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

Abstract

This specification provides 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, ACC is a monomer. In some embodiments, ACC is a complex of two, three, four, or more constructs. In some embodiments, ACC is a dimer complex comprising a first monomer construct comprising a first cytokine polypeptide, a first masking moiety, and a first dimerization domain, and a second monomer construct comprising a second cytokine polypeptide and / or an agonist of the first cytokine polypeptide, a second dimerization domain, and optionally a second masking moiety, wherein the first masking moiety and / or the second masking moiety is a novel masking moiety.

Description

[Technical Field]

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

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy, created on 12 April 2024, is named "4862-146.xml" and has a size of 819,200 bytes.

[0003] This disclosure relates to the field of biotechnology, and more specifically to isolated polypeptides and activatable cytokine constructs (including activatable interleukin 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 stimuli. Interleukins are a different subclass of cytokines. Interleukins regulate cell growth, differentiation, and motility. Interleukins are particularly important in stimulating immune responses, such as inflammation. Interleukins are used in the treatment of cancer, autoimmune disorders, and other disorders. For example, interleukin-2 (IL-2) is indicated for the treatment of melanoma, graft-versus-host disease (GVHD), neuroblastoma, and renal cell carcinoma (RCC), and is also considered useful for conditions including acute coronary syndrome, acute 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 disorders. Interleukin-15 (IL-15) is known to enhance the antitumor response by promoting the differentiation and proliferation of T cells, B cells, and natural killer (NK) cells. IL-15 has been identified as a promising candidate for anticancer therapy and is being tested in numerous clinical trials. Despite its promising potential, IL-15 is known to exhibit undesirable pro-inflammatory effects and is associated with the pathogenesis of several autoimmune diseases. Recombinant IL-15 has been reported to have a maximum tolerated dose of 2 micrograms / 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). Recombinant soluble IL-15 has also been reported to have a short half-life in vivo, which hinders its use as a therapeutic agent.Berraondo, P., et al. “Cytokines in clinical cancer immunotherapy.” Br J Cancer 120, 6-15 (2019). Other interleukins, particularly IL-4, IL-6, IL-7, IL-9, IL-12, and IL-21, also have potential as treatments for cancer and other disorders. However, interleukin therapy is often accompanied by undesirable side effects, including flu-like symptoms, nausea, vomiting, diarrhea, hypotension, and arrhythmias. Therefore, there is a continued need for cytokine therapy that minimizes the undesirable side effects of existing cytokine therapies. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] 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 [Non-Patent Document 2] Berraondo, P., et al. “Cytokines in clinical cancer immunotherapy.” Br J Cancer 120,6-15(2019) [Overview of the project] [Means for solving the problem]

[0006] This disclosure provides isolated polypeptides and activatable cytokine constructs (ACCs) comprising one or more novel masking moieties.

[0007] In one aspect, this disclosure is, (a) A first masking subunit encoded by a first partial sequence of the amino acid sequence encoding the first receptor polypeptide of the cytokine receptor, (b)(i) A second subsequence of an amino acid sequence encoding a cytokine receptor polypeptide, 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 of a cytokine receptor, wherein the first subsequence and the second subsequence are not adjacent within the amino acid sequence encoding the first receptor polypeptide, and (ii) A cytokine masking moiety (MM) comprising a second masking subunit encoded by a second amino acid sequence selected from the group consisting of (ii) an amino acid sequence encoding a blocking moiety (BM).

[0008] In one embodiment, the disclosure includes an activatable cytokine construct (ACC) comprising a masking portion (MM), a cleavable portion (CM), and a cytokine polypeptide (CP), wherein the MM is (a) A first masking subunit encoded by a first partial sequence of the amino acid sequence encoding the first receptor polypeptide of the CP receptor, (b)(i) A second partial sequence of an amino acid sequence encoding a cytokine receptor polypeptide 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 of the receptor for CP, wherein the first partial sequence and the second partial sequence are not adjacent within the amino acid sequence encoding the first receptor polypeptide, and (ii) a second masking subunit encoded by a second amino acid sequence selected from the group consisting of (ii) an amino acid sequence encoding a blockage portion (BM).

[0009] In one aspect, the present disclosure includes an isolated polypeptide comprising a polypeptide sequence that includes a linked sequence of two or more receptor sub - sequences, where each receptor sub - sequence is derived from a receptor of a cytokine polypeptide (CP), and the two or more receptor sub - sequences are i) not adjacent within the sequence of the receptor, ii) directly or indirectly linked to each other.

[0010] In one aspect, the present disclosure includes an ACC comprising a masking moiety (MM), a cleavable moiety (CM), and a cytokine polypeptide (CP), a) The MM comprises a polypeptide sequence that includes a linked sequence of two or more receptor sub - sequences, b) Each receptor sub - sequence is derived from a receptor of the CP, c) The two or more receptor sub - sequences are i) not adjacent within the sequence of the receptor, ii) directly or indirectly linked to each other.

[0011] In one aspect, each of the two or more receptor sub - sequences includes contiguous amino acids in a receptor having at least one atom (C, O, N, or S) within 1.0 - 8.0 angstroms, 2.0 - 8.0 angstroms, or 2.0 - 7.0 angstroms of any atom (C, O, N, or S) of the cytokine amino acids in the co - crystal structure of the complex of the receptor and the cytokine (cytokine - receptor complex co - crystal structure). In one aspect, the isolated polypeptide further includes a linker disposed between at least two of the two or more receptor sub - sequences. In one aspect, each of the receptor sub - sequences includes two, three, four, or more than four amino acids. In one aspect, at least one of the two or more receptor sub - sequences includes a conservative substitution of at least one amino acid as compared to the sequence of the receptor. In one aspect, the two or more receptor sub - sequences include a first receptor sub - sequence and a second receptor sub - sequence, the second receptor sub - sequence is on the C - terminal side of the first receptor sub - sequence in the sequence of the receptor, the linker includes X amino acids, and X = n / y, where n is the distance in angstroms between the C - terminal of the N - terminal peptide of the first receptor sub - sequence and the N - terminal of the C - terminal peptide of the second receptor sub - sequence in the cytokine - receptor complex co - crystal structure, and y is a number within the range of 1.5 - 3.5 or 2 - 3.5 or 2 - 3 or 2 - 2.5. When X is not an integer, X is rounded up to the next integer. In some aspects, the two or more receptor sub - sequences include a first receptor sub - sequence and a second receptor sub - sequence, the second receptor sub - sequence is on the C - terminal side of the first receptor sub - sequence in the sequence of the receptor, the linker includes X amino acids, and X = n / 2.5. When X is not an integer, X is rounded up to the next integer.

[0012] Masking moieties having a chain - like receptor sub - sequence have been found to exhibit excellent masking properties with respect to their ability to interfere with the binding of cytokines to their receptors, as described herein. Further, ACCs containing such MMs show a significantly better recovery of cytokine activity after activation.

[0013] In one embodiment, the disclosure comprises an isolated polypeptide having the amino acid sequence ALTTVX-linker-ASHYFE (SEQ ID NO: 508) (MM), where X is D, K, or R, and the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid (such as those found in SEQ ID NOs: 515-518). In some embodiments, the N-terminal alanine residue is substituted with lysine. In some embodiments, 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 certain embodiments, the isolated polypeptide contains a sequence selected from SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737). In some embodiments, the linker consists of 1 to 20 amino acids. In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from 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), ALTTVRGGGGSASHYFE (SEQ ID NO: 514), ALTTVRGGGGSASHYFER (SEQ ID NO: 240), or ALTTVRGGGGSASHYFEK (SEQ ID NO: 241), or the terminal alanine residue in each sequence is optionally absent or optionally substituted with any other amino acid. In some embodiments, the N-terminal alanine residue is substituted with lysine. In some embodiments, the N-terminus, C-terminus, or both of any MM sequence in this specification 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 region that inhibits the binding of cytokines to their receptors.

[0015] In one embodiment, the disclosure includes an isolated polypeptide further comprising a cytokine. In several embodiments, the isolated polypeptide is arranged in a complex comprising two or more polypeptides, the complex comprising a cytokine. In one embodiment, the disclosure includes a complex comprising a polypeptide comprising a cytokine complexed with the isolated polypeptide of the disclosure. In several embodiments, the cytokine is arranged in a polypeptide complexed with the isolated polypeptide. In several embodiments, the cytokine is a cytokine that binds to IL2 / IL15 receptor beta and / or IL2 / IL15 receptor gamma. In several embodiments, the cytokine is a cytokine that binds to IL-15Rα. In several embodiments, the cytokine is a cytokine that binds to IL-2Rα.

[0016] In one embodiment, the Disclosure comprises an activatable cytokine construct (ACC) comprising a cytokine polypeptide (CP), a cleavable moiety (CM), and an isolated polypeptide (MM) of the Disclosure, wherein the MM is coupled with the CP via the CM and inhibits the binding of the CP to its receptor.

[0017] In one embodiment, the Disclosure comprises an ACC comprising a first monomer construct and a second monomer construct, the first monomer construct comprising a first cytokine polypeptide (CP1), a first cleavable moiety (CM1), a first dimerization domain (DD1) coupled to CP1 via CM1, and a first masking moiety (MM1), the second monomer construct comprising a second cytokine polypeptide (CP2), a second cleavable moiety (CM2), a second dimerization domain (DD2) coupled to CP2 via CM2, and a second masking moiety (MM2), wherein DD1 and DD2 are bound to each other to form dimers of the first monomer construct and the second monomer construct, and MM1 and / or MM2 comprises an isolated polypeptide of the Disclosure.

[0018] In one embodiment, the Disclosure comprises an ACC comprising a first monomer construct and a second monomer construct, the first monomer construct comprising a first cytokine polypeptide (CP1), a first dimerization domain (DD1), and a first masking moiety (MM1), the second monomer construct comprising a second cytokine polypeptide (CP2), a first cleavable moiety (CM1), a second dimerization domain (DD2) coupled to CP2 via CM1, and a second masking moiety (MM2), wherein MM1 and / or MM2 are isolated polypeptides of the Disclosure, and DD1 and DD2 are bound to each other, thereby forming dimers of the first monomer construct and the second monomer construct.

[0019] In one embodiment, the Disclosure comprises an ACC comprising a first monomer construct and a second monomer construct, the first monomer construct comprising a first cytokine polypeptide (CP1), a first dimerization domain (DD1), and a first masking moiety (MM1), the second monomer construct comprising a second cytokine polypeptide (CP2), a second dimerization domain (DD2), and a second masking moiety (MM2), wherein CP1 and / or CP2 comprises an amino acid sequence that functions as a substrate for a protease, DD1 and / or DD2 are coupled to CP1 and / or CP2 via the amino acid sequence, MM1 and / or MM2 are isolated polypeptides of the Disclosure, and DD1 and DD2 are bound to each other, thereby forming dimers of the first monomer construct and the second monomer construct.

[0020] In one embodiment, the Disclosure comprises an ACC including a first monomer construct and a second monomer construct, the first monomer construct comprising a cytokine polypeptide (CP), a first dimerization domain (DD1), a first cleavable moiety (CM1), a second cleavable moiety (CM2), and an isolated polypeptide or masking moiety (MM) of the Disclosure, the isolated polypeptide or MM being coupled to CP via CM1, and DD1 being coupled to CP via CM2, the second monomer construct comprising an agonist of CP, a third cleavable moiety (CM3), and a second dimerization domain (DD2) coupled to the agonist via CM3, wherein DD1 and DD2 are bound to each other, thereby forming dimers of the first monomer construct and the second monomer construct.

[0021] In one embodiment, the Disclosure includes an isolated polypeptide of the Disclosure or a polynucleotide encoding a monomer construct of the Disclosure. The Disclosure also includes vectors, host cells, compositions, methods of preparation, and therapeutic methods as disclosed below. [Brief explanation of the drawing]

[0022] [Figure 1A]Figure 1A, left shows the IL-15 complex with its receptor or the IL-2 complex (Figure 1B, left), and an example of a multiplicative molecule (MM) that binds to IL-15 (Figure 1A, right) or IL-2 (Figure 1B, right) and interferes with the binding between interleukins and their receptors. [Figure 1B] Figure 1A, left shows the IL-15 complex with its receptor or the IL-2 complex (Figure 1B, left), and an example of a multiplicative molecule (MM) that binds to IL-15 (Figure 1A, right) or IL-2 (Figure 1B, right) and interferes with the binding between interleukins and their receptors. [Figure 2] This is a schematic diagram of an exemplary activatable cytokine construct, comprising, in N-terminus to C-terminus, (1) a first monomer construct 110 having optionally MM1 119, optionally CM3 117, CP1 115, CM1 113, and DD1 111; (2) a second monomer construct 120 having optionally MM2 129, optionally CM4 127, CP2 125, CM2 123, and DD2 121; and (3) one or more covalent or non-covalent bonds (←→) connecting the first monomer construct 110 to the second monomer construct 120. The ACC may further include one or more optionally linkeders 112, 114, 116, 118, 122, 124, 126, and 128 between its components. In one example, DD1 111 and DD2 121 are identical. In another example, DD1 111 and DD2 121 are different. [Figure 3] A-E schematically illustrate examples of additional ACCs. Exemplary ACCs having MM (e.g., beta peptide), CM ("substrate"), CP (e.g., IL-15(A)), and in B, DD1 and DD2(Fc). Exemplary ACCs (C-E) having cytokine agonists (e.g., sucoid domains) and optional histidine tags ("His tags"). [Figure 4] An embodiment of ACC is schematically shown with its connecting region (LR) illustrated. [Figure 5]A schematic diagram of the exemplary ACC ProC2970 structure (top left), the tertiary structure of the monomer construct including the interleukin, cleavable moiety, and MM (top right), and the tertiary structure of the monomer construct complexed with its receptor (alpha, beta, and gamma chains of the receptor shown) (bottom) are shown. [Figure 6] The electrophoretic results of the exemplary cleavage of ACC ProC2970 by uPA are shown. [Figure 7] The masking efficiency of the masking portion of exemplary ACC ProC2970, tested by a reporter assay, is shown in comparison to that of ProC1879. [Figure 8-1] The exemplary activity of ACC ProC2970 against PMBC proliferation is shown in comparison to that of ProC1879. [Figure 8-2] The exemplary activity of ACC ProC2970 against PMBC proliferation is shown in comparison to that of ProC1879. [Figure 9A] This shows the activation of ACC. The electrophoresis of ACC before and after uPA cleavage is shown. [Figure 9B] This shows the activation of ACC. The electrophoresis of ACC before and after uPA cleavage is shown. [Figure 9C] This indicates ACC activation. The EC50 of ACC in the HEK-Blue reporter assay decreases after uPA-mediated activation. [Figure 9D] This indicates ACC activation. The EC50 of ACC in the HEK-Blue reporter assay decreases after uPA-mediated activation. [Figure 9E] This indicates ACC activation. The EC50 of ACC in the HEK-Blue reporter assay decreases after uPA-mediated activation. [Figure 10] Schematic diagrams of the structure of an ACC without MM (top row) and the structure of an exemplary ACC component with MM (bottom row) are shown. [Modes for carrying out the invention]

[0023] The subject matter of this disclosure can be embodied in various forms, and the following description is intended merely 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 intended to be limited to the forms or aspects described herein.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. This specification describes methods and materials for use herein. Other preferred methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification shall prevail, including definitions.

[0025] Other features and advantages of the present invention will become apparent from the embodiments and drawings for carrying out the invention and from the claims.

[0026] The terms "a" and "an" refer to one or more (i.e., at least one) grammatical objects of the article. For example, "a cell" includes one or more cells.

[0027] As used herein, the terms “about” and “approximately,” when used to modify a quantity specified by a number or range, indicate not only that number but also a reasonable deviation from that value, as known to those skilled in the art. For example, where appropriate, the intended meaning of the stated value includes ±20%, ±10%, or ±5%.

[0028] In this specification, concentrations, quantities, and other numerical data may be expressed or indicated in range form. Such range form is used solely for convenience and simplicity, and should therefore be interpreted flexibly to include not only the numerical values ​​explicitly listed as limits to the range, but also all individual numerical values ​​or partial ranges contained within that range, as if each numerical value and partial range were explicitly listed. For example, the numerical range "approximately 0.01 to 2.0" should be interpreted to include not only the explicitly listed values ​​of approximately 0.01 to approximately 2.0, but also the individual values ​​and partial ranges within the indicated range. Therefore, this numerical range includes individual values, e.g., 0.5, 0.7, and 1.5, as well as partial ranges, e.g., 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5. Furthermore, such interpretation should apply regardless of the width or characteristics of the range described. In addition, it should be noted that all percentages are weight percent unless otherwise specified.

[0029] For understanding the scope of this disclosure, the terms “including” or “comprising” and their derivatives are intended to be open-ended terms, as used herein, to specify the existence of described features, elements, components, groups, integers and / or steps, but not to exclude the existence of other undescribed features, elements, components, groups, integers and / or steps. The foregoing also applies to similarly meaninged words, such as “including” and “having” and their derivatives. As used herein, the term “consisting” and its derivatives are intended to be restrictive terms, specifying the existence of described features, elements, components, groups, integers and / or steps, but excluding the existence of other undescribed features, elements, components, groups, elements and / or steps. The term "consisting essentially of," as used herein, is intended to specify the existence of a feature, element, component, group, component, and / or step described herein, in addition to specifying the existence of a feature, element, component, group, component, and / or step that does not substantially affect the basic and novel features(plural) of the feature, element, component, group, component, and / or step. Any reference to any one of these transitional phrases (i.e., "comprising," "consisting," or "consisting essentially") is understood to provide direct support for substitution with any of the other transitional phrases not specifically used. For example, the modification of the term "comprising" to "consisting essentially of" or "consisting of" finds direct support for any element disclosed through this disclosure to be defined in this way. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0030] For convenience, as used herein, multiple compounds, elements, or steps may be presented in a general list. However, these lists should be interpreted as if each member of the list were individually identified as a distinct and unique member. Therefore, no individual member of such a list should be interpreted as a de facto equivalent of any other member of the same list based solely on its presentation in a common group, unless otherwise specified.

[0031] Furthermore, certain molecules, constructs, compositions, elements, parts, excipients, diseases, conditions, properties, processes, etc., may be considered in the context of specific embodiments or aspects of this disclosure, or in separate paragraphs or sections. This is solely for convenience and brevity, and it should be understood that any such disclosure is equally applicable to and intended to be combined with any other embodiments or aspects found in this disclosure and claims that form the inventions contained herein as of the filing date. For example, any list of constructs, molecules, method steps, kits, or compositions described in relation to a construct, composition, or method is intended, and indeed is intended, to directly support any embodiments relating to constructs, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context of that embodiment or aspect or section.

[0032] The terms “cleavable moiety” and “CM” are used interchangeably herein to refer to polypeptides whose amino acid sequence contains a substrate for a sequence-specific protease. Suitable cleavable moieties for use in ACC herein include any of the protease substrates known in the art. Exemplary cleavable moieties are described in more detail below.

[0033] The terms “masking moiety” and “MM” are used interchangeably herein to refer to a peptide or protein that reduces or inhibits the activity of one or more cytokine polypeptides. In some embodiments, when positioned 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, containing any number or range of amino acids between 1 and 50. In some embodiments, the MM has an amino acid length of 40 or less. In preferred embodiments, the MM has an amino acid length of 20 or less. In some embodiments, the MM has an amino acid length of 19, 18, 17, 16, or 15 or less. In some embodiments, the MM has at least 1, 2, 3, or 4 amino acids. In some embodiments, the MM has 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids.

[0034] As used herein, the term “masking efficiency” refers to the activity of an uncleaved ACC (e.g., EC50) divided by the activity of a control interleukin, where the control interleukin may be either a cleavage product of the ACC or a cytokine used as a CP of the ACC. An ACC with reduced levels of at least one interleukin activity has a masking efficiency greater than 10. In some embodiments, the ACCs described herein have masking efficiencies greater than 10, greater than 100, greater than 1000, or greater than 5000. In some embodiments, when measured by the ratio of the EC50 of an uncleaved ACC to the EC50 of the cleavage product of the ACC in IL-2 / IL-15 responsive HEK293 cells, 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.

[0035] As used herein, the terms “blocking moiety” or “BM” refer to a moiety that reduces or inhibits the interaction between a cytokine polypeptide and its binding partner, but does not contain a receptor sequence or subsequence derived from a cytokine receptor sequence.

[0036] As used herein, the term “subsequence” means that the moiety does not contain the full amino acid sequence of the cytokine receptor sequence, but instead has fewer amino acids than all the amino acids in the amino acid sequence of the cytokine receptor sequence. Thus, as used herein, a subunit, monomer, construct, polypeptide, or amino acid sequence “encoded by” a subsequence does not contain the full amino acid sequence of the cytokine receptor sequence, but instead has fewer amino acids than all the amino acids in the amino acid sequence of the cytokine receptor sequence.

[0037] As used herein, "consecutive" in the context of receptor subsequences means that two or more amino acids are adjacent to each other in the same order from the N-terminus to the C-terminus within the subsequence.

[0038] When used in reference to a cytokine construct, the term "activatable" means that the cytokine construct exhibits a first level of activity of one or more segments, and upon exposure to conditions that cause cleavage of one or more cleavable segments, leads to the formation of a cytokine construct exhibiting a second level of activity of one or more segments, where the second level of activity is greater than the first level of activity. Non-limiting examples of activity include any exemplary activity of cytokines described herein or cytokines known in the art.

[0039] The term “mature cytokine polypeptide” as used herein refers to a cytokine polypeptide lacking a signal sequence. A cytokine polypeptide (e.g., an interleukin polypeptide) may be a mature cytokine polypeptide or a cytokine polypeptide having a signal peptide. Thus, the ACC of this disclosure may, in some embodiments, include a mature cytokine polypeptide sequence. In some embodiments, the ACC of this disclosure may include a mature cytokine polypeptide sequence, in addition to a signal sequence. In some embodiments, the ACC of this disclosure may include sequences disclosed herein that include or lack the signal sequence described herein.

[0040] The terms “dimerization domain” and “DD” are used interchangeably herein to refer to one member of a pair of dimerization domains, each member of which can be bound to the other via one or more covalent or noncovalent interactions. The first DD and the second DD may be the same or different. Illustrative DDs suitable for use as DD1 and / or DD2 are described below in more detail herein.

[0041] As used herein, polypeptides such as cytokines or Fc domains may be wild-type polypeptides (e.g., naturally occurring polypeptides) or variants of wild-type polypeptides. A variant may be a polypeptide modified by substitution, insertion, deletion, and / or addition of one or more amino acids to a wild-type polypeptide, provided that the variant retains the basic function or activity of the wild-type polypeptide. In some examples, a variant may have a change in function or activity (e.g., an increase or decrease) compared to a wild-type polypeptide. In some embodiments, a variant may be a functional fragment of a wild-type polypeptide. The term “functional fragment” means that the sequence of a polypeptide (e.g., a cytokine) may contain fewer amino acids than the full-length polypeptide sequence, but may contain a sufficient polypeptide chain length to confer activity (e.g., cytokine activity).

[0042] In the context of two or more nucleic acid sequences or amino acid sequences, the term "at least [a certain]% identical" means that, when compared and aligned to achieve maximum match across a nucleic acid or amino acid comparison window or specified sequence, the two or more sequences share a given percentage of common nucleotides or amino acid residues (i.e., the sequences have at least 90 percent (%) identity). The percentage of 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 verification (see, for example, blast.ncbi.nlm.nih.gov / Blast.cgi). A person skilled in the art can determine parameters suitable for sequence alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. For example, the percentage of sequence identity of a given amino acid sequence A to, when compared with, or to a given amino acid sequence B (this can be alternatively rephrased as a given amino acid sequence A having or containing a certain percentage of sequence identity to, when compared with, or to a given amino acid sequence B) is calculated as follows: 100×X / Y ratio In the formula, X is the number of amino acid residues scored as identical sequences in the alignment of A and B by the program, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the sequence identity percentage of A to B will not be equal to the sequence identity percentage of B to A.

[0043] As used herein, “isolated polypeptide” means a polypeptide of cDNA origin, recombinant RNA origin, synthetic origin, or any combination thereof, which, depending on its origin or source, is substantially free of endogenously expressed host (e.g., mammalian cells) components, or, in the case of a cell-free expression system, substantially free of cell-free expression reagents, which are not found in nature. By using conventional isolation methods (e.g., chromatography), isolated polypeptides are substantially free of endogenously expressed host cell components and can substantially not contain cell-free expression reagents. According to aspects of this disclosure, isolated polypeptides can be placed in complexes containing two or more polypeptides, including cases where the complex contains cytokines. Unless otherwise specified, “protein-coding nucleic acid sequence” includes all nucleotide sequences that code for the same amino acid sequence because they are degenerate versions of each other.

[0044] Unless otherwise specified, a "protein-coding nucleic acid sequence" includes all nucleotide sequences that code for the same amino acid sequence because they are degenerate versions of each other.

[0045] The term "N-terminal" refers to the position of a first domain or sequence relative to a second domain or sequence in the polypeptide primary amino acid sequence, meaning that the first domain or sequence is located closer to the N-terminus of the polypeptide primary amino acid sequence than the second domain or sequence. In some embodiments, additional sequences and / or domains may exist between the first domain or sequence and the second domain or sequence.

[0046] The term "C-terminal" refers to the position of a first domain or sequence relative to a second domain or sequence in the polypeptide primary amino acid sequence, meaning that the first domain or sequence is located closer to the C-terminus of the polypeptide primary amino acid sequence than the second domain or sequence. In some embodiments, additional sequences and / or domains may exist between the first domain or sequence and the second domain or sequence.

[0047] The term "exogenous" means any substance that is introduced from or originates from outside a cell, tissue, or organism, and that is not produced or originates from the same cell, tissue, or organism into which it is introduced.

[0048] The terms “transduced,” “transfected,” or “transformed” refer to the process by which exogenous nucleic acids are introduced or transferred into cells. A “transduced,” “transfected,” or “transformed” cell (e.g., mammalian cell) is a cell that has been transduced, transfected, or transformed using an exogenous nucleic acid (e.g., a vector) containing an exogenous nucleic acid encoding one of the activatable cytokine constructs described herein.

