FMS-like tyrosine kinase 3 ligand (FLT3L)-based chimeric protein

Abstract

FMS-like tyrosine kinase 3L (FLT3L) fused to a human cytokine is described, which can be used, for example, in cancer therapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 825,579, filed March 28, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION FMS-like tyrosine kinase 3 ligand (FLT3L) fused to signal transduction agents, including but not limited to human IFNα2, IFNβ, and IL1β, is described, which can be used, for example, in cancer therapy.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on March 23, 2020, is named ORN-062PC_A_Sequence_Listing_ST25.txt and is 28,672 bytes in size. [Background technology]

[0004] FMS-like tyrosine kinase 3 (FLT3) is expressed on the surface of many hematopoietic progenitor cells. FLT3 signaling is important for the normal development of hematopoietic stem and progenitor cells. The FLT3 gene is one of the most frequently mutated genes in acute myeloid leukemia (AML). Furthermore, FMS-like tyrosine kinase 3 ligand (FLT3L) agents are used to stimulate the immune system, for example, to alter the number of dendritic cells.

[0005] Cytokines are naturally occurring substances that can regulate cell growth and differentiation. Cytokines play important roles in a variety of physiological processes, including metabolism, respiration, sleep, excretion, healing, movement, reproduction, mood, stress, tissue function, immune function, sensory perception, and growth and development.

[0006] Clinically, cytokines appear to be applicable to the treatment of various diseases and disorders, including cancer. However, the administration of these soluble substances is not without risks. The therapeutic use of cytokines is often associated with systemic toxicity and adverse side effects, thus limiting the dosage levels at which these agents can be used. Summary of the Invention

[0007] Thus, in some aspects, the present invention relates to chimeric proteins comprising a targeting moiety comprising a single copy of FMS-like tyrosine kinase 3 ligand (FLT3L) or a portion thereof. In various embodiments, the targeting moiety functionally modulates an antigen or receptor of interest. In some embodiments, the targeting moiety binds but does not functionally modulate an antigen or receptor of interest. In some embodiments, the targeting moiety comprises a single copy of the extracellular domain of FLT3L, or a respective portion thereof. Chimeric proteins according to embodiments of the present invention also comprise a signal transduction agent or modified version thereof, such as a signal transduction agent described herein, for example, but not limited to, human IFNα2, IFNβ, and IL1β. The chimeric protein also comprises one or more flexible linkers connecting the chimeric protein and the signal transduction agent.

[0008] In some embodiments, the signaling agent can be a wild-type signaling agent described herein, such as, but not limited to, human IFNα2, IFNβ, and IL1β. In other embodiments, the signaling agent can be modified to include one or more mutations. The one or more mutations introduced into the signaling agent can confer various improved properties to the chimeric protein compared to a chimeric protein having an unmodified (e.g., wild-type) signaling agent. For example, the signaling agent can be a mutant human signaling agent described herein, such as, but not limited to, human IFNα2, IFNβ, and IL1β, having one or more mutations that confer improved safety compared to a wild-type signaling agent described herein, such as, but not limited to, human IFNα2, IFNβ, and IL1β. In various embodiments, the one or more mutations can confer improved safety, reduced affinity of the signaling agent for its receptor, or reduced biological activity of the signaling agent for its receptor compared to the wild-type signaling agent. In some embodiments, one or more mutations can attenuate the activation of the signal transduction substance, for example, the agonist or antagonist activity of the signal transduction substance can be attenuated. In some embodiments, one or more mutations in the modified signal transduction substance convert the activity of the signal transduction substance from agonist activity to antagonist activity. In various embodiments, the mutations confer reduced affinity or activity that can be restored by binding to one or more targeting moieties. Furthermore, in various embodiments, the mutations confer reduced or eliminated affinity or activity that cannot be substantially restored by attachment to a targeting moiety.

[0009] In various embodiments, the targeting moiety is directed to immune cells, thereby directly or indirectly recruiting immune cells to tumor cells or the tumor microenvironment. Non-limiting examples of immune cells include dendritic cells, T cells, B cells, macrophages, neutrophils, myeloid-derived suppressor cells, or NK cells. In some embodiments, the targeting moiety is directed to hematopoietic stem cells (HSCs), early progenitor cells, immature thymocytes, or steady-state dendritic cells (DCs). In some embodiments, targeting is directed to dendritic cells, such as conventional dendritic cells (cDCs) or plasmacytoid dendritic cells (pDCs). In some embodiments, targeting is directed to cDCs, optionally cDC-1, migratory DCs, and Flt3+ DCs. In some embodiments, the targeting moiety can increase the number of dendritic cells. In some embodiments, the targeting moieties of the present invention enhance tumor antigen presentation, optionally by dendritic cells.

[0010] In various embodiments, the chimeric proteins of the present invention are used in patients with various diseases or disorders, such as one or more of cancer, infectious diseases, immune disorders, autoimmune diseases, and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, metabolic diseases, and / or many other diseases and disorders. The present invention encompasses various methods of treating and preventing diseases and disorders, for example, various types of cancer and autoimmune diseases and / or neurodegenerative diseases. In some embodiments, the cancer is acute myeloid leukemia (AML). [Brief explanation of the drawings]

[0011] [Figure 1] Tumor growth curves in humanized mice after treatment with buffer or Flt3L-AcTaferon (i.e., a chimera of Flt3L ECD and IFNα2, R149A mutant). Mean values ​​(in mm3) (+SEM) from 5 or 6 animals per time point are plotted. [Figure 2] SEC (size exclusion chromatography) profile of purified FLT3L-AFN (dark line). Protein markers are indicated by gray lines. [Figure 3] SDS-PAGE gel of fractions 2, 3, and 4 from the SEC column under reducing conditions. DETAILED DESCRIPTION OF THE INVENTION

[0012] In some embodiments, chimeric proteins are provided that include a targeting moiety comprising a single copy of FMS-like tyrosine kinase 3 ligand (FLT3L) or a portion thereof. The chimeric protein also includes a wild-type signal transduction agent or a modified version thereof, where the signal transduction agent is one of the signal transduction agents described herein, such as, but not limited to, human IFNα2, IFNβ, and IL1β, which in various embodiments can be wild-type human or mutant forms. In the chimeric protein, one or more flexible linkers connect the targeting moiety and the signal transduction agent.

[0013] In some embodiments, the targeting moiety comprises a single copy of a portion of FLT3L. In other embodiments, the targeting moiety comprises a single copy of the extracellular domain of FLT3L, or a portion thereof. In some embodiments, the targeting moiety comprises an amino acid sequence that is a truncated version of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 (full length Flt3L) is: [ka] where bold = leader sequence, underlined letters: extracellular region not part of the receptor binding domain, italic letters = transmembrane and intracellular domains.

[0014] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2-5, or an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-5.

[0015] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2-5, or a single copy of an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-5.

[0016] The amino acid sequence of SEQ ID NO:2 (mature Flt3L-ec (extracellular domain)) is: TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQP.

[0017] The amino acid sequence of SEQ ID NO:3 (mature Flt3L-ec (extracellular domain) functional, shorter variant, commercially available (Prospecbio)) is: TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTA.

[0018] The amino acid sequence of SEQ ID NO: 4 (Flt3L-ec (extracellular domain) minimal functional domain (Savvides et al., 2000, Nature Structural Biology) is as follows: TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQP.

[0019] The amino acid sequence of the mature Flt3L-ec (extracellular domain) minimal functional domain (Savvides et al., 2000, Nature Structural Biology), truncated by starting with the first cysteine ​​and ending with the last cysteine ​​of SEQ ID NO: 5, is as follows: CSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQC.

[0020] In some embodiments, the chimeric proteins of the present invention are dimers. In some embodiments, the chimeric proteins are non-covalently linked dimers. In some embodiments, the chimeric proteins of the present invention comprise an amino acid sequence having at least about 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 9, or a variant.

[0021] In some embodiments, the signaling agent may comprise an amino acid sequence having at least 95% identity to one of SEQ ID NOs: 6, 7, 38, or 39, or may comprise the amino acid sequence of one of SEQ ID NOs: 6, 7, 38, or 39.

[0022] In various embodiments, the signal transduction agent is a modified (e.g., mutant) signal transduction agent having one or more mutations. In various embodiments, the mutations enable the modified signal transduction agent to have one or more attenuated activities, such as reduced binding affinity, reduced intrinsic activity, and one or more reduced specific biological activities, compared to the unmodified or non-mutated, i.e., wild-type, form of the signal transduction agent (e.g., comparing the wild-type and modified (e.g., mutant) forms of the same signal transduction agent). In various embodiments, the mutations enable the modified signal transduction agent to have one or more attenuated activities, such as reduced binding affinity, reduced intrinsic activity, and one or more reduced specific biological activities, compared to the unmodified or non-mutated form, e.g., wild-type IFNα2, IFNβ, or IL1β. In some embodiments, mutations that weaken or reduce binding or affinity include mutations that substantially reduce or eliminate binding or activity. In some embodiments, mutations that weaken or reduce binding or affinity are different from mutations that substantially reduce or eliminate binding or activity. As a result, in various embodiments, the mutations allow the signaling agent to be safer, e.g., have reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the non-mutated, i.e., wild-type, signaling agent (e.g., comparing the wild-type form with the modified (e.g., mutated) form of the same signaling agent). In various embodiments, the mutations allow the signaling agent to be safer, e.g., have reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the non-mutated sequence of a non-mutated interferon, e.g., IFNα2, IFNβ, or IL1β.

