monomeric construct comprising protein moieties of CP2 and CM2, and optionally MM2 and CM4, followed by conjugating the protein moieties to DD1 and DD2 moieties, respectively.

[0272] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that allow disulfide bonds to form between dimerizing domains to form and maintain the dimerization of ACC.

[0273] In some embodiments of any of the methods described herein, the method further includes formulating the isolated ACC into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any isolated ACC described herein can be formulated for use via any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., transsurface), transmucosal, or intramuscular).

[0274] This document also provides ACCs generated by any of the methods described herein. Compositions (e.g., pharmaceutical compositions) comprising any ACC generated by any of the methods described herein are also provided. A kit comprising at least one dose of any of the compositions described herein (e.g., pharmaceutical compositions) is also provided.

[0275] In some embodiments, the ACC disclosed herein comprises a mutant of a cytokine. For example, a mutant with advantageous properties compared to a wild-type cytokine may be used, such as exhibiting less aggregation compared to a control ACC containing a wild-type cytokine peptide or a cytokine peptide without the mutation. In some embodiments, this disclosure provides a method for producing ACC, comprising: culturing cells containing a polynucleotide encoding the ACC herein in a liquid culture medium under conditions sufficient for ACC production; purifying the ACC using affinity chromatography, wherein the purified peptide has a monomeric purity of at least about 40%; and recovering the ACC from the cells or the liquid culture medium. In some embodiments, the purified peptide has a monomeric purity of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0276] In another aspect, this disclosure also provides a composition (e.g., a composition produced during the manufacture of ACC) wherein at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the purified ACC is in monomeric form.

[0277] Treatment This article provides a method for treating a subject’s disease (e.g., cancer (e.g., any cancer described herein)), the method comprising administering a therapeutically effective amount of any ACC described herein to the subject.

[0278] As used herein, the term "subject" refers to any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, rat, hamster, or guinea pig), a non-human primate (e.g., an ape (e.g., a monkey (e.g., a baboon, marmoset) or an ape (e.g., a chimpanzee, gorilla, or gorilla, or gibbon)) or a human. In some embodiments, the subject is a human.

[0279] In some implementations, the subject has been previously identified or diagnosed with a disease (e.g., cancer, such as any cancer described herein).

[0280] As used herein, the term "treatment" includes reducing the severity, frequency, or number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., cancer) in a subject (e.g., any subject described herein). In some implementations where the disease is cancer, the treatment results in a reduction in cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduction in the risk of cancer recurrence in a subject with cancer.

[0281] In some embodiments of any of the methods described herein, the disease is cancer. Methods for treating a subject in need (e.g., any exemplary subject described herein or known in the art) are also provided herein, the methods 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).

[0282] In some implementations of these methods, the subject has been identified or diagnosed with cancer. Non-limiting examples of cancer include: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell tumors, multiple myeloma, and lymphomas (e.g., B-cell lymphoma, B-cell non-Hodgkin's lymphoma). (e.g., hairy cell leukemia, Hodgkin's lymphoma, cutaneous T-cell lymphoma), leukemia (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL)), myelodysplastic syndrome (MDS), Kaposi's sarcoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colon cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, endometrial cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some implementations, the cancer is lymphoma. In some implementations, the lymphoma is Burkitt's lymphoma.) In some respects, the subjects have been identified or diagnosed with familial cancer syndromes, such as Li-Fraumeni Syndrome, familial breast-ovarian cancer (BRCA1 or BRAC2 mutation) syndrome, etc. The disclosed methods can also be used to treat non-solid cancers. Exemplary solid tumors include malignancies of various organ systems (e.g., sarcomas, adenocarcinomas, and carcinomas), such as those of the lungs, breasts, lymph nodes, gastrointestinal tract (e.g., colon), and genitourinary tracts (e.g., kidney, urothelial, or testicular tumors), pharynx, prostate, and ovaries. Exemplary adenocarcinomas include colorectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, and small bowel cancer.

[0283] Exemplary cancers described by the National Cancer Institute include: adult acute lymphoblastic leukemia; childhood acute lymphoblastic leukemia; adult acute myeloid leukemia; adrenocortical carcinoma; childhood adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; childhood cerebellar astrocytoma; childhood cerebral astrocytoma; extrahepatic bile duct cancer; bladder cancer; childhood bladder cancer; osteosarcoma / malignant fibrous histiocytoma bone cancer; childhood brainstem glioma; adult brain tumors; and childhood brainstem glioma. Tumors; Pediatric cerebellar astrocytoma brain tumors; Pediatric cerebral astrocytoma / malignant glioma brain tumors; Pediatric ependymoma brain tumors; Pediatric medulloblastoma brain tumors; Pediatric supratentorial primitive neuroectodermal tumors; Pediatric visual pathway and hypothalamic glioma brain tumors; Pediatric brain tumors (other); Breast cancer; Breast cancer and pregnancy; Pediatric breast cancer; Male breast cancer; Pediatric bronchial adenoma / carcinoid; Pediatric carcinoid tumors; Gastrointestinal carcinoid tumors; Adrenocortical carcinoma; Pancreatic islet cell carcinoma; Carcinoma of unknown primary origin. Unknown Primary); Primary Central Nervous System Lymphoma; Childhood Cerebellar Astrocytoma; Childhood Brain Astrocytoma / Malignant Glioma; Cervical Cancer; Childhood Cancer; Chronic Lymphocytic Leukemia; Chronic Myeloid Leukemia; Chronic Myeloproliferative Disorder; Tenosynovial Clear Cell Sarcoma; Colon Cancer; Childhood Colorectal Cancer; Cutaneous T-cell Lymphoma; Endometrial Cancer; Childhood Ependymoma; Ovarian Epithelial Cancer; Esophageal Cancer; Childhood Esophageal Cancer; Ewing Family Cancer; Childhood Extracranial Germ Cell Tumors; Gonadal Extragerminal Germ Cell Tumors; Extrahepatic Bile Duct Cancer; Intraocular Melanoma Ocular Cancer; Retinoblastoma Ocular Cancer; Gallbladder Cancer; Gastric (Stomach) Cancer; Childhood Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumors; Childhood Extracranial Germ Cell Tumors; Gonadal Extragerminal Germ Cell Tumors; Ovarian Germ Cell Tumors; Gestational Trophoblastic Tumors; Childhood Brainstem Glioma; Childhood Visual Pathway and Hypothalamic Glioma; Hairy Cell Leukemia; Head and Neck Cancer; Adult (Primary) Hepatocellular carcinoma; Primary hepatocellular carcinoma in children; Hodgkin's lymphoma in adults; Hodgkin's lymphoma in children; Hodgkin's lymphoma during pregnancy; Hypopharyngeal carcinoma; Hypothalamic and optic pathway glioma in children; Intraocular melanoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Renal cell carcinoma; Laryngeal carcinoma; Laryngeal carcinoma in children; Acute lymphoblastic leukemia in adults; Acute lymphoblastic leukemia in children; Acute myeloid leukemia in adults; Acute myeloid leukemia in children; Chronic lymphocytic leukemia; chronic myeloid leukemia; hairy cell leukemia; lip and oral cavity cancer; adult (primary) liver cancer; pediatric (primary) liver cancer; non-small cell lung cancer; small cell lung cancer; adult acute lymphoblastic leukemia; pediatric acute lymphoblastic leukemia; chronic lymphocytic leukemia; AIDS-related lymphoma; central nervous system (primary) lymphoma; cutaneous T-cell lymphoma; adult Hodgkin's lymphoma; pediatric Hodgkin's lymphoma;Hodgkin's lymphoma during pregnancy; non-Hodgkin's lymphoma in adults; non-Hodgkin's lymphoma in children; primary central nervous system lymphoma; Waldenstrom's macroglobulinemia; male breast cancer; malignant mesothelioma in adults; malignant mesothelioma in children; malignant thymoma; medulloblastoma in children; melanoma; intraocular melanoma; Merkel cell carcinoma. Carcinoma; malignant mesothelioma; metastatic squamous neck cancer with occult primary lesions; multiple endocrine neoplasia syndrome in children; multiple myeloma / plasma cell vegetations; mycosis fungoides; myelodysplastic syndrome; chronic myeloid leukemia; acute myeloid leukemia in children; multiple myeloma; chronic myeloproliferative disorders; nasal cavity and paranasal sinus carcinoma; nasopharyngeal carcinoma; nasopharyngeal carcinoma in children; neuroblastoma; non-Hodgkin's lymphoma in adults; non-Hodgkin's lymphoma in children; non-Hodgkin's lymphoma during pregnancy; non-small cell lung cancer; oral cancer in children; oral and lip cancer; oropharyngeal cancer; bone Sarcoma / Malignant osteofibrous histiocytoma; Pediatric ovarian cancer; Ovarian epithelial cancer; Ovarian germ cell tumors; Low-potential ovarian tumors; Pancreatic cancer; Pediatric pancreatic cancer; Islet cell pancreatic cancer; Paranasal sinus and nasal cavity cancer; Parathyroid carcinoma; Penile cancer; Pheochromocytoma; Pediatric pineal and supratentorial primitive neuroectodermal tumors; Pituitary tumors; Plasma cell vegetations / multiple myeloma; Pleural pulmonary blastoma; Pregnancy and breast cancer; Pregnancy and Hodgkin's lymphoma; Pregnancy and non-Hodgkin's lymphoma; Primary central nervous system lymphoma; Primary liver cancer in adults; Primary liver cancer in children; Prostate cancer; Rectal cancer; Renal cell (kidney) carcinoma; Pediatric renal cell carcinoma; Transitional cell carcinoma of the renal pelvis and ureter; Retinoblastoma; Pediatric rhabdomyosarcoma; Salivary gland carcinoma; Pediatric salivary gland carcinoma; Ewing family neoplastic sarcoma; Kaposi's sarcoma; Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; Pediatric rhabdomyosarcoma; Adult soft tissue sarcoma; Pediatric soft tissue sarcoma; Sezary syndrome; Skin cancer; Pediatric skin cancer; Skin cancer (melanoma); Merkel cell skin cancer; Small cell lung cancer; Small bowel cancer; Adult soft tissue sarcoma; Pediatric soft tissue sarcoma Tumors; metastatic squamous cell carcinoma of the neck with occult primary lesions; gastric (stomach) cancer; pediatric gastric (stomach) cancer; pediatric supratentorial primitive neuroectodermal tumors; cutaneous T-cell lymphoma; testicular cancer; pediatric thymoma; malignant thymoma; thyroid cancer; pediatric thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic tumor; pediatric cancer of unknown primary site; unusual pediatric cancer; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; pediatric optic pathway and hypothalamic glioma; vulvar cancer; Waldenström's macroglobulinemia; and Wilms' tumor.