[0049] The term “nucleic acid” means deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or a combination thereof, in single-stranded or double-stranded form. Unless otherwise specified, the term includes nucleic acids containing well-known analogues of natural nucleotides having binding properties similar to those of the reference nucleotide. Unless otherwise specified, a particular nucleic acid sequence implicitly includes complementary sequences in addition to the explicitly indicated sequence. 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.

[0050] As used herein, the phrase "specifically binds" means that ACC binds to its receptor or target and does not react with other polypeptides, or has a much lower affinity (e.g., about 10%). -6 This means joining (M or more).

[0051] The term "treatment" refers to improving at least one symptom of a disorder. In some embodiments, the disorder being treated is cancer. In some embodiments, the disorder being treated is an autoimmune disorder. In some embodiments, the disorder being treated is an inflammatory disorder.

[0052] Polypeptide containing new masking areas This specification provides isolated polypeptides containing amino acid sequences that can be used as masking moieties in activatable cytokine constructs.

[0053] In one aspect, this disclosure is, (a) A first masking subunit encoded by a first partial sequence of the amino acid sequence encoding the first receptor polypeptide of the cytokine receptor, (b)(i) A second subsequence of an amino acid sequence encoding a cytokine receptor polypeptide, 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 of a cytokine receptor, wherein the first subsequence and the second subsequence are not adjacent within the amino acid sequence encoding the first receptor polypeptide, and (ii) A cytokine masking moiety (MM) comprising a second masking subunit encoded by a second amino acid sequence selected from the group consisting of (ii) an amino acid sequence encoding a blocking moiety (BM).

[0054] The cytokine MMs provided herein are molecules of non-natural origin. In some embodiments, one or both of the first and second masking subunits have an amino acid length of 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or less. Cytokine MMs may be expressed in the form of polypeptides comprising cytokine MMs directly or indirectly coupled to a cleavable moiety (CM) directly or indirectly coupled to a cytokine polypeptide (CP).

[0055] In some embodiments, the Disclosure provides isolated polypeptides comprising a masking moiety (MM) that interferes with the binding of an interleukin to its binding partner. For example, the MM may comprise a chain peptide derived from a cytokine receptor, comprising amino acid residues in the receptor-cytokine interaction site. The chain peptide of the Disclosure comprises one or more receptor subsequences derived from a cytokine receptor sequence. The receptor subsequence is an amino acid sequence derived from the receptor of the CP. The receptor subsequences in the MM comprising the chain peptide may be different from or identical to each other. Thus, in some examples, the receptor subsequences are not identical. In some examples, the receptor sequences may be identical.

[0056] If the cytokine receptor has multiple subunits, the MM may include receptor sub-sequences derived from different subunits.

[0057] Masking moieties having linked receptor sub-arrays have been found to exhibit excellent masking properties with respect to their ability to interfere with the binding of cytokines to their receptors, as described herein. Furthermore, ACCs containing such MMs show remarkably good recovery of cytokine activity after activation.

[0058] By referring to a specific amino acid sequence, the MM described below is an example of an MM containing the chain amino acid sequence of this disclosure.

[0059] In some embodiments, each of the two or more receptor subsequences contains a sequence of amino acids in the receptor having at least one atom (C, O, N, or S) within 1.0–8.0 angstroms, 2.0–8.0 angstroms, or 2.0–7.0 angstroms of any of the cytokine amino acids in the cocrystal structure of the receptor-cytokine complex (cytokine-receptor complex cocrystal structure). In some embodiments, the ACC further includes a linker positioned between at least two of the two or more receptor subsequences. In some embodiments, each of the receptor subsequences contains two, three, four, or more amino acids. In some embodiments, at least one of the two or more subsequences contains a conserved substitution of at least one amino acid compared to the receptor sequence.

[0060] Conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), non-charged amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine), and hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include aliphatic hydroxyamino acids (e.g., serine and threonine), amide families (e.g., asparagine and glutamine), aliphatic families (e.g., alanine, valine, leucine, and isoleucine), and aromatic families (e.g., phenylalanine, tryptophan, and tyrosine).

[0061] In some embodiments, two or more receptor subsequences include a first receptor subsequence and a second receptor subsequence, the second receptor subsequence being C-terminal to the first receptor subsequence in the receptor sequence, the linker containing X amino acids, where X = n / y, where n is the distance in angstroms 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 cytokine-receptor complex cocrystal structure, y is a number in the range of 1.5 to 3.5, 2 to 3.5, 2 to 3, or 2 to 2.5, or y is 2.5, and if X is not an integer, X is rounded up to the next integer. In some embodiments, the receptor subsequences are different from each other. In some embodiments, two or more receptor subsequences are different or identical.

[0062] In one embodiment, the disclosure provides a polypeptide (e.g., an isolated polypeptide) comprising one or more masking moieties (MMs).

[0063] In some embodiments, the cytokine-receptor complex (MM) is generated by chaining peptides derived from receptor proteins that form at least a portion of the cytokine-receptor interface. The receptor peptides forming at least a portion of the cytokine-receptor interface (e.g., 3–20 α-lengths) are identified from the co-crystal structure of the receptor-cytokine complex as sequential amino acids in the receptor located within 2.0–7.0 angstroms of any atom (C, O, N, or S) of the cytokine amino acids. The distance can also be calculated based on the C-alpha atom distance within 1–20 angstroms. Several receptor / cytokine crystal structures have been reported in the literature and can be used herein to identify receptor peptides. Examples of such receptor / cytokine crystal structures include: 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-2 and 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. “Crystal structure 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. The structural basis for receptor recognition of human interleukin-18. Nat Commun 5,5340(2014).Structures reported in (doi.org / 10.1038 / ncomms6340)(pdb:3WO4,IL-18), Mendoza, JL, Escalante, NK, Jude, KMet al. Structure of the IFNγ receptor complex guides design of biased agonists. Nature 567,56-60(2019)(doi.org / 10.1038 / s41586-019-0988-7)(pdb:6E3K,IFNγ), and similar works are among others, and these documents are incorporated herein by reference in their entirety. Preferred peptides can also be identified by homology modeling and molecular docking protocols using BIOVIA Discovery Studio (Dassault Systems), Schrodinger (Schrodinger, Inc.), and similar software. These receptor peptides can be chained together with linkers (e.g., mobile linkers containing Gly, Ser, Thr, Asn, Pro (such as those disclosed below) and similar linkers). The linker length can be determined based on the distance between the individual peptides in the cocrystal. In some examples, if the distance between the C-terminus of one N-terminal peptide and the N-terminus of the other C-terminal peptide is n angstroms, the linker length is selected to be greater than or equal to n / 2.5 (rounded up to the next integer) (since the average C-alpha distance between amino acids is approximately 2.5 angstroms).

[0064] In some embodiments, MM comprises the sequence ALTTVX-linker-ASHYFE (SEQ ID NO: 508), where X is D, K, or R, and the N-terminal alanine residue in each sequence is optionally absent or optionally substituted with any other amino acid. In some embodiments, the N-terminal alanine residue is substituted with lysine. In some embodiments, 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 a particular embodiment, MM includes a sequence selected from SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0065] The MM sequence is linked with one or more linker sequences (e.g., a mobile linker, a linker containing Gly, Ser, Thr, Asn, Pro (such as those disclosed below), a linker designed to give a specific structure, and the like).

[0066] 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 yet another example, the linker consists of 3 amino acids. In yet another example, the linker consists of 4 amino acids. In yet another example, the linker consists of 5 amino acids. In yet another example, the linker consists of 6 amino acids. In yet another example, the linker consists of 7 amino acids. In yet another example, the linker consists of 8 amino acids. In yet another example, the linker consists of 9 amino acids. In yet another example, the linker consists of 10 amino acids. In yet another example, the linker consists of 11 amino acids. In yet another example, the linker consists of 12 amino acids. In yet another example, the linker consists of 13 amino acids. In yet another example, the linker consists of 14 amino acids. In yet another example, the linker consists of 15 amino acids. In yet another example, the linker consists of 16 amino acids. In yet another example, the linker consists of 17 amino acids. In yet another example, the linker consists of 18 amino acids. In another example, the linker consists of 19 amino acids. In yet another example, the linker consists of 20 amino acids. In yet another example, the linker consists of 21 amino acids. In yet another example, the linker consists of 22 amino acids. In some examples, the linker consists of 21 to 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).

[0067] Examples of linkers include the sequences listed below (e.g., sequence numbers 2 and 210-235, 245, or 250). In one example, the linker is GGGGS (sequence number 216).

[0068] In some embodiments, MM comprises the sequence ALTTVD-linker-ASHYFE (SEQ ID NO: 509), the sequence ALTTVD-linker-ASHYFER (SEQ ID NO: 242), or the sequence ALTTVD-linker-ASHYFEK (SEQ ID NO: 243), or in the sequence, the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some embodiments, MM comprises the sequence ALTTVK-linker-ASHYFE (SEQ ID NO: 510), the sequence ALTTVK-linker-ASHYFER (SEQ ID NO: 244), or the sequence ALTTVK-linker-ASHYFEK (SEQ ID NO: 246), or in the sequence, the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some embodiments, MM comprises the sequence ALTTVR-linker-ASHYFE (SEQ ID NO: 511), the sequence ALTTVR-linker-ASHYFER (SEQ ID NO: 247), or the sequence ALTTVR-linker-ASHYFEK (SEQ ID NO: 248), or in the sequence, the N-terminal alanine residue is optionally absent or substituted with any other amino acid. In some embodiments, the N-terminal alanine residue is substituted with lysine. In some embodiments, 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 a particular embodiment, MM includes a sequence selected from SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0069] In some embodiments, MM consists of the sequence ALTTVD-linker-ASHYFER (SEQ ID NO: 242) or the sequence ALTTVD-linker-ASHYFE(R / K) (SEQ ID NO: 502), or the N-terminal alanine residue in the sequence is optionally absent or substituted with any other amino acid. In some embodiments, MM consists of the sequence ALTTVK-linker-ASHYFE (SEQ ID NO: 510) or the sequence ALTTVK-linker-ASHYFE(R / K) (SEQ ID NO: 503), or the N-terminal alanine residue in the sequence is optionally absent or substituted with any other amino acid. In some embodiments, MM consists of the sequence ALTTVR-linker-ASHYFE (SEQ ID NO: 511) or the sequence ALTTVR-linker-ASHYFE(R / K) (SEQ ID NO: 504), or the N-terminal alanine residue in the sequence is optionally absent or substituted with any other amino acid. In some embodiments, the N-terminal alanine residue is substituted with lysine. In some embodiments, 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 certain embodiments, MM comprises a sequence selected from SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0070] In one example, MM contains the sequence ALTTVDGGGGSASHYFE (SEQ ID NO: 512) or the sequence ALTTVDGGGGSASHYFE(R / K) (SEQ ID NO: 505), or the N-terminal alanine residue in the sequence is optionally absent or substituted with any other amino acid. In another example, MM contains the sequence ALTTVKGGGGSASHYFE (SEQ ID NO: 513) or the sequence ALTTVKGGGGSASHYFE(R / K) (SEQ ID NO: 506), or the N-terminal alanine residue in the sequence is optionally absent or substituted with any other amino acid. In yet another example, MM contains the sequence ALTVRGGGGSASHYFE (SEQ ID NO: 514) or the sequence ALTVRGGGGSASHYFE(R / K) (SEQ ID NO: 507), or the N-terminal alanine residue in the sequence is optionally absent or substituted with any other amino acid. In some embodiments, the N-terminal alanine residue is substituted with lysine. In some embodiments, 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 certain embodiments, MM comprises a sequence selected from SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0071] In one example, MM consists of the sequence ALTTVDGGGGSASHYFE (SEQ ID NO: 512) or the sequence ALTTVDGGGGSASHYFE(R / K) (SEQ ID NO: 505), or the N-terminal alanine residue in the sequence is optionally absent or substituted with any other amino acid. In another example, MM consists of the sequence ALTTVKGGGGSASHYFE (SEQ ID NO: 513) or the sequence ALTTVKGGGGSASHYFE(R / K) (SEQ ID NO: 506), or the N-terminal alanine residue in the sequence is optionally absent or substituted with any other amino acid. In yet another example, MM consists of the sequence ALTVRGGGGSASHYFE (SEQ ID NO: 514) or the sequence ALTVRGGGGSASHYFE(R / K) (SEQ ID NO: 507), or the N-terminal alanine residue in the sequence is optionally absent or substituted with any other amino acid. In some embodiments, the N-terminal alanine residue is substituted with lysine. In some embodiments, 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 certain embodiments, MM comprises a sequence selected from SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0072] The linkers in the MM may contain any one or more amino acids and any combination of amino acid sequences. In some embodiments, the linkers are movable linkers. In some embodiments, the linkers are designed to impart a desired two-dimensional and / or three-dimensional structure to the MM.

[0073] In some embodiments, the masking moiety (MM) “masks,” reduces, or otherwise inhibits the activity of the cytokine polypeptide. In some embodiments, the MM masks, reduces, or otherwise inhibits the binding of the cytokine polypeptide to its receptor. In some embodiments, coupling the cytokine polypeptide with the MM, or modifying the cytokine polypeptide with the MM, inhibits the cytokine polypeptide’s ability to specifically bind to its receptor by inhibitory means known to those skilled in the art (e.g., structural changes and competition for receptor binding). In some embodiments, coupling the cytokine polypeptide with the MM, or modifying the cytokine polypeptide with the MM, can result in a structural change that reduces or inhibits the protein’s ability to specifically bind to its receptor. In some embodiments, coupling the cytokine polypeptide with the MM, or modifying the cytokine polypeptide with the MM, sterically blocks, reduces, or inhibits the cytokine polypeptide’s ability to specifically bind to its receptor.

[0074] Activatable cytokine components In one embodiment, the present disclosure provides an activatable cytokine construct (ACC) comprising a cytokine polypeptide (CP) (e.g., an interleukin polypeptide), a cleavable moiety (CM), and a cytokine molecule (MM) described herein that is coupled to the CP via the CM.

[0075] In some examples, ACC comprises cytokine polypeptide-CM-MM or MM-CM-cytokine polypeptide in the order of N-terminus to C-terminus. Where used herein, unless otherwise noted, each dash (-) between ACC components represents either direct linkage or linkage via, for example, one or more linkers.

[0076] In one embodiment, the disclosure includes an ACC comprising a masking portion (MM), a cleavable portion (CM), and a cytokine polypeptide (CP). a) MM comprises a polypeptide sequence containing a chain of two or more receptor subsequences, b) Each receptor subsequence is derived from the CP receptor, c) Two or more receptor subsequences i) Not adjacent within the receptor sequence, ii) They are directly or indirectly connected to each other.

[0077] In some embodiments, the disclosure provides activatable cytokine constructs (ACCs) in various forms that incorporate MM.

[0078] In one embodiment, the disclosure includes an activatable cytokine construct (ACC) comprising a masking portion (MM), a cleavable portion (CM), and a cytokine polypeptide (CP), wherein the MM is (a) A first masking subunit encoded by a first partial sequence of the amino acid sequence encoding the first receptor polypeptide of the CP receptor, (b)(i) A second partial sequence of an amino acid sequence encoding a cytokine receptor polypeptide 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 of the receptor for CP, wherein the first partial sequence and the second partial sequence are not adjacent within the amino acid sequence encoding the first receptor polypeptide, and (ii) a second masking subunit encoded by a second amino acid sequence selected from the group consisting of (ii) an amino acid sequence encoding a blockage portion (BM).

[0079] In some embodiments, the Disclosure provides an activatable cytokine construct (ACC) that gives a cytokine product exhibiting a reduced level of activity of at least one of the corresponding cytokines but having substantially restored activity after exposure to activation conditions. In some embodiments, the ACC comprises a cytokine polypeptide (CP), a cleavable moiety (CM), and a masking moiety (MM) according to the 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 binds to IL-15 (Figure 1A) or IL-2 (Figure 1B) and interferes with the binding of an interleukin to its receptor.

[0080] The inventors have surprisingly found that ACC containing MM as disclosed herein has improved properties (such as increased masking efficiency) compared to its counterpart ACC that does not contain such MM.

[0081] The ACCs of this disclosure can be selectively activated upon exposure to diseased tissue but not in normal tissue. Following activation of the ACC upon cleavage of the cleavable portion, cytokine activity is restored, indicating that the released masking portion is not thought to remain bound to the cytokine after cleavage and does not interfere with or compete with the cytokine for binding to its target. Therefore, the ACCs have the potential to provide the benefits of cytokine-based therapy, with potentially lower toxicity and improved pharmacokinetics compared to certain cytokine-based therapies.

[0082] This specification also provides related intermediates, compositions, kits, nucleic acids, and recombinant cells, as well as related methods including methods for using, producing, and delivering any of the ACCs described herein.

[0083] In some embodiments, ACC is characterized by having cytokine activity at a level at least 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or 6000-fold lower than that of the corresponding recombinant wild-type cytokine. For example, ACC is characterized by having an EC50 at least 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or 6000-fold higher than that of recombinant wild-type IL-15 when measured in IL-2 / IL15-responsive HEK293 cells.

[0084] In some embodiments, the ACC further comprises an agonist of CP (e.g., a sucoid domain). For example, the ACC may include the MM, CM, CP (e.g., IL-15 or its variants) described herein, and an agonist of CP (e.g., a sucoid domain). In some examples, the agonist (e.g., a sucoid domain) may be coupled to CP via a linker.

[0085] As used herein, the term “sucoid domain” has its general meaning in the art and refers to the domain of IL-15Rα beginning at the first cysteine ​​residue (C1) after the signal peptide and ending at the fourth cysteine ​​residue (C4) after the signal peptide. The sucoid domain, corresponding to a portion of the extracellular region of IL-15Rα, is necessary for its binding to IL-15 (Wei et al., J.Immunol., vol.167(1), p:277-282, 2001 (the whole of which is incorporated herein by reference)). In one example, the sucoid domain contains the sequence of Sequence ID No. 520. In another example, the sucoid domain contains a functional fragment of the sequence of Sequence ID No. 520. The sucoid domain of IL-15Rα or its derivatives have at least 10% (e.g., at least 25%, more preferably at least 50%) of the binding activity of the sucoid domain of human IL-15Rα to human interleukin-15. The binding activity can be easily determined by the method disclosed in Wei et al. 2001.

[0086] In some embodiments, the sucoid domain is covalently linked to an interleukin polypeptide, MM, DD1, or DD2. In some embodiments, the covalent bond is a non-alpha carbon covalent bond (e.g., an isopeptide bond). In some embodiments, the isopeptide bond is present between lysine and a glutamic acid residue or an aspartic acid residue. In some embodiments, the non-alpha carbon covalent bond is present between MM and a functional group substituted onto the alpha carbon in the cytokine. In some embodiments, the isopeptide bond is present between the gammacarboxamide group of glutamine and the epsilonamino group of the lysine side chain. In some embodiments, the non-alpha carbon covalent bond is an ester bond between threonine and glutamine. In some embodiments, the non-alpha carbon covalent bond is a thioester bond between cysteine ​​and glutamine. In some embodiments, the non-alpha carbon covalent bond is a thioether bond between cysteine ​​and tyrosine. In some embodiments, the non-alpha carbon covalent bond is formed by bridging between histidine and tyrosine (for example, this type of histidine-tyrosine bridge is known to be present in cytochrome c oxidase enzymes). In some embodiments, the non-alpha carbon covalent bond is a nitrogen-oxygen-sulfur (NOS) bond formed between lysine and cysteine. In some embodiments, the non-alpha carbon covalent bond is a disulfide bond.

[0087] In ACC, MM may be coupled to cytokine polypeptides by CM and one or more optional linkers (described in more detail herein). In some embodiments, when ACC is not activated, MM prevents cytokine polypeptides from binding to their receptors, but when ACC is activated (the CM between MM and cytokine polypeptides is cleaved by a protease), MM does not substantially or significantly interfere with cytokine polypeptides binding to their receptors.

[0088] In ACC, MM may be directly or indirectly coupled to a cytokine polypeptide (e.g., via one or more linkers). Alternatively, MM may be directly or indirectly coupled to a component of ACC that is not a cytokine polypeptide. For example, MM may be directly or indirectly coupled to a different cytokine polypeptide. In another example, MM may be directly or indirectly coupled to DD. In any case, in the tertiary or quaternary structure of the activatable structure, MM may be located in a position that allows MM to mask the cytokine polypeptide (e.g., proximal to the cytokine polypeptide to be masked).

[0089] In some embodiments, the ACC further comprises a CP agonist (such as the sucoid domain described below). For example, the ACC may include the MM, CM, CP (e.g., IL-15 or its variants) described herein, and a CP agonist (e.g., the sucoid domain). In some examples, the agonist (e.g., the sucoid domain) is coupled to the CP via a linker.

[0090] Interleukin polypeptides In some cases, CP is an interleukin polypeptide. Examples of interleukin polypeptides of ACC as used herein 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, and IL-17, as well as IL-21.

[0091] In some examples, CP is IL-15. For example, CP may contain sequence numbers 348, 129, or 130, or a functional fragment thereof. In some examples, CP may contain a sequence that is at least 85%, 90%, or 95% identical to sequence numbers 348, 129, or 130.

[0092] In some embodiments, the interleukin polypeptide is wild-type IL-15. In some embodiments, the interleukin polypeptide is wild-type human IL-15. In some embodiments, the interleukin polypeptide is mutant IL-15. In some embodiments, the interleukin polypeptide is mutant human IL-15. In some embodiments, the interleukin polypeptide is at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 348. In some embodiments, the interleukin polypeptide is at least 85% identical to IL-15 (SEQ ID NO: 348), and the amino acid at position 45 of the interleukin polypeptide is not leucine. In some examples, the interleukin polypeptide is at least 90% identical to SEQ ID NO: 348, and the amino acid at position 45 of the interleukin polypeptide is not leucine. In some examples, the interleukin polypeptide is at least 95% identical to SEQ ID NO: 348, and the amino acid at position 45 of the interleukin polypeptide is not leucine. In some cases, the interleukin polypeptide is at least 99% identical to SEQ ID NO: 348, and the amino acid at position 45 of the interleukin polypeptide is not leucine. The location of the mutation is relative to the reference sequence. Therefore, for example, if the mutation is located at position 45, it is relative to the reference sequence. For example, if the reference sequence is, [ka] In that case, the amino acids at positions 45 and 52 are shown in bold.

[0093] In some cases, the amino acid at position 45 of SEQ ID NO: 348 in the interleukin polypeptide is aspartic acid. In some cases, the amino acid at position 45 of SEQ ID NO: 348 in the interleukin polypeptide is asparagine. In some cases, the amino acid at position 45 of SEQ ID NO: 348 in the interleukin polypeptide is threonine.

[0094] In some embodiments, the interleukin polypeptide is at least 85% identical to SEQ ID NO: 348, and the amino acid at position 52 of the interleukin polypeptide is not leucine. In some examples, the interleukin polypeptide is at least 90% identical to SEQ ID NO: 348, and the amino acid at position 52 of the interleukin polypeptide is not leucine. In some examples, the interleukin polypeptide is at least 95% identical to SEQ ID NO: 348, and the amino acid at position 52 of the interleukin polypeptide is not leucine. In some examples, the interleukin polypeptide is at least 99% identical to SEQ ID NO: 348, and the amino acid at position 52 of the interleukin polypeptide is not leucine.

[0095] In some cases, the amino acid at position 52 of SEQ ID NO: 348 in the interleukin polypeptide is aspartic acid. In some cases, the amino acid at position 52 of SEQ ID NO: 348 in the interleukin polypeptide is asparagine. In some cases, the amino acid at position 52 of SEQ ID NO: 348 in the interleukin polypeptide is threonine.

[0096] In some embodiments, the interleukin polypeptide is at least 85% identical to SEQ ID NO: 348, the amino acid at position 45 of the interleukin polypeptide is not leucine, and the amino acid at position 52 is not leucine. In some examples, the interleukin polypeptide is at least 90% identical to SEQ ID NO: 348, the amino acid at position 45 of the interleukin polypeptide is not leucine, and the amino acid at position 52 is not leucine. In some embodiments, the interleukin polypeptide is at least 95% identical to SEQ ID NO: 348, the amino acid at position 45 of the interleukin polypeptide is not leucine, and the amino acid at position 52 is not leucine.

[0097] In some cases, the amino acids at positions 45 and 52 of SEQ ID NO: 348 in the interleukin polypeptide are aspartic acid. In some cases, the amino acids at positions 45 and 52 of SEQ ID NO: 348 in the interleukin polypeptide are asparagine. In some cases, the amino acids at positions 45 and 52 of SEQ ID NO: 348 in the interleukin polypeptide are threonine.

[0098] In some cases, the amino acids at positions 45 and 52 of SEQ ID NO: 348 in the interleukin polypeptide are neither aspartic acid, asparagine, nor threonine.

[0099] In some embodiments, the interleukin polypeptide includes one of SEQ ID NOs: 402 to 422. In one example, the interleukin polypeptide includes SEQ ID NOs: 402. In another example, the interleukin polypeptide includes SEQ ID NOs: 403. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 404. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 405. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 406. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 407. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 408. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 409. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 410. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 411. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 412. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 413. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 414. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 415. In yet another example, the interleukin polypeptide includes SEQ ID NOs: 416. In another example, the interleukin polypeptide includes SEQ ID NO: 417. In yet another example, the interleukin polypeptide includes SEQ ID NO: 418. In yet another example, the interleukin polypeptide includes SEQ ID NO: 419. In yet another example, the interleukin polypeptide includes SEQ ID NO: 420. In yet another example, the interleukin polypeptide includes SEQ ID NO: 421. In yet another example, the interleukin polypeptide includes SEQ ID NO: 422.