[0023] In various embodiments, the signaling agent is modified to have one or more mutations that reduce binding affinity or activity for one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or eliminate binding affinity or activity for the receptor. In some embodiments, the activity conferred by the wild-type signaling agent is agonism for the receptor (e.g., activation of a cellular effect at the site of treatment). For example, the wild-type signaling agent may activate its receptor. In such embodiments, the mutation results in the signaling agent being modified to reduce or eliminate the activating effect on the receptor. For example, the mutation may result in the signaling agent being modified to send a reduced activation signal to the target cell, or the activation signal may be eliminated. In some embodiments, the effect conferred by the wild-type signaling agent is antagonism for the receptor (e.g., blocking or suppressing a cellular effect at the site of treatment). For example, the wild-type signaling agent may antagonize or inhibit the receptor. In these embodiments, the mutation results in the signaling agent being modified to reduce or eliminate antagonizing activity for the receptor. For example, the mutations can result in the signal transduction agent being altered to send a reduced inhibitory signal to the target cell, or the inhibitory signal can be eliminated. In various embodiments, the signal transduction agent is an antagonist due to one or more mutations, e.g., an agonist signal transduction agent is converted to an antagonist signal transduction agent (e.g., as described in WO 2015 / 007520, the entire contents of which are incorporated herein by reference), and such converted signal transduction agent optionally also has one or more mutations that reduce its binding affinity or activity to one or more of its receptors, or reduce or eliminate its binding affinity or activity to one or more of its receptors.

[0024] In some embodiments, the reduced affinity or activity for the receptor is recoverable by binding of one or more targeting moieties, while in other embodiments, the reduced affinity or activity for the receptor is not substantially recoverable by the activity of one or more targeting moieties.

[0025] In various embodiments, the signaling agent is active on the target cell because the targeting moiety compensates for missing / insufficient binding (e.g., without limitation, and / or avidity) required for substantial activation. In various embodiments, the modified signaling agent is substantially inactive en route to the site of therapeutic action and exerts its effect substantially on the specifically targeted cell type, thereby greatly reducing undesirable side effects.

[0026] In some embodiments, a signaling agent may contain one or more mutations that weaken or reduce binding or affinity for one receptor (i.e., a therapeutic receptor) and one or more mutations that substantially reduce or eliminate binding or activity for a second receptor. In such embodiments, these mutations may be in the same or different positions (i.e., the same mutation or mutations). In some embodiments, the mutation(s) that reduce binding and / or activity for one receptor are different from the mutation(s) that substantially reduce or eliminate binding and / or activity for another receptor. In some embodiments, the mutation(s) that reduce binding and / or activity for one receptor are the same as the mutation(s) that substantially reduce or eliminate binding and / or activity for another receptor. In some embodiments, the chimeric protein has an altered signaling agent with both mutations that weaken binding and / or activity for a therapeutic receptor, thereby allowing a more controlled, on-target therapeutic effect (e.g., compared to a wild-type signaling agent), and mutations that substantially reduce or eliminate binding and / or activity for another receptor, thereby reducing side effects (e.g., compared to a wild-type signaling agent).

[0027] In some embodiments, the substantial reduction or elimination of binding or activity is substantially irreversible with the targeting moiety. In some embodiments, the substantial reduction or elimination of binding or activity is reversible with the targeting moiety. In various embodiments, the substantial reduction or elimination of binding or activity to a second receptor may also prevent adverse effects mediated by other receptors. Alternatively, or in addition, the substantial reduction or elimination of binding or activity to other receptors may reduce or eliminate sequestering of the therapeutic chimeric protein away from the therapeutic site of action, thereby improving therapeutic efficacy. For example, in some embodiments, this eliminates the need for high doses of the chimeric protein of the present invention to compensate for losses at other receptors. The ability to reduce such dosages further reduces the potential for side effects.

[0028] In various embodiments, the modified signaling agent may be modified to increase the affinity, e.g., binding (e.g., K D ) and / or activation (e.g., when the altered signal transduction agent is an agonist of that receptor, e.g., K A and / or EC 50 ) and / or inhibition (e.g., if the altered signal transduction agent is an antagonist of that receptor, e.g., K I and / or IC 50 The signaling agent may comprise one or more mutations that reduce, substantially reduce, or eliminate affinity (measurable as affinity, agonism, or antagonism) for the receptor. In various embodiments, the reduced affinity of the signaling agent for the receptor allows for attenuated activity (including agonism or antagonism). In such embodiments, the modified signaling agent has about 1%, or about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 10% to 20%, or about 20% to 40%, or about 50%, or about 40% to 60%, or about 60% to 80%, or about 80% to 100% affinity for the receptor compared to the wild-type signaling agent. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower compared to the wild-type signaling agent (including but not limited to, compared to non-mutated IFNα2, IFNβ, or IL1β).

[0029] In some embodiments where a chimeric protein has mutations that reduce binding to one receptor and substantially reduce or eliminate binding to a second receptor, the binding affinity of the altered signal transduction agent for one receptor is attenuated or reduced less than the affinity for the other receptor. In some embodiments, the binding affinity of the altered signal transduction agent for one receptor is attenuated or reduced by about 1%, or about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95% less than the affinity for the other receptor. In various embodiments, substantial reduction or elimination refers to a reduction in binding affinity and / or activity that is greater than a reduction or reduction.

[0030] In various embodiments, the modified signaling agent comprises one or more mutations that reduce the intrinsic activity of the signaling agent to, for example, about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1% compared to the wild-type signaling agent (including but not limited to, compared to non-mutated IFNα2, IFNβ, or IL1β).

[0031] In various embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and / or activity for a receptor for any one of cytokines, growth factors, and hormones, as described herein.

[0032] In some embodiments, the modified signaling agent contains one or more mutations that cause the signaling agent to have a reduced affinity for its receptor that is lower than the binding affinity of the targeting moiety for that receptor. In some embodiments, this difference in binding affinity exists between the signaling agent / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity allows the signaling agent, e.g., the mutant signaling agent, to have a localized on-target effect and minimize off-target effects that underlie side effects observed with wild-type signaling agents. In some embodiments, the binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold lower, or at least about 25-fold, or at least about 50-fold lower, or at least about 100-fold, or at least about 150-fold lower.

[0033] Receptor binding activity can be measured by using known methods in the art.For example, affinity and / or binding activity can be evaluated by Scatchard plot analysis and computer fitting of binding data (for example, Scatchard, 1949 Annals of the New York Academy of Sciences.51(4):660-672) or by reflectance interferometry under flow-through conditions, as described by Brecht et al.(1993), Biosens Bioelectron 1993;8:387-392.The entire contents of these documents are incorporated herein by reference.

[0034] In some embodiments, the wild-type or modified signaling agent is an interferon type I. In some embodiments, the wild-type or modified signaling agent is selected from IFNα2, IFNα1, IFNβ, IFNγ, consensus IFN, IFNε, IFNκ, IFNτ, IFNδ, and IFNν.

[0035] In some embodiments, the wild-type or modified signaling agent is interferon alpha. In such embodiments, the modified IFNα2 agent has reduced affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFNα2 agent has substantially reduced or eliminated affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0036] Mutant forms of interferon alpha 2 are known to those of skill in the art. In one exemplary embodiment, the modified signal transducer is an allelic IFNα2a having the following amino acid sequence: CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 6).

[0037] In an exemplary embodiment, the wild-type or modified signal transducer is an allelic IFNα2b having the following amino acid sequence: CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEWRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 7, which differs from IFNα2a at amino acid position 23).

[0038] In some embodiments, the IFNα2 mutant (IFNα2a or IFNα2b) has one or more amino acid mutations introduced at positions 144-154, e.g., amino acid positions 148, 149, and / or 153. In some embodiments, the IFNα2 mutant contains one or more mutations selected from L153A, R149A, and M148A. Such mutants are described, for example, in WO 2013 / 107791 and Piehler et al. (2000) J. Biol. Chem. 275:40425-33, the entire contents of which are incorporated herein by reference.

[0039] In some embodiments, the IFNα2 mutant has reduced affinity and / or activity for IFNAR1. In some embodiments, the IFNα2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A, as described in WO 2010 / 030671, the entire contents of which are incorporated herein by reference.

[0040] In some embodiments, the IFNα2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A, as described in WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0041] In some embodiments, the IFNα2 mutant comprises one or more mutations selected from R120E and R120E / K121E, as described in WO 2015 / 007520 and WO 2010 / 030671, the entire contents of which are incorporated herein by reference. In such embodiments, the IFNα2 mutant antagonizes wild-type IFNα activity 2. In such embodiments, the mutant IFNα2 has reduced affinity and / or activity for IFNAR1 but retains activity for IFNR2.

[0042] In some embodiments, the human IFNα2 mutant comprises one or more mutations selected from (1) R120E and R120E / K121E (which, without wishing to be bound by theory, produce an antagonistic effect), and (2) one or more mutations selected from K133A, R144A, R149A, and L153A (which, without wishing to be bound by theory, enable, for example, an attenuating effect on IFNAR2). In certain embodiments, the human IFNα2 mutant comprises R120E and L153A.

[0043] In some embodiments, the human IFNα2 variant comprises one or more mutations selected from L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, as disclosed in WO 2013 / 059885, the entire contents of which are incorporated herein by reference. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L30A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or R33A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or M148A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L153A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations N65A, L80A, Y85A, and / or Y89A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations N65A, L80A, Y85A, Y89A, and / or D114A as disclosed in WO 2013 / 059885.