[0284] Other exemplary cancers include diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).

[0285] The metastasis of the cancers mentioned above can also be treated or prevented using the methods described in this article.

[0286] In some implementations, these methods may result in a reduction in the number, severity, or frequency of one or more symptoms of a subject's cancer (e.g., compared to the number, severity, or frequency of one or more symptoms of a subject's cancer before treatment).

[0287] In some embodiments of any of the methods described herein, the method further includes administering an additional therapeutic agent (e.g., one or more of the therapeutic agents listed in Table 4) to the subject.

[0288] Table 4. Additional Treatments Composition / Pharm Kit This document also provides compositions (e.g., pharmaceutical compositions) comprising any ACC described herein and one or more (e.g., 1, 2, 3, 4, or 5) pharmaceutically acceptable carriers (e.g., any pharmaceutically acceptable carrier described herein), diluents, or excipients.

[0289] In some implementations, compositions including any of the ACCs described herein (e.g., pharmaceutical compositions) may be placed in sterile vials or pre-loaded syringes.

[0290] In some implementations, compositions including any of the ACCs described herein (e.g., pharmaceutical compositions) may be formulated for different routes of administration (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, or intratumoral).

[0291] In some embodiments, any pharmaceutical composition described herein may include one or more buffers (e.g., neutral buffered saline, phosphate buffered saline (PBS), amino acids (e.g., glycine), one or more carbohydrates (e.g., glucose, mannose, sucrose, dextran, or mannitol), one or more antioxidants, one or more chelating agents (e.g., EDTA or glutathione), one or more preservatives, and / or pharmaceutically acceptable carriers (e.g., antibacterial water, PBS, or saline).

[0292] As used herein, the phrase "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial agents, antimicrobial agents, isotonics, and absorption delay agents that are compatible with drug administration. Suitable carriers include, but are not limited to: water, saline, Ringer's solution, dextran solution, and approximately 5% human serum albumin.

[0293] In some embodiments of any of the pharmaceutical compositions described herein, any ACC described herein is prepared together with a carrier that prevents rapid elimination from the body, said carrier being, for example, a sustained-release and controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such pharmaceutical compositions and formulations will be apparent to those skilled in the art.

[0294] This document also provides kits comprising any ACC described herein, any composition comprising any ACC described herein, or any pharmaceutical composition comprising any ACC described herein. Kits also include, in addition to the ACC described herein, one or more second therapeutic agents selected from Table 4. One or more second therapeutic agents may be provided in a separate dosage form from the ACC. Alternatively, one or more second therapeutic agents may be formulated together with the ACC. In some embodiments, the kit comprises (1) an ACC comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 347-356, and (2) a second therapeutic agent selected from Table 4.

[0295] Any pillbox described herein may include instructions for using any of the compositions described herein (e.g., pharmaceutical compositions) and / or any ACC. In some embodiments, the pillbox may include instructions for performing any of the methods described herein. In some embodiments, the pillbox may include at least one dose of any of the compositions described herein (e.g., pharmaceutical compositions). In some embodiments, the pillbox may provide a syringe for administering any of the pharmaceutical compositions described herein. Example

[0296] The invention is further described in the following embodiments, which do not limit the scope of the invention as set forth in the claims.

[0297] Example 1: Generation of an IL-15 cytokine construct A recombinant cytokine construct (ProC2970) containing human IL-15 was prepared. The first and second monomeric constructs of ProC2970 are identical, each being a polypeptide having the amino acid sequence of SEQ ID NO: 523 and a signal sequence at its N-terminus. Each of the first and second monomeric constructs contains, from the N-terminus to the C-terminus, a signal sequence derived from the mouse IgG κ signal sequence (METDTLLLWVLLLWVPGSTG (SEQ ID NO: 345)), MM (SEQ ID NO: 236), a cleavable portion of SGRSDNI (SEQ ID NO: 655), a mature cytokine protein corresponding to amino acid residues 49-161 of human IL-15 (SEQ ID NO: 347), a cleavable portion of SGRSDNI (SEQ ID NO: 655), and a dimerization domain corresponding to human IgG4 Fc, truncated at Cys226 (according to EU designation), and includes the S228P mutation (SEQ ID NO: 3). Figure 5 The complete monomeric construct sequence (including the signal sequence) of ProC2970 is shown in SEQ ID NO: 524.

[0298] A peptide mask for IL-15 was designed based on the IL-2Rβ sequence in the crystal structure of the IL-15 quaternary complex (PDB ID: 4GS7). ALTTVDGGGGSASHYFER (SEQ ID NO: 236). Two peptide motifs from IL-2Rβ, KLTTVD (SEQ ID NO: 720) and ASHYFER (SEQ ID NO: 721), interact with IL-15. They are linked together via a linker to form a single tandem peptide as a cytokine masking motif (MM).

[0299] Peptides were prepared by transforming host cells with a polynucleotide having the sequence SEQ ID NO: 529, followed by culturing the resulting recombinant host cells. The dimerization of the expressed peptide yielded the cytokine construct ProC2970. ProC2970 was purified from the culture supernatant by protein A and size exclusion chromatography, and determined to be >95% of the desired species.

[0300] Example 2: Exemplary IL-15 cytokine construct in vitro Characterization To cleave structure-guided peptide tandem masking, MM, and dimerization domains, ACC containing IL-15 was treated overnight at 37°C with recombinant human protease urokinase plasminogen activator (uPA). A mixture of protease inhibitors was added to neutralize the protease before activity testing. Cleavage at expected sites in the cleavable moiety using uPA was confirmed by electrophoresis. Figure 6 The results showed that the uPA protease cleaved the cleavable portions (CM) in ProC2970 and ProC1879.

[0301] The activity of ProC2970 and ProC1879 was evaluated using IL-2 / IL-15-responsive HEK293 cells before and after uPA lysis. in vitro Testing. IL-2 / IL-15 responsive HEK293 cells were generated by stably transfecting human CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ) genes, as well as human JAK3 and STAT5 genes, to obtain fully functional IL-2 / IL-15 signaling pathways. The cells were also characterized by a STAT5-inducible SEAP (secretory embryonic alkaline phosphatase) reporter gene. To maintain transgene expression, cells were supplemented with 10% FBS, penicillin / streptomycin (Pen / Strep), 10 μg / ml puromycin, and 100 μg / mL Normocin. TM DMEM GlutaMax TM Cells were cultured in a culture medium. Adding IL-2 and IL-15 to these cells activated STAT5 and subsequently induced SEAP production, which could be easily assessed in the supernatant using QUANTI-Blue solution (a colorimetric assay for alkaline phosphatase activity).

[0302] IL-2 / IL-15 responsive HEK293 cells were prepared in DMEM medium supplemented with 10% FBS at a concentration of 280,000 cells / mL, and 180 μL aliquots were transferred to the wells of white flat-bottomed 96-well plates (50,000 cells / well). The test cytokines were diluted in DMEM medium supplemented with 10% FBS. Two 3-fold serial dilutions were prepared, and 20 μL of each was added to each well. After incubation at 37°C for 20–24 hours, 20 μL of induced reporter cell supernatant was transferred to the wells of a flat-bottomed 96-well plate. 180 μL of resuspended QUANTI-Blue solution was added to each well. After incubation at 37°C for 1–3 hours, SEAP levels were measured using a spectrophotometer at 620 nm. Dose-response curves were generated, and EC50 values ​​were obtained using GraphPad Prism software via sigmoid fitting and nonlinear regression.

[0303] In reporter gene assays, ProC2970 showed at least a 6000-fold (6000-fold) reduction in activity compared to PeproTech IL-15 (recombinant human IL-15 (rhIL-15), available from PeproTech, catalog number 200-15), and a 9.5-fold (9.5-fold) reduction in activity compared to Fc-masked IL-15 ProC1879 (SEQ ID NO: 356). Figure 7 This indicates that the MM fusion according to this disclosure provides additional masking for IL-15 in the ACC construct. Activation of the protease with uPA partially restored the activity of ProC2970, bringing it close to but below the level of recombinant IL-15. Table 5 below provides the EC50 values ​​for rhIL-15, ProC1879, ProC2970, ProC1879+uPA, and ProC2970+uPA.