[0100] In some embodiments, the interleukin polypeptide consists of one of the sequence numbers 402 to 422. In one example, the interleukin polypeptide consists of sequence number 402. In another example, the interleukin polypeptide consists of sequence number 403. In yet another example, the interleukin polypeptide consists of sequence number 404. In yet another example, the interleukin polypeptide consists of sequence number 405. In yet another example, the interleukin polypeptide consists of sequence number 406. In yet another example, the interleukin polypeptide consists of sequence number 407. In yet another example, the interleukin polypeptide consists of sequence number 408. In yet another example, the interleukin polypeptide consists of sequence number 409. In yet another example, the interleukin polypeptide consists of sequence number 410. In yet another example, the interleukin polypeptide consists of sequence number 411. In yet another example, the interleukin polypeptide consists of sequence number 412. In yet another example, the interleukin polypeptide consists of sequence number 413. In yet another example, the interleukin polypeptide consists of sequence number 414. In yet another example, the interleukin polypeptide consists of sequence number 415. In yet another example, the interleukin polypeptide consists of sequence number 416. In another example, the interleukin polypeptide consists of SEQ ID NO: 417. In yet another example, the interleukin polypeptide consists of SEQ ID NO: 418. In yet another example, the interleukin polypeptide consists of SEQ ID NO: 419. In yet another example, the interleukin polypeptide consists of SEQ ID NO: 420. In yet another example, the interleukin polypeptide consists of SEQ ID NO: 421. In yet another example, the interleukin polypeptide consists of SEQ ID NO: 422.

[0101] In some examples, CP is IL-2 or a functional fragment thereof. For example, CP may contain sequence number 119 or 120 or a functional fragment thereof. In some examples, CP may contain a sequence that is at least 85%, 90%, or 95% identical to sequence number 119 or 120.

[0102] In some examples, CP is IL-4 or a functional fragment thereof. For example, CP may contain sequence numbers 121 or 122 or a functional fragment thereof. In some examples, CP may contain sequences that are at least 85%, 90%, or 95% identical to sequence numbers 121 or 122.

[0103] In some examples, CP is IL-7 or a functional fragment thereof. For example, CP may contain sequence numbers 123 or 124 or a functional fragment thereof. In some examples, CP may contain sequences that are at least 85%, 90%, or 95% identical to sequence numbers 123 or 124.

[0104] In some examples, CP is IL-9 or a functional fragment thereof. For example, CP may contain sequence numbers 125 or 126 or a functional fragment thereof. In some examples, CP may contain sequences that are at least 85%, 90%, or 95% identical to sequence numbers 125 or 126.

[0105] In some examples, CP is IL-21 or a functional fragment thereof. For example, CP may contain sequence numbers 521 or 522 or a functional fragment thereof. In some examples, CP may contain sequences that are at least 85%, 90%, or 95% identical to sequence numbers 521 or 522.

[0106] ACC having a dimerizing domain In some embodiments, the ACC as used herein is a dimerized complex comprising a first monomer construct and a second monomer construct. Dimerization of the monomer components is facilitated by a pair of dimerizing domains. In one embodiment, each monomer construct comprises a cytokine polypeptide, the MM as described herein, and a dimerizing domain (DD).

[0107] In a particular embodiment, the present invention provides an ACC comprising a first monomer construct and a second monomer construct, The first monomer construct comprises a first cytokine polypeptide (CP1), a first cleavable moiety (CM1), a first dimerization domain (DD1) coupled to CP1 via CM1, and a first masking moiety (MM1). The second monomer construct comprises a second cytokine polypeptide (CP2), a second cleavable moiety (CM2), a second dimerization domain (DD2) coupled to CP2 via CM2, and a second masking moiety (MM2). DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer construct and the second monomer construct. MM1 and / or MM2 include MM as described herein.

[0108] In some embodiments, the ACC is characterized by having a reduced level of at least one CP1 and / or CP2 activity compared to a control level of at least one CP1 and / or CP2 activity.

[0109] In some embodiments, the first monomer structure 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 monomer structure includes a fourth cleavable portion (CM4), and MM2 is coupled to CP2 via CM4. In some embodiments, MM2 is coupled to CP2 via CM2.

[0110] In some embodiments, ACC further comprises a third monomer comprising a sucoid domain comprising the sequence of SEQ ID NO: 520. In some embodiments, ACC further comprises a fourth monomer comprising a sucoid domain comprising 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 or a myc tag). In some embodiments, the fourth monomer further comprises a tag (e.g., a peptide tag such as a His tag or a myc tag).

[0111] In some embodiments, the ACC comprises a first monomer construct and a second monomer construct, the first monomer construct comprising a first cytokine polypeptide (CP1), a first dimerization domain (DD1), and a first masking moiety (MM1), The second monomer construct comprises a second cytokine polypeptide (CP2), a first cleavable moiety (CM1), a second dimerization domain (DD2) coupled to CP2 via CM1, and a second masking moiety (MM2). MM1 and / or MM2 are MM as defined herein. DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer construct and the second monomer construct.

[0112] In some embodiments, the first monomer structure further includes 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 monomer structure includes a third cleavable portion (CM3), and MM2 is coupled to CP2 via CM3.

[0113] In some embodiments, ACC comprises a first monomer construct and a second monomer construct. The first monomer construct comprises a first cytokine polypeptide (CP1), a first dimerization domain (DD1), and a first masking moiety (MM1), The second monomer construct comprises a second cytokine polypeptide (CP2), a second dimerization domain (DD2), and a second masking moiety (MM2), CP1 and / or CP2 contain amino acid sequences that function as substrates for proteases, and DD1 and / or DD2 are coupled to CP1 and / or CP2 via amino acid sequences. MM1 and / or MM2 are MM as defined herein. DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer construct and the second monomer construct.

[0114] In some embodiments, CP1 comprises an amino acid sequence that functions as a substrate for a 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 that functions as a substrate for a 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.

[0115] In some embodiments, ACC comprises a first monomer construct and a second monomer construct. The first monomer construct comprises a cytokine polypeptide (CP), a first dimerization domain (DD1), a first cleavable portion (CM1), a second cleavable portion (CM2), and an MM as 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 a CP agonist, a third cleavable moiety (CM3), and a second dimerization domain (DD2) coupled to the agonist via CM3. DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer construct and the second monomer construct.

[0116] In some embodiments, CP is IL-15, and the agonist is a sucoid domain containing the sequence of SEQ ID NO: 520. In some embodiments, ACC includes a linker between the sucoid domain and CM3. In some embodiments, the linker may contain 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). In some embodiments, the linker may contain 1 to 3 amino acids (e.g., 1, 2, or 3 amino acids), or in some embodiments, it consists of 2 amino acids.

[0117] In the dimer complex ACC, the first and second monomer constructs may further contain additional elements (e.g., one or more linkers) and the like. These additional elements are described in more detail below. The composition 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, for example, with respect to the molecular weight, size, amino acid sequence, and the like of the CP and CM components (and the DD component in embodiments where the DD component is a polypeptide). Thus, the resulting dimer may have symmetric or asymmetric monomer constructs.

[0118] In some embodiments, the first monomer construct includes CP1, CM1, and DD1 directly or indirectly (via linkers) linked from the N-terminus to the C-terminus of the CP and CM components. In other embodiments, the first monomer construct includes CP1, CM1, and DD1 directly or indirectly (via linkers) linked from the C-terminus to the N-terminus of the CP and CM components. In some embodiments, the second monomer construct includes CP2, CM2, and DD2 directly or indirectly (via linkers) linked from the N-terminus to the C-terminus of the CP and CM components. In other embodiments, the second monomer construct includes CP2, CM2, and DD2 directly or indirectly (via linkers) linked 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 monomers (MMs). In some embodiments, the ACC further comprises a monomer (CM) between the MM and the CP.

[0119] In some embodiments, the activatable cytokine construct (ACC) comprises a first monomer construct and a second monomer construct, characterized in that (a) the first monomer construct comprises a first masking moiety (MM1), a first mature cytokine polypeptide (CP1), a first cleavable moiety and a third cleavable moiety (CM1 and CM3), and a first dimerization 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 moiety (CM2), and a second dimerization domain (DD2), wherein CM2 is located between CP2 and DD2, and DD1 and DD2 are bound to each other, thereby forming a dimer of the first monomer construct and the second monomer construct, and the ACC is characterized in that the level of at least one CP1 and / or CP2 activity is reduced compared to a control level of at least one CP1 and / or CP2 activity.

[0120] In some embodiments, the second monomer construct further comprises a second masking portion (MM2) and a fourth cleavable portion (CM4), where 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.

[0121] The ACC structure was found to be highly effective in reducing the activity of mature cytokine polypeptide components in a manner that substantially preserves the cytokine activity after activation. The activity of CP in ACC can be reduced by both the structure of ACC (e.g., dimeric structure) and the masking moiety(s) within ACC. In some embodiments, the activation conditions of ACC described herein are exposure to one or more proteases capable of dissociating 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 ACC resulted in a substantial recovery of cytokine activity. The results suggest that the conformation of cytokine components did not irreversibly change in the presence of ACC.

[0122] In some embodiments, when cytokine polypeptides are coupled to MM and a natural binding partner (e.g., its receptor) of the cytokine polypeptide is present, the binding time measured in the mask efficiency assay is at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150 hours, compared to the binding of cytokine polypeptides not coupled to MM. Over 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer, binding of the cytokine polypeptide to its binding partner is absent or substantially absent, or its binding to the binding partner is 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% or less. For example, a mask efficiency assay may involve measuring the affinity of an ACC to the cell surface displaying a candidate masking moiety, for example, by FACS. Another non-limiting exemplary assay includes evaluating the ability of a masking moiety to inhibit the binding of an ACC to its binding partner at therapeutically appropriate concentrations and times. For this second method, an immunoadsorption assay for measuring the time-dependent binding of a proprotein to its binding partner has been developed as described in US20200308243, which is incorporated herein by reference. In one embodiment, a mask efficiency assay may be accompanied by measuring the level of secreted alkaline phosphatase (SEAP) production in IL-2 / IL15-responsive HEK293 cells.

[0123] In certain embodiments, the first and second monomer constructs are oriented such that the components in each member of the dimer are configured such that the CP and CM components are in the same order from N-terminus to C-terminus. Figure 2 is a schematic diagram of an exemplary activatable cytokine construct, comprising, in N-terminus to C-terminus, (1) a first monomer construct 110 having optionally MM1 119, optionally CM3 117, CP1 115, CM1 113, and DD1 111; (2) a second monomer construct 120 having optionally MM2 129, optionally CM4 127, CP2 125, CM2 123, and DD2 121; and (3) one or more covalent or non-covalent bonds (←→) connecting the first monomer construct 110 to the second monomer construct 120. ACC may further include one or more optional linkers 112, 114, 116, 118, 122, 124, 126, and 128 between its components. In one example, DD1 111 and DD2 121 are identical. In another example, DD1 111 and DD2 121 are different. In some examples, DD1 111 and DD2 121 are different polypeptides that bind to each other.

[0124] In an alternative embodiment, either of the two portions represented as CP1 115 and CP2 125 is a mutant cytokine polypeptide lacking cytokine activity. In an alternative embodiment, either of the two portions represented as CP1 115 and CP2 125 is a polypeptide sequence lacking cytokine activity (e.g., a signaling portion and / or a stub sequence). In an alternative embodiment, the first portion of the two portions represented as CP1 115 and CP2 125 is a polypeptide sequence that binds with high affinity to the second portion of the two portions represented as CP1 115 and CP2 125, reducing the cytokine activity of the second portion compared to a control level of the second portion.

[0125] Figures 3A–3E show additional exemplary embodiments of ACC. The substrates added to the masked portions in these figures are all optional and exemplary embodiments of the present invention, and the CM or CM-MM features are optional and non-limiting. An example of the construct in Figure 3C is ProC2982 (sequence number 525 complexed with sequence number 526, dimerized with a second identical monomer construct complex), and an example of the construct in Figure 3D is ProC3571 (sequence number 528 dimerized with sequence number 527 using a knob-hole Fc dimer). In some embodiments, ACC may comprise a cytokine polypeptide (e.g., IL-15) or its biologically active fragment, CM, and MM. In some embodiments, ACC may comprise a first monomer construct and a second monomer construct, each monomer construct comprising a cytokine polypeptide (e.g., IL-15) or a biologically active fragment thereof, CM, MM, and DD, and the first and second monomer constructs are dimerized via DD. In some embodiments, ACC may comprise a first monomer construct comprising a cytokine polypeptide (e.g., IL-15) or a biologically active fragment thereof, CM, MM, and DD, and a second monomer construct comprising a sucoid domain or a fragment thereof, CM, and DD, and the first and second monomer constructs are dimerized via DD. In some embodiments, ACC may comprise a first monomer construct comprising a cytokine polypeptide (e.g., IL-15) or a biologically active fragment thereof linked to DD1 via CM1, and a second monomer construct comprising MM linked to DD2, and the first and second monomer constructs are dimerized via DD1 and DD2. In some embodiments, MM is linked to DD2 via CM2 on a second monomer construct. In some embodiments, the cytokine polypeptide is linked to CM1 via a sucoid domain on a first monomer construct.In some embodiments, the ACC may comprise a cytokine polypeptide (e.g., IL-15) or a biologically active fragment thereof, CM, MM, where IL-15 is linked to a sucoid domain or a fragment thereof. In some embodiments, the ACC may comprise a first monomer construct and a second monomer construct, each monomer construct comprising a cytokine polypeptide (e.g., IL-15) or a biologically active fragment thereof, CM, MM, and DD, where the first and second monomer constructs are dimerized via DD, and each IL-15 is linked to its sucoid domain or fragment.

[0126] The activation conditions for ACC described herein are exposure to a protease capable of cleaving at least one of the cleavable moieties (CMs) in ACC. As shown in the examples, ACC activation resulted in a substantial recovery of cytokine activity. The results suggest that the conformation of cytokine components did not irreversibly change in the ACC environment.

[0127] The mature cytokine polypeptides CP1 and CP2 may be the same or different. In certain embodiments, CP1 and CP2 are the same. In other embodiments, CP1 and CP2 are different. ACC may contain additional amino acid residues at one or both of the N-terminus and / or C-terminus of CP1 and / or CP2.

[0128] Dimerization domain (DD) In each monomeric construct of the ACC dimer complex, any of a variety of dimerizing domains (DDs) may be used. Preferred DDs include both polymeric moieties (e.g., synthetic polymers, polypeptides, polynucleotides, and the like) and small molecular weight moieties (non-polymeric moieties having a molecular weight of less than about 1 kilodalton, and possibly less than about 800 daltons). A pair of DDs may be any pair of moieties known in the art to bind to each other.

[0129] For example, in some embodiments, DD1 and DD2 are members of a pair selected from the following: sucoid domain and soluble IL-15 derived from the alpha chain of the human IL-15 receptor (IL15Rα); barnase and barnstar; PKA and AKAP; adapter / docking tag molecules based on mutant RnaseI fragments; a pair of antigen-binding domains (e.g., a pair of single-domain antibodies); a soluble N-ethyl-maleimide-sensitive factor-adhering protein receptor (SNARE) module based on the interaction of protein syntaxin, synaptotagmin, synaptobrevinn, and SNAP25; a single-domain antibody (sdAb) and its corresponding epitope; an antigen Binding domains (e.g., single-chain antibodies (e.g., single-chain variable fragments (scFv)), single-domain antibodies, and similar) 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 (e.g., biotin and avidin or streptavidin), amines / aldehydes, lectins / carbohydrates; pairs of polymers that can bind to each other (e.g., pairs of sulfur-containing polymers or pairs of thiol-containing polymers (e.g., pairs of Fc domains, pairs of thiolated human serum albumin polypeptides, and similar)); and similar.

[0130] In some embodiments, DD1 and DD2 are non-polypeptide polymers. Non-polypeptide polymers may be covalently bonded to each other. In some examples, the non-polypeptide polymer may be a sulfur-containing polymer (e.g., sulfur-containing polyethylene glycol). In such cases, DD1 and DD2 are covalently bonded to each other via one or more disulfide bonds.

[0131] If the paired DD1 and DD2 are members of the paired epitope and antigen-binding domain, the epitope may be of natural or non-natural origin. Exemplary non-natural epitopes include non-natural peptides such as polyHis peptides (e.g., His tags).

[0132] In certain embodiments, DD1 and DD2 are a pair of Fc domains. As used herein, “Fc domain” means a sequence of amino acids in a single heavy chain of immunoglobulin. The pair of Fc domains are linked together to form the Fc region of the immunoglobulin.

[0133] In some embodiments, the paired Fc domains are a pair of human Fc domains (e.g., a pair of wild-type human Fc domains). In some embodiments, the human Fc domains are human IgG1 Fc domains (e.g., wild-type human IgG1 Fc domains), human IgG2 Fc domains (e.g., wild-type human IgG2 Fc domains), human IgG3 Fc domains (e.g., wild-type human IgG3 Fc domains), or human IgG4 Fc domains (e.g., wild-type human IgG4 Fc domains). In some embodiments, the human Fc domains contain a sequence that is at least 80% identical to Sequence ID No. 3 (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical).

[0134] In some embodiments, a pair of Fc domains may include a knob variant and a hole variant of the Fc domains. The knob and hole variants may interact with each other to promote dimerization. In some embodiments, the knob and hole variants may include one or more amino acid modifications within the interface between two Fc domains (e.g., CH3 domains). For example, modifications may include the amino acid substitution T366W and optionally S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other of the antibody heavy chain (numbered according to the EU index of the Kabat numbering system). Examples of knob and hole variants include the Fc variants of SEQ ID NOs. 315 and 316, and those described in U.S. Patents 5,731,168, 7,695,936, and 10,683,368, which are incorporated herein by reference in their entirety. In some embodiments, the dimerization domains each contain sequences that are at least 80% identical to sequence numbers 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 includes mutations N297Q, N297A, or N297G; in some embodiments, the human Fc domain includes mutations at positions 234 and / or 235 (e.g., L235E, or L234A and L235A (in IgG1), or F234A and L235A (in IgG4)); and in some embodiments, the human Fc domain is an IgG2Fc domain including mutations V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or combinations thereof (all according to EU numbering). Additional examples of genetically modified human Fc domains are well known to those skilled in the art.Examples of Ig heavy chain constant region amino acids in which a mutation in at least one amino acid results in 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 (in accordance with EU numbering). Examples of combinations of mutant amino acids are also known in the art, including, but are not limited to, combinations of mutations in amino acids 234, 235, and 331 such as L234F, L235E, and P331S, or combinations of mutations in amino acids 318, 320, and 322 such as E318A, K320A, and K322A.

[0135] Further examples of genetically modified Fc domains include F243L / R292P / Y300L / V305I / P396 IgG1;S239D / I332E IgG1;S239D / I332E / A330L IgG1;S298A / E333A / K334A;L234Y / L235Q / G236W / S239M / H268D / D270E / S298A IgG1 in one heavy chain, and D270E / K326D, A330M / K334E IgG;G236A / S239D / I332E IgG1;K326W / E333S IgG1;S267E / H268F / S324T in the opposite heavy chain. Examples include IgG1;E345R / E430G / S440Y, IgG1;N297A or N297Q or N297G, IgG1;L235E, IgG1;L234A / L235A, IgG1;F234A / L235A, IgG4;H268Q / V309L / A330S / P331S, IgG2;V234A / G237A / P238S / H268A / V309L / A330S / P331S, IgG2;M252Y / S254T / T256E, IgG1;M428L / N434S, IgG1;S267E / L328F, IgG1;N325S / L328F, and IgG1. In some embodiments, the manipulated Fc domain includes one or more substitutions selected from the group consisting of N297A IgG1, N297Q IgG1, and S228P IgG4.

[0136] In some embodiments, the dimerization domain is an IgG Fc region lacking an upper hinge residue. For example, Fc is a variant lacking the N-terminal sequence EPKSCDKTHT (SEQ ID NO: 387), ERK, ELKTPLGDTTHT (SEQ ID NO: 388), or ESKYGPP (SEQ ID NO: 389).

[0137] In some embodiments, DD, or DD1 and / or DD2, may further comprise serum half-life extension moieties (e.g., polypeptides that bind to serum proteins such as immunoglobulins (e.g., IgG) or serum albumin (e.g., human serum albumin(I))). Examples of half-life extension moieties include hexa-hat GST (glutathione S-transferase) glutathione affinity, calmodulin-binding peptide (CBP), Strep tag, cellulose-binding domain, maltose-binding protein, S-peptide tag, chitin-binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, and VSV epitope.

[0138] In some embodiments, DD1 and / or DD2 each contain a total of approximately 5 to 250 amino acids, approximately 5 to 200 amino acids, approximately 5 to 180 amino acids, approximately 5 to 160 amino acids, approximately 5 to 140 amino acids, approximately 5 to 120 amino acids, approximately 5 to 100 amino acids, approximately 5 to 80 amino acids, approximately 5 to 60 amino acids, approximately 5 to 40 amino acids, and approximately 5 to 20 amino acids. Approximately 5 amino acids to approximately 10 amino acids, approximately 10 amino acids to approximately 250 amino acids, approximately 10 amino acids to approximately 200 amino acids, approximately 10 amino acids to approximately 180 amino acids, approximately 10 amino acids to approximately 160 amino acids, approximately 10 amino acids to approximately 140 amino acids, approximately 10 amino acids to approximately 120 amino acids, approximately 10 amino acids to approximately 100 amino acids, approximately 10 amino acids to approximately 80 amino acids, approximately 10 amino acids to approximately 60 amino acids, approximately 10 amino acids to approximately 40 amino acids, approximately 10 amino acids to approximately 20 amino acids, approximately 2 0 amino acids to approximately 250 amino acids, approximately 20 amino acids to approximately 200 amino acids, approximately 20 amino acids to approximately 180 amino acids, approximately 20 amino acids to approximately 160 amino acids, approximately 20 amino acids to approximately 140 amino acids, approximately 20 amino acids to approximately 120 amino acids, approximately 20 amino acids to approximately 100 amino acids, approximately 20 amino acids to approximately 80 amino acids, approximately 20 amino acids to approximately 60 amino acids, approximately 20 amino acids to approximately 40 amino acids, approximately 40 amino acids to approximately 250 amino acids, approximately 40 amino acids to approximately 200 amino acids, approximately 40 amino acids to approximately 180 amino acids, approximately 40 amino acids to approximately 160 amino acids, approximately 40 amino acids to approximately 140 amino acids, approximately 40 amino acids to approximately 120 amino acids, approximately 40 amino acids to approximately 100 amino acids, approximately 40 amino acids to approximately 80 amino acids, approximately 40 amino acids to approximately 60 amino acids, approximately 60 amino acids to approximately 250 amino acids, approximately 60 amino acids to approximately 200 amino acids, approximately 60 amino acids to approximately 180 amino acids, approximately 60 amino acids to approximately 160 amino acids, approximately 60 amino acids to approximately 140 amino acids,Approximately 60 amino acids to approximately 120 amino acids, approximately 60 amino acids to approximately 100 amino acids, approximately 60 amino acids to approximately 80 amino acids, approximately 80 amino acids to approximately 250 amino acids, approximately 80 amino acids to approximately 200 amino acids, approximately 80 amino acids to approximately 180 amino acids, approximately 80 amino acids to approximately 160 amino acids, approximately 80 amino acids to approximately 140 amino acids, approximately 80 amino acids to approximately 120 amino acids, approximately 80 amino acids to approximately 100 amino acids, approximately 100 amino acids to approximately 250 amino acids, approximately 100 amino acids to approximately 200 amino acids, approximately 100 amino acids to approximately 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 160 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, approximately 140 amino acids to approximately 180 amino acids, approximately 140 amino acids to approximately 160 amino acids, approximately 160 amino acids to approximately 250 amino acids, approximately 160 amino acids It contains amino acids ~ approximately 200 amino acids, approximately 160 amino acids ~ approximately 180 amino acids, approximately 180 amino acids ~ approximately 250 amino acids, approximately 180 amino acids ~ approximately 200 amino acids, approximately 200 amino acids ~ approximately 250 amino acids, approximately 210 ~ approximately 220 amino acids, approximately 215 ~ approximately 225 amino acids, approximately 215 ~ approximately 220 amino acids, approximately 217 ~ approximately 200 amino acids, or approximately 218 ~ approximately 200 amino acids. In some embodiments, DD1 and DD2 are Fc domains containing a portion of a hinge region containing two cysteine ​​residues, a CH2 domain, and a CH3 domain, respectively. In some embodiments, DD1 and DD2 are Fc domains where, when read from the N-terminus to the C-terminus, the N-terminus is the first cysteine ​​residue in the hinge region (e.g., cysteine ​​226 of human IgG1 or IgG4, using EU numbering).

[0139] In some embodiments, the first monomer and / or the second monomer each contain, in total, approximately 150 to 800 amino acids, approximately 150 to 750 amino acids, approximately 150 to 700 amino acids, approximately 150 to 650 amino acids, approximately 150 to 600 amino acids, approximately 150 to 550 amino acids, approximately 150 to 500 amino acids, approximately 150 to 450 amino acids, approximately 150 to 400 amino acids, and approximately 150 amino acids. Mino acids ~ approximately 350 amino acids, approximately 150 amino acids ~ approximately 300 amino acids, approximately 150 amino acids ~ approximately 250 amino acids, approximately 150 amino acids ~ approximately 200 amino acids, approximately 200 amino acids ~ approximately 800 amino acids, approximately 200 amino acids ~ approximately 750 amino acids, approximately 200 amino acids ~ approximately 700 amino acids, approximately 200 amino acids ~ approximately 650 amino acids, approximately 200 amino acids ~ approximately 600 amino acids, approximately 200 amino acids ~ approximately 550 amino acids, approximately 200 amino acids ~ approximately 500 amino acids, approximately 200 amino acids ~ approximately 450 amino acids, approximately 200 amino acids ~ approximately 400 amino acids, approximately 200 amino acids ~ approximately 350 amino acids, approximately 200 amino acids ~ approximately 300 amino acids, approximately 200 amino acids ~ approximately 250 amino acids, approximately 250 amino acids ~ approximately 800 amino acids, approximately 250 amino acids ~ approximately 750 amino acids, approximately 250 amino acids ~ approximately 700 amino acids, approximately 250 amino acids ~ approximately 650 amino acids, approximately 250 amino acids ~ approximately 600 amino acids, approximately 250 amino acids ~ approximately 550 amino acids, approximately 250 amino acids ~ approximately 500 amino acids Amino acids, approximately 250 amino acids to approximately 450 amino acids, approximately 250 amino acids to approximately 400 amino acids, approximately 250 amino acids to approximately 350 amino acids, approximately 250 amino acids to approximately 300 amino acids, approximately 300 amino acids to approximately 800 amino acids, approximately 300 amino acids to approximately 750 amino acids, approximately 300 amino acids to approximately 700 amino acids, approximately 300 amino acids to approximately 650 amino acids, approximately 300 amino acids to approximately 600 amino acids, approximately 300 amino acids to approximately 550 amino acids, approximately 300 amino acids to approximately 500 amino acids,Approximately 300 amino acids to approximately 450 amino acids, approximately 300 amino acids to approximately 400 amino acids, approximately 300 amino acids to approximately 350 amino acids, approximately 350 amino acids to approximately 800 amino acids, approximately 350 amino acids to approximately 750 amino acids, approximately 350 amino acids to approximately 700 amino acids, approximately 350 amino acids to approximately 650 amino acids, approximately 350 amino acids to approximately 600 amino acids, approximately 350 amino acids to approximately 550 amino acids, approximately 350 amino acids to approximately 500 amino acids, approximately 350 amino acids to approximately 450 amino acids, approximately 350 Approximately 400 amino acids, approximately 400 amino acids, approximately 800 amino acids, approximately 400 amino acids, approximately 750 amino acids, approximately 400 amino acids, approximately 700 amino acids, approximately 400 amino acids, approximately 650 amino acids, approximately 400 amino acids, approximately 600 amino acids, approximately 400 amino acids, approximately 550 amino acids, approximately 400 amino acids, approximately 500 amino acids, approximately 400 amino acids, approximately 450 amino acids, approximately 450 amino acids, approximately 800 amino acids, approximately 450 amino acids, approximately 750 amino acids, approximately 450 amino acids 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 The 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,It may contain approximately 700 to 800 amino acids, approximately 700 to 750 amino acids, or approximately 750 to 800 amino acids.