[0044] In various embodiments, the signal transducer is mutant human IFNα2. In some embodiments, the mutant human IFNα2 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the mutant human IFNα2 has one or more mutations that confer improved safety relative to wild-type IFNα2 having the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, the IFNα2 has one or more mutations at positions 144-154 relative to SEQ ID NO: 6 or 7. In some embodiments, the human IFNα2 has one or more mutations at positions L15, A19, R22, R23, L26, F27, L30, K31, D32, R33, H34, D35, Q40, H57, E58, Q61, F64, N65, T69, L80, Y85, Y89, D114, L117, R120, R125, K133, K134, R144, A145, M148, R149, S152, L153, ​​and N156 relative to SEQ ID NO: 6 or 7. In some embodiments, the mutant IFNα2 has one or more mutations at positions R149, M148, or L153 relative to SEQ ID NO: 6 or 7. In some embodiments, the one or more mutations are one or more of L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57Y, E58N, Q61S, F64A, N65A, T69A, L80A, Y85A, Y89A, D114R, L117A, R120A, R125A, K133A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A relative to SEQ ID NO: 6 or 7. In some embodiments, the mutant human IFNα2 has a R149A mutation relative to SEQ ID NO:6 or 7.

[0045] In some embodiments, the mutant human IFNα2 has one or more mutations at positions R33, R144, A145, M148, R149, and L153 relative to SEQ ID NO: 6 or 7. In some embodiments, the mutant human IFNα2 has R33A, R144A, R144I, R144L, R144S, R144T, R144Y, A145D, A145G, A145H, A145K, A145Y, M148A, R149A, and L153A mutations relative to SEQ ID NO: 6 or 7.

[0046] In some embodiments, the mutant human IFNα2 has one or more mutations at positions R33, T106, R144, A145, M148, R149, and L153 relative to SEQ ID NO: 6 or 7. In some embodiments, the mutant human IFNα2 has one or more mutations selected from R33A, T106X3, R120E, R144X1, A145X2, M148A, R149A, and L153A relative to the amino acid sequence of SEQ ID NO: 6 or 7, wherein X1 is selected from A, S, T, Y, L, and I; X2 is selected from G, H, Y, K, and D; and X3 is selected from A and E.

[0047] In some embodiments, the wild-type or modified signal transducer is IFNβ. In some embodiments, the IFNβ is human, having the sequence shown below: MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSL HLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN (SEQ ID NO: 38).

[0048] In various embodiments, IFNβ includes functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of IFNβ. In various embodiments, IFNβ includes IFNβ from any species. In one embodiment, the chimeric protein includes a modified mouse IFNβ. In one embodiment, the chimeric protein includes a modified human IFNβ. Human IFNβ is a polypeptide containing 166 amino acid residues and having a molecular weight of approximately 22 kDa. The amino acid sequence of human IFNβ is SEQ ID NO:38.

[0049] In some embodiments, the human IFNβ is IFNβ1a, a glycosylated form of human IFNβ. In some embodiments, the IFNβ is IFNβ1b, a non-glycosylated form of human IFNβ with a Met-1 deletion and a Cys-17 to Ser mutation.

[0050] In various embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity to the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFNβ has reduced affinity and / or activity for IFNAR1. In various embodiments, the modified IFNβ is human IFNβ and has one or more mutations at positions F67, R71, L88, Y92, I95, N96, K123, and R124. In some embodiments, the one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G. In some embodiments, the modified IFNβ comprises an F67G mutation. In some embodiments, the modified IFNβ comprises a K123G mutation. In some embodiments, the modified IFNβ comprises an F67G and an R71A mutation. In some embodiments, the modified IFNβ comprises L88G and Y92G mutations. In some embodiments, the modified IFNβ comprises Y92G, I95A, and N96G mutations. In some embodiments, the modified IFNβ comprises K123G and R124G mutations. In some embodiments, the modified IFNβ comprises F67G, L88G, and Y92G mutations. In some embodiments, the modified IFNβ comprises F67S, L88S, and Y92S mutations.

[0051] In some embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity to the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFNβ has reduced affinity and / or activity for IFNAR2. In various embodiments, the modified IFNβ is human IFNβ and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. In some embodiments, the one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In certain embodiments, the modified IFNβ comprises a W22G mutation. In certain embodiments, the modified IFNβ comprises an L32A mutation. In certain embodiments, the modified IFNβ comprises an L32G mutation. In some embodiments, the modified IFNβ comprises an R35A mutation. In some embodiments, the modified IFNβ comprises an R35G mutation. In some embodiments, the modified IFNβ comprises a V148G mutation. In some embodiments, the modified IFNβ comprises an R152A mutation. In some embodiments, the modified IFNβ comprises an R152G mutation. In some embodiments, the modified IFNβ comprises a Y155G mutation. In some embodiments, the modified IFNβ comprises a W22G and R27G mutation. In some embodiments, the modified IFNβ comprises an L32A and R35A mutation. In some embodiments, the modified IFNβ comprises an L151G and R152A mutation. In some embodiments, the modified IFNβ comprises a V148G and R152A mutation.

[0052] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with C17S or C17A.

[0053] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with other IFNβ mutations described herein.

[0054] The crystal structure of human IFNβ is known and is described in Karpusas et al., (1998) PNAS, 94(22):11813-11818. In particular, the structure of human IFNβ has been shown to contain five α-helices (i.e., A, B, C, D, and E) and four loop regions (i.e., AB, BC, CD, and DE loops) connecting these helices. In various embodiments, the modified IFNβ has one or more mutations in the A, B, C, D, and E helices and / or the AB, BC, CD, and DE loops that reduce its binding affinity or activity to a therapeutic receptor such as IFNAR. Representative mutations are described in International Publication No. 2000 / 023114 and U.S. Patent Application Publication No. 20150011732, the entire contents of which are incorporated herein by reference. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 28-30, 32, and 33. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 36, 37, 39, and 42. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 71-73. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 92, 96, 99, and 100. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 128, 130, 131, and 134. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 149, 153, 156, and 159.

[0055] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at W22, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0056] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R27, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0057] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at W22, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R27, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0058] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L32, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0059] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R35, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0060] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L32, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R35, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0061] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R67, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0062] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R71, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0063] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at F67, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R71, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0064] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L88, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0065] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at Y92, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0066] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at F67, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at L88, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0067] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at Y92, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0068] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at I95, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and further comprises a mutation at Y92, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0069] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at N96, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0070] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at Y92, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a mutation at I95, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and a mutation at N96, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0071] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at K123, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0072] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R124, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0073] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at K123, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R124, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0074] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L151, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0075] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R152, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0076] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L151, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0077] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at V148, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0078] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at V148, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0079] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at Y155, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0080] In some embodiments, the wild-type or modified signal transducer is IL1β. In certain embodiments, the wild-type IL1β has the following amino acid sequence: APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS (SEQ ID NO: 39). IL1β is a pro-inflammatory cytokine and an important regulator of the immune system. It is a potent activator of CD4 T cell responses, promoting the proportion of Th17 cells and increasing the proliferation of IFNγ- and IL4-producing cells. IL1β also mediates the proliferation of CD8 + A potent regulator of T cells, antigen-specific CD8 +Enhances T cell proliferation, differentiation, peripheral migration, and memory. IL1β receptors include IL1R1 and IL1R2. Binding to and signaling through IL1R1 constitutes the mechanism by which IL1β mediates many of its biological (and pathological) effects. IL1R2 can function as a decoy receptor, reducing the availability of IL1β to interact and signal through IL1R1.

[0081] In some embodiments, the wild-type or modified signal transducer IL1β has reduced affinity and / or activity (e.g., agonist activity) for IL1R1. In some embodiments, the modified IL1β has substantially reduced or eliminated affinity and / or activity for IL1R2. Such embodiments result in restorable IL1β / IL1R1 signaling and prevention of loss of the therapeutic chimeric protein to ILR2 and consequently reduced dosage of IL1β (e.g., compared to a chimeric protein having only wild-type or attenuating mutations for ILR1). Such constructs are used, e.g., in methods for treating cancer, including, e.g., stimulating the immune system to mount an anti-cancer response.

[0082] In such embodiments, the modified signal transducer has a deletion of amino acids 52-54, which produces a modified human IL1β with reduced binding affinity to type I IL1R and reduced biological activity. See, e.g., WO 1994 / 000491, the entire contents of which are incorporated herein by reference. In some embodiments, the modified human IL1β comprises A117G / P118G, R120X, L122A, T125G / L126G, R127G, Q130X, Q131G, K132A, S137G / Q138Y, L145G, H146X, L145A / L147A, Q148X, Q148G / Q150G, Q150G / D151A, M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209X, K209A / K210A, K219X, E221X, and having one or more substitution mutations selected from E221S / N224A, N224S / K225S, E244K, N245Q (where X can be any amino acid change, e.g., a non-conservative change), which exhibit reduced binding to IL1R, as described, for example, in WO 2015 / 007542 and WO 2015 / 007536, the entire contents of which are incorporated herein by reference (GenBank Accession No. NP_000567, version NP-000567.1, G1:10835145, numbering based on the human IL1β sequence). In some embodiments, the modified human IL1β may have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S, and E221K. In certain embodiments, the modified human IL1β comprises the mutations Q131G and Q148G. In certain embodiments, the modified human IL1β comprises the mutations Q148G and K208E. In certain embodiments, the modified human IL1β comprises the mutations R120G and Q131G. In certain embodiments, the modified human IL1β comprises the mutations R120G and H146A. In one embodiment, the modified human IL1β comprises the mutations R120G and H146N.In some embodiments, the modified human IL1β comprises the mutations R120G and H146R. In some embodiments, the modified human IL1β comprises the mutations R120G and H146E. In some embodiments, the modified human IL1β comprises the mutations R120G and H146G. In some embodiments, the modified human IL1β comprises the mutations R120G and K208E. In some embodiments, the modified human IL1β comprises the mutations R120G, F162A, and Q164E. The modified human IL1β mutations are relative to SEQ ID NO: 39.

