FMS-like tyrosine kinase 3 ligand (FLT3l)-based chimeric proteins

Abstract

To provide FMS-like tyrosine kinase 3L (FLT3L) fused to human cytokines, which is use in, e.g., cancer treatment.SOLUTION: The present invention provides a chimeric protein comprising a targeting moiety which comprises 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 the antigen or receptor of interest. In some embodiments, the targeting moiety comprises a single copy of the extracellular domain of FLT3L, or respective portions thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 825,579, filed on Mar. 28, 2019. The content of this provisional application is hereby incorporated by reference in its entirety into this specification.

[0002] Field of the Invention Described is an FMS - like tyrosine kinase 3 ligand (FLT3L) fused to a signaling substance, such as, but not limited to, human IFNα2, IFNβ, and IL1β, which is used, for example, in cancer treatment.

[0003] Sequence Listing This application includes a sequence listing filed in ASCII format via EFS - Web, which is hereby incorporated by reference in its entirety into this specification. The above - mentioned ASCII copy created on Mar. 23, 2020 is named ORN - 062PC_A_Sequence_Listing_ST25.txt and is 28,672 bytes in size.

Background Art

[0004] FMS - like tyrosine kinase 3 (FLT3) is expressed on the surface of many hematopoietic progenitor cells. The signal transduction of FLT3 is important for the normal development of hematopoietic stem cells and progenitor cells. The FLT3 gene is one of the genes most frequently mutated 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 natural - origin substances that can regulate cell growth and differentiation. Cytokines play important roles in various physiological processes, such as, for example, metabolism, respiration, sleep, excretion, healing, movement, reproductive functions, mood, stress, tissue function, immune function, sensory perception, and growth and development.

[0006] Clinically, cytokines would appear to be applicable, for example, to the treatment of various diseases and disorders including cancer. However, the administration of these soluble substances is not without risk. 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] Accordingly, in some aspects, the present invention relates to chimeric proteins comprising a targeting moiety that includes 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 includes the extracellular domain of FLT3L, or a single copy of a portion thereof. Chimeric proteins according to embodiments of the present invention also include a signaling agent or a modified form thereof, signaling agents described herein, for example, but not limited to, human IFNα2, IFNβ, and IL1β. The chimeric proteins also include one or more flexible linkers that link the chimeric protein and the signaling agent.

[0008] In some embodiments, the signaling molecule can be a wild-type signaling molecule described herein, such as, but not limited to, human IFNα2, IFNβ, and IL1β. In other embodiments, the signaling molecule may be modified to include one or more mutations. One or more mutations introduced into the signaling molecule can confer various improved properties to the chimeric protein as compared to a chimeric protein having an unmodified (e.g., wild-type) signaling molecule. For example, the signaling molecule can be a mutant human signaling molecule described herein having one or more mutations that confer improved safety as compared to the wild-type signaling molecules described herein, such as, but not limited to, human IFNα2, IFNβ, and IL1β. In various embodiments, one or more mutations can confer improved safety, reduced affinity for the receptor of the signaling molecule, or reduced bioactivity for the receptor of the signaling molecule as compared to the wild-type signaling molecule. In some embodiments, one or more mutations enable attenuation of activation of the signaling molecule, such that, for example, the agonist activity or antagonist activity of the signaling molecule can be attenuated. In some embodiments, one or more mutations of the modified signaling molecule convert the activity of the signaling molecule from agonist activity to antagonist activity. In various embodiments, the mutation confers reduced affinity or activity that is recoverable by binding to one or more targeting moieties. Further, in various embodiments, the mutation confers reduced or eliminated affinity or activity that is not substantially recoverable 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 to 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, the targeting is to dendritic cells, such as conventional dendritic cells (cDCs) or plasmacytoid dendritic cells (pDCs). In some embodiments, the targeting is 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 moiety of the present invention enhances tumor antigen presentation by dendritic cells as needed.

[0010] In various embodiments, the chimeric protein of the present invention is used in patients having 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 for treating and preventing diseases and disorders, such as various types of cancer and autoimmune diseases and / or neurodegenerative diseases. In some embodiments, the cancer is acute myeloid leukemia (AML). BRIEF DESCRIPTION OF THE DRAWINGS

[0011]

Figure 1

Figure 2

Figure 3

[0012] In some embodiments, chimeric proteins are provided that include a targeting moiety that includes a single copy of a portion of FMS-like tyrosine kinase 3 ligand (FLT3L). The chimeric protein also includes a wild-type signaling molecule or a modified form thereof, and the signaling molecule is one of the signaling molecules described herein, for example, but not limited to, human IFNα2, IFNβ, and IL1β, which can be wild-type human type or mutant type in various embodiments. In the chimeric protein, one or more flexible linkers link the targeting moiety and the signaling molecule.

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

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

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

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

[0017] The amino acid sequence of SEQ ID NO: 3 (mature Flt3L-ec (extracellular domain) function, shorter variant, commercially available (Prospecbio)) is as follows: 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), which is shortened by starting with the first cysteine of SEQ ID NO: 5 and ending with the last cysteine, is as follows: CSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQC。

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

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

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

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

[0024] In some embodiments, the reduced affinity or activity for the receptor can be restored by the binding of one or more targeting moieties. In other embodiments, the reduced affinity or activity for the receptor is not substantially restorable by the activity of one or more targeting moieties.

[0025] In various embodiments, the signaling agent is active against the target cell. The reason is that the targeting moiety compensates for the missing / inadequate binding (e.g., but not limited to, and / or binding force) required for substantial activation. In various embodiments, the modified signaling agent is substantially inactive en route to the site of therapeutic action and has its effect substantially on specifically targeted cell types, thereby greatly reducing undesirable side effects.