[0140] Cuttable part (CM) In some embodiments, the ACC includes one or more CMs. The CMs may be located between two components in the ACC (e.g., between a cytokine polypeptide and an MM, between a cytokine polypeptide and a DD, and / or between a cytokine polypeptide and another component in the ACC). In some embodiments, the MM is coupled to the cytokine polypeptide via the CM, i.e., the CM is located between the interleukin and the MM.

[0141] In some embodiments, the CM is located directly or indirectly (e.g., via a linker) between the MM and the cytokine polypeptide. In some embodiments, the CM is located directly or indirectly (e.g., via a linker) between the cytokine polypeptide and the DD.

[0142] In some embodiments, the CMs used herein may include substrates of proteases reported in cancer or certain cancers. See, for example, La Roca et al., British J. Cancer 90(7):1414-1421, 2004. Suitable substrates for use in the CM components used herein include those more commonly found in cancerous cells and tissues. Therefore, in certain embodiments, the CMs include substrates of proteases more commonly found in cancer-associated disease tissues. In some embodiments, the cancer is selected from the group of gastric cancer, breast cancer, osteosarcoma, and esophageal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is HER2-positive cancer. In some embodiments, the cancers include Kaposi's sarcoma, pilocytic cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, malignant cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, breast cancer, ovarian cancer, bladder cancer, BCG-resistant nonmuscle-invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer, or testicular cancer. In some of the embodiments described above, the CM component includes a substrate of a protease(s) more commonly found in tumor tissue. For example, the protease(s) may be produced by the tumor in the subject.

[0143] Suitable CMs for use in ACC as described herein include any of the protease substrates known in the art. In some examples, CMs may include substrates of serine proteases (e.g., u-plasminogen activator (uPA, also known as urokinase)) and matryptases (also known herein as MT-SP1 or MTSP1). In some examples, CMs may include substrates of matrix metalloproteinases (MMPs). In some examples, CMs may include substrates of cysteine ​​proteases (CPs) (e.g., regmine).

[0144] In some embodiments, CM is a disintegrin and metalloproteinase (ADAM) or a disintegrin and metalloproteinase (ADAMTS) having a thrombospongin motif (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDE C1, ADAMTS1, ADAMTS4, ADAMTS5), aspartate protease (e.g., BACE, renin), aspartate cathepsin (e.g., cathepsin D) Cathepsin E), caspase (e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14), cysteine ​​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 ​​proteinase (e.g., cruzipain, regmain, otubai n)-2) Chymase, DESC1, DPP-4, FAP, Elastase, FVIIa, FiXA, Fxa, FXIa, FXIIa, Granzyme B, Guanidinobenzoate, Hepsin, HtrA1, Human neutrophil elastase, KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14), Metalloproteinase (e.g., Meprin, Neprilysin, PSMA, BMP-1), Lactoferrin, Marapsin, It may contain substrates for matryptase-2, MT-SP1 / matryptase, 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, tryptase, and uPA.

[0145] In some embodiments, the protease substrate in the CM may include polypeptide sequences that are not substantially identical to any polypeptide sequence that is spontaneously cleaved by the same protease (e.g., having an identity of 90%, 80%, 70%, 60%, or 50% or less).

[0146] In some embodiments, CM includes or consists of the sequence LSGRSDNH (SEQ ID NO: 552) or PLGLAG (SEQ ID NO: 615). In some embodiments, CM includes or consists of a consensus of any one of the sequences 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 (an application that is incorporated herein by reference in its entirety).

[0147] In some embodiments, CM is disclosed in WO2015048329, with sequence numbers 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, WO2015116933, with sequence numbers 163-267, 307-384, 402-445, 665-683, WO2016118629 (the entire application is disclosed herein by reference). (to be incorporated herein by reference) the sequences of SEQ ID NOs. 20-21, 411, 480-482, 351-369, 18, 71, 370-380, 412-415, 468, 547-554, 319-346 disclosed in (to be incorporated herein by reference), and one of the sequences of SEQ ID NOs. 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 (the application which is incorporated herein by reference in its entirety). In some examples, the CM of cysteine ​​protease contains or consists of the sequence of AAN, SAN, or GPTN (SEQ ID NO. 152). Examples of CMs include WO2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165 Also listed are those described in 143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974, which are incorporated herein by reference in their entirety.

[0148] In some embodiments, the CM includes, consists of, or is encompassed by a consensus of, any one of the sequences in Table 1, which include the following CM sequences.

[0149] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0150] In some embodiments, the CM includes or consists of a combination of the sequence examples discussed above, a C-terminal shortened variant, or an N-terminal shortened variant. Shortened variants of the aforementioned amino acid sequences suitable for use in the CM are any that retain the recognition site of the corresponding protease. These include C-terminal and / or N-terminal shortened variants containing at least three consecutive amino acids of the above amino acid sequences or at least four, at least five, at least six, or at least seven amino acids of the above amino acid sequences that retain the protease recognition site. In certain embodiments, the truncated variants of the above amino acid sequences correspond to any of the above but are amino acid sequences in which 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 are truncated at the C-terminus and / or N-terminus, and (1) have at least three amino acid residues; and (2) retain the protease recognition site. In some of the embodiments described above, the shortened CM is a CM with the N-terminal end shortened. In some embodiments, the shortened CM is a CM with the C-terminal end shortened. In some embodiments, the shortened C is a CM with both the C-terminal and N-terminal ends shortened.

[0151] In some embodiments, CM may contain a total of 3 to 25 amino acids. In some embodiments, 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.

[0152] In some embodiments, CM is reduced by at least a protease to about 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 is specifically cleaved at a rate of -1 . The rate can be measured as the substrate cleavage kinetics (k cat / K m ) as disclosed in WO2016118629.

[0153] In some embodiments of any of the activatable cytokine constructs described herein, the CM comprises a total of from about 3 amino acids to about 25 amino acids. In some embodiments, the CM comprises a total of from about 3 amino acids to about 25 amino acids, from about 3 amino acids to about 20 amino acids, from about 3 amino acids to about 15 amino acids, from about 3 amino acids to about 10 amino acids, from about 3 amino acids to about 5 amino acids, from about 5 amino acids to about 25 amino acids, from about 5 amino acids to about 20 amino acids, from about 5 amino acids to about 15 amino acids, from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 25 amino acids, from about 10 amino acids to about 20 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 25 amino acids, from about 15 amino acids to about 20 amino acids, or from about 20 amino acids to about 25 amino acids.

[0154] In some embodiments, the ACC comprises a plurality of CMs that are substrates for different proteases. In some embodiments, CM1 and CM2 in the dimer construct comprise substrates for different proteases. In some embodiments, CM1 and CM2 in the dimer construct comprise substrates for the same protease.

[0155] ACC, or the first and second monomer constructs of ACC as a dimer complex, may contain one or more additional components, including one or more linkers and the like. In some embodiments, the first monomer may include a linker positioned between CP and CM. In some embodiments, CP and CM are directly adjacent to each other.

[0156] In some embodiments, in the dimeric complex ACC, the first monomer may include a linker positioned between CP1 and CM1. In some embodiments, CP1 and CM1 are directly adjacent to each other in the first monomer. In some embodiments, the first monomer includes a linker positioned between CM1 and DD1. In some embodiments, the linker has a total length of 1 to about 15 amino acids. In some embodiments, CM1 and DD1 are directly adjacent to each other in the first monomer. In some embodiments, CM and any linker positioned between CP1 and DD1 have a total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0157] In some embodiments, the second monomer includes a linker positioned between CP2 and CM2. In some embodiments, CP2 and CM2 are directly adjacent to each other in the second monomer. In some embodiments, the second monomer includes a linker positioned between CM2 and DD2. In some embodiments, the linker has a total length of 1 to about 15 amino acids. In some embodiments, the linker includes the sequence G, GG, or GGGS (SEQ ID NO: 2). In some embodiments, CM2 (e.g., any of the cleavable portions described herein) and DD2 (e.g., any of the DDs described herein) are directly adjacent to each other in the second monomer. In some embodiments, CM and any linker positioned between CP2 and DD2 have a total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0158] Cytokine masking portion (MM) and blocking portion (BM) In some embodiments, the cytokine masking portion (MM) includes a linker positioned between a first masking subunit and a second masking subunit.

[0159] In some embodiments, the second masking subunit is encoded by a second sub-sequence of the amino acid sequence encoding the receptor polypeptide, selected from the group consisting of an amino acid sequence encoding the first receptor polypeptide and an amino acid sequence encoding the second receptor polypeptide of the cytokine receptor, wherein the first and second sub-sequences are not adjacent within the amino acid sequence encoding the first receptor polypeptide.

[0160] In some embodiments, the receptor polypeptide of a cytokine receptor is the extracellular domain of the receptor. The extracellular domain of cytokine receptors has been identified in literature such as Wang, Xinquan, et al. “Structural biology of shared cytokine receptors.” Annual review of immunology 27(2009):29-60, which is incorporated in its entirety by reference.

[0161] In some embodiments, such peptides having binding affinity to cytokines include sequences disclosed in Table 2.

[0162] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0163] In some embodiments, the second masking subunit is encoded by a second amino acid sequence encoding a blocking moiety (BM). In some embodiments, the BM is a polypeptide that binds to a cytokine polypeptide but is neither a cytokine receptor polypeptide nor a subsequence of a cytokine receptor polypeptide. Thus, the BM binds specifically and / or selectively to cytokine polypeptides and does not bind to tumor antigens or tissue antigens. In some examples, the BM may be a binding domain based on an antibody or antibody fragment (e.g., Fab fragment, F(ab')2 fragment, scFv, scAb, variable domain of camel-type nanobody (VHH), dAb, single-domain heavy chain antibody, and single-domain light chain antibody), a non-immunoglobulin protein that mimics the binding and / or structure of an antibody (e.g., anticarin, affin, affibody molecule, affimer, affitin, alpha-body, avimer, DARPin, finomer, Knitz domain peptide, monobody), as well as other manipulated scaffolds that bind to cytokine polypeptides to interfere with their binding to their targets (e.g., SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffold). In some examples, the BM may be a peptide that binds to a cytokine polypeptide and interferes with its binding to its target.

[0164] This specification provides methods for screening candidate peptides to obtain BM peptides that selectively and / or specifically bind to cytokine polypeptides, such methods may include protein display methods and screening methods for candidate peptides to identify BM described, for example, US20200308243A1, WO2009025846A2, and WO2010081173 (these documents are incorporated herein by reference in their entirety).

[0165] In some embodiments, BM includes masking subsequences disclosed in WO2022 / 197764, WO2010096838A2, WO2020 / 069398A1, and WO2019 / 246392 (these documents are incorporated herein by reference in their entirety).

[0166] In some embodiments, the BM includes an scFv having binding affinity to a cytokine.

[0167] In some embodiments, the BM is a stereomask that inhibits the binding of cytokines to their binding partners via steric hindrance.

[0168] In some embodiments, the first and second subarrays are the same. In some embodiments, the first and second subarrays are different.

[0169] In some embodiments, the first receptor polypeptide and the second receptor polypeptide are the same. In some embodiments, the first receptor polypeptide and the second receptor polypeptide are different.

[0170] In some embodiments, cytokine MMs are functionally linked to cytokines. In some embodiments, cytokine MMs are linked to cytokines directly or via linkers and positioned to have a functional relationship, so that the cytokine MMs bind to cytokines and are located in a position in the target body that inhibits the interaction between cytokine polypeptides and their binding partners.

[0171] Additional masking area (MM) In some embodiments, the ACC as herein may include one or more MMs in addition to the MMs described above. The following disclosures and embodiments, which discuss additional MMs below, also apply to BMs according to any of the embodiments described herein. In some embodiments, the additional MMs interact with the cytokine polypeptide, thereby reducing or inhibiting the interaction between the cytokine polypeptide and its binding partner. In some embodiments, the additional MMs include at least a partial or complete amino acid sequence of the naturally occurring binding partner of the cytokine polypeptide. For example, the additional MMs may be fragments of the naturally occurring binding partner. The fragments may retain 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, or less than 20% nucleic acid homology or amino acid sequence homology with the naturally occurring binding partner. As used herein, the term “naturally occurring” means, when applied to an object, the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by a human in the laboratory or is otherwise of natural origin.

[0172] In some embodiments, the additional MM includes an amino acid sequence that is not of natural origin or does not include an amino acid sequence of the natural binding partner. In certain embodiments, the MM is not the natural binding partner of the cytokine polypeptide. The additional MM may be a modified binding partner of the cytokine polypeptide that contains amino acid changes that reduce the affinity and / or binding strength to the cytokine polypeptide. In some embodiments, the additional MM does not contain, or substantially does not contain, nucleic acid homology or amino acid homology with the natural binding partner of the cytokine polypeptide. In other embodiments, the additional MM is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% or less similar to the natural binding partner of the cytokine polypeptide.

[0173] In some embodiments, the additional MM does not specifically bind to the cytokine polypeptide but still interferes with its binding to the cytokine polypeptide's binding partner through nonspecific interactions (such as steric hindrance) ("steric masking"). For example, the additional MM may be positioned within the ACC such that its tertiary or quaternary structure allows it to mask the cytokine polypeptide via charge-based interactions, thereby keeping the additional MM in place to interfere with the approach of the binding partner to the cytokine polypeptide.

[0174] In some embodiments, the additional MM may have a dissociation constant for binding to the cytokine polypeptide that is less than or equal to the dissociation constant of the cytokine polypeptide for the binding partner. In some embodiments, the additional MM, in the cleaved state, neither interferes with nor competes with the cytokine polypeptide for binding to its binding partner.

[0175] The structural properties of MMs can be selected based on factors such as the minimum amino acid sequence required to interfere with protein binding to the binding partner, the target binding partner protein-protein binding pair, the size of the cytokine polypeptide, the presence or absence of linkers, and similar factors.

[0176] In some embodiments, additional MMs are specific to the coupled cytokine polypeptide. Examples of additional MMs include MMs that have been specifically screened to bind to the binding domain of the cytokine polypeptide or its fragment (e.g., affinity masks). The Specified herein provides methods for screening MMs to obtain cytokine polypeptide-specific MMs, as well as those that bind specifically and / or selectively to the binding domain of their binding partner, such methods may include protein display methods.

[0177] In some embodiments, the additional MM is a polypeptide with an amino acid length of approximately 2 to 50. For example, the additional MM may be a polypeptide with an amino acid 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. For example, an additional MM could be a polypeptide having an amino acid 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. In some examples, an additional MM could be a polypeptide with an amino acid length greater than 50 (e.g., 100, 200, 300, 400, 500, 600, 700, 800, or greater).

[0178] In some embodiments, in the inactive state of ACC with cytokine polypeptides and interfering MM, even in the presence of a binding partner for the cytokine polypeptide, when measured in an in vitro immunoadsorption assay (e.g., as described in US20200308243A1), binding was measured at least 0.1, 0.5, 1, 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60 hours, compared to binding of the corresponding antibody without interfering MM, when measured in an in vitro immunoadsorption assay (e.g., as described in US20200308243A1). , 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, there is no or substantially no binding of cytokine polypeptides to their binding partners, or the binding of cytokine polypeptides to their binding partners is 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% or less.

[0179] The binding affinity of cytokine polypeptides to their binding partners when interfering microorganisms (MMs) are present is 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, or 50,000,000 times lower than the binding affinity of cytokine polypeptides to their binding partners when interfering MMs are absent, or 5-10, 10-100, 10 ~1,000, 10~10,000, 10~100,000, 10~1,000,000, 10~10,000,000, 100~1,000, 100~10,000, 100~1,000,000, 100~10,000,000, 1,000~10,000, 1,000~10,000, 1,000~1 It can be 00,000, 1,000~1,000,000, 1,000~10,000,000, 10,000~100,000, 10,000~1,000,000, 10,000~10,000,000, 100,000~1,000,000, or 100,000~10,000,000 times lower.

[0180] The dissociation constant of MM for the cytokine polypeptide it masks may be greater than the dissociation constant of the cytokine polypeptide for its binding partner. The dissociation constant of MM for the masked cytokine polypeptide may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the dissociation constant of the cytokine polypeptide for its binding partner. Conversely, the binding affinity of MM for the masked cytokine polypeptide may be lower than the binding affinity of the cytokine polypeptide for its binding partner. The binding affinity of MM to cytokine polypeptides 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 lower than the binding affinity of cytokine polypeptides to their binding partners.

[0181] In some embodiments, the additional MM contains either genetically encoded or non-genetically encoded amino acids. Examples of non-genetically encoded amino acids include, but are not limited to, D-amino acids, β-amino acids, and γ-amino acids. In certain embodiments, the MM contains 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% or less of genetically encoded amino acids.

[0182] In some embodiments, once released from the ACC and in a free state, the additional MM possesses biological activity or therapeutic effects (such as binding ability). For example, the free peptide may bind to the same or different binding partners. In certain embodiments, the free MM exerts therapeutic effects, imparting secondary functions to the compositions disclosed herein. In some embodiments, once released from coupling with the ACC and in a free state, the MM may not advantageously exhibit biological activity. For example, in some embodiments, the free MM does not induce an immune response in the subject.

[0183] Suitable additional MMs can be identified and / or further optimized through a screening procedure from a library of candidate ACCs having variable MMs. For example, cytokine polypeptides and CMs may be selected to obtain a desired enzyme / target combination, and MMs that give a switchable phenotype can be identified by identifying the amino acid sequences of the additional MMs by the screening procedure described below. For example, suitable MMs can be identified by using a random peptide library (e.g., peptides containing 2 to 40 amino acids or more) in the screening method disclosed herein.

[0184] This specification provides methods for screening candidate peptides to obtain cytokine polypeptide-specific MMs, such methods may include protein display methods and screening methods for candidate peptides to identify masking (MM), blocking (BM), or additional MMs as described in, for example, US20200308243A1, WO2009025846A2, and WO2010081173 (these documents are incorporated herein by reference in their entirety).

[0185] Examples of additional MM and BM peptides include polypeptides that bind to IL-15 and / or IL-2 (e.g., any one of SEQ ID NOs. 358-374).

[0186] Linker In some embodiments of any ACC described herein, mobility is obtained by introducing one or more linkers (e.g., mobile linkers) into the activatable cytokine construct at one or more junctions between domains, between sub-substrates, between sub-substrates and domains, or at any other junction where a linker would be beneficial. In some embodiments, when the ACC is provided as a conformationally constrained construct, the insertion of a mobile linker facilitates the formation and maintenance of structure in the uncleaved activatable cytokine construct. Any of the linkers described herein can confer desired mobility to facilitate inhibition of binding to a binding partner (e.g., a cytokine receptor) or to facilitate cleavage of CM by a protease. In some embodiments, fully mobile or partially mobile linkers are included in the ACC, and as a result, the linker can confer a mobile linker and one or more sub-substrates that confer less mobile structures, thereby providing the desired ACC. Some linkers may contain cysteine ​​residues, which can form disulfide bonds and reduce the mobility of the construct. In some embodiments, shortening the length of the linker or linking region reduces the activity of mature cytokine polypeptides in the ACC. In most cases, the linker length is determined by counting the number of amino acids from the N-terminus to the C-terminus, from the C-terminal amino acid of the preceding component and the N-terminus of the adjacent linker to the N-terminal amino acid of the succeeding component and the C-terminus of the adjacent linker (i.e., the linker length does not include either the C-terminal amino acid of the preceding component or the N-terminal amino acid of the succeeding component). In embodiments in which a linker is used at the N-terminus of a DD containing an Fc domain, the linker length is determined by counting the number of amino acids from the C-terminal amino acid of the preceding component and the N-terminus of the adjacent linker to the first cysteine ​​of the Fc hinge region and the C-terminus of the adjacent linker (i.e., the linker length does not include either the C-terminal amino acid of the preceding component or the first cysteine ​​of the Fc hinge region).

[0187] In some embodiments, the ACC of the present disclosure includes an amino acid interval between the proximal point of interaction between the CP and the dimerization domain (see example in Figure 4). This amino acid interval may be referred to as the linking region (LR). As used herein, the terms “linking region” or “LR” mean an extension of amino acid residues between the C-terminus of the cytokine and the amino acid residue adjacent at the N-terminus to the proximal point of interaction between the dimerization domains (i.e., the linking region does not contain the C-terminal amino acid of the cytokine or the N-terminal amino acid of the DD that forms the proximal point of interaction with the DD of the corresponding second monomer). For example, if the DD is a pair of Fc domains, the linking region is an extension of amino acid residues between the C-terminus of the cytokine and the first N-terminal cysteine ​​residue that participates in the disulfide bond of the Fc (e.g., cysteine ​​226 of the Fc domain of IgG1 or IgG4, according to EU numbering). If the dimerization domain is not a peptide, the linking region is an extension of amino acid residues from the C-terminus of the cytokine to the last amino acid. For example, if DD is a biotin-streptavidin pair, the linking region of the biotin-containing monomer is an extension of amino acid residues between the C-terminus of the cytokine and the biotin molecule, and the linking region of the streptavidin-containing monomer is an extension of amino acid residues between the C-terminus of the cytokine and the streptavidin molecule. In some embodiments, the linking region may contain 24, 18, 14, 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer amino acids (e.g., 5-14, 7-12, 7-11, or 8-11 amino acids).

[0188] In some embodiments, the additional amino acid sequence is located either N-terminal or C-terminal to either domain of the ACC. Examples include, but are not limited to, a targeting site (e.g., a ligand for a cell receptor present in a target tissue) and a serum half-life extension portion (e.g., a polypeptide that binds to a serum protein such as immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin(I))).

[0189] In some embodiments of the activatable cytokine constructs described herein, the linker comprises, in total, about 1 to about 25 amino acids (e.g., about 1 to about 24 amino acids, about 1 to about 22 amino acids, about 1 to about 20 amino acids, about 1 to about 18 amino acids, about 1 to about 16 amino acids, about 1 to about 15 amino acids, about 1 to about 14 amino acids, about 1 to about 12 amino acids, about 1 to about 10 amino acids, about 1 1 amino acid ~ approximately 8 amino acids, approximately 1 amino acid ~ approximately 6 amino acids, approximately 1 amino acid ~ approximately 5 amino acids, approximately 1 amino acid ~ approximately 4 amino acids, approximately 1 amino acid ~ approximately 3 amino acids, approximately 1 amino acid ~ approximately 2 amino acids, approximately 2 amino acids ~ approximately 25 amino acids, approximately 2 amino acids ~ approximately 24 amino acids, approximately 2 amino acids ~ approximately 22 amino acids, approximately 2 amino acids ~ approximately 20 amino acids, approximately 2 amino acids ~ approximately 18 amino acids, approximately 2 amino acids ~ approximately 16 amino acids, approximately 2 amino acids ~ approximately 15 amino acids, approximately 2 amino acids ~ approximately 14 amino acids, about 2 amino acids ~ about 12 amino acids, about 2 amino acids ~ about 10 amino acids, about 2 amino acids ~ about 8 amino acids, about 2 amino acids ~ about 6 amino acids, about 2 amino acids ~ about 5 amino acids, about 2 amino acids ~ about 4 amino acids, about 2 amino acids ~ about 3 amino acids, about 4 amino acids ~ about 25 amino acids, about 4 amino acids ~ about 24 amino acids, about 4 amino acids ~ about 22 amino acids, about 4 amino acids ~ about 20 amino acids, about 4 amino acids ~ about 18 amino acids, about 4 amino acids ~ about 16 amino acids Mino 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 to approximately 18 amino acids, approximately 5 amino acids to approximately 16 amino acids,Approximately 5 amino acids to approximately 15 amino acids, approximately 5 amino acids to approximately 14 amino acids, approximately 5 amino acids to approximately 12 amino acids, approximately 5 amino acids to approximately 10 amino acids, approximately 5 amino acids to approximately 8 amino acids, approximately 5 amino acids to approximately 6 amino acids, approximately 6 amino acids to approximately 25 amino acids, approximately 6 amino acids to approximately 24 amino acids, approximately 6 amino acids to approximately 22 amino acids, approximately 6 amino acids to approximately 20 amino acids, approximately 6 amino acids to approximately 18 amino acids, approximately 6 amino acids to approximately 16 amino acids, approximately 6 amino acids to approximately 15 amino acids, approximately 6 1 amino acid ~ approximately 14 amino acids, approximately 6 amino acids ~ approximately 12 amino acids, approximately 6 amino acids ~ approximately 10 amino acids, approximately 6 amino acids ~ approximately 8 amino acids, approximately 8 amino acids ~ approximately 25 amino acids, approximately 8 amino acids ~ approximately 24 amino acids, approximately 8 amino acids ~ approximately 22 amino acids, approximately 8 amino acids ~ approximately 20 amino acids, approximately 8 amino acids ~ approximately 18 amino acids, approximately 8 amino acids ~ approximately 16 amino acids, approximately 8 amino acids ~ approximately 15 amino acids, approximately 8 amino acids ~ approximately 14 amino acids, approximately 8 amino acids ~ approximately 12 amino acids, approximately 8 Amino acids ~ approximately 10 amino acids, approximately 10 amino acids ~ approximately 25 amino acids, approximately 10 amino acids ~ approximately 24 amino acids, approximately 10 amino acids ~ approximately 22 amino acids, approximately 10 amino acids ~ approximately 20 amino acids, approximately 10 amino acids ~ approximately 18 amino acids, approximately 10 amino acids ~ approximately 16 amino acids, approximately 10 amino acids ~ approximately 15 amino acids, approximately 10 amino acids ~ approximately 14 amino acids, approximately 10 amino acids ~ approximately 12 amino acids, approximately 12 amino acids ~ approximately 25 amino acids, approximately 12 amino acids ~ approximately 24 amino acids, approximately 12 amino acids ~ approximately 2 2 amino acids, approximately 12 amino acids to approximately 20 amino acids, approximately 12 amino acids to approximately 18 amino acids, approximately 12 amino acids to approximately 16 amino acids, approximately 12 amino acids to approximately 15 amino acids, approximately 12 amino acids to approximately 14 amino acids, approximately 14 amino acids to approximately 25 amino acids, approximately 14 amino acids to approximately 24 amino acids, approximately 14 amino acids to approximately 22 amino acids, approximately 14 amino acids to approximately 20 amino acids, approximately 14 amino acids to approximately 18 amino acids, approximately 14 amino acids to approximately 16 amino acids, approximately 14 amino acids to approximately 15 amino acids,Approximately 15 amino acids to approximately 25 amino acids, approximately 15 amino acids to approximately 24 amino acids, approximately 15 amino acids to approximately 22 amino acids, approximately 15 amino acids to approximately 20 amino acids, approximately 15 amino acids to approximately 18 amino acids, approximately 15 amino acids to approximately 16 amino acids, approximately 16 amino acids to approximately 25 amino acids, approximately 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 It may contain amino acids, approximately 18 to 25 amino acids, approximately 18 to 24 amino acids, approximately 18 to 22 amino acids, approximately 18 to 20 amino acids, approximately 20 to 25 amino acids, approximately 20 to 24 amino acids, approximately 20 to 22 amino acids, approximately 22 to 25 amino acids, approximately 22 to 24 amino acids, or approximately 24 to 25 amino acids.