[0083] In various embodiments, one or more mutations in a signal transduction agent may confer improved safety to the chimeric protein compared to the wild-type signal transduction agent. The mutations may confer various other beneficial properties, including, but not limited to, reduced affinity of the signal transduction agent for its receptor and / or reduced biological activity of the signal transduction agent for its receptor. In some embodiments, one or more mutations in a signal transduction agent allow the activity of the signal transduction agent to be attenuated. For example, the agonist or antagonist activity of the signal transduction agent may be attenuated. Furthermore, in some embodiments, the modified signal transduction agent contains one or more mutations that convert its activity from agonist activity to antagonist activity.

[0084] In some embodiments, the signaling agent comprises one or more mutations that confer reduced affinity or activity that is reversible upon attachment to one or more targeting moieties, while in other embodiments, the signaling agent has one or more mutations that confer substantially reduced or eliminated affinity or activity that is not substantially reversible upon attachment to a targeting moiety.

[0085] In some embodiments, the targeting moiety is directed to an immune cell, which can be selected from a dendritic cell, a T cell, a B cell, a macrophage, a neutrophil, a myeloid-derived suppressor cell, or a NK cell. In some embodiments, the targeting moiety is directed to a hematopoietic stem cell (HSC), an early progenitor cell, an immature thymocyte, or a steady-state dendritic cell (DC). The targeting moiety can functionally modulate an antigen or receptor of interest. In some embodiments, the targeting moiety binds but does not functionally modulate an antigen or receptor of interest.

[0086] In various embodiments, the chimeric protein, among other features, directly or indirectly recruits one or more immune cells to diseased cells, for example, via the targeting moiety. Thus, in some embodiments, the targeting moiety directly or indirectly recruits immune cells to tumor cells or to the tumor microenvironment. In this manner, the targeting moiety can increase the number of dendritic cells. In some embodiments, the targeting moiety optionally enhances tumor antigen presentation by dendritic cells.

[0087] In various embodiments, the chimeric proteins are suitable for use in patients with one or more of cancer, infectious diseases, immune disorders, autoimmune and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases. In some aspects, methods for treating or preventing cancer are provided, the methods comprising administering to a patient in need thereof an effective amount of a chimeric protein according to various embodiments of the present disclosure.

[0088] In various embodiments, the cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney or renal cancer cancer); laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, tonsil, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland carcinoma; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; Hodgkin's lymphoma and non-Hodgkin's lymphoma; and lymphomas, including B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; The cancer is selected from one or more of the following: intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD); and abnormal vascular proliferation associated with phacomatosis; edema (for example, associated with brain tumor); and Meigs syndrome. In certain embodiments, the cancer is acute myeloid leukemia (AML).

[0089] Furthermore, in some aspects, the present invention includes methods for treating or preventing autoimmune and / or neurodegenerative diseases, comprising administering to a patient in need thereof an effective amount of a chimeric protein according to various embodiments of the present disclosure. The autoimmune and / or neurodegenerative disease can be selected from multiple sclerosis, diabetes, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barré syndrome, scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, primary sclerosing cholangitis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Menier's syndrome, transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjögren's syndrome, lupus erythematosus, myasthenia gravis, Reiter's syndrome, and Graves' disease.

[0090] In some embodiments, a chimeric protein is provided that comprises an amino acid sequence having at least 90% identity to SEQ ID NO:9, or an amino acid sequence having at least 95% identity to SEQ ID NO:9.

[0091] In some embodiments, the chimeric proteins of the invention optionally comprise one or more flexible linkers. In some embodiments, the chimeric proteins of the invention comprise a flexible linker connecting the targeting moiety and the signal transduction agent (e.g., IFNα2, IFNβ, or IL1β, or a variant thereof). In some embodiments, the chimeric proteins of the invention comprise a flexible linker within the signal transduction agent (e.g., IFNα2, IFNβ, or IL1β, or a variant thereof). In some embodiments, flexible linkers may be utilized to connect various functional groups, residues, or moieties described herein to the chimeric protein. In some embodiments, the flexible linker is a plurality of amino acids that does not affect or reduce the stability, orientation, binding, neutralization, and / or efflux properties of the binding region and the binding protein.

[0092] In some embodiments, the chimeric proteins include one or more additional signal transducing agents, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, as described herein, which may be wild-type or modified. In various embodiments, the chimeric proteins of the invention have modified signal transducing agents and provide improved safety compared to the unmodified wild-type. For clarity, the invention includes chimeric proteins with one, two, or three signal transducing agents in some embodiments.

[0093] In various embodiments, the chimeric protein comprises one or more targeting moieties (e.g., various antibody formats, including, but not limited to, single domain antibodies) that specifically bind to a target of interest (e.g., antigen, receptor). In various embodiments, the targeting moiety specifically binds to a target of interest (e.g., antigen, receptor), including those found on one or more immune cells, which may include, but are not limited to, T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendritic cells. In some embodiments, the targeting moiety specifically binds to a target of interest (e.g., antigen, receptor) and effectively recruits one or more immune cells. In some embodiments, the target of interest (e.g., antigen, receptor) may be found on one or more tumor cells. In some embodiments, the chimeric proteins of the present invention can recruit immune cells, e.g., immune cells that kill and / or suppress tumor cells, to a site of action (such as, by way of non-limiting example, the tumor microenvironment). In some embodiments, the targeting moiety specifically binds to a target of interest (e.g., an antigen, a receptor) that is part of a non-cellular structure. For clarity, the present invention includes chimeric proteins that, in some embodiments, have one, two, or three targeting moieties.

[0094] In some embodiments, vectors encoding the chimeric proteins of the present invention linked as a single nucleotide sequence to any of the flexible linkers described herein are provided and can be used to prepare such chimeric proteins.

[0095] In some embodiments, the length of the flexible linker allows for effective binding of the targeting moiety and signaling agent (e.g., IFNα2, IFNβ, or IL1β or variants thereof) to their receptors. For example, in some embodiments, the length of the flexible linker allows for effective binding of the targeting moiety and signaling agent to one receptor on the same cell.

[0096] In some embodiments, the length of the flexible linker is at least equal to the shortest distance between the binding sites of a targeting moiety and a signaling agent receptor on the same cell, hi some embodiments, the length of the flexible linker is at least 2, 3, 4, 5, 10, 20, 25, 50, 100, or more times the shortest distance between the binding sites of a targeting moiety and a signaling agent receptor on the same cell.

[0097] As described herein, the length of the flexible linker allows for the effective binding of a single targeting moiety and signaling agent to a receptor on the same cell, and the binding is sequential, e.g., targeting moiety / receptor binding precedes signaling agent / receptor binding.

[0098] In some embodiments, there are two flexible linkers in a single chimera, each linking a signaling agent to a targeting moiety. In various embodiments, the flexible linkers have a length that allows for the formation of a site with disease cells and effector cells without steric hindrance that could interfere with the regulation of either cell.

[0099] The present invention contemplates the use of various flexible linker sequences. In various embodiments, the flexible linker may be functional. For example, but not limited to, the flexible linker may function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve the biological activity of the chimeric protein of the present invention.

[0100] In some embodiments, the linker is a polypeptide. In some embodiments, the flexible linker is less than about 100 amino acids in length. For example, the flexible linker can be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the flexible linker is a polypeptide. In some embodiments, the flexible linker is more than about 100 amino acids in length. For example, the flexible linker can be greater than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length.

[0101] In various embodiments, the flexible linker is substantially composed of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine). For example, in some embodiments, the flexible linker is (Gly4Ser) nwherein n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NOs: 10 to 17, respectively). In one embodiment, the flexible linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 18). Examples of additional flexible linkers include, but are not limited to, the sequences: LE, GGGGS (SEQ ID NO: 10), (GGGGS) n (n = 1 to 4) (SEQ ID NOs: 10 to 13), (Gly) (SEQ ID NO: 19), (Gly) (SEQ ID NO: 20), (EAAAK) n (n = 1 to 3) (SEQ ID NOs: 21 to 23), A(EAAAK) n A (n = 2 to 5) (SEQ ID NOs: 24 to 27), AEAAAKEAAAKA (SEQ ID NO: 24), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 28), PAPAP (SEQ ID NO: 29), KESGSVSSEQLAQFRSLD (SEQ ID NO: 30), EGKSSGSGSESKST (SEQ ID NO: 31), GSAGSAAGSGEF (SEQ ID NO: 32), and (XP) n (X represents any amino acid, for example, Ala, Lys, or Glu). In various embodiments, the flexible linker is GGS.

[0102] In some embodiments, the flexible linker is one or more of GGGSE (SEQ ID NO: 33), GSESG (SEQ ID NO: 34), GSEGS (SEQ ID NO: 35), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 36), and a flexible linker of G, S, and E randomly spaced every four amino acids.