[0026] In some embodiments, the signaling agent can include one or more mutations that weaken or reduce the binding or affinity for one receptor (i.e., the therapeutic receptor) and one or more mutations that substantially reduce or eliminate the binding or activity for a second receptor. In such embodiments, these mutations can be at the same or different positions (i.e., the same mutation or multiple 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 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 for another receptor. In some embodiments, the chimeric protein has mutations that weaken the binding and / or activity for the therapeutic receptor and thus allow for a more controlled on-target therapeutic effect (e.g., as compared to the wild-type signaling agent) and mutations that substantially reduce or eliminate the binding and / or activity for another receptor and thus reduce side effects (e.g., as compared to the wild-type signaling agent), having a modified signaling agent with both.

[0027] In some embodiments, substantial reduction or elimination of binding or activity cannot be substantially restored in the targeting moiety. In some embodiments, substantial reduction or elimination of binding or activity can be restored using the targeting moiety. In various embodiments, substantial reduction or elimination of binding or activity to a second receptor can also prevent adverse effects mediated by other receptors. Alternatively, or in addition, substantial reduction or elimination of binding or activity to other receptors reduces or eliminates sequestration of the therapeutic chimeric protein away from the site of therapeutic action, thereby improving the therapeutic effect. For example, in some embodiments, this obviates the need for high doses of the chimeric protein of the invention to compensate for loss at other receptors. The ability to reduce such dosages further reduces the likelihood of side effects.

[0028] In various embodiments, the modified signaling molecule has reduced affinity, e.g., binding (e.g., K D ), and / or activation (e.g., if the modified signaling molecule is an agonist of its receptor, e.g., K A and / or EC 50 measurable as) and / or inhibition (e.g., if the modified signaling molecule is an antagonist of its receptor, e.g., K I and / or IC 50 measurable as) for one or more of its receptors, and contains one or more mutations that reduce, substantially reduce, or eliminate such affinity. In various embodiments, the reduced affinity of the signaling molecule for its receptor allows for attenuation of activity (including agonism or antagonism). In such embodiments, the modified signaling molecule has an affinity for the receptor that is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% - 20%, about 20% - 40%, about 50%, about 40% - 60%, about 60% - 80%, about 80% - 100% compared to the wild-type signaling molecule. 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- to 50-fold lower, about 50- to 100-fold lower, about 100- to 150-fold lower, about 150- to 200-fold lower, or more than 200-fold lower (including comparisons to non-mutated IFNα2, IFNβ, or IL1β, without limitation).

[0029] In some embodiments where the chimeric protein has a mutation that reduces binding to one receptor and substantially reduces or eliminates binding to a second receptor, the attenuation or reduction of the binding affinity of the modified signaling molecule to one receptor is less than the substantial reduction or elimination of the affinity to the other receptor. In some embodiments, the attenuation or reduction of the binding affinity of the modified signaling molecule to one receptor is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% less than the substantial reduction or elimination of the affinity to the other receptor. In various embodiments, substantial reduction or elimination refers to a reduction in binding affinity and / or activity that is greater than the attenuation or reduction.

[0030] In various embodiments, the modified signaling molecule includes one or more mutations that reduce the intrinsic activity of the signaling molecule, for example, to 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 molecule (including comparisons to non-mutated IFNα2, IFNβ, or IL1β, without limitation).

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

[0032] In some embodiments, the modified signaling agent comprises 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 enables the modified signaling agent, e.g., the mutant signaling agent, to have a localized on-target effect and to minimize the off-target effects underlying the side effects observed with the wild-type signaling agent. In some embodiments, this 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 using methods known in the art. For example, affinity and / or binding activity can be evaluated by Scatchard plot analysis and computer fitting of binding data (e.g., Scatchard, 1949 Annals of the New York Academy of Sciences. 51(4):660-672) or by reflectometric interference spectroscopy under flow-through conditions as described by Brecht et al. (1993), Biosens Bioelectron 1993;8:387-392. The entire contents of these references are incorporated herein by reference.

[0034] In some embodiments, the wild-type or modified signaling agent is a type I interferon of interferon. 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 a 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 a substantially reduced or eliminated affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0036] Mutant interferon alpha 2 is known to those skilled in the art. In one exemplary embodiment, the modified signaling agent is allelic IFNα2a having the following amino acid sequence: CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 6).

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

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

[0039] In some embodiments, the IFNα2 variant has a reduced affinity and / or activity for IFNAR1. In some embodiments, as described in WO 201 (0) 030671, the IFNα2 variant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A. The entire contents of this patent are incorporated herein by reference.

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

[0041] In some embodiments, as described in International Publication No. WO 2015 / 007520 and International Publication No. WO 2010 / 030671, the IFNα2 variant comprises one or more mutations selected from R120E and R120E / K121E. The entire contents of these patents are incorporated herein by reference. In such embodiments, the IFNα2 variant antagonizes wild-type IFNα activity 2. In such embodiments, the mutant IFNα2 has a reduced affinity and / or activity for IFNAR1, but retains its activity for IFNAR2.

[0042] In some embodiments, the human IFNα2 variant comprises (1) one or more mutations selected from R120E and R120E / K121E (which, although not wishing to be bound by theory, create an antagonist effect), and (2) one or more mutations selected from K133A, R144A, R149A, and L153A (which, although not wishing to be bound by theory, allow for, for example, a weakening effect on IFNAR2). In certain embodiments, the human IFNα2 variant 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 International Publication No. WO 2013 / 059885. The entire content of this patent is incorporated herein by reference. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or L30A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or R33A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or M148A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or L153A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations N65A, L80A, Y85A, and / or Y89A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations N65A, L80A, Y85A, Y89A, and / or D114A.