[0190] In some embodiments of any ACC described herein, the linker contains, in total, 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.

[0191] In some embodiments, the ACC does not contain any linker between the CP and DD. Such an ACC may exhibit the most significant reduction in cytokine activity compared to wild-type mature cytokines. Furthermore, even in configurations where a linker is present between the CP and DD, effective cleavage of the CM located between the CP and DD may be possible. Therefore, in some embodiments, the ACC does not contain any linker between the CP and DD, and the CM between the CP and DD contains 10, 9, 8, 7, 6, 5, 4, or 3 or fewer amino acids. In some embodiments, the total number of amino acids in the LR includes 25 or fewer amino acids (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer amino acids, or any range or a specific number of amino acids selected from the range encompassed by 3 to 10 amino acids, or 5 to 15 amino acids, or 7 to 12 amino acids, or 3 to 25 amino acids).

[0192] 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 linker 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 GGGSG sequences).

[0193] In some embodiments of the ACC described herein, the linker is 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), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 214), GGGGSGGGGS (SEQ ID NO: 215), GGGGS (SEQ ID NO: 216), GS, GGGGSGS (SEQ ID NO: 217), GG This includes one or more combinations of GGSGGGGSGGGGSGS (sequence number 218), GGSLDPKGGGGS (sequence number 219), PKSCDKTHTCPPCPAPELLG (sequence number 220), SKYGPPCPPCPAPEFLG (sequence number 221), GKSSGSGSESKS (sequence number 222), GTSTGSGKSSEGKG (sequence number 223), GTSTGSGKSSEGSGSTKG (sequence number 224), and GTSTGSGKPGSGEGSTKG (sequence number 225).

[0194] Non-restrictive examples of linkers may include sequences that are at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to GGGS (SEQ ID NO: 2), GSSGGSGGSGG (SEQ ID NO: 210), GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGS (SEQ ID NO: 217), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGGGSGGGGSGGGGGS (SEQ ID NO: 235), GGSLDPKGGGGS (SEQ ID NO: 219), and GTSTGSGKPGSSEGST (SEQ ID NO: 226).

[0195] In some embodiments, the linker contains an array selected from the group GGSLDPKGGGGS (SEQ ID NO: 219), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGGGSGS (SEQ ID NO: 217), GS, (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 227), and (GGGS)n (SEQ ID NO: 228), GGSG (SEQ ID NO: 229), GGSGG (SEQ ID NO: 230), GSGSG (SEQ ID NO: 231), GSGGG (SEQ ID NO: 232), GGGSG (SEQ ID NO: 233), GSSSG (SEQ ID NO: 234), GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 235), GTSTGSGKPGSSEGST (SEQ ID NO: 226), and (GGGGS)n (SEQ ID NO: 216), where n is at least an integer of 1. In some embodiments, the linker contains a sequence selected from the group consisting of GGSLDPKGGGGS (SEQ ID NO: 219), GGGGSGGGGSGGGGSGS (SEQ ID NO: 218), GGGGSGS (SEQ ID NO: 217), and GS. In some embodiments of any ACC described herein, the linker contains a sequence selected from the group consisting of GGGSGGGGSGGGGS (SEQ ID NO: 213), GGGSGGGGSGGGGSGGGGS (SEQ ID NO: 235), and GTSTGSGKPGSSEGST (SEQ ID NO: 226). In some embodiments of the activatable cytokine constructs described herein, the linker contains a sequence selected from the group consisting of GGGSGGGGSGGGGS (SEQ ID NO: 213) or GGGGS (SEQ ID NO: 216). In some embodiments, the linker contains the sequence GGGS (SEQ ID NO: 2). In some embodiments, the linker contains a sequence of a single glycine residue (G) or two glycine residues (GG).

[0196] In some embodiments, ACC may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (for example, linker sequences identical or different from any of the exemplary linker sequences described herein or known in the art). In some embodiments, the linker comprises sulfo-SIAB, SMPB, and sulfo-SMPB, and the linker reacts with a primary amine sulfhydryl.

[0197] In some embodiments, spacers are used in the polypeptides or constructs of the present disclosure. As used herein, the terms “spacer” or “header” refer to an amino acid residue or amino acid sequence incorporated into the free end of a mature ACC (e.g., between the signal peptide and the N-terminus of the mature ACC). In some embodiments, the spacer comprises one or more glutamine (Q) residues. In some embodiments, the residues in the spacer prevent cleavage of the N-terminal amino acid by minimizing the action of aminopeptidases and / or exopeptidases. Exemplary and non-limiting spacer amino acid sequences include, or may 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 removed.

[0198] In some embodiments of the ACC described herein, the ACC is characterized in that the activity of at least one of CP or, if the ACC is a dimeric complex, CP1 and / or CP2 is reduced compared to a control level of the activity of at least one 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, and so on). In some embodiments, the control level is the activity level of the cleaved (activated) form of the ACC. In certain embodiments, the control level is the activity level of pegylated CP or pegylated CP1 and / or CP2.

[0199] In some embodiments, at least one activity is the binding affinity (K) of CP or CP1 and / or CP2 to its corresponding receptor. D ) is determined using surface plasmon resonance (e.g., performed in phosphate-buffered saline at 25°C). In certain embodiments, at least one activity is the level of lymphoma cell proliferation. In other embodiments, at least one activity is the level of JAK / STAT / ISGF3 pathway activation in lymphoma cells. In some embodiments, at least one activity is the level of SEAP production in lymphoma cells. In some embodiments, at least one activity is the level of SEAP production in cell-based assays using HEK cells. In further embodiments, at least one activity of CP or CP1 and / or CP2 is the level of cytokine-stimulated gene induction using, for example, RNA sequencing (see, e.g., Zimmerer et al., Clin. Cancer Res. 14(18):5900-5906, 2008, Hilkens et al., J. Immunol. 171:5255-5263, 2003).

[0200] In some embodiments, the ACC is characterized in that the activity of at least one of CP or CP1 and / or CP2 is reduced by at least 2 times compared to a control level of the activity of at least one of CP or CP1 and / or CP2. In some embodiments, the ACC is characterized in that the activity of at least one of CP or CP1 and / or CP2 is reduced by at least 5 times compared to a control level of the activity of at least one of CP or CP1 and / or CP2. In some embodiments, the ACC is characterized in that the activity of at least one of CP or CP1 and / or CP2 is reduced by at least 10 times compared to a control level of the activity of at least one of CP or CP1 and / or CP2. In some embodiments, the ACC is characterized in that the activity of at least one of CP or CP1 and / or CP2 is reduced by at least 20 times compared to a control level of the activity of at least one of CP or CP1 and / or CP2. In some embodiments, the ACC is characterized in that the activity of at least one of CP or CP1 and / or CP2 is reduced by at least 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 times compared to a control level of the activity of at least one of CP or CP1 and / or CP2.In some embodiments, the ACC is such that the activity of at least one of CP or CP1 and / or CP2 is reduced by at least 1 to 20 times, 200 to 500 times, 300 to 500 times, 400 to 500 times, 500 to 600 times, 600 to 700 times, 150 to 1000 times, 100 to 1500 times, 200 to 1500 times, 300 to 1500 times, 400 to 1500 times, and 500 to 1500 times compared to a control level of the activity of at least one of CP or CP1 and / or CP2. It is characterized by being present, being reduced by 1000 to 1500 times, being reduced by 100 to 1000 times, being reduced by 200 to 1000 times, being reduced by 300 to 1000 times, being reduced by 400 to 1000 times, being reduced by 500 to 1000 times, being reduced by 100 to 500 times, being reduced by 20 to 50 times, being reduced by 30 to 50 times, being reduced by 40 to 50 times, being reduced by 100 to 400 times, being reduced by 200 to 400 times, or being reduced by 300 to 400 times, being reduced by 100 to 300 times, being reduced by 200 to 300 times, or being reduced by 100 to 200 times.

[0201] In some embodiments, the ACC is characterized by producing a cleavage product after exposure to a protease(s), the cleavage product containing the activity of at least one CP1 and / or CP2. In some embodiments, the activity of at least one CP1 and / or CP2 is antiproliferative activity. In some embodiments, the control level is the EC50 value of wild-type mature cytokine, and the ratio of EC50(cleavage product) to EC50(wild-type control level) is less than about 10, or less than about 9, or less than about 8, or less than about 7, or less than about 6, or less than about 5, or less than about 4, or less than about 3, or less than about 2, or less than 1.5, or equal to about 1. In some embodiments, the EC50 of the cleavage product is approximately the same as the EC50 of wild-type mature cytokine, which demonstrates that the activity of CP1 and / or CP2 is fully or nearly fully restored after cleavage. In some embodiments, the ratio of the EC50 of the cleavage product 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, which demonstrates a good recovery of cytokine activity after protease activation. In some embodiments, ACC is characterized by having a cleavage product after protease activation, and when measured in IL-2 / IL-15 responsive HEK293 cells, the ratio of the EC50 of the cleavage product to the EC50 of recombinant IL-15 is about 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.

[0202] In some embodiments, the control level of the activity of CP or at least one of CP1 and / or CP2 is the activity of CP or CP1 and / or CP2 ("cleavage products") released from ACC after cleavage of CM or CM1 and CM2 by a protease(s). In some embodiments, the control level of the activity of CP or at least one of CP1 and / or CP2 is the activity of the corresponding wild-type mature cytokine (e.g., recombinant wild-type mature cytokine).

[0203] In some embodiments, incubation of ACC with a protease yields one or more activated cytokine products, the activity of one or more CP or CP1 and / or CP2 in the activated cytokine product(s) is greater than the activity of one or more CP or CP1 and / or CP2 in intact ACC. In some embodiments, the activity of one or more CP or CP1 and / or CP2 in the activated cytokine product(s) is at least 1-fold greater than the activity of one or more CP or CP1 and / or CP2 in ACC. In some embodiments, the activity of one or more CP or CP1 and / or CP2 in the activated cytokine product(s) is at least 2-fold greater than the activity of one or more CP or CP1 and / or CP2 in ACC. In some embodiments, the activity of one or more CP or CP1 and / or CP2 in the activated cytokine product(s) is at least 5-fold greater than the activity of one or more CP or CP1 and / or CP2 in ACC. In some embodiments, the activity of one or more CPs or CP1 and / or CP2s of the activated cytokine product(s) is at least 10 times greater than the activity of one or more CPs or CP1 and / or CP2s of the ACC. In some embodiments, the activity of one or more CPs or CP1 and / or CP2s of the activated cytokine product(s) is at least 20 times greater than the activity of one or more CPs or CP1 and / or CP2s of the ACC. In some embodiments, the activity of one or more CP or CP1 and / or CP2 of the activated cytokine product(s) is at least 1 to 20 times greater, 2 to 20 times greater, 3 to 20 times greater, 4 to 20 times greater, 5 to 20 times greater, 10 to 20 times greater, 15 to 20 times greater, 1 to 15 times greater, 2 to 15 times greater, 3 to 15 times greater, 4 to 15 times greater, 5 to 15 times greater, 10 to 15 times greater, 1 to 10 times greater, 2 to 10 times greater, 3 to 10 times greater, 4 to 10 times greater, 5 to 10 times greater, 1 to 5 times greater, 2 to 5 times greater, 3 to 5 times greater, 4 to 5 times greater, 1 to 4 times greater, 2 to 4 times greater, 3 to 4 times greater, 1 to 3 times greater, 2 to 3 times greater, or 1 to 2 times greater.

[0204] In some embodiments, ACC may contain sequences that are at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to any one of sequence numbers 423-431. In some embodiments, ACC may contain sequences that are at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to sequence number 423. In some embodiments, ACC may contain sequences that are at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to sequence number 424. In some embodiments, ACC may contain 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%) the same sequence as sequence number 425. In some embodiments, ACC may contain 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%) the same sequence as sequence number 426. In some embodiments, ACC may contain 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%) the same sequence as sequence number 427.In some embodiments, ACC may contain 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%) sequences identical to sequence number 428. In some embodiments, ACC may contain 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%) sequences identical to sequence number 430. In some embodiments, ACC may contain sequences that are at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to sequence number 431.

[0205] In some embodiments, ACC may or may not include such sequences, but may or may not include signal sequences of such sequences. Signal sequences are not particularly limited. Some non-limiting examples of signal sequences include, for example, MRAWIFFLLCLAGRALA (sequence number 343), MALTFALLVALLVLSCKSSCSVG (sequence number 344), and METDTLLLWVLLLWVPGSTG (sequence number 345).

[0206] Various exemplary embodiments of these activatable cytokine constructs are described below. These embodiments can be used in any combination, without limitation, in the methods provided herein. Exemplary embodiments of activatable cytokine constructs and methods for producing activatable cytokine constructs are described below.

[0207] In some embodiments, ACC includes CP1 selected from sequence numbers 402-422, CM1 selected from sequence numbers 5-118, 131-209, 251-314, 432-499, 530-599, and 603-719, and DD1, dimerized with CP2 selected from sequence numbers 402-422, CM2 selected from sequence numbers 5-118, 131-209, 251-314, 432-499, 530-599, and 603-719, and DD2. In some embodiments, ACC may include linkers selected from SEQ ID NO: 2 and 210-235, 245, or 250 between CP1 and CM1 and / or between CM1 and DD1, and linkers selected from SEQ ID NO: 2 and 210-235, 245, or 250 between CP2 and CM2 and / or between CM2 and DD2. In some embodiments, ACC includes DD1 and / or DD2 having amino acid sequences that are at least 80% identical to SEQ ID NO: 3 or SEQ ID NO: 4 (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical). In some embodiments, ACC includes DD1 having an amino acid sequence that is at least 80% identical to SEQ ID NO: 315 or SEQ ID NO: 316 (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical). In some embodiments, ACC includes DD2 having an amino acid sequence that is at least 80% identical to SEQ ID NO: 315 or SEQ ID NO: 316 (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical).

[0208] Conjugation with drugs This disclosure also provides methods and materials for incorporating additional elements into any of the isolated polypeptides and ACCs described herein, including, for example, a targeted portion to facilitate delivery to a cell or tissue of interest, a drug (e.g., a therapeutic agent, an antineoplastic agent), a toxin, or a fragment thereof.

[0209] In some embodiments, ACC can be conjugated with a cytotoxic agent (including, but not limited to, toxins (e.g., enzymatically active toxins or fragments thereof originating from bacteria, fungi, plants, or animals) or radioisotopes). In some embodiments of any of the ACC described herein, the activatable cytokine construct can be conjugated with a cytotoxic agent (including, but not limited to, toxins (e.g., enzymatically active toxins or fragments thereof originating from bacteria, fungi, plants, or animals) or radioisotopes).

[0210] Non-limiting exemplary cytotoxic agents that can be conjugated with any of the ACCs described herein include: drastatin and its derivatives (e.g., auristatin E, AFP, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), desmethyl auristatin E (DMAE), auristatin F, desmethyl auristatin F (DMAF), drastatin 16 (DmJ), drastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone), metansinoids (e.g., DM-1, DM-4), metansinoid derivatives, duocalmycin, alpha-amanitin, turvostatin, fenstatin, hydroxyfen This includes statins, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocomstatin, pyrrolobenzimidazole (PBI), sibrostatin 6, doxaliform, semadin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracyclines, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecin, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaprysiatoxin, KahaMide-F, discodermolide, and ectinacidin.

[0211] Non-limiting, exemplary enzymatically active toxins that can be conjugated to any of the ACCs described herein include diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, lysine A chain, abrin A chain, modesin A chain, α-salsin, Aleuriies fordii protein, diansin protein, Phytoiaca Americana protein (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitors, curcin, crotirs, sapaonaria officinalis inhibitors, geionin, mitogeliin, restrictosin, phenomycin, neomycin, and trichothecenes.

[0212] Examples of non-exclusive, exemplary anti-cancer agents that can be conjugated to any of the ACCs described herein include adriamycin, serubidin, bleomycin, alkeran, vervan, oncovin, fluorouracil, methotrexate, thiotepa, bisanthren, novantrone, thioguanine, procarbazine, and cytarabine.

[0213] Examples of non-exclusive, exemplary antiviral agents that can be conjugated to any of the ACCs described herein include acyclovir, Villa A, and Symmetrel.

[0214] Examples of non-limiting, exemplary antifungal agents that can be conjugated to any of the ACCs described herein include nystatin.

[0215] Examples of non-limiting, exemplary conjugable detection reagents that can be conjugated to any of the ACCs described herein include fluorescein and its derivatives, and fluorescein isothiocyanate (FITC).

[0216] Non-limiting exemplary antibacterial agents that can be conjugated with any of the activatable cytokine constructs described herein include aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin.

[0217] Non-limiting exemplary 3-beta,16-beta,17-alpha-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-beta-D-xylopyranosyl)-(1->3)-(2-O-acetyl-alpha-L-arabinopyranoside)(OSW-1), which can be conjugated with any of the activatable cytokine constructs described herein, include s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterlin, cephalotaxin, homoharringionine, pyrrolobenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepines, calcicheamicins, podophyiitoxins, taxanes, and vinca alkaloids.

[0218] Non-limiting exemplary radiopharmaceuticals that can be conjugated with any of the activatable cytokine constructs described herein include: 123 I, 89 Zr, 125 I, 131 I, 99 mTC, 201 T1, 62 Cu, 18 F, 68 Ga, 13 N, 15 O, 38 K, 82 Rb, 111 In, 133 Xe, 11 C, and 99 It contains mTc (technetium).

[0219] Examples of non-limiting heavy metals that can be conjugated to any of the ACCs described herein include barium, gold, and platinum.

[0220] Examples of non-limiting, exemplary anti-mycoplasma agents that can be conjugated to any of the ACCs described herein include tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0221] Those skilled in the art will understand that a wide variety of possible parts can be conjugated to any of the activatable cytokine constructs described herein. The conjugate may involve any chemical reaction that results in the joining of two molecules, insofar as ACC and the other parts retain their respective activities. The conjugate may involve many chemical reaction mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complexation. In some embodiments, the preferred bond is covalent bonding. Covalent bonding can be achieved either by direct condensation of existing side chains or by the incorporation of external crosslinking molecules. Many divalent or polyvalent binders are useful for conjugating any of the activatable cytokine constructs described herein. For example, the conjugate may contain organic compounds, such as thioesters, carbodiimides, succinimides, glutaraldehyde, diazobenzene, and hexamethylenediamine. In some embodiments, the activatable cytokine construct may contain, or otherwise introduce, one or more non-natural amino acid residues to provide a site suitable for conjugation.

[0222] In some embodiments of the ACC described herein, the drug and / or conjugate is attached to the antigen-binding domain by disulfide bonds (e.g., disulfide bonds on a cysteine ​​molecule). Since many cancers spontaneously release high levels of glutathione, a reducing agent, glutathione present in the cancerous tissue microenvironment can reduce disulfide bonds, subsequently releasing the drug and / or conjugate at the delivery site.

[0223] In some embodiments of the ACC described herein, when the conjugate binds to the target within a target site (e.g., diseased tissue (e.g., cancerous tissue)) in the presence of complement, the amide or ester bond attaching the conjugate and / or drug to the linker is cleaved, resulting in the release of the conjugate and / or drug in an activated state. When these conjugates and / or drugs are administered to a subject, they achieve delivery and release at the target site (e.g., diseased tissue (e.g., cancerous tissue)). These conjugates and / or drugs are particularly effective for in vivo delivery of any of the conjugates and / or drugs described herein.

[0224] In some embodiments, the linker is not cleavable by complement system enzymes. For example, since complement activation ultimately lyses target cells, the conjugate and / or drug is released without complement activation. In such embodiments, the conjugate and / or drug is delivered to the target cell (e.g., hormone, enzyme, corticosteroid, neurotransmitter, or gene). Furthermore, the linker becomes more readily cleavable by serum proteases, and the conjugate and / or drug is slowly released at the target site.

[0225] In some embodiments of the ACC described herein, the conjugate and / or drug is designed so that the conjugate and / or drug is delivered to a target site (e.g., diseased tissue (e.g., cancerous tissue)) but the conjugate and / or drug is not released.

[0226] In some embodiments of any ACC described herein, the conjugate and / or agent is added to the ACC directly or via an inclementable linker. Exemplary inclementable linkers include amino acids (e.g., D-amino acids), peptides, or other organic compounds that can be modified to include functional groups that can be subsequently utilized in addition to the ACC by the methods described herein.

[0227] In some embodiments of any ACC described herein, the ACC includes at least one conjugate site to a drug. In some embodiments, all potential conjugate sites are available for conjugation with a drug. In some embodiments, one or more conjugate 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 sulfide bonds but not in intrachain disulfide bonds, and / or sulfur atoms of cysteine ​​or other sulfur atom-containing amino acid residues. In such cases, the residues may be of natural origin in the protein construct or may be incorporated into the protein construct by methods including, but not limited to, site-directed mutagenesis, chemical transformation, or accidental incorporation of non-natural amino acids.

[0228] This disclosure also provides methods and materials for preparing ACC for conjugates. In some embodiments of any ACC described herein, the ACC is modified to contain one or more interchain disulfide bonds. For example, the disulfide bonds in the ACC can be reduced after exposure to a reducing agent, for example, without limitation, TCEP, DTT, or β-mercaptoethanol. In some cases, the reduction of the disulfide bonds is only partial. As used herein, the term "partial reduction" means a situation in which an ACC comes into contact with a reducing agent and some of all available conjugate sites are reduced (e.g., not all disulfide bonds are reduced). In some embodiments, an activatable cytokine construct is partially reduced if, after contact with a reducing agent, less than 99% of all available conjugate sites (e.g., less than 98%, less than 97%, less than 96%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%) are reduced. In some embodiments, an ACC having reduction at one or more interchain disulfide bonds is conjugated to a drug that is reactive with free thiols.

[0229] This disclosure also provides methods and materials for conjugating therapeutic agents to specific locations on ACC. In some embodiments of ACC described herein, ACC is modified so that a therapeutic agent can be conjugated to ACC at specific locations on ACC. For example, ACC may be partially reduced to facilitate conjugation to ACC. In such cases, the partial reduction of ACC occurs such that the conjugation site of ACC is not reduced. In some embodiments, the conjugation site(s) on ACC are selected to facilitate the conjugation of the drug at specific locations on the protein structure. Various factors can affect the “level of reduction” of ACC during treatment with a reducing agent. For example, to achieve partial reduction of ACC using the methods and materials described herein, optimization of the ratio of reducing agent to ACC, incubation length, incubation temperature, and / or pH of the reduction reaction solution may be required, but are not limited to these. Partial reduction of ACC can be achieved using any suitable combination of factors (e.g., the ratio of reducing agent to ACC, the incubation period and temperature with the reducing agent, and / or the pH of the reducing agent) (e.g., overall reduction of available conjugate sites, or reduction of specific conjugate sites).

[0230] The effective ratio of a reducing agent to ACC can be any ratio that reduces ACC at least partially (e.g., overall reduction of available conjugation sites, or reduction of specific conjugation sites) in a manner that allows for conjugation to the drug. In some embodiments, the ratio of reducing agent to ACC is in the range of approximately 20:1 to 1:1, approximately 10:1 to 1:1, approximately 9:1 to 1:1, approximately 8:1 to 1:1, approximately 7:1 to 1:1, approximately 6:1 to 1:1, approximately 5:1 to 1:1, approximately 4:1 to 1:1, approximately 3:1 to 1:1, approximately 2:1 to 1:1, approximately 20:1 to 1:1.5, approximately 10:1 to 1:1.5, approximately 9:1 to 1:1.5, approximately 8:1 to 1:1.5, approximately 7:1 to 1:1.5, approximately 6:1 to 1:1.5, approximately 5:1 to 1:1.5, approximately 4:1 to 1:1.5, approximately 3:1 to 1:1.5, approximately 2:1 to 1:1.5, approximately 1.5:1 to 1:1.5, or approximately 1:1 to 1:1.5. In some embodiments, the ratio is in the range of approximately 5:1 to 1:1. In some embodiments, the ratio is in the range of approximately 5:1 to 1.5:1. In some embodiments, the ratio is in the range of approximately 4:1 to 1:1. In some embodiments, the ratio is in the range of approximately 4:1 to 1.5:1. In some embodiments, the ratio is in the range of approximately 8:1 to approximately 1:1. In some embodiments, the ratio is in the range of approximately 2.5:1 to 1:1.