[0103] In various embodiments, the flexible linker can be functional. For example, but not limited to, the flexible linker can function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve biological activity of the chimeric protein of the present invention. In another example, the flexible linker can function to target the chimeric protein to a particular cell type or site.

[0104] In various embodiments, the chimeric proteins of the present invention may contain one or more functional groups, residues, or moieties. In various embodiments, the one or more functional groups, residues, or moieties are attached to or genetically fused with any of the signal transduction agents or targeting moieties described herein. In some embodiments, such functional groups, residues, or moieties impart one or more desirable properties or functionalities to the chimeric proteins of the present invention. Examples of such functional groups and techniques for introducing them into chimeric proteins are known in the art; see, for example, Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

[0105] In various embodiments, each of the chimeric proteins may be conjugated and / or fused to another agent to extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties. In some embodiments, the chimeric proteins may be fused or conjugated to one or more of PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, etc.

[0106] In various embodiments, each individual chimeric protein is fused to one or more agents described in BioDrugs (2015) 29:215-239, the entire contents of which are incorporated herein by reference.

[0107] In some embodiments, the functional group, residue, or moiety comprises a suitable pharmaceutically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly(ethylene glycol) or mPEG). In some embodiments, the attachment of a PEG moiety extends the half-life and / or reduces the immunogenicity of the chimeric protein. Any suitable form of PEGylation is commonly used, such as PEGylation used in the art for antibodies and antibody fragments (including, but not limited to, single-domain antibodies such as VHHs); see, e.g., Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev., 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003); and WO 04 / 060965, the entire contents of which are incorporated herein by reference. Various reagents for protein PEGylation are also commercially available, for example, from Nektar Therapeutics, USA. In some embodiments, site-specific PEGylation, particularly via cysteine ​​residues, is used (see, e.g., Yang et al., Protein Engineering, 16, 10, 761-770 (2003) (the entire contents of which are incorporated herein by reference). In some embodiments, the chimeric protein of the present invention is modified to appropriately introduce one or more cysteine ​​residues for PEG attachment, or an amino acid sequence containing one or more cysteine ​​residues for PEG attachment can be fused to the amino and / or carboxy termini of the chimeric protein using techniques known in the art.

[0108] In some embodiments, the functional group, residue, or moiety comprises N-linked or O-linked glycosylation, hi some embodiments, the N-linked or O-linked glycosylation is introduced as part of a co-translational and / or post-translational modification.

[0109] In some embodiments, the functional group, residue, or moiety comprises one or more detectable labels or other signal-generating groups or moieties. Suitable labels and techniques for their attachment, use, and detection are known in the art and include, but are not limited to, fluorescent labels (e.g., fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine and fluorescent metals, e.g., Eu or other metals of the lanthanide series), phosphorescent labels, chemiluminescent labels, or bioluminescent labels (e.g., luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, dioxetane, or GFP and its analogs), radioisotopes, metals, metal chelates, or metal cations. or other metals or metal cations that are particularly suited for use in in vivo, in vitro, or in situ diagnostics and imaging, as well as chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase). Other suitable labels include moieties that can be detected using NMR or ESR spectroscopy. Polypeptides of the invention so labeled may be used, for example, for in vitro, in vivo, or in situ assays (themselves known as immunoassays such as ELISAs, RIAs, and EIAs and other "sandwich" assays) and in vivo diagnostic and imaging purposes, depending on the choice of the particular label.

[0110] In some embodiments, the functional group, residue, or moiety comprises a tag attached or genetically fused to the chimeric protein. In some embodiments, the chimeric protein can comprise a single tag or multiple tags. For example, the tag is a peptide, sugar, or DNA molecule that does not inhibit or interfere with binding of the chimeric protein to its target or any other antigen of interest, such as a tumor antigen. In various embodiments, the tag is at least about: 3-5 amino acids in length, 5-8 amino acids in length, 8-12 amino acids in length, 12-15 amino acids in length, or 15-20 amino acids in length. Examples of tags are described, for example, in U.S. Patent Application Publication No. 2013 / 0058962. In some embodiments, the tag is an affinity tag, such as a glutathione-S-transferase (GST) or histidine (His) tag. In certain embodiments, the chimeric protein comprises a histidine tag.

[0111] In some embodiments, the functional group, residue, or moiety comprises a chelating group, for example, for chelating a metal or metal cation. Suitable chelating groups include, for example, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0112] In some embodiments, the functional group, residue, or moiety comprises a functional group that is one half of a specific binding pair, such as a biotin-(streptavidin) binding pair. Such functional groups can be used to link the chimeric proteins of the present invention to another protein, polypeptide, or chemical compound that is bound to the other half of the binding pair, i.e., via binding of the binding pair. For example, the chimeric proteins of the present invention can be bound to biotin and then linked to another protein, polypeptide, compound, or carrier that is bound to avidin or streptavidin. Such conjugated chimeric proteins can be used, for example, as reporters in diagnostic systems in which a detectable signal-generating substance is bound to avidin or streptavidin. For example, such binding pairs can be used to bind the chimeric proteins to carriers, including carriers suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described in Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs can also be used to link therapeutically active agents to the chimeric proteins of the present invention.

[0113] Methods for producing the chimeric proteins of the present invention are described herein. For example, DNA sequences encoding the chimeric proteins of the present invention (e.g., DNA sequences encoding a signal transduction agent (e.g., IFNα2, IFNβ, or IL1β or a variant thereof) and a targeting moiety and a flexible linker) can be chemically synthesized using methods known in the art. The synthetic DNA sequence can be linked to other appropriate nucleotide sequences, including, for example, expression control sequences, to produce a gene expression construct encoding the chimeric protein of interest. Thus, in various embodiments, the present invention provides isolated nucleic acids comprising nucleotide sequences encoding the chimeric proteins of the present invention.

[0114] The nucleic acid encoding the chimeric protein of the present invention may be incorporated (linked) into an expression vector, which can be introduced into host cells by gene transfer, transformation, or transduction techniques. For example, the nucleic acid encoding the chimeric protein of the present invention can be introduced into host cells by retroviral transduction. Examples of host cells include E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, cultured monkey kidney (COS) cells, or human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. The transformed host cells can be grown under conditions that allow the host cells to express the gene encoding the chimeric protein of the present invention. Thus, in various embodiments, the present invention provides expression vectors containing nucleic acids encoding the chimeric protein of the present invention. In various embodiments, the present invention further provides host cells containing such expression vectors.

[0115] Specific expression and purification conditions vary depending on the expression system used.For example, if a gene is expressed in E. coli, the gene is first inserted into an expression vector by placing the engineered gene downstream of a bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence.In another example, if a gene is expressed in a eukaryotic host cell, such as CHO cell, the gene is first inserted into an expression vector that includes, for example, a suitable eukaryotic promoter, a secretion signal, a transcription factor, and various introns.The gene construct can be introduced into host cells using gene transfer, transformation, or transduction techniques.

[0116] The chimeric proteins of the invention can be produced by growing host cells transfected with an expression vector encoding the chimeric protein under conditions that allow expression of the protein. After expression, the protein can be harvested and purified using techniques well known in the art, for example, by affinity tags such as glutathione-S-transferase (GST) and histidine tags, or by chromatography.

[0117] Thus, in various embodiments, the invention provides nucleic acids encoding the chimeric proteins of the invention. In various embodiments, the invention provides host cells comprising nucleic acids encoding the chimeric proteins of the invention. In various embodiments, the invention provides nucleic acids encoding the chimeric proteins of the invention that are suitable for production in a cell-free system (e.g., in vitro transcription and / or in vitro translation).

[0118] In various embodiments, IFNα2, IFNβ, or IL1β, variants thereof, or chimeric proteins comprising IFNα2, IFNβ, or IL1β, or variants thereof, can be expressed in vivo, for example, in a patient. For example, in various embodiments, IFNα2, IFNβ, or IL1β, variants thereof, or chimeric proteins comprising IFNα2, IFNβ, or IL1β, or variants thereof, can be administered in the form of a nucleic acid encoding IFNα2, IFNβ, or IL1β, variants thereof, or chimeric proteins comprising IFNα2, IFNβ, or IL1β, or variants thereof. In various embodiments, the nucleic acid is DNA or RNA. In some embodiments, IFNα2, IFNβ, or IL1β, variants thereof, or chimeric proteins comprising IFNα2, IFNβ, or IL1β, or variants thereof, are encoded by modified mRNA, i.e., mRNA containing one or more modified nucleotides. In some embodiments, the modified mRNA comprises one or more modifications found in U.S. Patent No. 8,278,036, the entire contents of which are incorporated herein by reference. In some embodiments, the modified mRNA comprises one or more of m5C, m5U, m6A, s2U, Ψ, and 2'-O-methyl-U. In some embodiments, the invention relates to the administration of modified mRNA encoding one or more chimeric proteins of the invention. In some embodiments, the invention relates to gene therapy vectors comprising the modified mRNA. In some embodiments, the invention relates to gene therapy methods comprising the modified mRNA. In various embodiments, the nucleic acid is in the form of an oncolytic virus, such as adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus, or vaccinia.

[0119] The chimeric proteins described herein may have sufficiently basic functional groups that can react with inorganic or organic acids or carboxyl groups, or with inorganic or organic bases, to form pharmaceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids as is well known in the art. Such salts include, for example, the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich (Switzerland), 2002. These references are incorporated herein by reference in their entireties.