[0044] In various embodiments, the signaling substance is mutant human IFNα2. In some embodiments, the mutant human IFNα2 comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 6 or 7, and the mutant human IFNα2 has one or more mutations that confer improved safety compared to wild-type IFNα2 having the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, IFNα2 has one or more mutations at positions 144-154 relative to SEQ ID NO: 6 or 7. In some embodiments, 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 position 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, Q6 / S, 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 IFNα has the 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, where 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 signaling substance is IFNβ. In some embodiments, 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 having a molecular weight of about 22 kDa and containing 166 amino acid residues. The amino acid sequence of human IFNβ is SEQ ID NO: 38.

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

[0050] In various embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity for the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFNβ has a 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 one embodiment, the modified IFNβ includes the F67G mutation. In one embodiment, the modified IFNβ includes the K123G mutation. In one embodiment, the modified IFNβ includes the F67G and R71A mutations. In one embodiment, the modified IFNβ includes the L88G and Y92G mutations. In one embodiment, the modified IFNβ includes the Y92G, I95A, and N96G mutations. In one embodiment, the modified IFNβ includes the K123G and R124G mutations. In one embodiment, the modified IFNβ includes the F67G, L88G, and Y92G mutations. In one embodiment, the modified IFNβ includes the F67S, L88S, and Y92S mutations.

[0051] In some embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity for the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFNβ has a 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 one embodiment, the modified IFNβ comprises the W22G mutation. In one embodiment, the modified IFNβ comprises the L32A mutation. In one embodiment, the modified IFNβ comprises the L32G mutation. In one embodiment, the modified IFNβ comprises the R35A mutation. In one embodiment, the modified IFNβ comprises the R35G mutation. In one embodiment, the modified IFNβ comprises the V148G mutation. In one embodiment, the modified IFNβ comprises the R152A mutation. In one embodiment, the modified IFNβ comprises the R152G mutation. In one embodiment, the modified IFNβ comprises the Y155G mutation. In one embodiment, the modified IFNβ comprises the W22G and R27G mutations. In one embodiment, the modified IFNβ comprises the L32A and R35A mutations. In one embodiment, the modified IFNβ comprises the L151G and R152A mutations. In one embodiment, the modified IFNβ comprises the V148G and R152A mutations.

[0052] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G seventy-eight S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G seventy-eight S, 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 include five α helices (i.e., A, B, C, D, and E) and four loop regions (i.e., AB, BC, CD, and DE loops) that connect these helices. In various embodiments, the modified IFNβ has one or more mutations in the A, B, C, D, E helices and / or the AB, BC, CD, and DE loops that reduce its binding affinity or activity for a therapeutic receptor such as IFNAR. Representative mutations are described in WO 2000 / 023114 and US 20150011732 A1. The entire contents of these are incorporated herein by reference. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 28-30, 32, and 33. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 36, 37, 39, and 42. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 71-73. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 92, 96, 99, and 100. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 128, 130, 131, and 134. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 149, 153, 156, and 159.

[0055] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and contains a mutation at W22, and 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 contains a mutation at R27, and 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 contains a mutation at W22, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further contains a mutation at R27, and 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 contains a mutation at L32, and 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 contains a mutation at R35, and 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, comprises a mutation at L32, the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), further comprises a mutation at R35, and 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, comprises a mutation at R67, and 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, comprises a mutation at R71, and 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, comprises a mutation at F67, the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), further comprises a mutation at R71, and 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, comprises a mutation at L88, and 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 contains a mutation at Y92, and 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 contains a mutation at F67, the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), further contains a mutation at L88, the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further contains a mutation at Y92, 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 contains a mutation at L88, the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further contains a mutation at Y92, the mutation 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 contains a mutation at I95, the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and further contains a mutation at Y92, the mutation 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, comprises a mutation at N96, the mutation being 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, the mutation being 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, comprises a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), further comprises a mutation at I95, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and further comprises a mutation at N96, the mutation being 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, comprises a mutation at K123, the mutation being 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, comprises a mutation at R124, the mutation being 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, comprises a mutation at K123, the mutation being 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, the mutation being 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, comprises a mutation at L151, the mutation being 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, comprises a mutation at R152, the mutation being 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, comprises a mutation at L151, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, the mutation being 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, comprises a mutation at V148, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0078] In some embodiments, the variant IFNβ comprises SEQ ID NO: 38, comprises a mutation at V148, and 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, and 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 variant IFNβ comprises SEQ ID NO: 38, comprises a mutation at Y155, and 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 signaling substance is IL1β. In certain embodiments, wild-type IL1β has the following amino acid sequence: APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS (SEQ ID NO: 39). IL1β is a pro-inflammatory cytokine and an important immune system regulator. It is a potent activator of CD4 T cell responses, increases the proportion of Th17 cells, and enhances the proliferation of IFNγ- and IL4-producing cells. IL1β is also a potent regulator of CD8 + T cells and antigen-specific CD8 +Enhances T cell proliferation, differentiation, migration to the periphery, and memory. The IL1β receptor includes IL1R1 and IL1R2. Binding to IL1R1 and signal transduction via IL1R1 constitute 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 for interaction and signal transduction via IL1R1.