[0304] Table 5. EC50: HEK-Blue reporter gene assay rhIL-15 ProC1879 ProC2970 ProC1879+uPA ProC2970+uPA EC50 (pM) 2.133 1413 13817 30.83 43.71 Example 3: The activity of ACC containing IL-15 on the proliferation of human PBMCs In cell proliferation assays, human PBMCs were incubated for 3 days with recombinant IL-15 or IL-15-ACC (with or without prior protease activation). After incubation, the cells were immobilized with the viability-modifying dye eFlur. TM PBMCs were stained with antibodies against 780, anti-CD3-FITC (UCHTI), anti-CD4-BV608 (RPA-T4), anti-CD8-BV480 (RPA-T8), anti-CD56-BV421 (HCD56), and anti-Ki67-APC (Ki67). Various cell populations, including CD3-, CD56+ NK cells, CD3+, CD8+ T cells, and CD3+, CD4+ T cells, were analyzed. The proliferation of each cell population was determined based on the percentage of Ki67 expression. Figure 8 As shown in the table, the protease-treated IL-15-ACC exhibited stronger proliferative activity than the corresponding intact IL-15-containing ACC. Table 6 shows the EC50 of various IL-15-containing ACCs in PBMCs.

[0305] Table 6. EC50: Human PBMC proliferation Ki67 EC50 (nM) NK cells CD8 CD4 rhIL-15 0.1167 0.2225 0.1701 ProC1879+uPA 1.352 3.584 6.779 ProC2970+uPA 0.8067 3.644 3.673 ProC1879 71560 721308 28697 ProC2970 3392706 41289 780.3 Example 4: Activation of cytokine X Based on the examination of structural models of cytokine-receptor complexes, tandem masking peptides were designed for other cytokines. For example, an IL-10 mask (SEQ ID NO: 722) can be designed based on the sequence of IL-10R1 in the crystal structure of the IL-10 binary complex (PDB ID: 1J7V). Two peptide sequences from IL-10R1, TNTRFSVDEVT (SEQ ID NO: 723) and SVASRSNKG (SEQ ID NO: 724), which interact with the IL-10 dimer molecule, are tandemly linked by a linker to generate a candidate IL-10 masking motif, as shown below.

[0306] Similarly, the IL-18 masking motif (SEQ ID NO: 725) can be designed based on the sequence of IL-18α in the crystal structure of the IL-18 ternary complex (PDB ID: 3WO4). The peptide sequences VDEVYDYHQ (SEQ ID NO: 726) and LLLGSTG (SEQ ID NO: 727) from IL-18Rα, which interact with IL-18, are tandemly linked by a linker to obtain a potential IL-18 masking motif. Using the same strategy, an IFNγ peptide mask (SEQ ID NO: 728) can be designed based on the sequence of IFNGR1 in the crystal structure of the IFNγ ternary complex (PDB ID: 6E3K). The two peptide sequences EEFAVLRDGK (SEQ ID NO: 729) and GVLNVWGV (SEQ ID NO: 730) from IFNGR1, which interact with the IFNγ dimer molecule, are tandemly linked by a linker to obtain the IFNγ masking motif of SEQ ID NO: 728. Additional examples of concatenation masks are provided below and in full in this disclosure.

[0307] Table 7. Tandem Masked Peptides Example 5: An exemplary ACC containing IL-15 in vitro Characterization IL-15 WT ACC and the IL-15 mutant protein ACC were treated overnight at 37°C with recombinant uPA. A mixture of protease inhibitors was added to neutralize the protease before activity testing. Table 8 shows the masked sequence of IL-15. A schematic diagram of ACC is shown below. Figure 10 As shown. ACC was activated by incubating the construct overnight at 37°C with an ACC to uPA ratio of 1:5. Electrophoresis confirmed that the expected sites of the cleavable moiety were cleaved by uPA. Figure 9A and Figure 9B HEK293 reporter gene assay characterized the activity of intact and protease-treated IL-15ACC-containing proteins. Figures 9C to 9ETable 9 shows the average EC50 values ​​of ACC containing IL-15 from multiple experiments. The results indicate that structure-based peptide masking provides activity attenuation for WT and ACC containing the mutant protein IL-15.

[0308] Table 8. Table 9. EC50: HEK-Blue reporter gene assay Table 10. Instance Sequence Numbered Items 1. A cytokine masking component (MM) comprising: (a) A first masking subunit, the first masking subunit being encoded by a first subsequence of the amino acid sequence of a first receptor polypeptide encoding a cytokine receptor, and (b) A second masking subunit, which is encoded by a second amino acid sequence selected from the group consisting of: (i) a second subsequence encoding an amino acid sequence of a cytokine receptor polypeptide, said cytokine receptor polypeptide being selected from the group consisting of the amino acid sequence encoding the first receptor polypeptide and the amino acid sequence encoding the receptor for said cytokine, wherein the first subsequence and the second subsequence are discontinuous within the amino acid sequence encoding the first receptor polypeptide; and (ii) The amino acid sequence encoding the blocking portion (BM).

[0309] 2. The cytokine MM as described in Project 1, wherein the cytokine MM includes a connector disposed between the first masking subunit and the second masking subunit.

[0310] 3. The cytokine MM as described in any one or a combination of items 1-2, wherein the second masking subunit is encoded by a second amino acid sequence encoding BM.

[0311] 4. The cytokine MM as described in Project 3, wherein the BM comprises a peptide having binding affinity for the cytokine.

[0312] 5. The cytokine MM as described in Project 3, wherein the BM comprises scFv having binding affinity for the cytokine.

[0313] 6. The cytokine MM as described in any one or a combination of items 1-2, wherein the second masking subunit is encoded by a second subsequence of an amino acid sequence encoding a receptor polypeptide, the receptor polypeptide being selected from the group consisting of the amino acid sequence encoding the first receptor polypeptide and the amino acid sequence encoding the receptor of the cytokine, wherein the first subsequence and the second subsequence are discontinuous within the amino acid sequence encoding the first receptor polypeptide.

[0314] 7. The cytokine MM as described in Project 6, wherein the first subsequence and the second subsequence are identical, or wherein the first receptor polypeptide and the second receptor polypeptide are identical.

[0315] 8. The cytokine MM as described in Project 6, wherein the first subsequence and the second subsequence are different.

[0316] 9. The cytokine MM as described in any one or a combination of items 6 and 8, wherein the first receptor polypeptide and the second receptor polypeptide are different.

[0317] 10. The cytokine MM as described in any one or a combination of items 1-9, wherein the cytokine MM is operatively linked to the cytokine.

[0318] 11. An activatable cytokine construct (ACC) comprising a cleavable moiety (CM), a cytokine polypeptide (CP), and the cytokine MM described in any one or a combination of items 1-10.

[0319] 12. An activatable cytokine construct (ACC) comprising a masking motif (MM), a cleavable motif (CM), and a cytokine polypeptide (CP), wherein: a) The MM comprises a polypeptide sequence, the polypeptide sequence comprising a tandem sequence of two or more receptor subsequences; b) Each receptor from the CP in the receptor subsequence; and c) The two or more receptor subsequences: i) It is discontinuous in the sequence of the receptor; and ii) They are directly or indirectly connected to each other.

[0320] 13. The ACC as described in Item 12, wherein each of the two or more receptor subsequences comprises a continuous amino acid in the receptor, the continuous amino acid being in the range of 1.0 to 8.0 Å, 2.0 to 8.0 Å, or 2.0 to 7.0 Å for the cytokine amino acid in the co-crystal structure of the receptor and the cytokine complex (cytokine-receptor complex co-crystal structure).

[0321] 14. The ACC as described in item 12, further comprising a connector disposed between at least two of the two or more receptor subsequences.

[0322] 15. The ACC as described in any one or a combination of items 12-13, wherein each of the receptor subsequences comprises 2, 3, 4 or more amino acids.

[0323] 16. The ACC as described in any one or a combination of items 12-14, wherein at least one of the two or more receptor subsequences comprises a conserved substitution of at least one amino acid relative to the sequence of the receptor.

[0324] 17. The ACC as described in any one or a combination of items 12-15, wherein the two or more receptor subsequences comprise a first receptor subsequence and a second receptor subsequence. The second receptor subsequence is located at the C-terminus of the first receptor subsequence in the sequence of the receptor. The linker contains X amino acids. Where X = n / y, Where n = the distance between the C-terminus of the N-terminal peptide of the first receptor subsequence and the N-terminus of the C-terminal peptide of the second receptor subsequence in the co-crystal structure of the cytokine-receptor complex, in angstroms, and y is a number in the range of 1.5-3.5, or 2-3.5, or 2-3, or 2-2.5, or y is 2.5, and if X is not an integer, X is rounded to the next integer.

[0325] 18. The ACC as described in any one or a combination of items 12-16, wherein the MM comprises the amino acid sequence ALTTVX-linker-ASHYFE (SEQ ID NO: 508) (MM), wherein X is D, K, or R, wherein the N-terminal alanine residue is optionally absent or optionally substituted by any other amino acid, and the linker consists of 1 to 20 amino acids.