[0120] Pharmaceutically acceptable salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate. Acid salts include naphthalene-2-benzoate, isobutyrate, phenylbutyrate, alpha-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate.

[0121] The term "pharmaceutically acceptable salt" also refers to a salt of a composition of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, and unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, organic amines such as dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines) such as mono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

[0122] In some embodiments, the compositions described herein are in the form of pharmaceutically acceptable salts.

[0123] In various embodiments, the present invention relates to pharmaceutical compositions comprising a chimeric protein described herein and a pharmaceutically acceptable carrier or excipient. Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally contain a suitable amount of a pharmaceutically acceptable excipient to provide a suitable dosage form.

[0124] In various embodiments, pharmaceutical excipients can be liquids such as water and oils, including those of petroleum, animal, vegetable, or artificial origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Pharmaceutical excipients can be, for example, saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, and urea. Additionally, auxiliary agents, stabilizers, thickeners, lubricants, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Saline and aqueous dextrose and glycerin solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, etc. Any of the formulations described herein may contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Further examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0125] The present invention includes the described pharmaceutical compositions (and / or additional therapeutic agents) in a variety of formulations. Any of the pharmaceutical compositions (and / or additional therapeutic agents) of the present invention described herein may be in the form of a solution, suspension, emulsion, infusion, tablet, pill, pellet, capsule, liquid-containing capsule, gelatin capsule, powder, sustained-release formulation, suppository, emulsion, aerosol, spray, suspension, lyophilized powder, frozen suspension, dry powder, or any other form suitable for use. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In a further embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a liquid.

[0126] If necessary, the pharmaceutical compositions of the present invention (and / or additional agents) may also include a solubilizing agent. The agents may also be delivered using suitable vehicles or delivery devices known in the art. The combination therapeutic agents outlined herein may be co-delivered in a single delivery vehicle or carrier.

[0127] Formulations containing the pharmaceutical composition of the present invention (and / or additional agents) can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. Such methods typically include the step of combining the therapeutic agent with a carrier, which may constitute one or more accessory ingredients. Typically, formulations are prepared by uniformly and intimately admixing the therapeutic agent with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation (e.g., wet or dry granulation, powder blending, etc., followed by tabletting using conventional methods known in the art).

[0128] In various embodiments, any pharmaceutical composition (and / or additional agent) described herein is formulated in accordance with routine procedures as a composition adapted for the methods of administration described herein.

[0129] Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical. Administration can be local or systemic. In some embodiments, administration is oral. In other embodiments, administration is by parenteral injection. The method of administration is at the discretion of the practitioner and depends, in part, on the site of the medical condition. In most cases, administration results in the release of any agent described herein into the bloodstream.

[0130] In one embodiment, the chimeric proteins described herein are routinely formulated as compositions adapted for oral administration. Compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more agents to provide a pharmaceutically palatable formulation, such as sweeteners such as lactose, aspartame, or saccharin, flavorings such as peppermint, wintergreen oil, or cherry oil, coloring agents, and preservatives. Additionally, in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby enabling sustained action over an extended period of time. Selectively permeable membranes surrounding any of the osmotically activated chimeric proteins described herein are also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the carrier compound, which swells and expels the drug or drug composition through an opening. These delivery platforms can provide an essentially zero-order delivery profile, as opposed to the spiked release profiles of immediate-release formulations. Time-delay agents, such as glycerol monostearate or glycerol stearate, can also be used. Oral compositions can contain standard excipients, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are pharmaceutical grade. In addition to the active compound, the suspension can contain a suspending agent, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and the like, and mixtures thereof.

[0131] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may be prepared in the form of sterile solid compositions (e.g., lyophilized compositions), which can be dissolved or suspended in a sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art. Suitable formulation components for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetate, citrate, or phosphate; and agents for regulating osmolality such as sodium chloride or dextrose.

[0132] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier must be stable under the conditions of manufacture and storage and must be protected against microorganisms. The carrier may be a solvent or dispersion medium, for example, containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0133] The compositions provided herein, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., "nebulized" formulations) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0134] Any pharmaceutical composition of the present invention (and / or additional agent) described herein can be administered by controlled release or sustained release means or delivery device known to those skilled in the art.Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556.Each of these patents is incorporated herein by reference in its entirety. Such dosage forms are useful for enabling controlled or sustained release of one or more active ingredients using, for example, hydropropyl cellulose, hydropropyl methyl cellulose, polyvinylpyrrolidone, other polymer matrices, gels, osmotic membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, and can provide desired release profiles at various rates. Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the active ingredients of the pharmaceutical compositions described herein. The present invention thus provides unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel capsules, and caplets, adapted for controlled or sustained release.

[0135] Controlled or sustained release of an active ingredient can be stimulated by various conditions including, but not limited to, changes in pH, temperature, stimulation with light of a suitable wavelength, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0136] In another embodiment, a sustained-release system can be placed in the vicinity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review in Langer, 1990, Science 249:1527-1533, can be used.

[0137] The pharmaceutical preparation is preferably sterile. Sterilization can be achieved, for example, by filtration through a sterile filtration membrane. If the composition is lyophilized, filter sterilization can be performed before or after lyophilization and reconstitution.

[0138] It will be understood that the actual dose of the chimeric protein administered according to the present invention will vary depending on the specific dosage form and method of administration. Those skilled in the art can take into account many factors that may alter the action of the chimeric protein (e.g., body weight, sex, diet, administration time, administration route, excretion rate, condition of the subject, drug combinations, genetic predisposition, and reaction sensitivity). Administration can be carried out continuously or in one or more separate doses within the maximum tolerated dose range. The optimal administration rate for a given set of conditions can be ascertained by those skilled in the art using conventional dosage administration tests.

[0139] In some embodiments, a suitable dosage of the chimeric protein is in the range of about 0.01 μg / kg to about 100 mg / kg of the subject's body weight, about 0.01 μg / kg to about 10 mg / kg of the subject's body weight, or about 0.01 μg / kg to about 1 mg / kg of the subject's body weight, e.g., about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.06 μg / kg, about 0.07 μg / kg, about 0.08 μg / kg, about 0.09 μg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, 1.9 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg body weight, or about 100 mg / kg body weight (including all values ​​and ranges therebetween).

[0140] Individual doses of the chimeric protein can be administered as unit dosage forms (e.g., tablets, capsules, or liquid formulations) containing, for example, about 1 μg to about 100 mg, about 1 μg to about 90 mg, about 1 μg to about 80 mg, about 1 μg to about 70 mg, about 1 μg to about 60 mg, about 1 μg to about 50 mg, about 1 μg to about 40 mg, about 1 μg to about 30 mg, about 1 μg to about 20 mg, about 1 μg to about 10 mg, about 1 μg to about 5 mg, about 1 μg to about 3 mg, about 1 μg to about 1 mg, or about 1 μg to about 50 μg per unit dosage form. For example, the unit dosage form may contain about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 0.1 mg, about 0.2 mg g, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg (including all values ​​and ranges therebetween).

[0141] In one embodiment, the chimeric protein is administered in an amount of about 1 μg to about 100 mg daily, about 1 μg to about 90 mg daily, about 1 μg to about 80 mg daily, about 1 μg to about 70 mg daily, about 1 μg to about 60 mg daily, about 1 μg to about 50 mg daily, about 1 μg to about 40 mg daily, about 1 μg to about 30 mg daily, about 1 μg to about 20 mg daily, about 1 μg to about 10 mg daily, about 1 μg to about 5 mg daily, about 1 μg to about 3 mg daily, or about 1 μg to about 1 mg daily. In various embodiments, the chimeric protein is present in an amount of about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 0.1 mg, about 0 and / or about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg (including all values ​​and ranges therebetween).

[0142] In certain embodiments of the invention, a pharmaceutical composition comprising a chimeric protein may be administered, for example, two or more times daily (e.g., about twice, about three times, about four times, about five times, about six times, about seven times, about eight times, about nine times, or about ten times daily), about once daily, about every other day, about every third day, about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every six months, or about once a year. In certain embodiments, a pharmaceutical composition comprising a chimeric protein is administered about three times a week.

[0143] In various embodiments, the chimeric proteins of the present invention can be administered for an extended period of time. For example, the chimeric proteins can be administered for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, or at least about 12 weeks, as described herein. For example, the chimeric proteins can be administered for 12 weeks, 24 weeks, 36 weeks, or 48 weeks. In some embodiments, the chimeric proteins are administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months. In some embodiments, the chimeric proteins can be administered for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

[0144] In various embodiments, a pharmaceutical composition of the invention is co-administered with an additional therapeutic agent. Co-administration can be simultaneous or sequential.

[0145] In one embodiment, the additional therapeutic agent and the chimeric protein of the present invention are administered to a subject simultaneously. As used herein, the term "simultaneously" means that the additional therapeutic agent and the chimeric protein are administered at a time interval of about 60 minutes or less, for example, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, about 5 minutes or less, or about 1 minute or less. The administration of the additional therapeutic agent and the chimeric protein can be carried out by simultaneous administration of a single formulation (e.g., a formulation containing the additional therapeutic agent and the chimeric protein) or separate formulations (e.g., a first formulation containing the additional therapeutic agent and a second formulation containing the chimeric protein).