[0081] In some embodiments, wild-type or modified signaling substance IL1β has a reduced affinity and / or activity (e.g., agonist activity) for IL1R1. In some embodiments, modified IL1β has a substantially reduced or eliminated affinity and / or activity for IL1R2. In such embodiments, recoverable IL1β / IL1R1 signal transduction and prevention of loss of the therapeutic chimeric protein for ILR2 and consequent reduction in the required dose of IL1β administered (e.g., compared to a chimeric protein having only wild-type or attenuated mutations for ILR1) are provided. Such constructs are used, for example, in methods of treating cancer, including, for example, stimulating the immune system to initiate an anti-cancer response.

[0082] In such embodiments, the modified signaling substance has a deletion of amino acids 52-54, which results in the production of a modified human IL1β that has reduced binding affinity and reduced bioactivity for type I IL1R. See, for example, International Publication No. WO 1994 / 000491. The entire content of this patent is incorporated herein by reference. In some embodiments, the modified human IL1β has one or more substitution mutations selected from 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, E221S / N224A, N224S / K225S, E244K, N245Q (where X can be any change in an amino acid, e.g., a non-conservative change), which exhibit reduced binding to IL1R as described in, for example, International Publication No. WO 2015 / 007542 and International Publication No. WO 2015 / 007536, the entire contents of which are incorporated herein by reference (numbering based on GenBank accession number NP_000567, version NP-000567.1, GI:10835145, human IL1β sequence). In some embodiments, the modified human IL1β can 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 one embodiment, the modified human IL1β includes the mutations Q131G and Q148G. In one embodiment, the modified human IL1β includes the mutations Q148G and K208E. In one embodiment, the modified human IL1β includes the mutations R120G and Q131G. In one embodiment, the modified human IL1β includes the mutations R120G and H146A. In one embodiment, the modified human IL1β includes the mutations R120G and H146N.In certain embodiments, the modified human IL1β comprises the mutations R120G and H146R. In certain embodiments, the modified human IL1β comprises the mutations R120G and H146E. In certain embodiments, the modified human IL1β comprises the mutations R120G and H146G. In certain embodiments, the modified human IL1β comprises the mutations R120G and K208E. In certain 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, the mutation(s) of one or more signaling agents can confer improved safety to the chimeric protein as compared to the wild-type signaling agent. The mutation(s) can confer various other beneficial properties including, but not limited to, reduced affinity for the receptor of the signaling agent and / or reduced biological activity for the receptor of the signaling agent. In some embodiments, the mutation(s) of one or more signaling agents enable attenuation of the activity of the signaling agent. For example, the agonist activity or antagonist activity of the signaling agent can be attenuated. Further, in some embodiments, the modified signaling agent comprises 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 a reduced affinity or activity that is recoverable by attachment to one or more targeting moieties. In other embodiments, the mutation(s) of one or more signaling agents confer a substantially reduced or eliminated affinity or activity that is not substantially recoverable by attachment to a targeting moiety.

[0085] In some embodiments, the targeting moiety is directed to immune cells, which can be selected from 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). The targeting moiety can functionally regulate the antigen or receptor of interest. In some embodiments, the targeting moiety binds to the antigen or receptor of interest but does not functionally regulate it.

[0086] In various embodiments, the chimeric protein, among many 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 way, the targeting moiety can increase the number of dendritic cells. In some embodiments, the targeting moiety enhances tumor antigen presentation by dendritic cells as needed.

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

[0088] In various embodiments, the cancer is selected from 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; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer); melanoma; myeloma; neuroblastoma; oral cancer (lip, buccal, tongue, oral cavity, 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 lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; large tumor lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD); and abnormal blood vessel proliferation associated with nevus syndromes; edema (e.g., associated with brain tumors); and one or more of the MEGZ syndrome. In certain embodiments, the cancer is acute myeloid leukemia (AML).

[0089] Furthermore, in some embodiments, the invention includes a method for treating or preventing an autoimmune disease and / or a neurodegenerative disease, the method 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 disease 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 encephalitis, primary sclerosing cholangitis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Meniere's syndrome, transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, myasthenia gravis, Reiter's syndrome, and Graves' disease.

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

[0091] In some embodiments, the chimeric protein of the invention optionally comprises one or more flexible linkers. In some embodiments, the chimeric protein of the invention comprises a flexible linker that links a targeting moiety and a signaling agent (e.g., IFNα2, IFNβ, or IL1β or a variant thereof). In some embodiments, the chimeric protein of the invention comprises a flexible linker within a signaling agent (e.g., IFNα2, IFNβ, or IL1β or a variant thereof). In some embodiments, flexible linkers can be utilized to link 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 do not affect or decrease the stability, orientation, binding, neutralization, and / or excretion properties of the binding region and the binding protein.

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

[0093] In various embodiments, the chimeric protein comprises one or more targeting moieties that specifically bind to a target of interest (e.g., an antigen, a receptor), such as, but not limited to, various antibody formats including single domain antibodies. In various embodiments, the targeting moiety specifically binds to a target of interest (e.g., an antigen, a receptor) 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., an antigen, a receptor) and effectively recruits one or more immune cells. In some embodiments, the target of interest (e.g., an antigen, a receptor) may be found on one or more tumor cells. In some embodiments, the chimeric proteins of the invention may recruit immune cells, such as immune cells that kill and / or suppress tumor cells, to the site of action (non-limiting examples include the tumor microenvironment, etc.). 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 invention, in some embodiments, includes chimeric proteins having one, or two, or three targeting moieties.