[0326] 19. The ACC as described in Item 18, wherein the N-terminus, C-terminus, or both of the MM extend for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0327] 20. An isolated polypeptide comprising a polypeptide sequence comprising a tandem sequence of two or more receptor subsequences, wherein each of the receptor subsequences is derived from a receptor of a cytokine polypeptide (CP); and the two or more receptor subsequences: i) It is discontinuous in the sequence of the receptor; and ii) They are directly or indirectly connected to each other.

[0328] 21. The isolated polypeptide as described in Item 20, wherein each of the two or more receptor subsequences comprises a continuous amino acid of the receptor, the continuous amino acid being in the range of 1.0 to 8.0 Å, 2.0 to 8.0 Å, or 2.0 to 7.0 Å in the co-crystal structure of the receptor and the cytokine complex (cytokine-receptor complex co-crystal structure).

[0329] 22. The isolated polypeptide as described in Item 20, further comprising a linker disposed between at least two of the two or more receptor subsequences.

[0330] 23. The isolated polypeptide as described in any one or a combination of items 20-22, wherein each of the receptor subsequences comprises 2, 3, 4 or more amino acids.

[0331] 24. The isolated polypeptide as described in any one or a combination of items 20-23, wherein at least one of the two or more receptor subsequences comprises a conserved substitution of at least one amino acid relative to the sequence of the receptor.

[0332] 25. The isolated polypeptide as described in any one or a combination of items 21-24, wherein the two or more receptor subsequences comprise a first receptor subsequence and a second receptor subsequence. The second receptor subsequence is located at the C-terminus of the first receptor subsequence in the sequence of the receptor. The linker contains X amino acids. Where X = n / y, Where n = the distance between the C-terminus of the N-terminal peptide of the first receptor subsequence and the N-terminus of the C-terminal peptide of the second receptor subsequence in the co-crystal structure of the cytokine-receptor complex, in angstroms, and y is a number in the range of 1.5-3.5, or 2-3.5, or 2-3, or 2-2.5, or y is 2.5, and if X is not an integer, X is rounded to the next integer.

[0333] 26. An isolated polypeptide comprising the amino acid sequence ALTTVX-linker-ASHYFE (SEQ ID NO:508) (MM), wherein X is D, K, or R, wherein the N-terminal alanine residue is optionally absent or optionally substituted by any other amino acid, and the linker consists of 1 to 20 amino acids.

[0334] 27. The isolated polypeptide as described in Item 26, wherein the N-terminus, C-terminus, or both of the isolated polypeptide are extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0335] 28. The isolated polypeptide or ACC as described in any one of items 12-27, wherein the X is D.

[0336] 29. The isolated polypeptide or ACC as described in any one or a combination of items 12-28, wherein X is K.

[0337] 30. The isolated polypeptide or ACC as described in any one or combination of items 12-29, wherein X is R.

[0338] 31. The isolated polypeptide or ACC as described in any one or combination of items 12-30, wherein the linker consists of 4, 5, 6, 7 or 8 amino acids.

[0339] 32. The isolated polypeptide or ACC as described in any one or combination of items 12-31, wherein the linker consists of 6 amino acids.

[0340] 33. The isolated polypeptide or ACC as described in any one or combination of items 12-32, wherein the adapter is selected from the group consisting of SEQ ID NO: 2, 210-235, 245, 250 and 318-335.

[0341] 34. The isolated polypeptide or ACC as described in any one or combination of items 12-33, wherein the linker is GGGGS (SEQ ID NO: 216).

[0342] 35. The isolated polypeptide or ACC as described in any one or combination of items 12-34, wherein the amino acid sequence comprises ALTTVDGGGGSASHYFER (SEQ ID NO: 236) or ALTTVDGGGGSASHYFEK (SEQ ID NO: 237), or wherein the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid.

[0343] 36. The isolated polypeptide or ACC as described in any one or combination of items 12-35, wherein the amino acid sequence comprises ALTTVKGGGGSASHYFER (SEQ ID NO: 238) or ALTTVKGGGGSASHYFEK (SEQ ID NO: 239), or wherein the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid.

[0344] 37. The isolated polypeptide or ACC as described in any one or combination of items 12-36, wherein the amino acid sequence comprises ALTVRGGGGSASHYFER (SEQ ID NO: 240) or ALTVRGGGGSASHYFEK (SEQ ID NO: 241), or wherein the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid.

[0345] 38. The isolated polypeptide or ACC as described in any one or a combination of items 12-37, wherein the N-terminal alanine residue is replaced with lysine.

[0346] 39. The isolated polypeptide or ACC as described in any one or combination of items 12-37, wherein the isolated polypeptide comprises a sequence selected from the following: SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736) or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0347] 40. The isolated polypeptide or ACC as described in any of the foregoing items or combinations thereof, wherein the isolated polypeptide comprises cytokines.

[0348] 41. The isolated polypeptide or ACC as described in any one or a combination of items 12-40, wherein the isolated polypeptide is disposed in a complex comprising two or more polypeptides, and wherein the complex comprises cytokines.

[0349] 42. The isolated polypeptide or ACC as described in any one or combination of items 12-41, wherein the cytokine is contained in a polypeptide complexed with the isolated polypeptide.

[0350] 43. The isolated polypeptide or ACC as described in any one or a combination of items 12-42, wherein the amino acid sequence is a masking portion that inhibits the binding of the cytokine to its receptor.

[0351] 44. The isolated polypeptide or ACC as described in any one or combination of items 12-43, wherein the cytokine is a cytokine that binds to IL2 / IL15 receptor β and / or IL2 / IL15 receptor γ.

[0352] 45. The isolated polypeptide or ACC as described in any one or combination of items 12-44, wherein the cytokine binds to IL-15Rα.

[0353] 46. ​​The isolated polypeptide or ACC as described in any one of items 12-45, wherein the cytokine binds to IL-2Rα.

[0354] 47. An activatable cytokine construct (ACC) comprising a cytokine polypeptide (CP), a cleavable moiety (CM), and the isolated polypeptide, wherein the isolated polypeptide comprises a polypeptide sequence containing a tandem sequence or any of the foregoing items or combinations thereof, wherein the isolated polypeptide or the MM is coupled to the CP via the CM and inhibits the binding of the CP to its receptor.

[0355] 48. The ACC as described in item 47, wherein the CP is an interleukin polypeptide.

[0356] 49. The ACC as described in item 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 348, 129, or 130.

[0357] 50. The ACC as described in item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 348, 129 or 130.

[0358] 51. The ACC as described in item 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 119 or 120.

[0359] 52. The ACC as described in item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 119 or 120.

[0360] 53. The ACC as described in item 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 121 or 122.

[0361] 54. The ACC as described in item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 121 or 122.

[0362] 55. The ACC as described in item 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 123 or 124.

[0363] 56. The ACC as described in item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 123 or 124.

[0364] 57. The ACC as described in item 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 125 or 126.

[0365] 58. The ACC as described in item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 125 or 126.

[0366] 59. The ACC as described in item 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 521 or 522.

[0367] 60. The ACC as described in item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 521 or 522.

[0368] 61. The ACC as described in any one or a combination of items 47-60, wherein the CM comprises no more than 8 amino acids.

[0369] 62. The ACC as described in any one or a combination of items 47-61, wherein the CM is capable of being cleaved by urokinase (uPA) and / or matrix metalloproteinase (MMP).

[0370] 63. The ACC as described in any one or a combination of items 47-62, further comprising a connector (L1) between the CM and the CP.

[0371] 64. The ACC as described in any one or a combination of items 47-63, further comprising a connector (L2) between the CM and the MM.

[0372] 65. The ACC as described in any one or a combination of items 47-64, further comprising a first connector (L1) between the CM and the CP and a second connector (L2) between the CM and the MM.

[0373] 66. The ACC as described in any one or a combination of items 47-65, further comprising a spatial mask that further inhibits the binding of the CP to its receptor.

[0374] 67. An ACC comprising a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a first cytokine polypeptide (CP1), a first cleavable moiety (CM1), a first dimerizing domain (DD1) coupled to CP1 via CM1, and a first masking moiety (MM1). The second monomer construct comprises a second cytokine peptide (CP2), a second cleavable moiety (CM2), a second dimerizing domain (DD2) coupled to CP2 via the CM2, and a second masking moiety (MM2). The DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct, and The MM1 and / or the MM2 contain any of the isolated polypeptides described in the foregoing items or combinations thereof.

[0375] 68. The ACC as described in item 67, wherein the first monomeric construct comprises a third cleavable portion (CM3), and the MM1 is coupled to the CP1 via the CM3.

[0376] 69. The ACC as described in any one or a combination of items 67-68, wherein the MM1 is coupled to the CP1 via the DD1 and the CM1.

[0377] 70. The ACC as described in any one or a combination of items 67-69, wherein the second monomeric construct comprises a fourth cleavable portion (CM4), and the MM2 is coupled to the CP2 via the CM4.

[0378] 71. The ACC as described in any one or a combination of items 67-70, wherein the MM2 is coupled to the CP2 via the DD2 and the CM2.

[0379] 72. The ACC as described in any one or a combination of items 67-71, wherein each of the first monomeric construct and the second monomeric construct comprises a linker region containing no more than 18 amino acids.

[0380] 73. The ACC as described in any one or a combination of items 67-72, wherein CP1 and CM1 are directly adjacent to each other, and / or CM1 and DD1 are directly adjacent to each other.