[0231] Effective incubation times and temperatures for treating ACC with a reducing agent may be any time and temperature that at least partially reduces ACC in a manner that allows for conjugation of the agent with ACC (e.g., overall reduction of potential conjugation sites, or reduction at specific conjugation sites). In some embodiments, incubation times and temperatures for treating ACC are within the range of about 1 hour at 37°C to about 12 hours at 37°C (or any partial range therein).

[0232] The pH effective for the reduction reaction to treat ACC with a reducing agent may be any pH that at least partially reduces ACC in a manner that allows conjugation of ACC with the agent (e.g., overall reduction of potential conjugation sites, or reduction at specific conjugation sites).

[0233] When partially reduced ACC comes into contact with a thiol-containing agent, the agent can conjugate to the interchain thiols within the ACC. The agent can be modified in a manner that incorporates thiols using a thiol-containing reagent (e.g., cysteine ​​or N-acetylcysteine). For example, ACC may be partially reduced by incubation with a reducing agent (e.g., TCEP) at approximately 37°C for approximately 1 hour, with the ratio of reducing agent to ACC being a desired ratio. The effective ratio of reducing agent to ACC may be any ratio that partially reduces at least two interchain disulfide bonds located in the ACC (e.g., overall reduction of potential conjugation sites, or reduction at specific conjugation sites) in a manner that allows for the conjugation of the thiol-containing agent.

[0234] In some embodiments of ACC described herein, ACC is reduced by a reducing agent in a manner that avoids the reduction of any intrachain disulfide bonds. In some embodiments of ACC described herein, ACC is reduced by a reducing agent in a manner that avoids the reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0235] In some embodiments of the ACC described herein, the ACC may also contain a drug conjugated to the ACC. In some embodiments, the conjugated drug is a therapeutic agent.

[0236] In some embodiments, the agent (e.g., an agent conjugated to an activatable cytokine construct) is a detectable moiety, such as a label or other marker. For example, the agent is a radiolabeled amino acid, one or more biotinyl moieties detectable by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity that can be detected 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, or contains these. In some embodiments, the detectable moiety is attached by a spacer molecule.

[0237] In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is linked to the ACC using a carbohydrate moiety, sulfhydryl group, amino group or carboxylate group.

[0238] In some embodiments of any of the ACCs described herein conjugated to an agent, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to the ACC via a linker and / or CM (also referred to as a cleavable sequence). In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to a cysteine or lysine within the ACC. In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to another residue of the ACC, such as a residue disclosed herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a non-cleavable linker. Table 3 shows some non-limiting examples of cleavable moieties and / or linkers.

[0239] [Table 3]

[0240] Those skilled in the art will recognize that a variety of potential moieties can be coupled to the ACCs of the present disclosure. (See, e.g., “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr (eds), Carger Press, New York, (1989) (the entire contents of which are incorporated herein by reference)). In general, an effective conjugate of a drug (e.g., a cytotoxic agent) with an ACC can be achieved by any chemical reaction that binds the drug to the ACC while also allowing the drug and the ACC to retain functionality.

[0241] In some embodiments of any of the ACCs conjugated to a drug, various bifunctional protein coupling agents can be used to conjugate the drug to the ACC, including, without limitation, N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., tolylene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, a carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agent can be used to conjugate a radioactive nucleotide to the ACC. (See, e.g., WO94 / 11026).

[0242] Suitable linkers and CMs are described in the literature. (For example, see Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled with ACC via an oligopeptide linker. In some embodiments, preferred linkers include (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G)); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamide]hexanoate (Pierce Chem. Co., catalog no. 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamide]hexanoate (Pierce Chem. Co., catalog no. 2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimidide) conjugated with EDC: Pierce This includes Chem.Co., catalog number 24510). Additional linkers include, but are not limited to, SMCC, sulfoSMCC, SPDB, or sulfoSPDB.

[0243] The above-mentioned CMs and linkers contain components with different attributes, and therefore result in conjugates with different physiological and chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Linkers containing NHS esters are less soluble than sulfo-NHS esters. Furthermore, linker SMPTs contain sterically hindered disulfide bonds and can form highly stable conjugates. Since disulfide bonds are cleaved in vitro, they are generally less stable than other bonds, resulting in fewer usable conjugates. Sulfo-NHS can particularly enhance the stability of carbodiimide coupling. When a carbodiimide coupling agent (such as EDC) is used in combination with sulfo-NHS, an ester with increased hydrolysis resistance is formed compared to the carbodiimide coupling reaction alone.

[0244] In some embodiments of ACC, a drug can be conjugated to ACC using a modified amino acid sequence incorporated into the amino acid sequence of ACC. By inserting conjugable amino acids at specific positions within the amino acid sequence of ACC, the protein construct can be designed so that the arrangement and / or dosage of the conjugated drug (e.g., a cytotoxic agent) can be controlled. For example, ACC can be modified to include cysteine ​​amino acid residues at positions on a first monomer, second monomer, third monomer, and / or fourth monomer that provide a reactive thiol group, do not negatively affect protein folding and / or construction, and do not alter target properties. In some embodiments, ACC can be modified to include one or more non-native amino acid residues within its amino acid sequence to provide a site suitable for conjugation. In some embodiments, ACC can be modified to include an enzymatically activatable peptide sequence within its amino acid sequence.

[0245] nucleic acid This specification provides nucleic acids comprising a sequence encoding an isolated polypeptide or ACC, or, if the ACC is a dimeric complex, nucleic acids comprising a sequence encoding a first monomer construct (or the protein portion of the first monomer construct) (e.g., any of the first monomer constructs described herein) and a second monomer construct (or the protein portion of the second monomer construct) (e.g., any of the second monomer constructs described herein) of any of the ACCs described herein. In some embodiments, a pair of nucleic acids together encodes the first monomer construct (or the protein portion of the first monomer construct) and the second monomer construct (or the protein portion of the second monomer construct). In some embodiments, the nucleic acid sequence encoding the first monomer construct (or the protein portion of the first monomer construct) is at least 70% identical to the nucleic acid sequence encoding the second monomer construct (or the protein portion of the second monomer construct) (for example, 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).

[0246] 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 encodes the protein portion of the first monomer construct and the protein portion of the second monomer construct, and the protein portions are subsequently conjugated to DD1 and DD2 portions, respectively (in a subsequent conjugation step).

[0247] 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.

[0248] This disclosure includes polynucleotides encoding proteins or parts thereof as described herein, and the use of such polynucleotides for the production of proteins and / or for therapeutic purposes. Such polynucleotides may include DNA and RNA molecules encoding proteins as defined herein (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.). This disclosure includes compositions comprising such polynucleotides. In some embodiments, such compositions are used therapeutically or prophylactically.

[0249] Modifications can be introduced into nucleotide sequences using standard methods known in the field (such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis).

[0250] vector This specification provides vectors and sets of vectors containing any of the nucleic acids described herein. Those skilled in the art will be able to select a suitable vector or set of vectors (e.g., an expression vector) to produce any of the ACCs described herein, and to express any of the ACCs described herein using the vector or set of vectors. For example, in the selection of a vector or set of vectors, cells must be considered, since the vector(s) may need to be incorporated into and / or replicated within the chromosomes of a cell. Exemplary vectors usable for producing ACCs are also described below.

[0251] As used herein, the term “vector” means a polynucleotide capable of inducing the expression of recombinant proteins (e.g., the first or second monomer) within a cell (e.g., any of the cells described herein). A “vector” can deliver nucleic acids and their fragments into a host cell and contains regulatory sequences (e.g., promoters, enhancers, poly(A) signals). Exogenous polynucleotides may be inserted into an expression vector for expression. The term “vector” also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0252] Methods for constructing vectors containing any of the nucleic acids described herein and suitable for the transformation of cells (e.g., mammalian cells) are well known in the art. For example, Sambrook et al., Eds. "Molecular Cloning: A Laboratory Manual," 2 nd See Ed., Cold Spring Harbor Press, 1989 and Ausubel et., Eds., "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0253] Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenovirus vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector), as well as any Gateway® vector. A vector may, for example, contain a cis-acting element sufficient for expression, and other elements for expression may be supplied by host mammalian cells or in an in vitro expression system. Those skilled in the art will be able to select vectors and mammalian cells suitable for the preparation of any of the ACCs described herein.

[0254] In some embodiments of the ACC described herein, the ACC is biosynthetically produced using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0255] In some embodiments, the vector comprises nucleic acids encoding a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the vector is an expression vector.

[0256] In some embodiments, the paired vector contains together a pair of nucleic acids encoding a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the paired vector is a paired expression vector.

[0257] cell This specification also provides host cells containing any of the vectors or vector sets described herein, each containing one of the nucleic acids described herein.

[0258] Methods for introducing nucleic acids and vectors (e.g., any of the vectors or vector sets described herein) into cells are known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids into cells include lipofection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transfection (e.g., adenovirus transfection, lentivirus transfection), nanoparticle transfection, and electroporation.

[0259] In some embodiments, the introduction step includes introducing a vector (e.g., any of the vectors or vector sets described herein) containing nucleic acids encoding monomers that constitute any of the ACCs described herein into the cells.

[0260] In some embodiments of any of the methods described herein, ACC can be produced 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 can include, for example, mammalian cells, insect cells, fungal cells, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell (such as Saccharomyces cerevisiae). In some embodiments, the eukaryotic cell is a higher eukaryotic cell (such as a mammalian cell, avian cell, plant cell, 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 fetal kidney cells (e.g., HEK293 cells)).

[0261] In some embodiments, the cell contains nucleic acids encoding any one of the first monomer and the second monomer of any of the ACCs described herein. In some embodiments, the cell contains a pair of nucleic acids encoding the first monomer and the second monomer of any of the ACCs described herein together. In some aspects, the nucleic acids encoding the first monomer and the second monomer are integrated into the genomic DNA of the host cell.

[0262] Method for producing an activatable cytokine construct Provided herein is a method for making any of the ACCs described herein, the method comprising: (a) culturing any of the recombinant host cells described herein in a liquid medium under conditions sufficient to produce the ACC; and (b) recovering the ACC from the host cell and / or the liquid medium.

[0263] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions favorable for cell proliferation, cell differentiation, and cell growth. For example, cells can be cultured by contacting them (e.g., any of the cells described herein) with a cell medium containing the necessary growth factors and sufficient adjuvants to support cell viability and growth.

[0264] In some embodiments of any of the methods described herein, the method further includes isolating the recovered ACC. Non-limiting examples of isolation methods include ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand affinity chromatography, ion exchange chromatography (e.g., anionic or cationic), and hydrophobic interaction chromatography.

[0265] In some embodiments, the disclosure includes a method of inducing cells to produce a protein moiety of a first monomer construct comprising CP1, CM1, MM2, and CM3, and a protein moiety of a second monomer construct comprising CP2 and CM2 and optionally MM2 and CM4, and subsequently conjugating the protein moieties to DD1 and DD2 moieties, respectively.

[0266] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions, which enable the formation and maintenance of ACC dimerization by forming disulfide bonds between dimerizing domains.

[0267] In some embodiments of any of the methods described herein, the method further includes preparing isolated ACC into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any of the isolated ACC described herein can be prepared for any route of administration (e.g., intravenous, intratumor, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0268] This specification also provides ACC prepared by any of the methods described herein. Compositions (e.g., pharmaceutical compositions) containing any of the ACC prepared by any of the methods described herein are also provided. This specification also provides kits containing at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0269] In some embodiments, the ACC disclosed herein includes cytokine variants. For example, variants having advantageous properties compared to wild-type cytokines (e.g., less aggregation compared to a control ACC that does not contain wild-type cytokine polypeptide or mutant cytokine polypeptide) can be used. In some embodiments, the disclosure provides a method for producing ACC, the method comprising: culturing cells containing the polynucleotide encoding the ACC herein in a liquid medium under conditions sufficient for ACC production; purifying the ACC using affinity chromatography such that the purified polypeptide has a monomer purity of at least about 40%; and recovering the ACC from the cells or liquid culture medium. In some embodiments, the purified polypeptide has a monomer purity of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0270] In another aspect, the disclosure also provides compositions in which at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the purified ACC is in monomeric form (for example, compositions produced during the process of making ACC).

[0271] Treatment method This specification provides a method for treating a target disease (for example, cancer (for example, any of the cancers described herein)), comprising administering a therapeutically effective dose of any of the ACCs described herein to the target.

[0272] As used herein, the term “Subject” refers to any mammal. In some embodiments, the subject is a feline (e.g., cat), a canid (e.g., dog), an equid (e.g., horse), a rabbit, a pig, a rodent (e.g., mouse, rat, hamster, or guinea pig), a non-human primate (e.g., simian (e.g., monkey (e.g., baboon, marmoset)), or ape (e.g., chimpanzee, gorilla, orangutan, or gibbon), or a human. In some embodiments, the subject is a human.

[0273] In some embodiments, subjects are pre-identified or diagnosed with a disease (e.g., cancer (e.g., any of the cancers described herein)).

[0274] As used herein, the term “treat” includes reducing the severity, frequency, or number of one or more symptoms or signs of a disease (e.g., cancer (e.g., any of the cancers described herein)) in a subject (e.g., any of the subjects described herein). In some embodiments where the disease is cancer, treatment results in a reduction of cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduction in the risk of cancer recurrence in a subject with cancer.

[0275] In some embodiments of any of the methods described herein, the disease is cancer. Also provided herein are methods for treating a subject in need of treatment (e.g., any of the exemplary subjects described herein or known in the art), comprising administering to the subject a therapeutically effective amount of any of the ACCs described herein or any of the compositions described herein (e.g., pharmaceutical compositions).

[0276] In some embodiments of these methods, the subject is identified or diagnosed with cancer. Non-limiting examples of cancer include solid tumors, hematological malignancies, sarcomas, osteosarcomas, gliablastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, lymphomas (e.g., B-cell lymphoma, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, malignant cutaneous T-cell lymphoma), leukemias (e.g., pilocytic cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic Examples include myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), spinal dysplasia 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, colorectal cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), squamous cell head and neck cancer, endometrial cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is Burkitt lymphoma. In some embodiments, the subjects are identified or diagnosed with familial cancer syndromes (such as Lee-Fraumeni syndrome, familial breast-ovarian cancer (BRCA1 mutation or BRAC2 mutation) syndrome). The methods disclosed are also useful for the treatment of non-solid tumors. Exemplary solid tumors include malignant tumors 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 tract (e.g., tumors of the kidneys, urothelium, or testes), pharynx, prostate, and ovaries). Exemplary adenocarcinomas include colorectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, and small intestine cancer.

[0277] Exemplary cancers described by the National Cancer Institute include: acute lymphoblastic leukemia (adult); acute lymphoblastic leukemia (child); acute myeloid leukemia (adult); adrenocortical carcinoma; adrenocortical carcinoma (child); AIDS-associated lymphoma; AIDS-associated malignant tumor; anal cancer; astrocytoma (childhood cerebellar); astrocytoma (childhood cerebral); cholangiocarcinoma (extrahepatic); bladder cancer; bladder cancer (child); bone cancer, osteosarcoma / malignant fibrous histiocytoma; brainstem glioma (child); brain tumor (adult); brain tumor, brainstem glioma (child); brain tumor, cerebellar astrocytoma (child); brain tumor, cerebral astrocytoma / malignant glioma (child); brain tumor, ependymal Tumors (children); brain tumors, medulloblastoma (children); brain tumors, supratentorial primitive neuroectodermal tumors (children); brain tumors, optic tract and hypothalamic gliomas (children); brain tumors (children (other)); breast cancer; breast cancer and pregnancy; breast cancer (children); breast cancer (men); bronchial adenoma / carcinoid (children); carcinoid tumors (children); carcinoid tumors (gastrointestinal); adrenocortical cell carcinoma; islet cell carcinoma; cancer of unknown primary origin; primary central nervous system lymphoma; cerebellar astrocytoma (children); cerebral astrocytoma / malignant glioma (children); cervical cancer; childhood cancer; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorder; details of tendon sheaths Eusarcoma; Colon cancer; Colorectal cancer (childhood); Cutaneous T-cell lymphoma; Endometrial cancer; Ependymoma (childhood); Ovarian epithelial carcinoma; Esophageal cancer; Esophageal cancer (childhood); Ewing family tumors; Extracranial germ cell tumors (childhood); Extragonadal germ cell tumors; Extrahepatic bile duct cancer; Eye cancer, intraocular melanoma; Eye cancer, retinoblastoma; Gallbladder cancer; Stomach cancer; Stomach cancer (childhood); Gastrointestinal carcinoid tumors; Extracranial germ cell tumors (childhood); Extragonadal germ cell tumors; Germ cell tumors, ovary; Germ cell tumors, gestational trophoblastoma; Pediatric brainstem glioma; Pediatric optic tract and hypothalamic glioma; Hairy cell leukemia; Head and neck cancer; Adult hepatocellular carcinoma (primary) ); Primary hepatocellular carcinoma (hepatic cancer) in children; Hodgkin's lymphoma (adult); Hodgkin's lymphoma (child); Hodgkin's lymphoma during pregnancy; Hypopharyngeal cancer; Hypothalamic and optic tract glioma (child); Intraocular melanoma; Islet cell carcinoma (endocrine pancreatic); Kaposi's sarcoma; Renal cancer; Laryngeal cancer; Laryngeal cancer (child); Acute lymphoblastic leukemia (adult); Acute lymphoblastic leukemia (child); Acute myeloid leukemia (adult); Acute myeloid leukemia (child); Chronic lymphocytic leukemia; Chronic myeloid leukemia; Hairy cell leukemia; Lip and oral cancer; Primary liver cancer (adult); Primary liver cancer (child); Non-small cell lung cancer; Small cell lung cancer;Adult acute lymphoblastic leukemia; pediatric acute lymphoblastic leukemia; chronic lymphocytic leukemia; AIDS-associated lymphoma; primary central nervous system lymphoma; cutaneous T-cell lymphoma; Hodgkin lymphoma (adult); Hodgkin lymphoma (child); Hodgkin lymphoma during pregnancy; non-Hodgkin lymphoma (adult); non-Hodgkin lymphoma (child); non-Hodgkin lymphoma during pregnancy; primary central nervous system lymphoma; Waldenström macroglobulinemia; male breast cancer; malignant mesothelioma (adult); malignant mesothelioma (child); malignant thymoma; medulloblastoma (child); melanoma; intraocular melanoma; Merkel cell carcinoma; malignant Mesothelioma; Metastatic cervical squamous cell carcinoma of unknown primary origin; Multiple endocrine neoplasia syndrome (childhood); Multiple myeloma / plasmacytic neoplasm; Mycosis fungoides; Myelodysplastic syndrome; Chronic myeloid leukemia; Acute myeloid leukemia in children; Multiple myeloma; Chronic myeloproliferative disorder; Nasal cavity and paranasal sinus cancer; Nasopharyngeal cancer; Nasopharyngeal cancer (childhood); Neuroblastoma; Non-Hodgkin lymphoma (adult); Non-Hodgkin lymphoma (childhood); Non-Hodgkin lymphoma during pregnancy; Non-small cell lung cancer; Oral cancer (childhood); Oral and lip cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer (childhood); Ovarian epithelial carcinoma; Ovarian germ cell tumor; Low-grade latent ovarian cancer Primary tumors; pancreatic cancer; pancreatic cancer (childhood); islet cell pancreatic cancer; sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pheochromocytoma; pineal and supratentorial primitive neuroectodermal tumors (childhood); pituitary tumors; plasma cell neoplasms / multiple myeloma; pleuroblastoma; pregnancy and breast cancer; pregnancy and Hodgkin lymphoma; pregnancy and non-Hodgkin lymphoma; primary central nervous system lymphoma; primary liver cancer (adult); primary liver cancer (childhood); prostate cancer; rectal cancer; renal cell carcinoma (kidney); renal cell carcinoma (childhood); renal pelvis and ureter, transitional cell carcinoma; retinoblastoma; rhabdomyosarcoma (childhood); salivary gland cancer; salivary gland cancer (childhood); sarcoma, Ewing family tumors Scars; Kaposi's sarcoma; bone sarcoma (osteosarcoma) / malignant fibrous histiocytoma; sarcoma, rhabdomyosarcoma (childhood); soft tissue sarcoma (adult); soft tissue sarcoma (childhood); Sézary syndrome; skin cancer; skin cancer (childhood); skin cancer (melanoma); Merkel cell carcinoma; small cell lung cancer; small intestine cancer; soft tissue sarcoma (adult); soft tissue sarcoma (childhood); metastatic cervical squamous epithelial tumor of unknown primary origin; gastric cancer; gastric cancer (childhood); supratentorial primitive neuroectodermal tumor (childhood); cutaneous T-cell lymphoma; testicular cancer; thymoma (childhood); malignant thymoma; thyroid cancer; thyroid cancer (childhood); transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic neoplasm;This includes cancers of unknown primary site in children; abnormal cancers in children; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; optic tract and hypothalamic glioma (in children); vulvar cancer; Waldenström macroglobulinemia; and Wilms' tumor.

[0278] Further exemplary cancers include diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).

[0279] The metastases of the cancers described above can also be treated or prevented according to the methods described herein.

[0280] In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more cancer symptoms in a subject (for example, compared to the number, severity, or frequency of one or more cancer symptoms in the subject before treatment).

[0281] In some embodiments of any of the methods described herein, the method further includes administering an additional therapeutic agent (e.g., one or more therapeutic agents listed in Table 4) to the target.

[0282] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0283] Composition / kit This specification also provides compositions (e.g., pharmaceutical compositions) comprising any of the ACCs described herein, and one or more (e.g., 1, 2, 3, 4, or 5) pharmaceutically acceptable carriers (e.g., any of the pharmaceutically acceptable carriers described herein), diluents, or excipients.

[0284] In some embodiments, a composition containing any of the ACCs described herein (e.g., a pharmaceutical composition) can be placed in a sterile vial or a pre-filled syringe.

[0285] In some embodiments, compositions containing any of the ACCs described herein (e.g., pharmaceutical compositions) can be prepared for different routes of administration (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, or intratumor).

[0286] In some embodiments, any of the pharmaceutical compositions described herein may contain one or more buffering agents (e.g., neutral buffered saline, phosphate-buffered saline (PBS), amino acids (e.g., glycine), one or more carbohydrates (e.g., glucose, mannose, sucrose, dextran, or mannitol), one or more antioxidants, one or more chelating agents (e.g., EDTA or glutathione), one or more preservatives, and / or a pharmaceutically acceptable carrier (e.g., bacteriostatic water, PBS, or physiological saline).

[0287] As used herein, the term “pharmaceutically acceptable carrier” means any solvent, dispersion medium, coating, antibacterial agent, antimicrobial agent, isotonic agent, and absorption retarder that is compatible with the administration of a pharmaceutical product. Examples of such carriers include, but are not limited to, water, saline, Ringer’s solution, dextrose solution, and approximately 5% human serum albumin.

[0288] In some embodiments of any of the pharmaceutical compositions described herein, any of the ACCs described herein are prepared together with a carrier that protects against rapid elimination from the body, such as implants and microencapsulated delivery systems, as well as sustained-release and controlled-release formulations. Biodegradable and biocompatible polymers (e.g., 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.

[0289] This specification also provides kits comprising any of the ACCs described herein, any composition comprising any of the ACCs described herein, or any pharmaceutical composition comprising any of the ACCs described herein. Kits are also provided comprising, in addition to the ACCs described herein, one or more second therapeutic agents selected from Table 4. The second therapeutic agent may be provided in a dose-dosage form different from that of the ACC. Alternatively, the second therapeutic agent 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 NOs: 129 and SEQ ID NOs: 347-356, and (2) a second therapeutic agent selected from Table 4.

[0290] Any of the kits described herein may include instructions for using any of the compositions (e.g., pharmaceutical compositions) and / or any of the ACCs described herein. In some embodiments, the kit may include instructions for carrying out any of the methods described herein. In some embodiments, the kit may include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kit may provide a syringe for administering any of the pharmaceutical compositions described herein. [Examples]

[0291] The present invention will be further illustrated in the following embodiments, but these embodiments will not limit the scope of the present invention as defined in the claims.

[0292] Example 1: Production of activatable IL-15 cytokine constructs An activatable cytokine construct (ProC2970) containing human IL-15 was prepared by recombinant DNA. The first and second monomer constructs of ProC2970 were identical, and each monomer construct was a polypeptide having the amino acid sequence of SEQ ID NO: 523 and a signal sequence at its N-terminus. The first and second monomer constructs, in order from the N-terminus to the C-terminus, contained a signal sequence derived from the mouse IgG kappa signal sequence (METDTLLLWVLLLWVPGSTG (SEQ ID NO: 345)), MM (SEQ ID NO: 236), a cleavable moiety SGRSDNI (SEQ ID NO: 655), a mature cytokine protein corresponding to human IL-15 amino acid residues 49-161 (SEQ ID NO: 347), a cleavable moiety SGRSDNI (SEQ ID NO: 655), and a dimerization domain (SEQ ID NO: 3) shortened by Cys226 (according to EU numbering) and corresponding to human IgG4 Fc containing the S228P mutation (Figure 5). The complete monomeric sequence of ProC2970, including the signal sequence, is shown in SEQ ID NO: 524.

[0293] Peptide mask for IL-15 derived from the sequence of IL-2Rβ in the crystal structure of IL-15 quaternary complex (PDB ID: 4GS7) [ka] We designed a single chain peptide (MM) to serve as a cytokine masking portion by linking two peptide motifs derived from IL-2Rβ that interact with IL-15 (KLTTVD (SEQ ID NO: 720) and ASHYFER (SEQ ID NO: 721)) using a linker.