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

[0095] In some embodiments, the length of the flexible linker enables effective binding of the targeting moiety and the 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 enables effective binding of the targeting moiety and one of the signaling agents to receptors 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 one targeting moiety and the receptor of the signaling agent on the same cell. In some embodiments, the length of the flexible linker is at least 2-fold, or 3-fold, or 4-fold, or 5-fold, or 10-fold, or 20-fold, or 25-fold, or 50-fold, or 100-fold, or more than the shortest distance between the binding sites of one targeting moiety and the receptor of the signaling agent on the same cell.

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

[0098] In some embodiments, two flexible linkers are present in a single chimera, each linking the signaling agent to the targeting moiety. In various embodiments, the flexible linker has a length that enables the formation of a site having diseased cells and effector cells without steric hindrance that could interfere with the regulation of any cell.

[0099] The present invention contemplates the use of various flexible linker sequences. In various embodiments, the flexible linker can be functional. For example, without limitation, the flexible linker can function to improve folding and / or stability, to improve expression, to improve pharmacokinetics, and / or to improve the biological activity of the chimeric proteins 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 greater 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, such as 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NOs: 10-17, respectively). In certain embodiments, 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 = 1-4) (SEQ ID NOs: 10-13), (Gly)8 (SEQ ID NO: 19), (Gly)6 (SEQ ID NO: 20), (EAAAK) n (n = 1-3) (SEQ ID NOs: 21-23), A(EAAAK) n A(n = 2-5) (SEQ ID NOs: 24-27), 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 (where X represents any amino acid, such as Ala, Lys, or Glu). Flexible linkers having (XP) are included. 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 placed every four amino acid intervals.

[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, to improve expression, to improve pharmacokinetics, and / or to improve the biological activity of the chimeric proteins of the present invention. In another example, the flexible linker can function to direct the chimeric protein to a specific cell type or site.

[0104] In various embodiments, the chimeric proteins of the present invention may include one or more functional groups, residues, or moieties. In various embodiments, one or more functional groups, residues, or moieties are attached or genetically fused to any of the signaling agents or targeting moieties described herein. In some embodiments, such functional groups, residues or moieties confer one or more desirable properties or functional groups 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 complex and / or fuse with another substance to extend the half-life or otherwise improve the pharmacodynamic and pharmacokinetic properties. In some embodiments, the chimeric protein may be fused or complexed with one or more of PEG, XTEN (e.g., as rPEG), 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 substances described in BioDrugs (2015) 29:215-239. The entire contents of this document 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 the PEG moiety extends the half-life and / or reduces the immunogenicity of the chimeric protein. For example, any suitable form of pegylation, such as that used in the art for antibodies and antibody fragments (including, but not limited to, single domain antibodies such as VHH), is commonly used; see, for example, 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 these documents are incorporated herein by reference. Various reagents for the pegylation of proteins are also commercially available, for example, from Nektar Therapeutics, USA. In some embodiments, site-specific pegylation via cysteine residues is used in particular (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003). The entire contents of this document are incorporated herein by reference). In some embodiments, the chimeric protein of the invention is modified to appropriately introduce one or more cysteine residues for the attachment of PEG, or an amino acid sequence containing one or more cysteine residues for the attachment of PEG can be fused to the amino terminus and / or carboxy terminus 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. In 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 that is 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 the binding of the chimeric protein to its target or any other target antigen, 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 glutathione - S - transferase (GST) and histidine (His) tag. In one embodiment, the chimeric protein comprises a histidine tag.

[0111] In some embodiments, the functional group, residue, or moiety comprises, for example, a chelating group for chelating one kind of 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 part of one member of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group can be used to link the chimeric protein of the invention to another protein, polypeptide, or chemical compound that is bound to the other half of the binding pair, i.e., via the binding of the binding pair. For example, the chimeric protein of the invention can be linked to biotin and then to another protein, polypeptide, compound, or carrier that is bound to avidin or streptavidin. For example, in a diagnostic system in which a detectable signal generating substance is bound to avidin or streptavidin, such a complexed chimeric protein can be used, for example, as a reporter. For example, using such a binding pair, the chimeric protein can be bound to a carrier, including a carrier 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). Also, using such a binding pair, a therapeutically active agent can be linked to the chimeric protein of the invention.

[0113] Methods for producing the chimeric proteins of the invention are described herein. For example, a DNA sequence encoding the chimeric protein of the invention (e.g., a DNA sequence encoding a signaling substance (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 ligated, for example, to other appropriate nucleotide sequences, including expression control sequences, to produce a gene expression construct encoding the chimeric protein of interest. Thus, in various embodiments, the invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the chimeric protein of the 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 a host cell by gene transfer, transformation, or transduction techniques. For example, the nucleic acid encoding the chimeric protein of the present invention can be introduced into a host cell by retroviral transduction. Examples of host cells include Escherichia coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cultured cells (COS), 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 an expression vector comprising a nucleic acid encoding the chimeric protein of the present invention. In various embodiments, the present invention further provides a host cell comprising such an expression vector.

[0115] Specific expression and purification conditions vary depending on the expression system used. For example, when 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, when the engineered gene is expressed in a eukaryotic host cell, such as a CHO cell, the gene is first inserted into an expression vector containing, for example, a suitable eukaryotic promoter, a secretion signal, a transcription enhancer, and various introns. The gene construct can be introduced into a host cell using gene transfer, transformation, or transduction techniques.