[0381] 74. The ACC as described in any one or a combination of items 67-73, wherein CP2 and CM2 are directly adjacent to each other, and / or CM2 and DD2 are directly adjacent to each other.

[0382] 75. The ACC as described in any one or a combination of items 67-74, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO:348, 129, or 130.

[0383] 76. The ACC as described in any one or a combination of items 67-75, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO:348, 129, or 130.

[0384] 77. The ACC as described in any one or a combination of items 67-76, wherein the CP1 comprises SEQ ID NO: 348, 129 or 130.

[0385] 78. The ACC as described in any one or a combination of items 67-77, wherein the CP2 comprises SEQ ID NO: 348, 129 or 130.

[0386] 79. The ACC as described in any one or a combination of items 67-78, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 119 or 120.

[0387] 80. The ACC as described in any one or a combination of items 67-79, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 119 or 120.

[0388] 81. The ACC as described in any one or a combination of items 67-80, wherein the CP1 comprises SEQ ID NO: 119 or 120.

[0389] 82. The ACC as described in any one or a combination of items 67-81, wherein the CP2 comprises SEQ ID NO: 119 or 120.

[0390] 83. The ACC as described in any one or a combination of items 67-82, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 121 or 122.

[0391] 84. The ACC as described in any one or a combination of items 67-83, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 121 or 122.

[0392] 85. The ACC as described in any one or a combination of items 67-84, wherein the CP1 comprises SEQ ID NO: 121 or 122.

[0393] 86. The ACC as described in any one or a combination of items 67-85, wherein the CP2 comprises SEQ ID NO: 121 or 122.

[0394] 87. The ACC as described in any one or a combination of items 67-86, wherein the CP1 comprises a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 123 or 124.

[0395] 88. The ACC as described in any one or a combination of items 67-87, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 123 or 124.

[0396] 89. The ACC as described in any one or a combination of items 67-88, wherein the CP1 comprises SEQ ID NO: 123 or 124.

[0397] 90. The ACC as described in any one or a combination of items 67-89, wherein the CP2 comprises SEQ ID NO: 123 or 124.

[0398] 91. The ACC as described in any one or a combination of items 67-90, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 125 or 126.

[0399] 92. The ACC as described in any one or a combination of items 67-91, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 125 or 126.

[0400] 93. The ACC as described in any one or a combination of items 67-92, wherein the CP1 comprises SEQ ID NO: 125 or 126.

[0401] 94. The ACC as described in any one or a combination of items 67-93, wherein the CP2 comprises SEQ ID NO: 125 or 126.

[0402] 95. The ACC as described in any one or a combination of items 67-94, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 521 or 522.

[0403] 96. The ACC as described in any one or a combination of items 67-95, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 521 or 522.

[0404] 97. The ACC as described in any one or a combination of items 67-96, wherein the CP1 comprises SEQ ID NO: 521 or 522.

[0405] 98. The ACC as described in any one or a combination of items 67-97, wherein the CP2 comprises SEQ ID NO: 521 or 522.

[0406] 99. The ACC as described in any one or a combination of items 67-98, wherein the first monomeric construct and the second monomeric construct are identical.

[0407] 100. The ACC as described in any one or a combination of items 67-99, wherein the first monomeric construct and the second monomeric construct have 95% sequence homology.

[0408] 101. The ACC as described in any one or a combination of items 67-100, further comprising a third monomer containing a sushi domain having the sequence of SEQ ID NO: 520, wherein the third monomer is non-covalently or covalently bound to the ACC.

[0409] 102. The ACC as described in any one or a combination of items 67-101, further comprising a fourth monomer comprising a sushi domain containing the sequence of SEQ ID NO: 520, wherein the fourth monomer is non-covalently or covalently bound to the ACC.

[0410] 103. The ACC as described in item 100 or 101, wherein the third monomer and / or the fourth monomer further comprises a label.

[0411] 104. An ACC comprising a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a first cytokine peptide (CP1), a first dimerizing domain (DD1), and a first masking moiety (MM1). The second monomer construct comprises a second cytokine peptide (CP2), a first cleavable moiety (CM1), a second dimerizing domain (DD2) coupled to CP2 via said CM1, and a second masking moiety (MM2). The MM1 and / or MM2 are any of the isolated polypeptides described in the foregoing items or combinations thereof. The DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct.

[0412] 105. The ACC as described in item 104, wherein the first monomeric construct further comprises a second cleavable portion (CM2), and the MM1 is coupled to the CP1 via the CM2.

[0413] 106. The ACC as described in item 104 or 105, wherein the MM2 is coupled to the CP2 via the DD2 and the CM1.

[0414] 107. The ACC as described in any one or a combination of items 104-106, wherein the second monomeric construct further comprises a third cleavable portion (CM3), wherein the MM2 is coupled to the CP2 via the CM3.

[0415] 108. An ACC comprising a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a first cytokine peptide (CP1), a first dimerizing domain (DD1), and a first masking moiety (MM1). The second monomer construct comprises a second cytokine peptide (CP2), a second dimerization domain (DD2), and a second masking moiety (MM2). CP1 and / or CP2 contain an amino acid sequence that serves as a substrate for the protease, and DD1 and / or DD2 are coupled to CP1 or CP2 via the amino acid sequence. The MM1 and / or MM2 are any of the isolated polypeptides described in the foregoing items or combinations thereof. The DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct.

[0416] 109. The ACC as described in item 108, wherein the CP1 comprises the amino acid sequence used as a substrate for a protease, and the MM1 is coupled to the CP1 via the amino acid sequence.

[0417] 110. The ACC as described in any one or a combination of items 108-109, wherein the first monomeric construct further comprises a first cleavable portion (CM1), and the MM1 is coupled to the CP1 via the CM1.

[0418] 111. The ACC as described in any one or combination of items 108, wherein the CP2 comprises the amino acid sequence used as a substrate for a protease, and the MM2 is coupled to the CP2 via the amino acid sequence.

[0419] 112. The ACC as described in any one or a combination of items 108-111, wherein the second monomeric construct further comprises a second cleavable portion (CM2), and the CM2 is coupled to the CP2 via the CM2.

[0420] 113. The ACC as described in any one or a combination of items 67-112, wherein DD1 and DD2 are a pair of human IgG Fc domains.

[0421] 114. The ACC as described in item 113, wherein DD1 and DD2 are a pair of human IgG4 Fc domains.

[0422] 115. The ACC as described in item 114, wherein DD1 and DD2 are a pair of human IgG1 or IgG4 Fc domains truncated at the N-terminus to cysteine ​​226 as numbered according to EU designation.

[0423] 116. The ACC as described in item 114, wherein the human IgG4 Fc domain contains the S228P mutation as numbered according to the EU designation.

[0424] 117. The ACC as described in any one or a combination of items 67-116, wherein each of said DD1 and said DD2 contains a sequence that is at least 95% identical to SEQ ID NO: 3.

[0425] 118. The ACC as described in any one or a combination of items 67-117, wherein each of said DD1 and said DD2 contains the sequence of SEQ ID NO: 3.

[0426] 119. The ACC as described in any one or a combination of items 67-118, wherein the first monomer builder and the second monomer builder are covalently bonded to each other by at least one, two, three or four disulfide bonds.

[0427] 120. The ACC as described in any one or a combination of items 12-19 and 47-119, wherein each of the first monomeric construct and the second monomeric construct contains at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 523.

[0428] 121. The ACC as described in any one or a combination of items 12-19 and 47-120, wherein each of the first monomeric construct and the second monomeric construct comprises SEQ ID NO: 523.

[0429] 122. The ACC as described in any one or a combination of items 12-19 and 47-121, wherein the ACC is characterized by having a reduced level of interleukin activity compared to a control level of interleukin activity.

[0430] 123. The ACC as described in any one or a combination of items 12-19 and 47-122, wherein the ACC is characterized by a reduced level of interleukin activity compared to wild-type human IL-15.

[0431] 124. The ACC as described in any one of items 12-19 and 47-123, wherein the ACC is characterized by having a reduced level of IL-15 activity compared to recombinant human IL-15, as measured by the level of SEAP (secretory embryonic alkaline phosphatase) production in IL-2 / IL-15 responsive HEK293 cells.

[0432] 125. The ACC as described in any one of items 12-19 and 47-124, wherein the ACC is characterized by having a reduced level of IL-15 activity compared to the activity of recombinant human IL-15.

[0433] 126. The ACC as described in item 125, wherein the ACC is characterized by a reduction in IL-15 activity level of at least 6,000 times compared to recombinant human IL-15.

[0434] 127. The ACC as described in any one or a combination of items 12-19 and 47-126, wherein the ACC is characterized in that, after cleavage by the uPA protease, its EC50 is at least 1,000, 5,000, or 6,000 times that of the EC50 of recombinant wild-type IL-15, as measured in IL-2 / IL15-responsive HEK293 cells.

[0435] 128. An ACC comprising a cytokine polypeptide (CP), an agonist of the CP, the isolated polypeptide, and a cleavable portion (CM), wherein the isolated polypeptide comprises a polypeptide sequence containing a tandem sequence or any of the foregoing items or combinations thereof, and wherein the isolated polypeptide or the MM is coupled to the CP via the CM.

[0436] 129. The ACC as described in item 128, wherein the CP is IL-15 and the agonist is a sushi domain.