[0294] Polypeptides were prepared by transforming host cells with a polynucleotide containing the sequence of SEQ ID NO: 529, followed by culturing the resulting recombinant host cells. The cytokine construct ProC2970 was obtained by dimerization of the resulting expressed polypeptide. ProC2970 was purified from the culture supernatant by protein A and size exclusion chromatography, and assays confirmed that the desired species accounted for over 95%.

[0295] Example 2: In vitro characterization of IL-15 cytokine constructs. To cleave the MM and dimerization domain, which are peptide chain-like masks based on structural information, IL-15-containing ACC was treated overnight at 37°C with recombinant human protease urokinase-type plasminogen activator (uPA). Prior to activity testing, the proteases were neutralized by adding a cocktail of protease inhibitors. Electrophoresis confirmed that uPA cleavage occurred at the expected site within the cleavable region (Figure 6). The results suggest that uPA proteases cleave the cleavable region (CM) in ProC2970 and ProC1879.

[0296] The activity of ProC2970 and ProC1879 was tested in vitro using IL-2 / IL-15 responsive HEK293 cells before and after cleavage with uPA. IL-2 / IL-15 responsive HEK293 cells were generated by stable transfection using human CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ) genes along with human JAK3 and STAT5 genes, resulting in a fully functional IL-2 / IL-15 signaling pathway. These cells also feature a STAT5-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. To maintain transgene expression, cells were cultured in DMEM GlutaMax® medium supplemented with 10% FBS, Pen / Strep, 10 ug / ml puromycin, and 100 μg / mL Normocin®. When IL-2 and IL-15 are added to these cells, STAT5 is activated, and subsequently, SEAP production is induced. This SEAP can be easily evaluated in the supernatant using QUANTI-Blue solution, a colorimetric detector for alkaline phosphatase activity.

[0297] IL-2 / IL-15 reactive HEK293 cells were prepared in DMEM medium supplemented with 10% FBS at a concentration of 280,000 cells / mL. 180 μL aliquots were pipetteed into the wells (50,000 cells / well) of a white, flat-bottomed, 96-well plate. The cytokine to be tested was 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 the supernatant from the induced reporter cells was transferred to the wells of a flat-bottomed, 96-well plate. 180 μL of resuspended QUANTI-Blue solution was added per well. After incubating the plates in a 37°C incubator for 1–3 hours, SEAP levels were measured using a spectrophotometer at 620 nm. Dose-response curves were created and EC50 values ​​were obtained using sigmoid-fit nonlinear regression with GraphPad Prism software.

[0298] In reporter assays, the activity of ProC2970 was reduced by at least 6000X (6000-fold) compared to PeproTech IL-15 (recombinant human IL-15 (rhIL-15), catalog number 200-15, available from PeproTech) and by 9.5X (9.5-fold) compared to ProC1879 (sequence number 356), which is Fc-masked IL-15 (Figure 7). This indicates that the MM fusion described herein provides additional masking of IL-15 in the ACC construct. Protease activation with uPA partially restored the activity of ProC2970 to a level close to that of recombinant IL-15, but still lower. Table 5 below shows the EC50 values ​​for rhIL-15, ProC1879, ProC2970, ProC1879+uPA, and ProC2970+uPA.

[0299] [Table 5]

[0300] Example 3: Activity of IL-15-containing ACC on human PBMC proliferation In cell proliferation assays, human PBMCs were incubated for 3 days with recombinant IL-15 or IL-15-ACC (with or without pre-activation by protease). After incubation, PBMCs were stained with the fixable viability analysis dye eFlur® 780, anti-CD3-FITC (UCHTI) antibody, anti-CD4-BV608 (RPA-T4) antibody, anti-CD8-BV480 (RPA-T8) antibody, anti-CD56-BV421 (HCD56) antibody, and anti-Ki67-APC (Ki67) antibody. As shown in Figure 8, various cell populations, including CD3-, CD56+ NK cells, CD3+, CD8+ T cells, and CD3+, CD4+ T cells, were analyzed, and the proliferation of various cell populations was determined based on Ki67 expression levels. Protease-treated IL-15-ACC showed stronger proliferation activity than the corresponding intact IL-15-containing ACC. Table 6 shows the EC50 values ​​of various IL-15-containing ACCs in PBMCs.

[0301] [Table 6]

[0302] Example 4: Activatable cytokine X Based on the investigation of the structural model of cytokine-receptor complexes, chained mask peptides are designed for other cytokines as well. For example, the 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 derived from IL-10R1 that interact with the IL-10 dimer molecule (TNTRFSVDEVT (SEQ ID NO: 723) and SVASRSNKG (SEQ ID NO: 724)) are linked together using a linker to generate the candidate IL-10 masking moieties shown below.

[0303] Similarly, the IL-18 masking moiety (SEQ ID: 725) can be designed based on the sequence of IL-18Rα in the crystal structure of the IL-18 ternary complex (PDB ID: 3WO4). The potential IL-18 masking moiety is obtained by linking peptide sequences derived from IL-18Rα that interact with IL-18 (VDEVYDYHQ (SEQ ID: 726) and LLLGSTG (SEQ ID: 727)) using a linker. Using the same approach, the peptide mask for IFNγ (SEQ ID: 728) can be designed from the sequence of IFNGR1 in the crystal structure of the IFNγ ternary complex (PDB ID: 6E3K). The IFNγ masking moiety of SEQ ID: 728 is obtained by linking two peptide sequences derived from IFNGR1 that interact with the IFNγ dimer molecule (EEFAVLRDGK (SEQ ID: 729) and GVLNVWGV (SEQ ID: 730)) using a linker. Examples of additional chain-like masks are shown below and throughout this disclosure.

[0304] [Table 7]

[0305] Example 5: In vitro characterization of an IL-15-containing ACC example. Recombinant uPA was used to treat IL-15 WT ACC and IL-15 mutant protein ACC overnight at 37°C. Prior to activity testing, the protease was neutralized by adding a cocktail of protease inhibitors. Table 8 shows the mask sequence of IL-15. Figure 10 schematically shows ACC. ACC activation was carried out by incubating the ACC-uPA mixture overnight at 37°C with a ratio of 1:5. Electrophoresis confirmed that cleavage by uPA occurred at the expected site within the cleavable region (Figures 9A and 9B). The HEK293 reporter assay characterized the activity of intact IL-15-containing ACC and protease-treated IL-15-containing ACC (Figures 9C-9E). Table 9 shows the mean EC50 values ​​of IL-15-containing ACC obtained from multiple experiments. The results show that the structure-based peptide mask attenuates the activity of both WT IL-15-containing ACCs and mutant protein IL-15-containing ACCs.

[0306] [Table 8]

[0307] [Table 9]

[0308] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 10-11] [Table 10-12] [Table 10-13] [Table 10-14] [Table 10-15] [Table 10-16] [Table 10-17] [Table 10-18] [Table 10-19] [Table 10-20]

[0309] Numbered items 1. The cytokine masking portion (MM), (a) A first masking subunit encoded by a first partial sequence of the amino acid sequence encoding the first receptor polypeptide of the cytokine receptor, (b)(i) A second subsequence of an amino acid sequence encoding a cytokine receptor polypeptide, selected from the group consisting of the amino acid sequence encoding the first receptor polypeptide and the amino acid sequence encoding the second receptor polypeptide of the cytokine receptor, wherein the first subsequence and the second subsequence are not adjacent within the amino acid sequence encoding the first receptor polypeptide, and (ii) an amino acid sequence encoding a second masking subunit selected from the group comprising the amino acid sequence encoding the blockage portion (BM), The aforementioned MM, including.

[0310] 2. The cytokine MM according to item 1, wherein the cytokine MM includes a linker positioned between the first masking subunit and the second masking subunit.

[0311] 3. The cytokine MM described in any one or combination of items 1-2, wherein the second masking subunit is encoded by a second amino acid sequence encoding BM.

[0312] 4. The cytokine MM according to item 3, wherein the BM comprises a peptide having binding affinity to the cytokine.

[0313] 5. The cytokine MM according to item 3, wherein the BM comprises an scFv having binding affinity to the cytokine.

[0314] 6. The cytokine MM according to any one or combination of items 1 to 2, wherein 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 encoding the second receptor polypeptide of the cytokine receptor, and the first subsequence and the second subsequence are not adjacent within the amino acid sequence encoding the first receptor polypeptide.

[0315] 7. The cytokine MM described in item 6, wherein the first subsequence and the second subsequence are the same, or the first receptor polypeptide and the second receptor polypeptide are the same.

[0316] 8. The cytokine MM described in item 6, wherein the first and second subsequences are different.

[0317] 9. Cytokine MM as described in any one of items 6 and 8 or a combination thereof, wherein the first receptor polypeptide and the second receptor polypeptide are different.

[0318] 10. The cytokine MM described in any one or combination of items 1 to 9, wherein the cytokine MM is functionally linked to the cytokine.

[0319] 11. An activatable cytokine construct (ACC) comprising a cleavable portion (CM), a cytokine polypeptide (CP), and a cytokine MM described in any one or combination of items 1-10.

[0320] 12. An activatable cytokine construct (ACC) comprising a masking portion (MM), a cleavable portion (CM), and a cytokine polypeptide (CP), a) The MM comprises a polypeptide sequence containing a chain of two or more receptor subsequences, b) Each of the receptor subsequences is derived from the receptor of the CP, c) The two or more receptor subsequences are i) Not adjacent within the sequence of the receptor, ii) The ACCs, which are directly or indirectly connected to one another.

[0321] 13. The ACC as described in item 12, wherein each of the two or more receptor subsequences contains a sequence of amino acids in the receptor within 1.0 to 8.0 angstroms, 2.0 to 8.0 angstroms, or 2.0 to 7.0 angstroms of cytokine amino acids in the cocrystal structure of the receptor-cytokine complex (cytokine-receptor complex cocrystal structure).

[0322] 14. The ACC according to item 12, further comprising a linker positioned between at least two of the two or more receptor subsequences.

[0323] 15. The ACC described in any one of items 12-13 or any combination thereof, wherein each of the receptor subsequences comprises two, three, four, or more amino acids.

[0324] 16. An ACC according to any one or combination of items 12-14, wherein at least one of the two or more receptor subsequences includes a conserved substitution of at least one amino acid compared to the sequence of the receptor.

[0325] 17. The two or more receptor partial sequences include a first receptor partial sequence and a second receptor partial sequence, The second receptor subsequence is located on the C-terminal side of the first receptor subsequence in the receptor sequence. The linker contains X amino acids, X = n / y, The ACC described in any one of items 12-15 or any combination thereof, where n is the distance in angstroms between the C-terminus of the N-terminal peptide of the first receptor sub-sequence and the N-terminus of the C-terminal peptide of the second receptor sub-sequence in the cytokine-receptor complex cocrystal structure, y is a number in the range of 1.5 to 3.5 or 2 to 3.5 or 2 to 3 or 2 to 2.5, or if y is 2.5 and X is not an integer, X is rounded up to the next integer.

[0326] 18. The ACC described in any one or combination of items 12 to 16, wherein the MM comprises the amino acid sequence ALTTVX-linker-ASHYFE (SEQ ID NO: 508) (MM), where X is D, K, or R, the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid, and the linker consists of 1 to 20 amino acids.

[0327] 19. The ACC described in item 18, wherein the N-terminus, C-terminus, or both of the MM 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.

[0328] 20. An isolated polypeptide comprising a polypeptide sequence comprising a chain of two or more receptor subsequences, wherein each of the receptor subsequences is derived from a cytokine polypeptide (CP) receptor, and the two or more receptor subsequences are i) Not adjacent within the sequence of the receptor, ii) The isolated polypeptides linked to each other directly or indirectly.

[0329] 21. The isolated polypeptide according to item 20, wherein each of the two or more receptor subsequences contains a sequence of amino acids in the receptor within 1.0 to 8.0 angstroms, 2.0 to 8.0 angstroms, or 2.0 to 7.0 angstroms of cytokine amino acids in the cocrystal structure of the receptor-cytokine complex (cytokine-receptor complex cocrystal structure).

[0330] 22. The isolated polypeptide according to item 20, further comprising a linker positioned between at least two of the two or more receptor subsequences.

[0331] 23. Isolated polypeptides according to any one or combination of items 20-22, wherein each of the receptor subsequences comprises two, three, four, or more amino acids.

[0332] 24. An isolated polypeptide according to any one or combination of items 20-23, wherein at least one of the two or more receptor subsequences comprises at least one conserved amino acid substitution compared to the receptor sequence.

[0333] 25. The two or more receptor partial sequences include a first receptor partial sequence and a second receptor partial sequence, The second receptor subsequence is located on the C-terminal side of the first receptor subsequence in the receptor sequence. The linker contains X amino acids, X = n / y, An isolated polypeptide according to any one or combination of items 21-24, where n is the distance in angstroms between the C-terminus of the N-terminal peptide of the first receptor sub-sequence and the N-terminus of the C-terminal peptide of the second receptor sub-sequence in the cytokine-receptor complex cocrystal structure, y is a number in the range of 1.5 to 3.5 or 2 to 3.5 or 2 to 3 or 2 to 2.5, or if y is 2.5 and X is not an integer, X is rounded up to the next integer.

[0334] 26. An isolated polypeptide comprising the amino acid sequence ALTTVX-linker-ASHYFE (SEQ ID NO: 508) (MM), wherein in the sequence X is D, K, or R, the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid, and the linker consists of 1 to 20 amino acids.

[0335] 27. The isolated polypeptide as described in item 26, wherein the N-terminus, C-terminus, or both of the isolated polypeptide are elongated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0336] 28. An isolated polypeptide or ACC as described in any one of items 12 to 27, wherein X is D.

[0337] 29. An isolated polypeptide or ACC according to any one or a combination of items 12 to 28, wherein X is K.

[0338] 30. An isolated polypeptide or ACC described in any one or combination of items 12-29, wherein X is R.

[0339] 31. The linker is an isolated polypeptide or ACC as described in any one or combination of items 12-30, comprising 4, 5, 6, 7, or 8 amino acids.

[0340] 32. The linker is an isolated polypeptide or ACC described in any one or a combination of items 12 to 31, comprising six amino acids.

[0341] 33. The linker is an isolated polypeptide or ACC as described in any one or combination of items 12 to 32, selected from the group consisting of Sequence ID No. 2, 210-235, 245, 250, and 318-335.

[0342] 34. An isolated polypeptide or ACC described in any one or a combination of items 12 to 33, wherein the linker is GGGGS (Sequence ID 216).

[0343] 35. An isolated polypeptide or ACC according to any one or combination of items 12 to 34, wherein the amino acid sequence includes ALTTVDGGGGSASHYFER (SEQ ID NO: 236) or ALTTVDGGGGSASHYFEK (SEQ ID NO: 237), or the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid.

[0344] 36. An isolated polypeptide or ACC according to any one or combination of items 12 to 35, wherein the amino acid sequence includes ALTTVKGGGGSASHYFER (SEQ ID NO: 238) or ALTTVKGGGGSASHYFEK (SEQ ID NO: 239), or the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid.

[0345] 37. An isolated polypeptide or ACC according to any one or combination of items 12 to 36, wherein the amino acid sequence includes ALTTVRGGGGSASHYFER (SEQ ID NO: 240) or ALTTVRGGGGSASHYFEK (SEQ ID NO: 241), or the N-terminal alanine residue is optionally absent or optionally substituted with any other amino acid.

[0346] 38. An isolated polypeptide or ACC according to any one or combination of items 12-37, wherein the N-terminal alanine residue is substituted with lysine.

[0347] 39. An isolated polypeptide or ACC described in any one or combination of items 12-37, wherein the isolated polypeptide contains a sequence selected from SQKLTTVDGGGGSASHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGSASHYFERHLE (SEQ ID NO: 734), SQALTTVRGGGGSASHYFERHLE (SEQ ID NO: 736), or SQALTTVDGGGGSASHYFERHLE (SEQ ID NO: 737).

[0348] 40. The isolated polypeptide comprising a cytokine, an isolated polypeptide or ACC described in any of the preceding items or combinations of items.

[0349] 41. The isolated polypeptide is located in a complex comprising two or more polypeptides, wherein the complex comprises a cytokine, the isolated polypeptide or ACC described in any one or combination of items 12 to 40.

[0350] 42. An isolated polypeptide or ACC according to any one or combination of items 12 to 41, wherein the cytokine is located in the polypeptide complexed with the isolated polypeptide.

[0351] 43. An isolated polypeptide or ACC according to any one or combination of items 12 to 42, wherein the amino acid sequence is a masking portion that inhibits the binding of the cytokine to its receptor.

[0352] 44. An isolated polypeptide or ACC according to any one or combination of items 12 to 43, wherein the cytokine is a cytokine that binds to IL2 / IL15 receptor beta and / or IL2 / IL15 receptor gamma.

[0353] 45. An isolated polypeptide or ACC described in any one or combination of items 12 to 44, wherein the cytokine binds to IL-15Rα.

[0354] 46. ​​The cytokine is an isolated polypeptide or ACC described in any one of items 12 to 45, which binds to IL-2Rα.

[0355] 47. An activatable cytokine construct (ACC) comprising a cytokine polypeptide (CP), a cleavable moiety (CM), and an isolated polypeptide or cytokine masking moiety (MM) comprising a polypeptide sequence including a chain sequence as described in any of the preceding 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 a receptor.

[0356] 48. The ACC described in item 47, wherein the CP is an interleukin polypeptide.

[0357] 49. The ACC according to item 48, wherein the interleukin polypeptide contains a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 348, 129, or 130.

[0358] 50. The ACC according to item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 348, 129, or 130.

[0359] 51. The ACC according to item 48, wherein the interleukin polypeptide contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 119 or 120.

[0360] 52. The ACC according to item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 119 or 120.

[0361] 53. The ACC according to item 48, wherein the interleukin polypeptide contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 121 or 122.

[0362] 54. The ACC according to item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 121 or 122.

[0363] 55. The ACC according to item 48, wherein the interleukin polypeptide contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 123 or 124.

[0364] 56. The ACC according to item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 123 or 124.

[0365] 57. The ACC according to item 48, wherein the interleukin polypeptide contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 125 or 126.

[0366] 58. The ACC according to item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 125 or 126.

[0367] 59. The ACC according to item 48, wherein the interleukin polypeptide contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 521 or 522.

[0368] 60. The ACC according to item 48, wherein the interleukin polypeptide comprises SEQ ID NO: 521 or 522.

[0369] 61. The ACC described in any one of items 47-60 or any combination thereof, wherein the CM contains eight or fewer amino acids.

[0370] 62. The ACC according to any one of items 47-61 or a combination thereof, wherein the CM can be cleaved by urokinase (uPA) and / or matrix metalloproteinase (MMP).

[0371] 63. An ACC according to any one of items 47 to 62 or a combination thereof, further comprising a linker (L1) between the CM and the CP.

[0372] 64. An ACC as described in any one of items 47 to 63 or a combination thereof, further comprising a linker (L2) between the CM and the MM.

[0373] 65. An ACC according to any one of items 47 to 64 or a combination thereof, further comprising a first linker (L1) between the CM and the CP, and a second linker (L2) between the CM and the MM.

[0374] 66. The ACC according to any one of items 47-65 or a combination thereof, further comprising a stereomask that further inhibits the binding of the CP to its receptor.

[0375] 67. An ACC comprising a first monomer construct and a second monomer construct, The first monomer construct comprises a first cytokine polypeptide (CP1), a first cleavable portion (CM1), a first dimerization domain (DD1) coupled to the CP1 via the CM1, and a first masking portion (MM1). The second monomer construct comprises a second cytokine polypeptide (CP2), a second cleavable portion (CM2), a second dimerization domain (DD2) coupled to the CP2 via the CM2, and a second masking portion (MM2), The DD1 and DD2 are bonded to each other, thereby forming dimers of the first monomer construct and the second monomer construct. The ACC wherein MM1 and / or MM2 comprises an isolated polypeptide described in either of the preceding items or combinations of items.

[0376] 68. The ACC according to item 67, wherein the first monomer structure includes a third cleavable portion (CM3), and the MM1 is coupled to the CP1 via the CM3.

[0377] 69. The ACC described in any one or combination of items 67-68, wherein the MM1 is coupled with the CP1 via the DD1 and the CM1.

[0378] 70. The ACC according to any one of items 67-69 or a combination thereof, wherein the second monomer construct includes a fourth cleavable portion (CM4), and the MM2 is coupled with the CP2 via the CM4.

[0379] 71. The ACC described in any one or combination of items 67-70, wherein the MM2 is coupled with the CP2 via the DD2 and the CM2.

[0380] 72. The ACC according to any one or combination of items 67 to 71, wherein the first monomer construct and the second monomer construct each include a linking region containing 18 or fewer amino acids.

[0381] 73. The CP1 and CM1 are directly adjacent to each other, and / or The ACC described in any one of items 67 to 72 or any combination thereof, wherein the CM1 and the DD1 are directly adjacent to each other.

[0382] 74. The CP2 and CM2 are directly adjacent to each other, and / or The ACC described in any one of items 67 to 73 or any combination thereof, wherein the CM2 and the DD2 are directly adjacent to each other.

[0383] 75. The ACC according to any one or combination of items 67-74, wherein CP1 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 348, 129, or 130.

[0384] 76. The ACC according to any one of items 67-75 or a combination thereof, wherein the CP2 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 348, 129, or 130.

[0385] 77. The ACC described in any one of items 67-76 or any combination thereof, wherein the CP1 includes sequence number 348, 129, or 130.

[0386] 78. The ACC described in any one of items 67-77 or any combination thereof, wherein the CP2 includes sequence numbers 348, 129, or 130.

[0387] 79. The ACC according to any one of items 67-78 or a combination thereof, wherein CP1 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 119 or 120.

[0388] 80. The ACC according to any one or combination of items 67-79, wherein CP2 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 119 or 120.

[0389] 81. The ACC described in any one of items 67-80 or any combination thereof, wherein the CP1 includes sequence number 119 or 120.

[0390] 82. The ACC described in any one of items 67-81 or any combination thereof, wherein the CP2 includes sequence number 119 or 120.

[0391] 83. The ACC according to any one of items 67-82 or any combination thereof, wherein CP1 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 121 or 122.

[0392] 84. The ACC according to any one or combination of items 67-83, wherein CP2 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 121 or 122.

[0393] 85. The ACC described in any one of items 67-84 or any combination thereof, wherein the CP1 includes sequence number 121 or 122.

[0394] 86. The ACC described in any one of items 67-85 or any combination thereof, wherein the CP2 includes sequence number 121 or 122.

[0395] 87. The ACC according to any one or combination of items 67-86, wherein CP1 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 123 or 124.

[0396] 88. The ACC according to any one or combination of items 67 to 87, wherein CP2 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 123 or 124.

[0397] 89. The ACC described in any one of items 67-88 or any combination thereof, wherein the CP1 includes sequence number 123 or 124.

[0398] 90. The ACC described in any one of items 67-89 or any combination thereof, wherein the CP2 includes sequence number 123 or 124.

[0399] 91. The ACC according to any one of items 67-90 or a combination thereof, wherein CP1 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 125 or 126.

[0400] 92. The ACC according to any one or combination of items 67-91, wherein CP2 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 125 or 126.

[0401] 93. The ACC described in any one of items 67-92 or any combination thereof, wherein the CP1 includes sequence number 125 or 126.

[0402] 94. The ACC described in any one of items 67-93 or any combination thereof, wherein the CP2 includes sequence number 125 or 126.

[0403] 95. The ACC according to any one or combination of items 67-94, wherein CP1 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 521 or 522.

[0404] 96. The ACC according to any one or combination of items 67-95, wherein CP2 contains a sequence that is at least 85%, 90%, or 95% identical to sequence number 521 or 522.

[0405] 97. The ACC described in any one or combination of items 67-96, wherein the CP1 includes sequence number 521 or 522.

[0406] 98. The ACC described in any one of items 67-97 or any combination thereof, wherein the CP2 includes sequence number 521 or 522.

[0407] 99. An ACC according to any one of items 67 to 98 or any combination thereof, wherein the first monomer construct and the second monomer construct are the same.

[0408] 100. The ACC according to any one or combination of items 67 to 99, wherein the first monomer construct and the second monomer construct have 95% sequence homology.

[0409] 101. The ACC according to any one of items 67 to 100 or a combination thereof, further comprising a third monomer containing a sucoid domain containing the sequence of sequence number 520, wherein the third monomer is bonded to the ACC non-covalently or covalently.

[0410] 102. The ACC according to any one of items 67 to 101 or a combination thereof, further comprising a fourth monomer comprising a sucoid domain having the sequence of sequence number 520, wherein the fourth monomer is bonded to the ACC non-covalently or covalently.

[0411] 103. The ACC described in item 100 or 101, wherein the third monomer and / or the fourth monomer further include a tag.

[0412] 104. An ACC comprising a first monomer construct and a second monomer construct, The first monomer construct comprises a first cytokine polypeptide (CP1), a first dimerization domain (DD1), and a first masking moiety (MM1), The second monomer construct comprises a second cytokine polypeptide (CP2), a first cleavable portion (CM1), a second dimerization domain (DD2) coupled to the CP2 via the CM1, and a second masking portion (MM2), The MM1 and / or MM2 are isolated polypeptides described in either of the preceding items or combinations thereof. The ACC, wherein DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer construct and the second monomer construct.

[0413] 105. The ACC according to item 104, wherein the first monomer construct further comprises a second cleavable portion (CM2), and the MM1 is coupled to the CP1 via the CM2.

[0414] 106. The ACC according to item 104 or 105, wherein the MM2 is coupled to the CP2 via the DD2 and the CM1.

[0415] 107. The ACC according to any one of items 104 to 106 or a combination thereof, wherein the second monomer construct further comprises a third cleavable portion (CM3), and the MM2 is coupled with CP2 via the CM3.