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

[0117] Thus, in various embodiments, the invention provides a nucleic acid encoding a chimeric protein of the invention. In various embodiments, the invention provides a host cell comprising a nucleic acid encoding a chimeric protein of the invention. In various embodiments, the invention provides a nucleic acid encoding a chimeric protein of the invention that is 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 a chimeric protein comprising IFNα2, IFNβ, or IL1β, variants thereof, or 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 a modified mRNA, i.e., an mRNA comprising 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 content of this patent is 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 a modified mRNA encoding one or more chimeric proteins of the invention. In some embodiments, the invention relates to a gene therapy vector comprising a modified mRNA. In some embodiments, the invention relates to a gene therapy method comprising a modified mRNA. In various embodiments, the nucleic acid is in the form of an oncolytic virus, such as an adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus, or vaccinia.

[0119] The chimeric proteins described herein may have sufficiently basic functional groups which can react with inorganic or organic acids or carboxyl groups and which can react with inorganic or organic bases to form pharmaceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids, as are 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 documents are incorporated herein by reference in their entirety.

[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, gentisinate, fumarate, gluconate, glucuronate, saccharinate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprinate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, terephthalate, 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 salts of the compositions of the present invention having acidic functional groups such as carboxylic acid functional groups, and bases. 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 organic amines such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine such as mono-, bis-, or tris-(2-OH-lower alkylamine), N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amine such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine and lysine, etc.

[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 the chimeric proteins 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 may optionally contain an appropriate amount of a pharmaceutically acceptable excipient so as to obtain a suitable form for administration.

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

[0125] The present invention includes the pharmaceutical composition (and / or additional therapeutic agent) described in various formulations. Any of the pharmaceutical compositions (and / or additional therapeutic agents) of the present invention described herein may take the form of a solution, suspension, emulsion, drip agent, 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 solution.

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

[0127] Formulations containing the pharmaceutical composition (and / or additional agent) of the present invention can be conveniently provided in unit dosage form and can be prepared by any method well known in the pharmaceutical art. Such methods generally include the step of mixing the therapeutic agent with a carrier, which comprises one or more accessory ingredients. Usually, the formulation involves uniformly and completely mixing the therapeutic agent with a liquid carrier, a micronized solid carrier, or both, and then, if necessary, shaping the product into the dosage form of the desired formulation (e.g., wet or dry granulation, powder blend, etc., followed by tableting using conventional methods known in the art).

[0128] In various embodiments, any of the pharmaceutical compositions (and / or additional agents) described herein are formulated according to routine procedures as compositions adapted to 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 effected orally. In another embodiment, administration is by parenteral injection. The method of administration can be left to the discretion of the attending physician and partly depends on the site of the medical condition. In most cases, administration results in the release of any of the agents described herein into the bloodstream.

[0130] In one embodiment, the chimeric proteins described herein are formulated according to conventional methods as compositions adapted for oral administration. Compositions for oral delivery may be in the form of tablets, troches, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Compositions for oral administration may contain one or more agents, such as sweeteners like lactose, aspartame or saccharin, flavorings like peppermint, wintergreen or cherry oil, coloring agents and preservatives, to provide a pharmaceutically palatable formulation. Further, in tablet or pill form, the composition can be coated to enable sustained action over a long period by delaying disintegration and absorption in the gastrointestinal tract. Selectively permeable membranes surrounding any of the osmotic active driving chimeric proteins described herein are also suitable as oral administration compositions. In these latter platforms, liquid from the environment around the capsule is absorbed by the transport compound, which swells and expels the drug or drug composition through the opening. These delivery platforms can provide essentially a zero-order delivery profile, in contrast to the rapid rise profile of immediate release formulations. Time delay substances such as glycerol monostearate or glycerol stearate can also be used. Oral compositions may contain standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are of pharmaceutical grade. In addition to the active compound, the suspension may contain, for example, precipitation inhibitors such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc., and mixtures thereof.

[0131] Formulations suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injections and infusions) include, for example, solutions, suspensions, dispersions, emulsions, etc. They may be manufactured 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, known suspending or dispersing agents in the art. Suitable formulation ingredients for parenteral administration include sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as EDTA, buffering agents such as acetate, citrate, or phosphate, and osmotic pressure regulators such as sodium chloride or dextrose.

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

[0133] The compositions provided herein can be used alone or in combination with other suitable ingredients to prepare aerosol formulations (i.e., "nebulized" formulations) for administration by inhalation. The aerosol formulations can be placed in acceptable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, etc.

[0134] Any pharmaceutical composition (and / or additional agent) of the present invention described herein can be administered by controlled release known to those skilled in the art or by sustained release means or delivery devices. 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 hereby incorporated by reference in its entirety. Such dosage forms are useful for enabling controlled or sustained release of one or more active ingredients, for example, using hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, 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 readily selected for use with the active ingredients of the agents described herein. The present invention thus provides, without limitation, unit dosage forms suitable for oral administration such as tablets, capsules, gel capsules, and caplets adapted for controlled or sustained release.

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

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

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

[0138] It will be understood that the actual dosage of the chimeric protein administered according to the present invention will vary depending on the particular dosage form and method of administration. One of ordinary skill in the art can take into account many factors that can alter the action of the chimeric protein (e.g., body weight, gender, diet, time of administration, route of administration, rate of excretion, condition of the subject, combination of drugs, genetic factors, and sensitivity to the response). Administration can be carried out continuously or in one or more separate dosages within the range of the maximum tolerated dose. The optimal rate of administration for a given set of conditions can be determined by one of ordinary skill in the art using conventional dose administration tests.