[0437] 130. The ACC as described in item 128 or 129, wherein the agonist is coupled to the CP via a connector.

[0438] 131. The ACC as described in item 130, wherein the agonist is coupled to the CP via a cleavable connector.

[0439] 132. The ACC as described in item 120, wherein the agonist is coupled to the CP via a non-lytic linker.

[0440] 133. The ACC as described in item 128 or 129, wherein the agonist is non-covalently bound to the CP.

[0441] 134. An ACC comprising a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a cytokine peptide (CP), a first dimerizing domain (DD1), a first cleavable moiety (CM1), a second cleavable moiety (CM2), and the isolated peptide, wherein the isolated peptide comprises a peptide sequence containing a tandem sequence or any of the foregoing items or combinations thereof, and wherein the isolated peptide or the MM is coupled to the CP via the CM1, and the DD1 is coupled to the CP via the CM2. The second monomer construct comprises the agonist of the CP, a third cleavable moiety (CM3), and a second dimerizing domain (DD2) coupled to the agonist via the CM3, and The DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct.

[0442] 135. The ACC as described in item 134, wherein the CP is IL-15 and the agonist is a sushi domain comprising the sequence of SEQ ID NO: 520.

[0443] 136. The ACC as described in item 134 or 135, comprising a linker consisting of two amino acids between the sushi domain and the CM3.

[0444] 137. A polynucleotide encoding an isolated polypeptide, an ACC, or a monomeric construct as described in any of the foregoing items or combinations thereof.

[0445] 138. A vector comprising the polynucleotide described in item 137.

[0446] 139. The carrier as described in item 138, wherein the carrier is an expression carrier.

[0447] 140. A host cell comprising the polynucleotide described in item 137 or the vector described in item 138 or 139.

[0448] 141. The host cell as described in the project, wherein the host cell is a mammalian cell.

[0449] 142. A composition comprising any isolated polypeptide described in any of the preceding items or combinations thereof, or any ACC described in any of the preceding items or combinations thereof, or the polynucleotide described in item 137.

[0450] 143. The composition as described in item 142, wherein the composition is a pharmaceutical composition.

[0451] 144. A container, vial, syringe, pen, or kit containing at least one dose of the composition described in item 142 or 143.

[0452] 145. A method of treating a subject in need, comprising administering to the subject a therapeutically effective amount of any of the isolated polypeptides described in any of the foregoing items or combinations thereof, any of the ACCs described in any of the foregoing items or combinations thereof, or the composition described in item 142 or 143.

[0453] 146. The method as described in item 145, wherein the subject has been identified or diagnosed with cancer.

[0454] 147. The method as described in item 146, wherein the cancer is leukemia, lymphoma, or a solid tumor.

[0455] 148. The method as described in item 147, wherein the subject has been identified or diagnosed with an inflammatory or autoimmune disease, condition, or disorder.

[0456] 149. A method for generating ACC, comprising: Cells containing the polynucleotides described in item 137 are cultured in a liquid culture medium to produce the ACC; and The ACC is recovered from the cells or the liquid culture medium.

[0457] 150. The method of claim 149, further comprising isolating the ACC recovered from the cells or the liquid culture medium.

[0458] 151. The method of item 149 or 150, further comprising formulating the isolated ACC into a pharmaceutical composition.

[0459] 152. A complex comprising a polypeptide, said polypeptide comprising a cytokine complexed with the isolated polypeptide described in combination with any of the foregoing items or items.

[0460] Other implementation plans It should be understood that although the invention has been described in conjunction with a detailed description of the invention, the foregoing description is intended to illustrate, and not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A cytokine masking component (MM) comprising: (a) A first masking subunit, the first masking subunit being encoded by a first subsequence of the amino acid sequence of a first receptor polypeptide encoding a cytokine receptor, and (b) A second masking subunit, which is encoded by a second amino acid sequence selected from the group consisting of: (i) a second subsequence encoding an amino acid sequence of a cytokine receptor polypeptide, said cytokine receptor polypeptide being selected from the group consisting of the amino acid sequence encoding the first receptor polypeptide and the amino acid sequence encoding the receptor for said cytokine, wherein the first subsequence and the second subsequence are discontinuous within the amino acid sequence encoding the first receptor polypeptide; and (ii) The amino acid sequence encoding the blocking portion (BM).

2. The cytokine MM of claim 1, wherein the cytokine MM comprises a connector disposed between the first masking subunit and the second masking subunit.

3. The cytokine MM as claimed in any one of claims 1-2, wherein the second masking subunit is encoded by a second amino acid sequence encoding BM.

4. The cytokine MM of claim 3, wherein the BM comprises a peptide having binding affinity for the cytokine.

5. The cytokine MM of claim 3, wherein the BM comprises scFv having binding affinity for the cytokine.

6. The cytokine MM as claimed in any one of claims 1-2, wherein the second masking subunit is encoded by a second subsequence of an amino acid sequence encoding a receptor polypeptide, the receptor polypeptide being selected from the group consisting of the amino acid sequence encoding the first receptor polypeptide and the amino acid sequence of the second receptor polypeptide encoding the receptor for the cytokine, wherein the first subsequence and the second subsequence are discontinuous within the amino acid sequence encoding the first receptor polypeptide.

7. The cytokine MM of claim 6, wherein the first subsequence and the second subsequence are identical, or wherein the first receptor polypeptide and the second receptor polypeptide are identical.

8. The cytokine MM of claim 6, wherein the first subsequence and the second subsequence are different.

9. The cytokine MM of any one of claims 6 and 8, wherein the first receptor polypeptide and the second receptor polypeptide are different.

10. The cytokine MM as claimed in any one of claims 1-9, wherein the cytokine MM is operatively linked to the cytokine.

11. An activatable cytokine construct (ACC) comprising a cleavable moiety (CM), a cytokine polypeptide (CP), and the cytokine MM of any one of claims 1-10.

12. An activatable cytokine construct (ACC) comprising a masking motif (MM), a cleavable motif (CM), and a cytokine polypeptide (CP), wherein: a) The MM comprises a polypeptide sequence, the polypeptide sequence comprising a tandem sequence of two or more receptor subsequences; b) Each receptor from the CP in the receptor subsequence; and c) The two or more receptor subsequences: i) It is discontinuous in the sequence of the receptor; and ii) They are directly or indirectly connected to each other.

13. The ACC of claim 12, wherein each of the two or more receptor subsequences comprises a continuous amino acid from the receptor, the continuous amino acid being in the range of 1.0 to 8.0 Å, 2.0 to 8.0 Å, or 2.0 to 7.0 Å for the cytokine amino acid in the co-crystal structure of the receptor and the cytokine complex (cytokine-receptor complex co-crystal structure).

14. The ACC of claim 12, further comprising a connector disposed between at least two of the two or more receptor subsequences.

15. The ACC of any one of claims 12-13, wherein each of the receptor subsequences comprises 2, 3, 4 or more amino acids.

16. The ACC of any one of claims 12-14, wherein at least one of the two or more receptor subsequences comprises a conserved substitution of at least one amino acid relative to the sequence of the receptor.

17. The ACC of any one of claims 12-15, wherein the two or more receptor subsequences comprise a first receptor subsequence and a second receptor subsequence. The second receptor subsequence is located at the C-terminus of the first receptor subsequence in the sequence of the receptor. The connector described herein contains X amino acids. Where X = n / 2.5, Where n = the distance between the C-terminus of the N-terminal peptide of the first receptor subsequence and the N-terminus of the C-terminal peptide of the second receptor subsequence in the co-crystal structure of the cytokine-receptor complex, in angstroms.

18. The ACC of any one of claims 12-16, wherein the MM comprises the amino acid sequence ALTTVX-linker-ASHYFE (SEQ ID NO: 508) (MM), wherein X is D, K, or R, wherein the N-terminal alanine residue is optionally absent or optionally substituted by any other amino acid, and the linker consists of 1 to 20 amino acids.

19. The ACC of claim 18, wherein the N-terminus, C-terminus, or both of the MM extend for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

20. An isolated polypeptide comprising a polypeptide sequence comprising a tandem sequence of two or more receptor subsequences, wherein each of the receptor subsequences is derived from a receptor of a cytokine polypeptide (CP); and the two or more receptor subsequences: i) It is discontinuous in the sequence of the receptor; and ii) They are directly or indirectly connected to each other.

21. The isolated polypeptide of claim 20, wherein each of the two or more receptor subsequences comprises a continuous amino acid of the receptor, the continuous amino acid being in the range of 1.0 to 8.0 Å, 2.0 to 8.0 Å, or 2.0 to 7.0 Å of the cytokine amino acid in the co-crystal structure of the receptor and the cytokine complex (cytokine-receptor complex co-crystal structure).

22. The isolated polypeptide of claim 20, further comprising a linker disposed between at least two of the two or more receptor subsequences.

23. The isolated polypeptide according to any one of claims 20-22, wherein each of the receptor subsequences comprises 2, 3, 4 or more amino acids.

24. The isolated polypeptide according to any one of claims 20-23, wherein at least one of the two or more receptor subsequences comprises a conserved substitution of at least one amino acid relative to the sequence of the receptor.