[0146] Co-administration does not require simultaneous administration of the therapeutic agents, provided that the timing of their administration is such that the pharmacological activities of the additional therapeutic agent and the chimeric protein overlap over time, thereby achieving a combined therapeutic effect. For example, the additional therapeutic agent and the chimeric protein can be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and the chimeric protein are administered at a time interval of more than about 60 minutes. For example, the time interval between the sequential administration of the additional therapeutic agent and the chimeric protein can be more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, more than about 1 week, more than about 2 weeks, or more than about 1 month. The optimal administration time will depend on the metabolism, excretion rate, and / or pharmacodynamic activity of the administered additional therapeutic agent and chimeric protein. Either the additional therapeutic agent or the chimeric protein may be administered first.

[0147] Co-administration also does not require that the therapeutic agents be administered to a subject by the same route of administration. Rather, each therapeutic agent can be administered by any suitable route, e.g., parenterally or non-parenterally.

[0148] In some embodiments, the chimeric proteins described herein act synergistically when co-administered with another therapeutic agent, and in such embodiments, the chimeric protein and the additional therapeutic agent may be administered at lower doses than would be employed if the therapeutic agent were used in monotherapy.

[0149] In some embodiments, the present invention relates to a chemotherapeutic agent as an additional therapeutic agent. For example, but not limited to, such combinations of chimeric proteins of the present invention and chemotherapeutic agents are used to treat cancer, as described elsewhere herein. Examples of chemotherapeutic agents include alkylating agents such as thiotepa, CYTOXAN (cyclophosphamide), alkyl sulfonates such as busulfan, improsulfan, and piposulfan, aziridines such as benzodopa, carboquone, mesuredopa, and uredopa, ethylenimines, methylmelamines such as altretamine, triethylenemelamine, triethylenenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine, acetogenins (e.g., bullatacin, bullatatinone), canthotecins (including the synthetic analog topotecan), bryostatin, kallistatin (cally statin), CC-1065 (including synthetic analogs of adozelesin, carzelesin, and bizelesin), cryptophycins (e.g., cryptophycin 1, cryptophycin 8, etc.), dolastatins, duocarmycins (including synthetic analogs KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictine, spongistatin, nitrogen mustards, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine, antibiotics such as enediyne antibiotics (calicheamicin, especially calicheamicin gamma II and calicheamicin omega II (see Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodol Bicine, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine , azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal substances such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as florinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate, demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine;Maytansinoids, e.g., maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (ara-C); cyclophosphamide; thiotepa; taxoids, e.g., taxol, paclitaxel (Bristol-Myers Squibb Company, Squibb Oncology, Princeton, NJ), Abraxane Cremophor-free, albumin-treated nanoparticle-formed paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), Taxotere doxetaxel (Rhone-Poulenc Rorer, Antony, France), chlorambucil, Gemzar (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine, platinum, etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (cancer ptsar, CPT-11) (including irinotecan plus 5-FU and leucovorin therapy); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin therapy (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A, which reduce cell proliferation;and pharmaceutically acceptable salts, acids, or derivatives of any of the above-mentioned agents. Furthermore, the method of treatment may further include the use of radiation. Furthermore, the method of treatment may further include the use of photodynamic therapy.

[0150] In some embodiments, the chimeric proteins described herein include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition, where the covalent attachment does not interfere with the activity of the composition. For example, without limitation, derivatives include compositions that are modified by glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, conjugation to cellular ligands or other proteins, among others. Any of a number of chemical modifications can be performed using known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

[0151] In still other embodiments, the chimeric proteins described herein further comprise cytotoxic agents, which in exemplary embodiments include toxins, chemotherapeutic agents, radioisotopes, and agents that cause apoptosis or cell death. Such agents may be conjugated to the compositions described herein.

[0152] The chimeric proteins described herein can be post-translationally modified in this manner to add effector moieties such as chemical linkers, detectable moieties such as fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as streptavidin, avidin, biotin, cytotoxins, cytotoxic drugs, and radioactive materials.

[0153] Examples of cytotoxic agents include methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, These include, but are not limited to, cis-dichlorodiamineplatinum(II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines (including daunorubicin (formerly daunomycin) and doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone, and bisantrene); antibiotics (including dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC)); antimytotic agents (e.g., vinca alkaloids, vincristine, and vinblastine). Other cytotoxic agents include paclitaxel (Taxol), ricin, Pseudomonas aeruginosa exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicine, dihydroxyanthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (O,P'-(DDD)), interferon, and mixtures of these cytotoxic agents.

[0154] Additional cytotoxic agents include chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platins, taxol, irinotecan, 5-fluorouracil, gemcytabine, leucovorin, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine). ), mustine, tyrosine kinase inhibitor, radiation therapy, sex hormone antagonist, selective androgen receptor modulator, selective estrogen receptor modulator, PDGF antagonist, TNF antagonist, IL1 antagonist, interleukin (e.g., IL12 or IL2), IL12R antagonist, toxin-conjugated monoclonal antibody, tumor antigen-specific monoclonal antibody, erbitux, avastin, pertuzumab, anti-CD20 antibody, rituxan, ocrelizumab, ofatumumab, DXL625, Herceptin®, or any combination thereof. Toxic enzymes from plants and bacteria, such as ricin, diphtheria toxin, and Pseudomonas toxin, can be conjugated with therapeutic agents (e.g., antibodies) to produce cell-type-specific killing agents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).

[0155] Other cytotoxic agents include cytotoxic ribonucleases, as described in U.S. Patent No. 6,653,104 to Goldenberg. Embodiments of the present invention also relate to radioimmunoconjugates in which alpha- or beta-particle-emitting radionuclides are stably attached to chimeric proteins, with or without the use of complexing agents. Such radionuclides include beta-emitters, such as phosphorus-32, scandium-47, copper-67, gallium-67, yttrium-88, yttrium-90, iodine-125, iodine-131, samarium-153, lutetium-177, rhenium-186, or rhenium-188, and alpha-emitters, such as astatine-211, lead-212, bismuth-212, bismuth-213, or actinium-225.

[0156] Examples of detectable moieties include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase, and luciferase. Further examples of fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Further examples of chemiluminescent moieties include, but are not limited to, luminol. Further examples of bioluminescent materials include, but are not limited to, luciferin and aequorin. Further examples of radioactive materials include, but are not limited to, iodine-125, carbon-14, sulfur-35, tritium, and phosphorus-32.

[0157] In some embodiments, including but not limited to autoimmune applications, the additional therapeutic agent is an immunosuppressant that is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a nonsteroidal anti-inflammatory drug (NSAID). Steroids, particularly corticosteroids, and their synthetic analogs are well known in the art. Examples of corticosteroids useful in the present invention include hydroxyltriamcinolone, α-methyldexamethasone, β-methyl β-methasone, beclomethasone dipropionate, β-methasone benzoate, β-methasone dipropionate, β-methasone valerate, clobetasol valerate, desonide, desoximetasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluchlorolone acetonide, flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortine butyl ester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortolone, hydroxypropyltriamcin ... These include, but are not limited to, flumethasone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolon acetonide, medrysone, amcinafide, amcinafide, betamethasone and its remaining esters, chloroprednisone, clocortelone, clesinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, and beclomethasone dipropionate. NSAIDS that may be used in the present invention include, but are not limited to, salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate, salicylamide, benzyl-2,5-diacetoxybenzoate, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin.In some embodiments, the immunosuppressant may be a cytostatic agent such as an alkylating agent, antimetabolite (e.g., azathioprine, methotrexate), cytotoxic antibiotic, antibody (basiliximab, daclizumab, and muromonab), anti-immunophilin (e.g., cyclosporine, tacrolimus, sirolimus), interferon, opioid, TNF-binding protein, mycophenolate, and small molecule biologic (e.g., fingolimod, myriocin). Additional anti-inflammatory agents are described, for example, in U.S. Pat. No. 4,537,776, the entire contents of which are incorporated herein by reference.

[0158] In some embodiments, the chimeric protein is used in methods for treating multiple sclerosis in combination with one or more disease-modifying therapeutics (DMTs) described herein (e.g., the agents in Table A). In some embodiments, the present invention provides an improved therapeutic effect compared to the use of one or more DMTs described herein (e.g., the agents listed in Table A below) without one or more disclosed binding agents. In certain embodiments, the combination of the chimeric protein and one or more DMTs results in a synergistic therapeutic effect. Examples of disease-modifying therapeutic agents include, but are not limited to: [Table 1] TIFF0007749186000003.tif216162TIFF0007749186000004.tif217162TIFF0007749186000005.tif225162TIFF0007749186000006.tif202162

[0159] The present invention also provides kits for administering any of the agents described herein (e.g., chimeric proteins, with or without various additional therapeutic agents). A kit is a collection of materials or components, including at least one pharmaceutical composition of the invention described herein. Thus, in some embodiments, the kit includes at least one pharmaceutical composition described herein.

[0160] The precise nature of the components comprising the kit will depend on its intended purpose, hi one embodiment, the kit is configured for the purpose of treating a human subject.

[0161] Instructions for use may be included in the kit. The instructions typically include clear language describing techniques to be employed in using the components of the kit to achieve a desired result, such as treating cancer. Optionally, the kit also includes other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings, or other useful accessories as would be readily apparent to one of ordinary skill in the art.