[0416] 108. An ACC comprising a first monomer construct and a second monomer construct, The first monomer construct comprises a first cytokine polypeptide (CP1), a first dimerization domain (DD1), and a first masking moiety (MM1), The second monomer construct comprises a second cytokine polypeptide (CP2), a second dimerization domain (DD2), and a second masking moiety (MM2), The CP1 and / or CP2 comprises an amino acid sequence that functions as a substrate for a protease, and the DD1 and / or DD2 are coupled to the CP1 and / or CP2 via the amino acid sequence. The MM1 and / or MM2 are isolated polypeptides described in either of the preceding items or combinations thereof. The ACC, wherein DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer construct and the second monomer construct.

[0417] 109. The ACC according to item 108, wherein CP1 comprises the amino acid sequence that functions as a substrate for a protease, and MM1 is coupled to CP1 via the amino acid sequence.

[0418] 110. The ACC according to any one of items 108 to 109 or a combination thereof, wherein the first monomer construct further comprises a first cleavable portion (CM1), and the MM1 is coupled to the CP1 via the CM1.

[0419] 111. The ACC according to any one or combination of item 108, wherein CP2 comprises the amino acid sequence that functions as a substrate for a protease, and MM2 is coupled to CP2 via the amino acid sequence.

[0420] 112. The ACC according to any one of items 108 to 111 or a combination thereof, wherein the second monomer construct further comprises a second cleavable portion (CM2), the MM2 being coupled to the CP2 via the CM2.

[0421] 113. The ACC according to any one or combination of items 67 to 112, wherein DD1 and DD2 are a pair of human IgG Fc domains.

[0422] 114. The ACC described in item 113, wherein DD1 and DD2 are a pair of human IgG4 Fc domains.

[0423] 115. The ACC as described in item 114, wherein DD1 and DD2 are a pair of shortened human IgG1 Fc domains or human IgG4 Fc domains at the N-terminal end of cysteine ​​226, as numbered by EU numbering.

[0424] 116. The ACC described in item 114, including the S228P mutation, when the human IgG4 Fc domain is numbered according to EU numbering.

[0425] 117. The ACC according to any one or combination of items 67 to 116, wherein DD1 and DD2 each contain at least 95% identical sequences to sequence number 3.

[0426] 118. The ACC described in any one or combination of items 67 to 117, wherein DD1 and DD2 each include the sequence of sequence number 3.

[0427] 119. The ACC according to any one of items 67-118 or any combination thereof, wherein the first monomer construct and the second monomer construct are covalently bonded to each other via at least one, two, three, or four disulfide bonds.

[0428] 120. The ACC according to any one or a combination of items 12-19 and 47-119, wherein the first monomer construct and the second monomer construct each contain a sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 523.

[0429] 121. The ACC according to any one or a combination of items 12-19 and 47-120, wherein the first monomer construct and the second monomer construct each include Sequence ID No. 523.

[0430] 122. The ACC according to any one of items 12-19 and 47-121 or a combination thereof, characterized in that the level of interleukin activity is reduced compared to a control level of interleukin activity.

[0431] 123. The ACC according to any one or a combination of items 12-19 and 47-122, characterized in that the ACC has a reduced level of interleukin activity compared to wild-type human IL-15.

[0432] 124. The ACC described in item 12-19 and item 1 of items 47-123, characterized in that, when measured by the level of SEAP (secreted embryonic alkaline phosphatase) production in IL-2 / IL-15 responsive HEK293 cells, the level of IL-15 activity is reduced compared to recombinant human IL-15.

[0433] 125. The ACC according to item 12-19 and item 47-124, characterized in that the ACC has a reduced level of IL-15 activity compared to the activity of recombinant human IL-15.

[0434] 126. The ACC according to item 125, characterized in that the ACC has a level of IL-15 activity reduced by at least 6,000 times compared to recombinant human IL-15.

[0435] 127. The ACC according to any one or a combination of items 12-19 and 47-126, characterized in that, when measured in IL-2 / IL15-responsive HEK293 cells, the EC50 of the ACC after cleavage by uPA protease is at least 1000 times, 5000 times, or 6000 times that of recombinant wild-type IL-15.

[0436] 128. An ACC comprising a cytokine polypeptide (CP), an agonist of the CP, an isolated polypeptide or cytokine masking moiety (MM) comprising a polypeptide sequence including a chain sequence as described in any of the preceding items or a combination of the items, and a cleavable moiety (CM), wherein the isolated polypeptide or the MM is coupled to the CP via the CM.

[0437] 129. The ACC described in item 128, wherein the CP is IL-15 and the agonist is sucrose.

[0438] 130. The ACC described in item 128 or item 129, wherein the agonist is coupled to the CP via a linker.

[0439] 131. The ACC according to item 130, wherein the agonist is coupled to the CP via a severable linker.

[0440] 132. The ACC as described in item 120, wherein the agonist is coupled to the CP via an inseparable linker.

[0441] 133. The ACC described in item 128 or item 129, wherein the agonist is bonded to the CP by a non-covalent bond.

[0442] 134. An ACC comprising a first monomer construct and a second monomer construct, The first monomer construct comprises a cytokine polypeptide (CP), a first dimerization domain (DD1), a first cleavable portion (CM1), a second cleavable portion (CM2), and an isolated polypeptide or cytokine masking portion (MM) comprising a polypeptide sequence including a chain sequence as described in any of the preceding items or combinations thereof, wherein the isolated polypeptide or 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 an agonist of CP, a third cleavable portion (CM3), and a second dimerization domain (DD2) coupled to the agonist via the CM3. The ACC, wherein DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer construct and the second monomer construct.

[0443] 135. The ACC according to item 134, wherein the CP is IL-15 and the agonist is a sucrose domain containing the sequence of sequence number 520.

[0444] 136. The ACC according to item 134 or 135, comprising a linker consisting of two amino acids between the sucrose domain and the CM3.

[0445] 137. A polynucleotide encoding an isolated polypeptide as described in any of the preceding items or combinations thereof, an ACC as described in any of the preceding items or combinations thereof, or a monomer construct as described in any of the preceding items or combinations thereof.

[0446] 138. A vector containing polynucleotides as described in item 137.

[0447] 139. The vector described in item 138, wherein the vector is an expression vector.

[0448] 140. Host cells containing polynucleotides as described in item 137 or vectors as described in item 138 or 139.

[0449] 141. The host cell described in the item, wherein the host cell is a mammalian cell.

[0450] 142. A composition comprising an isolated polypeptide described in any of the preceding items or combinations thereof, or an ACC described in any of the preceding items or combinations thereof, or a polynucleotide described in item 137.

[0451] 143. The composition described in item 142, wherein the composition is a pharmaceutical composition.

[0452] 144. A container, vial, syringe, injection pen, or kit comprising at least one dose of the composition described in item 142 or 143.

[0453] 145. A method for administering a treatment to a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an isolated polypeptide described in any of the preceding items or combinations thereof, an ACC described in any of the preceding items or combinations thereof, or a composition described in item 142 or 143.

[0454] 146. The method of item 145, wherein the subject has been identified or diagnosed with cancer.

[0455] 147. The method according to item 146, wherein the cancer is leukemia, lymphoma, or a solid tumor.

[0456] 148. The method of item 147, wherein the subject is identified or diagnosed with an inflammatory or autoimmune disease, disorder, or condition.

[0457] 149. A method for producing ACC, To produce the ACC by culturing cells containing the polynucleotide described in item 137 in a liquid culture medium, The method comprising recovering the ACC from the cells or the liquid culture medium.

[0458] 150. The method according to item 149, further comprising isolating the ACC recovered from the cells or the liquid culture medium.

[0459] 151. The method according to item 149 or 150, further comprising formulating the isolated ACC into a pharmaceutical composition.

[0460] 152. A complex comprising a polypeptide containing a cytokine complexed with an isolated polypeptide described in any of the preceding items or combinations thereof.

[0461] Other Embodiments While the present invention has been described in relation to embodiments for carrying out the invention described above, the foregoing description is intended to be illustrative and not to limit the scope of the invention, and it should be understood that the scope of the invention is defined by the appended claims. The following claims also include other embodiments, advantages, and modifications.

Claims

1. The cytokine masking portion (MM), (a) A first masking subunit encoded by a first partial sequence of the amino acid sequence encoding the first receptor polypeptide of the cytokine receptor, (b) (i) A second partial sequence of an amino acid sequence encoding a cytokine receptor polypeptide, selected from the group consisting of the amino acid sequence encoding the first receptor polypeptide and the amino acid sequence encoding the second receptor polypeptide of the cytokine receptor, wherein the first partial sequence and the second partial sequence are not adjacent within the amino acid sequence encoding the first receptor polypeptide, and (ii) A second masking subunit encoded by a second amino acid sequence selected from the group consisting of an amino acid sequence encoding a blockage portion (BM), The MM including the above.

2. The cytokine MM according to claim 1, wherein the cytokine MM includes a linker disposed between the first masking subunit and the second masking subunit.

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

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

5. The cytokine MM according to claim 3, wherein the BM comprises an scFv having binding affinity to the cytokine.

6. The cytokine MM according to any one of claims 1 to 2, wherein 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 encoding the second receptor polypeptide of the cytokine receptor, and the first subsequence and the second subsequence are not adjacent within the amino acid sequence encoding the first receptor polypeptide.

7. The cytokine MM according to claim 6, wherein the first partial sequence and the second partial sequence are the same, or the first receptor polypeptide and the second receptor polypeptide are the same.

8. The cytokine MM according to claim 6, wherein the first sub-sequence and the second sub-sequence are different.

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

10. The cytokine MM according to any one of claims 1 to 9, wherein the cytokine MM is functionally linked to the cytokine.

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

12. An activatable cytokine construct (ACC) comprising a masking portion (MM), a cleavable portion (CM), and a cytokine polypeptide (CP), a) The MM comprises a polypeptide sequence containing a chain of two or more receptor subsequences, b) Each of the receptor subsequences is derived from the receptor of the CP, c) The two or more receptor sub-sequences i) Not adjacent within the sequence of the receptor, ii) The ACCs, which are directly or indirectly connected to one another.

13. The ACC according to claim 12, wherein each of the two or more receptor subsequences contains a sequence of cytokine amino acids in the receptor within 1.0 to 8.0 angstroms, 2.0 to 8.0 angstroms, or 2.0 to 7.0 angstroms of the cytokine amino acids in the cocrystal structure of the receptor-cytokine complex (cytokine-receptor complex cocrystal structure).

14. The ACC according to claim 12, further comprising a linker positioned between at least two of the two or more receptor subsequences.

15. The ACC according to any one of claims 12 to 13, wherein each of the receptor subsequences comprises two, three, four, or more amino acids.

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

17. The two or more receptor partial sequences include a first receptor partial sequence and a second receptor partial sequence, The second receptor partial sequence is located on the C-terminal side of the first receptor partial sequence in the sequence of the receptor. The linker contains X amino acids, X = n / 2.5, The ACC according to any one of claims 12 to 15, wherein n is the distance in angstroms between the C-terminus of the N-terminal peptide of the first receptor sub-sequence and the N-terminus of the C-terminal peptide of the second receptor sub-sequence in the cytokine-receptor complex cocrystal structure.

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

19. The ACC according to claim 18, wherein the N-terminus, C-terminus, or both of the MM 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.

20. An isolated polypeptide comprising a polypeptide sequence comprising a chain of two or more receptor subsequences, wherein each of the receptor subsequences is derived from a cytokine polypeptide (CP) receptor, and the two or more receptor subsequences are i) Not adjacent within the sequence of the receptor, ii) The isolated polypeptides linked to each other directly or indirectly.

21. The isolated polypeptide according to claim 20, wherein each of the two or more receptor subsequences contains a sequence of cytokine amino acids in the receptor within 1.0 to 8.0 angstroms, 2.0 to 8.0 angstroms, or 2.0 to 7.0 angstroms of the cytokine amino acids in the cocrystal structure of the receptor-cytokine complex (cytokine-receptor complex cocrystal structure).

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

23. The isolated polypeptide according to any one of claims 20 to 22, wherein each of the receptor subsequences comprises two, three, four, or more amino acids.

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

25. The two or more receptor partial sequences include a first receptor partial sequence and a second receptor partial sequence, The second receptor partial sequence is located on the C-terminal side of the first receptor partial sequence in the sequence of the receptor. The linker contains X amino acids, X = n / 2.5, The isolated polypeptide according to any one of claims 21 to 24, wherein n is the distance in angstroms between the C-terminus of the N-terminal peptide of the first receptor sub-sequence and the N-terminus of the C-terminal peptide of the second receptor sub-sequence in the cytokine-receptor complex cocrystal structure.

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

27. The isolated polypeptide according to claim 26, wherein the N-terminus, C-terminus, or both of the isolated polypeptide are elongated 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 according to any one of claims 12 to 27, wherein X is D.

29. The isolated polypeptide or ACC according to any one of claims 12 to 28, wherein X is K.

30. The isolated polypeptide or ACC according to any one of claims 12 to 29, wherein X is R.

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

32. The isolated polypeptide or ACC according to any one of claims 12 to 31, wherein the linker consists of six amino acids.

33. The isolated polypeptide or ACC according to any one of claims 12 to 32, wherein the linker is selected from the group consisting of SEQ ID NOs: 2, 210 to 235, 245, 250, and 318 to 335.

34. The isolated polypeptide or ACC according to any one of claims 12 to 33, wherein the linker is GGGGS (Sequence ID 216).

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

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

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

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

39. The isolated polypeptide or ACC according to any one of claims 12 to 37, wherein the isolated polypeptide comprises a sequence selected from SQKLTTVDGGGGGSASHHYFERHLE (SEQ ID NO: 731), SQKLTTVRGGGGGSASHHYFERHLE (SEQ ID NO: 734), SQALTTTVRGGGGGSASHHYFERHLE (SEQ ID NO: 736), or SQALTTTVDGGGGGSASHHYFERHLE (SEQ ID NO: 737).

40. The isolated polypeptide is an isolated polypeptide or ACC according to any prior claim, wherein the isolated polypeptide comprises a cytokine.

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

42. The isolated polypeptide or ACC according to any one of claims 12 to 41, wherein the cytokine is arranged in the polypeptide complexed with the isolated polypeptide.

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

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

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

47. An activatable cytokine construct (ACC) comprising a cytokine polypeptide (CP), a cleavable portion (CM), and an isolated polypeptide or cytokine masking portion (MM) comprising a polypeptide sequence including a chain sequence as described in any prior claim, wherein the isolated polypeptide or the MM is coupled with the CP via the CM, thereby inhibiting the binding of the CP to a receptor.

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

49. The ACC according to claim 48, wherein the interleukin polypeptide comprises a sequence that is at least 85%, 90%, or 95% identical to sequence number 348, 129, or 130.

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

51. The ACC according to claim 48, wherein the interleukin polypeptide comprises a sequence that is at least 85%, 90%, or 95% identical to sequence number 119 or 120.

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

53. The ACC according to claim 48, wherein the interleukin polypeptide comprises a sequence that is at least 85%, 90%, or 95% identical to sequence number 121 or 122.

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

55. The ACC according to claim 48, wherein the interleukin polypeptide comprises a sequence that is at least 85%, 90%, or 95% identical to sequence number 123 or 124.

56. The ACC according to claim 48, wherein the interleukin polypeptide comprises sequence number 123 or 124.

57. The ACC according to claim 48, wherein the interleukin polypeptide comprises a sequence that is at least 85%, 90%, or 95% identical to sequence number 125 or 126.

58. The ACC according to claim 48, wherein the interleukin polypeptide comprises sequence number 125 or 126.

59. The ACC according to claim 48, wherein the interleukin polypeptide comprises a sequence that is at least 85%, 90%, or 95% identical to sequence number 521 or 522.

60. The ACC according to claim 48, wherein the interleukin polypeptide comprises sequence number 521 or 522.

61. The ACC according to any one of claims 47 to 60, wherein the CM comprises eight or fewer amino acids.

62. The ACC according to any one of claims 47 to 61, wherein the CM is cleavable by urokinase (uPA) and / or matrix metalloproteinase (MMP).

63. The ACC according to any one of claims 47 to 62, further comprising a linker (L1) between the CM and the CP.

64. The ACC according to any one of claims 47 to 63, further comprising a linker (L2) between the CM and the MM.

65. The ACC according to any one of claims 47 to 64, further comprising a first linker (L1) between the CM and the CP, and a second linker (L2) between the CM and the MM.

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

67. An ACC comprising a first monomer construct and a second monomer construct, The first monomer construct comprises a first cytokine polypeptide (CP1), a first cleavable portion (CM1), a first dimerization domain (DD1) coupled to the CP1 via the CM1, and a first masking portion (MM1). The second monomer construct comprises a second cytokine polypeptide (CP2), a second cleavable portion (CM2), a second dimerization domain (DD2) coupled to the CP2 via the CM2, and a second masking portion (MM2). The DD1 and DD2 are bonded to each other, thereby forming dimers of the first monomer structure and the second monomer structure. The ACC wherein MM1 and / or MM2 comprises an isolated polypeptide as described in any of the prior claims.

68. The ACC according to claim 67, wherein the first monomer structure includes a third cleavable portion (CM3), and the MM1 is coupled to the CP1 via the CM3.

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

70. The ACC according to any one of claims 67 to 69, wherein the second monomer structure includes a fourth cleavable portion (CM4), and the MM2 is coupled to the CP2 via the CM4.

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

72. The ACC according to any one of claims 67 to 71, wherein the first monomer construct and the second monomer construct each include a linking region containing 18 or fewer amino acids.

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

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

75. The ACC according to any one of claims 67 to 74, wherein the CP1 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 348, 129, or 130.

76. The ACC according to any one of claims 67 to 75, wherein the CP2 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 348, 129, or 130.

77. The ACC according to any one of claims 67 to 76, wherein the CP1 includes sequence number 348, 129, or 130.

78. The ACC according to any one of claims 67 to 77, wherein the CP2 includes sequence number 348, 129, or 130.

79. The ACC according to any one of claims 67 to 78, wherein the CP1 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 119 or 120.

80. The ACC according to any one of claims 67 to 79, wherein the CP2 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 119 or 120.

81. The ACC according to any one of claims 67 to 80, wherein the CP1 includes sequence number 119 or 120.

82. The ACC according to any one of claims 67 to 81, wherein the CP2 includes sequence number 119 or 120.

83. The ACC according to any one of claims 67 to 82, wherein the CP1 comprises an array that is at least 85%, 90%, or 95% identical to sequence number 121 or 122.

84. The ACC according to any one of claims 67 to 83, wherein the CP2 comprises an array that is at least 85%, 90%, or 95% identical to sequence number 121 or 122.

85. The ACC according to any one of claims 67 to 84, wherein the CP1 includes sequence number 121 or 122.

86. The ACC according to any one of claims 67 to 85, wherein the CP2 includes sequence number 121 or 122.

87. The ACC according to any one of claims 67 to 86, wherein CP1 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 123 or 124.

88. The ACC according to any one of claims 67 to 87, wherein the CP2 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 123 or 124.

89. The ACC according to any one of claims 67 to 88, wherein the CP1 includes sequence number 123 or 124.

90. The ACC according to any one of claims 67 to 89, wherein the CP2 includes sequence number 123 or 124.

91. The ACC according to any one of claims 67 to 90, wherein the CP1 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 125 or 126.

92. The ACC according to any one of claims 67 to 91, wherein the CP2 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 125 or 126.

93. The ACC according to any one of claims 67 to 92, wherein the CP1 includes sequence number 125 or 126.

94. The ACC according to any one of claims 67 to 93, wherein the CP2 includes sequence number 125 or 126.

95. The ACC according to any one of claims 67 to 94, wherein the CP1 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 521 or 522.

96. The ACC according to any one of claims 67 to 95, wherein the CP2 comprises an sequence that is at least 85%, 90%, or 95% identical to sequence number 521 or 522.

97. The ACC according to any one of claims 67 to 96, wherein the CP1 includes sequence number 521 or 522.

98. The ACC according to any one of claims 67 to 97, wherein the CP2 includes sequence number 521 or 522.

99. The ACC according to any one of claims 67 to 98, wherein the first monomer structure and the second monomer structure are the same.

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

101. The ACC according to any one of claims 67 to 100, further comprising a third monomer containing a sucoid domain containing the sequence of sequence number 520, wherein the third monomer is bonded to the ACC by non-covalent or covalent bonds.

102. The ACC according to any one of claims 67 to 101, further comprising a fourth monomer containing a sucoid domain containing the sequence of sequence number 520, wherein the fourth monomer is bonded to the ACC by non-covalent or covalent bonds.

103. The ACC according to claim 100 or 101, wherein the third monomer and / or the fourth monomer further comprises a tag.

104. An ACC comprising a first monomer construct and a second monomer construct, The first monomer construct comprises a first cytokine polypeptide (CP1), a first dimerization domain (DD1), and a first masking moiety (MM1), The second monomer construct comprises a second cytokine polypeptide (CP2), a first cleavable portion (CM1), a second dimerization domain (DD2) coupled to the CP2 via the CM1, and a second masking portion (MM2). The MM1 and / or the MM2 is an isolated polypeptide according to any of the prior claims, The ACC, wherein DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer structure and the second monomer structure.

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

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

107. The ACC according to any one of claims 104 to 106, wherein the second monomer structure further comprises a third cleavable portion (CM3), and the MM2 is coupled with the CP2 via the CM3.

108. An ACC comprising a first monomer construct and a second monomer construct, The first monomer construct comprises a first cytokine polypeptide (CP1), a first dimerization domain (DD1), and a first masking moiety (MM1), The second monomer construct comprises a second cytokine polypeptide (CP2), a second dimerization domain (DD2), and a second masking moiety (MM2), The CP1 and / or CP2 comprises an amino acid sequence that functions as a substrate for a protease, and the DD1 and / or DD2 are coupled to the CP1 and / or CP2 via the amino acid sequence. The MM1 and / or the MM2 is an isolated polypeptide according to any of the prior claims, The ACC, wherein DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer structure and the second monomer structure.

109. The ACC according to claim 108, wherein the CP1 includes the amino acid sequence that functions as a substrate for a protease, and the MM1 is coupled with the CP1 via the amino acid sequence.

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

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

112. The ACC according to any one of claims 108 to 111, wherein the second monomer structure further comprises a second cleavable portion (CM2), and the MM2 is coupled to the CP2 via the CM2.

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

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

115. The ACC according to claim 114, wherein, when DD1 and DD2 are numbered according to EU numbering, the N-terminal side of cysteine ​​226 is a pair of shortened human IgG1 Fc domains or human IgG4 Fc domains.

116. The ACC according to claim 114, wherein the human IgG4 Fc domain is numbered according to EU numbering, and includes the S228P mutation.

117. The ACC according to any one of claims 67 to 116, wherein each of DD1 and DD2 contains at least 95% the same sequence as sequence number 3.

118. The ACC according to any one of claims 67 to 117, wherein DD1 and DD2 each include the sequence of sequence number 3.

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

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

523.

121. The ACC according to any one of claims 12 to 19 and 47 to 120, wherein the first monomer construct and the second monomer construct each include SEQ ID NO:

523.

122. The ACC according to any one of claims 12 to 19 and 47 to 121, characterized in that the level of interleukin activity is reduced compared to a control level of interleukin activity.

123. The ACC according to any one of claims 12 to 19 and 47 to 122, characterized in that the ACC has a reduced level of interleukin activity compared to wild-type human IL-15.

124. The ACC according to claims 12 to 19 and 47 to 123, characterized in that when the ACC is measured by the level of SEAP (secreted embryonic alkaline phosphatase) production in IL-2 / IL-15 responsive HEK293 cells, the level of IL-15 activity is reduced compared to recombinant human IL-15.

125. The ACC according to claims 12 to 19 and 47 to 124, characterized in that the ACC has a reduced level of IL-15 activity compared to the activity of recombinant human IL-15.

126. The ACC according to claim 125, characterized in that the level of IL-15 activity is reduced by at least 6,000 times compared to recombinant human IL-15.

127. The ACC according to any one of claims 12 to 19 and 47 to 126, characterized in that, when measured in IL-2 / IL-15 responsive HEK293 cells, the EC50 of the ACC after cleavage by uPA protease is at least 1000 times, 5000 times, or 6000 times that of recombinant wild-type IL-15.

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

129. The ACC according to claim 128, wherein the CP is IL-15 and the agonist is sucrose domain.

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

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

132. The ACC according to claim 120, wherein the agonist is coupled to the CP via an inseparable linker.

133. The ACC according to claim 128 or claim 129, wherein the agonist is bonded to the CP by a non-covalent bond.

134. An ACC comprising a first monomer construct and a second monomer construct, The first monomer construct comprises a cytokine polypeptide (CP), a first dimerization domain (DD1), a first cleavable portion (CM1), a second cleavable portion (CM2), and an isolated polypeptide or cytokine masking portion (MM) comprising a polypeptide sequence including a chain sequence as described in any of the prior claims, wherein the isolated polypeptide or 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 CP, a third cleavable portion (CM3), and a second dimerization domain (DD2) coupled to the agonist via the CM3. The ACC, wherein DD1 and DD2 are bonded to each other, thereby forming a dimer of the first monomer structure and the second monomer structure.

135. The ACC according to claim 134, wherein the CP is IL-15 and the agonist is a sucoid domain containing the sequence of sequence number 520.

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

137. A polynucleotide encoding an isolated polypeptide according to any of the prior claims, an ACC according to any of the prior claims, or a monomer construct according to any of the prior claims.

138. A vector comprising a polynucleotide as described in claim 137.

139. The vector according to claim 138, wherein the vector is an expression vector.

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

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

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

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

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

145. A method for administering a treatment to a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an isolated polypeptide according to any of the prior claims, an ACC according to any of the prior claims, or a composition according to claim 142 or 143.

146. The method according to claim 145, wherein the subject is identified or diagnosed with cancer.

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

148. The method according to claim 147, wherein the subject is identified or diagnosed with an inflammatory or autoimmune disease, disorder, or condition.

149. A method for producing ACC, To produce the ACC by culturing cells containing the polynucleotide described in claim 137 in a liquid culture medium, The method comprising recovering the ACC from the cells or the liquid culture medium.

150. The method according to claim 149, further comprising isolating the ACC recovered from the cells or the liquid culture medium.

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

152. A complex comprising a polypeptide comprising a cytokine complexed with an isolated polypeptide according to any prior claim.

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