[0139] In some embodiments, a suitable dosage of the chimeric protein ranges from about 0.01 μg / kg to about 100 mg / kg of the subject's body weight, from about 0.01 μg / kg to about 10 mg / kg of the subject's body weight, or from about 0.01 μg / kg to about 1 mg / kg of the subject's body weight, for example, 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] The individual dosages of the chimeric protein can be administered, for example, as unit dosage forms (e.g., tablets, capsules, or liquid formulations) containing from about 1 μg to about 100 mg, from about 1 μg to about 90 mg, from about 1 μg to about 80 mg, from about 1 μg to about 70 mg, from about 1 μg to about 60 mg, from about 1 μg to about 50 mg, from about 1 μg to about 40 mg, from about 1 μg to about 30 mg, from about 1 μg to about 20 mg, from about 1 μg to about 10 mg, from about 1 μg to about 5 mg, from about 1 μg to about 3 mg, from about 1 μg to about 1 mg, or from about 1 μg to about 50 μg per unit dosage form. For example, the unit dosage form can be 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, 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, 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 per day, about 1 μg to about 90 mg per day, about 1 μg to about 80 mg per day, about 1 μg to about 70 mg per day, about 1 μg to about 60 mg per day, about 1 μg to about 50 mg per day, about 1 μg to about 40 mg per day, about 1 μg to about 30 mg per day, about 1 μg to about 20 mg per day, about 01 μg to about 10 mg per day, about 1 μg to about 5 mg per day, about 1 μg to about 3 mg per day, or about 1 μg to about 1 mg per day. In various embodiments, the chimeric protein is 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, 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, 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) per day.

[0142] In certain embodiments of the present invention, the pharmaceutical composition comprising the chimeric protein may be administered, for example, more than twice a day (e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times per day), about once a day, about once every other day, about once every three days, 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 one embodiment, the pharmaceutical composition comprising the chimeric protein is administered about three times a week.

[0143] In various embodiments, the chimeric protein of the invention can be administered over a long period of time. For example, the chimeric protein 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 protein can be administered for 12 weeks, 24 weeks, 36 weeks or 48 weeks. In some embodiments, the chimeric protein is 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 protein 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, the pharmaceutical composition of the invention is co-administered with an additional therapeutic agent. The co-administration can be simultaneous or sequential.

[0145] In one embodiment, the additional therapeutic agent and the chimeric protein of the invention are administered to the subject simultaneously. As used herein, the term "simultaneously" means that the additional therapeutic agent and the chimeric protein are administered at an 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 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 that the therapeutic agents be administered simultaneously, provided that the timing of their administrations overlaps over time with the pharmacological activities of the additional therapeutic agent and the chimeric protein, such that the combined therapeutic effect is exerted. 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 time intervals greater than about 60 minutes. For example, the time interval between sequential administrations of the additional therapeutic agent and the chimeric protein can be opened by 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 additional therapeutic agent and the chimeric protein being administered. Either the additional therapeutic agent or the chimeric protein can be administered first.

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

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

[0149] In some embodiments, the invention relates to chemotherapeutic agents as additional therapeutic agents. For example, but not limited to, such combinations of the chimeric proteins of the invention and chemotherapeutic agents are used for the treatment of 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, carbocone, meturedopa, and uredopa, ethyleneimine, methylmelamine such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine, acetogenins (e.g., bullatacin, bullatacinone), camptothecins (including the synthetic analogue topotecan), bryostatin, callystatin, CC-1065 (including adozelesin, carzelesin and bizelesin synthetic analogues), cryptophycins (e.g., cryptophycin 1, cryptophycin 8, etc.), dolastatin, duocarmycins (including the synthetic analogue KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobenbitin, phenesterine, prednimustine, trophosphamide, uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine, antibiotics such as enediyne antibiotics (see calicheamicin, particularly calicheamicin gamma II and calicheamicin omega II (Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein-engineered antibiotics (chromophore), actinomycin, actinomycin, aclacinomycin, azaserine, bleomycin, calicheamicin, carabicin, calminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin doxorubicin (including morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, queramycin, rhodomycin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyruridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate, demeclocycline; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;Maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; lysocine; sizofiran; spirigermanium; tenuazonic acid; 2,2’,2”-trichloro-triethylamine; trichothecene (e.g., T2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gasitocin; arabinoside (ara-C); cyclophosphamide; thiotepa; taxoids, such as taxol, paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), abraxane cremophor-free, albumin-processed nanoparticle-forming paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), taxotere docetaxel (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 (camptosar, CPT-11) (including the treatment of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS2,000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin including oxaliplatin treatment (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva) and VEGF-A that reduces cell proliferation;Also included are pharmaceutically acceptable salts, acids or derivatives of any of the above-mentioned agents, but not limited thereto. Further, the treatment method may further include the use of radiation. Further, the treatment method may further include the use of photodynamic therapy.;

[0150] In some embodiments, the chimeric proteins described herein include derivatives that are modified, i.e., derivatives that are modified by covalent attachment of any type of molecule to the composition such that the covalent bond does not interfere with the activity of the composition. For example, but not limited to, derivatives include compositions that are modified, in particular, by glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, binding to cell ligands or other proteins, etc. Any of a number of chemical modifications can be carried out using known techniques, for example, 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 include, in exemplary embodiments, cytotoxins including toxins, chemotherapeutic agents, radioisotopes, and substances that cause apoptosis or cell death. Such substances can be conjugated to the compositions described herein.