[0162] The materials and components incorporated into the kit can be stored and provided to the practitioner in any convenient and appropriate manner that maintains their operability and usefulness. For example, the components can be provided at room temperature, refrigerated, or frozen. The components are typically contained in suitable packaging. In various embodiments, the packaging is constructed in a well-known manner, preferably one that provides a sterile, contaminant-free environment. The packaging may have an exterior label indicating the contents and / or the purpose of the kit and / or its components.

[0163] definition As used herein, "a," "an," or "the" may mean one (one) or more than one (one). Unless otherwise stated or clear from context, as used herein, the term "or" includes and covers both "or" and "and." Additionally, the term "about" when used in connection with a reference numerical designation means the reference numerical designation plus or minus up to 10% of the reference numerical designation, e.g., a value within (±) 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the indicated value. For example, the term "about 50" covers a range of 45 to 55.

[0164] The term "effective amount," when used in relation to medical use, is an amount that is effective to measurably treat, prevent, or reduce the rate of onset of the disease of interest.

[0165] As used herein, something is "reduced" when, in the presence of a substance or stimulus, the activity and / or effect output value is reduced by a significant amount, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to at least about 100% (including about 100%), compared to the absence of such modulation. As will be understood by one of skill in the art, in some embodiments, activity is decreased and some downstream output values ​​are decreased, while others may increase.

[0166] Conversely, activity is "higher" if the activity and / or effect output value increases by a significant amount, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to at least about 100% (including about 100%) or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, or at least about 100-fold, in the presence of a substance or stimulus compared to the absence of such substance or stimulus.

[0167] When referenced herein, percentages for all compositions are by weight of the total composition unless otherwise specified. As used herein, the word "include" and variations thereof are intended to be non-limiting, such that the description of items in a list is not intended to exclude other similar items that may also be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and variations thereof are intended to be non-limiting, such that a description that an embodiment "can" or "may" include certain elements or features does not exclude other embodiments of the present technology that do not include these elements or features.

[0168] Although the invention is described and claimed herein using the open-ended term "comprising" as a synonym for terms such as including, containing, or having, the invention, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

[0169] As used herein, the terms "preferred" and "preferably" refer to embodiments of the present technology that offer certain advantages, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present technology.

[0170] The amount of the compositions described herein required to achieve a therapeutic effect may be empirically determined according to conventional procedures for a particular purpose. Generally, when a therapeutic agent is administered for therapeutic purposes, the therapeutic agent is administered in a pharmacologically effective amount. A "pharmacologically effective amount," "pharmacologically effective dose," "therapeutically effective amount," or "effective amount" refers to an amount sufficient to produce a desired physiological effect or to achieve a desired result, particularly for treating a disorder or disease. As used herein, an effective amount may include, for example, an amount sufficient to slow the progression of symptoms of a disorder or disease, alter the course of symptoms of a disorder or disease (e.g., slow the progression of symptoms of a disease), reduce or eliminate one or more symptoms or onset of a disorder or disease, and ameliorate symptoms of a disorder or disease. A therapeutic effect also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is achieved.

[0171] Effective doses, toxicity, and therapeutic effects can be determined by standard pharmaceutical procedures, for example, in cell cultures or experimental animals, to determine the LD50 (the dose lethal to approximately 50% of the population) and ED50 (the dose therapeutically effective in approximately 50% of the population). Dosages can vary depending on the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. In some embodiments, compositions and methods exhibiting large therapeutic indices are preferred. Therapeutically effective doses can be initially estimated from in vitro assays, including, for example, cell culture assays. Alternatively, doses can be formulated in animal models to achieve a circulating plasma concentration range, such as the IC50, determined in cell culture or an appropriate animal model. Plasma levels of the described compositions can be measured, for example, by high-performance liquid chromatography. The effects of any particular dosage can be monitored by an appropriate bioassay. Dosages can be determined by a physician and, if necessary, adjusted to accommodate observed therapeutic effects.

[0172] In certain embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. A therapeutic effect also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether an improvement is realized.

[0173] As used herein, "method of treatment" is equally applicable to compositions for the treatment of a disease or disorder described herein and / or compositions for use in the manufacture of a medicament and / or multiple uses for the treatment of a disease or disorder described herein. The present invention is further illustrated by the following non-limiting examples.

[0174] Example "AFN" or "AcTaferon" are used where appropriate to refer to the interferon-based chimeric proteins described herein. Example 1: In vivo testing of Flt3-targeted AcTaferon To evaluate the in vivo efficacy of Flt3-targeting AcTaferon, the Flt3L_linker_human IFNα2(R149A)_GGS-his9 fusion protein was expressed in HEKT cells and purified by metal affinity chromatography. The purified protein was then evaluated in a humanized mouse tumor model. Briefly, neonatal NSG mice (1-2 days old) were sublethally irradiated with 100 cGy, followed by 10 5 CD34+ human stem cells (HLA-A2 positive cord blood derived) were delivered intrahepatically. Thirteen weeks after stem cell transfer, mice received 25x10 5Human RL follicular lymphoma cells (ATCC CRL-2261; not susceptible to the direct antiproliferative effects of IFN) were inoculated subcutaneously. Mice were treated intraperitoneally daily with 30 μg of human Flt3L protein from days 8 to 18 after tumor inoculation. Once palpable tumors were observed, daily intravenous injections of buffer or Flt3L-AFN (30 μg) were initiated on day 10 after tumor inoculation (n = 5 or 6 mice per group). Tumor size (caliper measurements), body weight, and body temperature were assessed daily. Data in Figure 1 show tumor growth up to 2 days after the last treatment, demonstrating that Flt3-targeted AcTaferon potently suppresses tumor growth. Body weight and body temperature data did not show any significant differences between buffer and AFN treatments, confirming that AFN treatment was well tolerated. The sequence of mature Flt3L (bold)_linker (italic)_human IFNα2(R149A) (italic and bold)GGS_his9 used in this example is as follows: [ka] Without His9 the sequence is: [ka]

[0175] Example 2: FLT3L-AFN fusion forms dimers The Flt3L_linker_humanIFNα2(R149A)_GGS_his9 fusion protein from Example 1 was recloned into the pcDNA3.4 vector for expression in CHO cells, resulting in plasmid P-2373. Production was performed in ExpiCHO cells (ThermoFisher) according to the manufacturer's guidelines. Seven days after transfection, the supernatant was collected and the cells were removed by centrifugation. The protein was purified on a 1 ml HisTrap Excel column (GE Healthcare) using an AKTA pure instrument (GE Healthcare). The eluted protein was desalted against PBS-H8.0 buffer on Sephadex G25 (5 ml column). Finally, the sample was further analyzed using size exclusion chromatography on a Superdex 75 Increase 10 / 30 column (GE Healthcare) in 10 mM NH4-acetate pH 5.0 buffer containing 123.5 mM NaCl. The SEC profile and subsequent SDS-PAGE analysis of the peak fractions show that the protein behaves as a protein of approximately 150 kD by SEC (i.e., eluting at the 158 kD marker) (Figure 2), whereas it behaves as a protein of approximately 55 kD by SDS-PAGE (Figure 3). These data support the formation of a non-covalently linked dimer. Such a dimer is not observed in VHH-based AFNs and is therefore the result of FLT3L dimerization, which is required for FLT3L receptor binding and signaling.

Claims

1. (i) a targeting moiety that is a single copy of the extracellular domain of FMS-like tyrosine kinase 3 ligand (FLT3L) comprising an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 2-5; (ii) one or more flexible linkers connecting elements (i) and (iii); and (iii) a modified human IFNα2 comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 6 or 7 and having an R149A mutation that confers reduced affinity for the human IFNα2 receptor and / or reduced biological activity; A chimeric protein comprising: The reduced affinity and / or biological activity can be restored by attachment to a targeting moiety; Chimeric proteins.

2. the targeting moiety is directed against an immune cell, and / or the targeting moiety is directed against a dendritic cell, and / or The chimeric protein of claim 1, wherein the dendritic cells are conventional dendritic cells (cDCs) selected from cDC-1, migratory DCs, and Flt3+ DCs.

3. 3. The chimeric protein of claim 2, wherein the targeting moiety is directed to hematopoietic stem cells (HSCs), early progenitor cells, immature thymocytes, or steady-state dendritic cells (DCs).

4. The chimeric protein of claim 1 , wherein the targeting moiety increases the number of dendritic cells.

5. The chimeric protein of claim 1 , wherein the targeting moiety enhances tumor antigen presentation by dendritic cells.

6. The chimeric protein of claim 1 , comprising two targeting moieties, which may or may not be identical.

7. The chimeric protein of claim 1 , comprising an additional signal transduction substance.

8. the flexible linker comprises glycine and serine residues, and / or The flexible linker (Gly ４ Ser) ｎ and / or n is 1 to 8; the flexible linker comprises one or more of SEQ ID NOs: 10 to 17; The chimeric protein of claim 1.

9. The chimeric protein of claim 1 , wherein the protein is a dimer and / or the protein is a non-covalently linked dimer.

10. The chimeric protein of claim 1, comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 90% identity thereto.

11. A recombinant nucleic acid encoding one or more chimeric proteins according to any one of claims 1 to 10.

12. A host cell comprising the nucleic acid of claim 11.

13. Use of the chimeric protein according to any one of claims 1 to 10 for the manufacture of a medicament for treating or preventing cancer.

14. 14. The use of claim 13, wherein the cancer is acute myeloid leukemia (AML).

15. Use of a chimeric protein according to any one of claims 1 to 10 in the manufacture of a medicament for treating or preventing an autoimmune disease and / or a neurodegenerative disease.

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