25. The isolated polypeptide according to any one of claims 21-24, wherein the two or more receptor subsequences comprise a first receptor subsequence and a second receptor subsequence. The second receptor subsequence is located at the C-terminus of the first receptor subsequence in the sequence of the receptor. The connector described herein contains X amino acids. Where X = n / 2.5, Where n = the distance between the C-terminus of the N-terminal peptide of the first receptor subsequence and the N-terminus of the C-terminal peptide of the second receptor subsequence in the co-crystal structure of the cytokine-receptor complex, in angstroms.

26. An isolated polypeptide comprising the amino acid sequence ALTTVX-linker-ASHYFE (SEQ ID NO: 508)(MM), wherein X is D, K, or R, wherein the N-terminal alanine residue is optionally absent or optionally substituted by any other amino acid, and the linker consists of 1 to 20 amino acids.

27. The isolated polypeptide of claim 26, wherein the N-terminus, C-terminus, or both of the isolated polypeptide are extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

28. The isolated polypeptide or ACC as claimed in any one of claims 12-27, wherein X is D.

29. The isolated polypeptide or ACC as claimed in any one of claims 12-28, wherein X is K.

30. The isolated polypeptide or ACC as claimed in any one of claims 12-29, wherein X is R.

31. The isolated polypeptide or ACC according to any one of claims 12-30, wherein the linker consists of 4, 5, 6, 7 or 8 amino acids.

32. The isolated polypeptide or ACC as described in any one of claims 12-31, wherein the linker consists of 6 amino acids.

33. The isolated polypeptide or ACC according to any one of claims 12-32, wherein the adapter is selected from the group consisting of SEQ ID NO: 2, 210-235, 245, 250 and 318-335.

34. The isolated polypeptide or ACC according to any one of claims 12-33, wherein the linker is GGGGS (SEQ ID NO: 216).

35. The isolated polypeptide or ACC of any one of claims 12-34, wherein the amino acid sequence comprises ALTTVDGGGGSASHYFER (SEQ ID NO: 236) or ALTTVDGGGGSASHYFEK (SEQ ID NO: 237), or wherein the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid.

36. The isolated polypeptide or ACC of any one of claims 12-35, wherein the amino acid sequence comprises ALTTVKGGGGSASHYFER (SEQ ID NO: 238) or ALTTVKGGGGSASHYFEK (SEQ ID NO: 239), or wherein the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid.

37. The isolated polypeptide or ACC of any one of claims 12-36, wherein the amino acid sequence comprises ALTVRGGGGSASHYFER (SEQ ID NO: 240) or ALTVRGGGGSASHYFEK (SEQ ID NO: 241), or wherein the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid.

38. The isolated polypeptide or ACC of any one of claims 12-37, wherein the N-terminal alanine residue is replaced with lysine.

39. The isolated polypeptide or ACC according to any one of claims 12-37, wherein the isolated polypeptide comprises a sequence selected from the following: SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736) or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

40. The isolated polypeptide or ACC as described in any of the preceding claims, wherein the isolated polypeptide comprises cytokines.

41. The isolated polypeptide or ACC of any one of claims 12-40, wherein the isolated polypeptide is disposed in a complex comprising two or more polypeptides, and wherein the complex comprises cytokines.

42. The isolated polypeptide or ACC as described in any one of claims 12-41, wherein the cytokine is contained in a polypeptide complexed with the isolated polypeptide.

43. The isolated polypeptide or ACC as described in any one of claims 12-42, wherein the amino acid sequence is a masking portion that inhibits the binding of the cytokine to its receptor.

44. The isolated polypeptide or ACC according to any one of claims 12-43, wherein the cytokine is a cytokine that binds to IL2 / IL15 receptor β and / or IL2 / IL15 receptor γ.

45. The isolated polypeptide or ACC according to any one of claims 12-44, wherein the cytokine binds to IL-15Rα.

46. ​​The isolated polypeptide or ACC according to any one of claims 12-45, wherein the cytokine binds to IL-2Rα.

47. An activatable cytokine construct (ACC) comprising a cytokine polypeptide (CP), a cleavable moiety (CM), and the isolated polypeptide, wherein the isolated polypeptide comprises a polypeptide sequence containing a tandem sequence or any cytokine masking moiety (MM) as described in the preceding claims, wherein the isolated polypeptide or the MM is coupled to the CP via the CM and inhibits the binding of the CP to its receptor.

48. The ACC of claim 47, wherein the CP is an interleukin polypeptide.

49. The ACC of claim 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 348, 129, or 130.

50. The ACC of claim 48, wherein the interleukin polypeptide comprises SEQ ID NO: 348, 129 or 130.

51. The ACC of claim 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 119 or 120.

52. The ACC of claim 48, wherein the interleukin polypeptide comprises SEQ ID NO: 119 or 120.

53. The ACC of claim 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 121 or 122.

54. The ACC of claim 48, wherein the interleukin polypeptide comprises SEQ ID NO: 121 or 122.

55. The ACC of claim 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 123 or 124.

56. The ACC of claim 48, wherein the interleukin polypeptide comprises SEQ ID NO: 123 or 124.

57. The ACC of claim 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 125 or 126.

58. The ACC of claim 48, wherein the interleukin polypeptide comprises SEQ ID NO: 125 or 126.

59. The ACC of claim 48, wherein the interleukin polypeptide comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO: 521 or 522.

60. The ACC of claim 48, wherein the interleukin polypeptide comprises SEQ ID NO: 521 or 522.

61. The ACC of any one of claims 47-60, wherein the CM comprises no more than 8 amino acids.

62. The ACC of any one of claims 47-61, wherein the CM is capable of being cleaved by urokinase (uPA) and / or matrix metalloproteinase (MMP).

63. The ACC as claimed in any one of claims 47-62, further comprising a connector (L1) between the CM and the CP.

64. The ACC as claimed in any one of claims 47-63, further comprising a connector (L2) between the CM and the MM.

65. The ACC as claimed in any one of claims 47-64, further comprising a first connector (L1) between the CM and the CP and a second connector (L2) between the CM and the MM.

66. The ACC of any one of claims 47-65, further comprising a spatial mask that further inhibits the binding of the CP to its receptor.

67. An ACC comprising a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a first cytokine polypeptide (CP1), a first cleavable moiety (CM1), a first dimerizing domain (DD1) coupled to CP1 via CM1, and a first masking moiety (MM1). The second monomer construct comprises a second cytokine peptide (CP2), a second cleavable moiety (CM2), a second dimerizing domain (DD2) coupled to CP2 via the CM2, and a second masking moiety (MM2). The DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct, and The MM1 and / or the MM2 contain any of the isolated polypeptides described in the preceding claims.

68. The ACC of claim 67, wherein the first monomeric construct comprises a third cleavable portion (CM3), and the MM1 is coupled to the CP1 via the CM3.

69. The ACC of any one of claims 67-68, wherein the MM1 is coupled to the CP1 via the DD1 and the CM1.

70. The ACC of any one of claims 67-69, wherein the second monomeric construct comprises a fourth cleavable portion (CM4), and the MM2 is coupled to the CP2 via the CM4.

71. The ACC of any one of claims 67-70, wherein the MM2 is coupled to the CP2 via the DD2 and the CM2.

72. The ACC of any one of claims 67-71, wherein each of the first monomer construct and the second monomer construct comprises a linker region containing no more than 18 amino acids.

73. The ACC as claimed in any one of claims 67-72, wherein CP1 and CM1 are directly adjacent to each other, and / or CM1 and DD1 are directly adjacent to each other.

74. The ACC as claimed in any one of claims 67-73, wherein CP2 and CM2 are directly adjacent to each other, and / or CM2 and DD2 are directly adjacent to each other.

75. The ACC of any one of claims 67-74, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 348, 129, or 130.

76. The ACC of any one of claims 67-75, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 348, 129, or 130.

77. The ACC of any one of claims 67-76, wherein the CP1 comprises SEQ ID NO: 348, 129 or 130.

78. The ACC of any one of claims 67-77, wherein the CP2 comprises SEQ ID NO: 348, 129 or 130.

79. The ACC of any one of claims 67-78, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 119 or 120.

80. The ACC of any one of claims 67-79, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 119 or 120.

81. The ACC of any one of claims 67-80, wherein the CP1 comprises SEQ ID NO: 119 or 120.

82. The ACC of any one of claims 67-81, wherein the CP2 comprises SEQ ID NO: 119 or 120.

83. The ACC of any one of claims 67-82, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 121 or 122.

84. The ACC of any one of claims 67-83, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 121 or 122.

85. The ACC of any one of claims 67-84, wherein the CP1 comprises SEQ ID NO: 121 or 122.

86. The ACC of any one of claims 67-85, wherein the CP2 comprises SEQ ID NO: 121 or 122.

87. The ACC of any one of claims 67-86, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 123 or 124.

88. The ACC of any one of claims 67-87, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 123 or 124.

89. The ACC of any one of claims 67-88, wherein the CP1 comprises SEQ ID NO: 123 or 124.

90. The ACC of any one of claims 67-89, wherein the CP2 comprises SEQ ID NO: 123 or 124.

91. The ACC of any one of claims 67-90, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 125 or 126.

92. The ACC of any one of claims 67-91, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 125 or 126.

93. The ACC of any one of claims 67-92, wherein the CP1 comprises SEQ ID NO: 125 or 126.

94. The ACC of any one of claims 67-93, wherein the CP2 comprises SEQ ID NO: 125 or 126.

95. The ACC of any one of claims 67-94, wherein the CP1 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 521 or 522.

96. The ACC of any one of claims 67-95, wherein the CP2 comprises at least 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 521 or 522.

97. The ACC of any one of claims 67-96, wherein the CP1 comprises SEQ ID NO: 521 or 522.

98. The ACC of any one of claims 67-97, wherein the CP2 comprises SEQ ID NO: 521 or 522.

99. The ACC of any one of claims 67-98, wherein the first monomeric construct and the second monomeric construct are identical.

100. The ACC of any one of claims 67-99, wherein the first monomeric construct and the second monomeric construct have 95% sequence homology.

101. The ACC of any one of claims 67-100, further comprising a third monomer comprising a sushi domain containing the sequence of SEQ ID NO: 520, wherein the third monomer is non-covalently or covalently bound to the ACC.

102. The ACC of any one of claims 67-101, further comprising a fourth monomer comprising a sushi domain containing the sequence of SEQ ID NO: 520, wherein the fourth monomer is non-covalently or covalently bound to the ACC.

103. The ACC of claim 100 or 101, wherein the third monomer and / or the fourth monomer further comprises a label.

104. An ACC comprising a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a first cytokine peptide (CP1), a first dimerizing domain (DD1), and a first masking moiety (MM1). The second monomer construct comprises a second cytokine peptide (CP2), a first cleavable moiety (CM1), a second dimerizing domain (DD2) coupled to CP2 via said CM1, and a second masking moiety (MM2). The MM1 and / or MM2 are any of the isolated polypeptides described in the preceding claims. The DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct.

105. The ACC of claim 104, wherein the first monomeric construct further comprises a second cleavable portion (CM2), and the MM1 is coupled to the CP1 via the CM2.

106. The ACC as claimed in claim 104 or 105, wherein the MM2 is coupled to the CP2 via the DD2 and the CM1.

107. The ACC of any one of claims 104-106, wherein the second monomeric construct further comprises a third cleavable portion (CM3), wherein the MM2 is coupled to the CP2 via the CM3.

108. An ACC comprising a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a first cytokine peptide (CP1), a first dimerizing domain (DD1), and a first masking moiety (MM1). The second monomer construct comprises a second cytokine peptide (CP2), a second dimerization domain (DD2), and a second masking moiety (MM2). CP1 and / or CP2 contain an amino acid sequence that serves as a substrate for the protease, and DD1 and / or DD2 are coupled to CP1 or CP2 via the amino acid sequence. The MM1 and / or MM2 are any of the isolated polypeptides described in the preceding claims. The DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct.

109. The ACC of claim 108, wherein the CP1 comprises the amino acid sequence used as a substrate for the protease, and the MM1 is coupled to the CP1 via the amino acid sequence.

110. The ACC of any one of claims 108-109, wherein the first monomeric construct further comprises a first cleavable portion (CM1), and the MM1 is coupled to the CP1 via the CM1.

111. The ACC of any one of claims 108, wherein the CP2 comprises the amino acid sequence used as a substrate for the protease, and the MM2 is coupled to the CP2 via the amino acid sequence.

112. The ACC of any one of claims 108-111, wherein the second monomeric construct further comprises a second cleavable portion (CM2), and the CM2 is coupled to the CP2 via the CM2.

113. The ACC of any one of claims 67-112, wherein DD1 and DD2 are a pair of human IgG Fc domains.

114. The ACC of claim 113, wherein DD1 and DD2 are a pair of human IgG4 Fc domains.

115. The ACC of claim 114, wherein DD1 and DD2 are a pair of human IgG1 or IgG4 Fc domains truncated at the N-terminus to cysteine ​​226 as designated by EU numbering.

116. The ACC of claim 114, wherein the human IgG4 Fc domain comprises the S228P mutation as numbered according to EU designation.

117. The ACC of any one of claims 67-116, wherein each of DD1 and DD2 comprises a sequence that is at least 95% identical to SEQ ID NO:

3.

118. The ACC of any one of claims 67-117, wherein each of DD1 and DD2 comprises the sequence of SEQ ID NO:

3.

119. The ACC of any one of claims 67-118, wherein the first monomer builder and the second monomer builder are covalently bonded to each other by at least one, two, three or four disulfide bonds.

120. The ACC of any one of claims 12-19 and 47-119, wherein each of the first monomeric construct and the second monomeric construct comprises at least 85%, 90%, or 95% of the same sequence as SEQ ID NO:

523.

121. The ACC of any one of claims 12-19 and 47-120, wherein each of the first monomeric construct and the second monomeric construct comprises SEQ ID NO:

523.

122. The ACC of any one of claims 12-19 and 47-121, wherein the ACC is characterized by having a reduced level of interleukin activity compared to a control level of interleukin activity.

123. The ACC of any one of claims 12-19 and 47-122, wherein the ACC is characterized by having a reduced level of interleukin activity compared to wild-type human IL-15.

124. The ACC of any one of claims 12-19 and 47-123, wherein the ACC is characterized by having a reduced level of IL-15 activity compared to recombinant human IL-15, as measured by the level of SEAP (secretory embryonic alkaline phosphatase) production in IL-2 / IL-15 responsive HEK293 cells.

125. The ACC of any one of claims 12-19 and 47-124, wherein the ACC is characterized by having a reduced level of IL-15 activity compared to the activity of recombinant human IL-15.

126. The ACC of claim 125, wherein the ACC is characterized in that its IL-15 activity level is reduced by at least 6000 times compared with recombinant human IL-15.

127. The ACC of any one of claims 12-19 and 47-126, wherein the ACC is characterized in that, after cleavage by the uPA protease, its EC50 is at least 1,000, 5,000, or 6,000 times that of the EC50 of recombinant wild-type IL-15, as measured in IL-2 / IL15-responsive HEK293 cells.

128. An ACC comprising a cytokine polypeptide (CP), an agonist of the CP, the isolated polypeptide, and a cleavable moiety (CM), wherein the isolated polypeptide comprises a polypeptide sequence containing a tandem sequence or any cytokine masking moiety (MM) as described in the preceding claims, wherein the isolated polypeptide or the MM is coupled to the CP via the CM.

129. The ACC of claim 128, wherein the CP is IL-15 and the agonist is a sushi domain.

130. The ACC of claim 128 or claim 129, wherein the agonist is coupled to the CP via a connector.

131. The ACC of claim 130, wherein the agonist is coupled to the CP via a pyrolytic linker.

132. The ACC of claim 120, wherein the agonist is coupled to the CP via a non-lytic linker.

133. The ACC of claim 128 or claim 129, wherein the agonist is non-covalently bound to the CP.

134. An ACC comprising a first monomeric construct and a second monomeric construct, wherein The first monomer construct comprises a cytokine peptide (CP), a first dimerizing domain (DD1), a first cleavable moiety (CM1), a second cleavable moiety (CM2), and the isolated peptide, wherein the isolated peptide comprises a peptide sequence containing a tandem sequence or any cytokine masking moiety (MM) as described in the preceding claims, wherein the isolated peptide or the MM is coupled to the CP via the CM1, and the DD1 is coupled to the CP via the CM2. The second monomer construct comprises the agonist of the CP, a third cleavable moiety (CM3), and a second dimerizing domain (DD2) coupled to the agonist via the CM3, and The DD1 and DD2 combine with each other to form a dimer of the first monomer construct and the second monomer construct.

135. The ACC of claim 134, wherein the CP is IL-15 and the agonist is a sushi domain comprising the sequence of SEQ ID NO:

520.

136. The ACC of claim 134 or 135, comprising a linker consisting of two amino acids between the sushi domain and the CM3.

137. A polynucleotide encoding any isolated polypeptide as claimed in any of the preceding claims, any ACC as claimed in any of the preceding claims, or any monomeric construct as claimed in any of the preceding claims.

138. A vector comprising the polynucleotide of claim 137.

139. The carrier of claim 138, wherein the carrier is an expression carrier.

140. A host cell comprising the polynucleotide of claim 137 or the vector of claim 138 or 139.

141. The host cell of claim 141, wherein the host cell is a mammalian cell.

142. A composition comprising any isolated polypeptide as described in any of the preceding claims, or any ACC as described in any of the preceding claims, or the polynucleotide as described in claim 137.

143. The composition of claim 142, wherein the composition is a pharmaceutical composition.

144. A container, vial, syringe, pen, or kit containing at least one dose of the composition of claim 142 or 143.

145. A method of treating a subject in need, comprising administering to the subject a therapeutically effective amount of any isolated polypeptide as described in any of the preceding claims, any ACC as described in any of the preceding claims, or the composition as described in claim 142 or 143.

146. The method of claim 145, wherein the subject has been identified or diagnosed with cancer.

147. The method of claim 146, wherein the cancer is leukemia, lymphoma, or a solid tumor.

148. The method of claim 147, wherein the subject has been identified or diagnosed with an inflammatory or autoimmune disease, condition, or disorder.

149. A method for generating ACC, comprising: Cells comprising the polynucleotide of claim 137 are cultured in a liquid culture medium to produce the ACC; and The ACC is recovered from the cells or the liquid culture medium.

150. The method of claim 149, further comprising separating the ACC recovered from the cells or the liquid culture medium.

151. The method of claim 149 or 150, further comprising formulating the isolated ACC into a pharmaceutical composition.

152. A complex comprising a polypeptide, said polypeptide comprising a cytokine complexed with the isolated polypeptide of any of the preceding claims.

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