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

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

[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, platinum, taxol, irinotecan, 5-fluorouracil, gemcitabine, leucovorin, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine and vinorelbine), mustine, tyrosine kinase inhibitors, radiation therapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL1 antagonists, interleukins (e.g., IL12 or IL2), IL12R antagonists, toxin-conjugated monoclonal antibodies, tumor antigen-specific monoclonal antibodies, Arbitux, Avastin, Pertuzumab, anti-CD20 antibodies, Rituxan, Ofatumumab, Ofatumumab, DXL625, Herceptin®, or any combination thereof, but not limited thereto. Toxic enzymes derived from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin can complex 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 the cytotoxic ribonucleases described in U.S. Patent No. 6,653,104 by Goldenberg. Embodiments of the present invention also relate to radioimmunoconjugates, in which a radionuclide that emits alpha or beta particles is stably bound to a chimeric protein, with or without the use of a complexing agent. Such radionuclides include, for example, 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, without limitation, additional therapeutic agents, including but not limited to autoimmune applications, are immunosuppressive agents that are anti-inflammatory agents such as corticosteroid anti-inflammatory agents or non-steroidal anti-inflammatory agents (NSAIDs). Steroids, particularly corticosteroid agents and their synthetic analogs, are well known in the art. Examples of corticosteroids useful in the present invention include, but are not limited to, hydroxytriamcinolone, α-methyl dexamethasone, β-methyl β-methasone, beclomethasone dipropionate, β-methasone benzoate, β-methasone dipropionate, β-methasone valerate, clobetasol valerate, desonide, desoxymethasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, flucloronide acetonide, flumethasone pivalate, fluocinonide acetonide, fluocinonide, flucortin butyl ester, fludrocortisone, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, flurandrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and its remaining esters, chloroprednisone, corticosterone, crescinolone, dichlorisone, difluprednate, flucoronide, flunisolide, fluorometholone, fluparolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, 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-diacetoxybenzoic acid, ibuprofen, sulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin.In some embodiments, the immunosuppressive agent may be a cytostatic agent such as an alkylating agent, an antimetabolite (e.g., azathioprine, methotrexate), a cytotoxic antibiotic, an antibody (basiliximab, daclizumab, and muromonab), an anti-immunophilin agent (e.g., cyclosporine, tacrolimus, sirolimus), interferon, opioid, TNF binding protein, mycophenolate, and small molecule biologics (e.g., fingolimod, myriocin). Additional anti-inflammatory agents are described, for example, in U.S. Patent No. 4,537,776, the entire contents of which are incorporated herein by reference.

[0158] In some embodiments, the chimeric protein is used in a method of treating multiple sclerosis in combination with one or more disease-modifying therapeutics (DMTs) described herein (e.g., the substances in Table A). In some embodiments, the present invention provides an improved therapeutic effect as compared to the use of one or more DMTs described herein (e.g., the substances listed in Table A below) that do not include one or more of the disclosed binding substances. In certain embodiments, the combination of the chimeric protein and one or more DMTs results in a synergistic therapeutic effect. Examples of disease-modifying therapeutics include, but are not limited to: [Table 1] TIFF2025111546000004.tif216162TIFF2025111546000005.tif217162TIFF2025111546000006.tif225162TIFF2025111546000007.tif202162

[0159] The present invention also provides a kit for the administration of any of the agents described herein (e.g., a chimeric protein with or without various additional therapeutics). The kit is an assembly of materials or components that includes at least one pharmaceutical composition of the present invention described herein. Thus, in some embodiments, the kit includes at least one pharmaceutical composition described herein.

[0160] The exact nature of the components that make up the kit depends on its intended purpose. In 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 for use typically include clear statements explaining the techniques to be employed when using the components of the kit to achieve a desired result, such as the treatment of cancer. Optionally, the kit may also include other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressing materials, or other useful sets of equipment that 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 practicing physician in any convenient and appropriate manner that maintains their operability and usefulness. For example, the components can be provided at room temperature, refrigerated temperature, or frozen temperature. The components are typically housed in suitable packaging materials. In various embodiments, the packaging materials are constructed in well-known ways, preferably ways that provide a sterile, contaminant-free environment. The packaging materials may have external labels that display the contents and / or the purpose of the kit and / or its components.

[0163] Definitions As used herein, "a", "an", or "the" can mean one or more than one. Unless otherwise specified or clear from the context, as used herein, the term "or" includes both "or" and "and". Furthermore, the term "about" when used in connection with a numerical indication means a value within ± up to 10% of the numerical indication, e.g., 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" is intended to cover the range of 45 to 55.

[0164] As used in connection with medical use, the term "effective amount" is an amount effective to bring about treatment, prevention, or a reduction in the rate of onset of the targeted disease to a measurable extent.

[0165] As used herein, something is "reduced" when, in the presence of a substance or stimulus, the output value of an activity and / or effect 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 those skilled in the art, in some embodiments, the activity decreases and some downstream output values decrease, while others may increase.

[0166] Conversely, an activity is "high" when, in the presence of a substance or stimulus, the output value of an activity and / or effect 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, at least about 100-fold compared to the absence of such substance or stimulus.

[0167] In this specification, when referenced, all percentages regarding compositions are by weight of the total composition unless otherwise specified. As used herein, the word "include" and its variants are intended to be non-limiting, such that the recitation of items in a list is not intended to exclude other similar items that may be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and their variants are intended to be non-limiting, such that a statement that a particular embodiment "can" or "may" include a particular element or feature does not exclude other embodiments of the technology of the invention that do not include these elements or features.

[0168] The open-ended term "comprising", used herein as a synonym for terms such as "including", "containing", or "having", is used to describe and claim the present invention, but the present invention, or embodiments thereof, can 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 provide certain advantages under certain circumstances. However, in the same or other circumstances, other embodiments may also be preferred. Further, the recitation of one or more preferred embodiments does not indicate that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present technology.

[0170] The amount of the composition described herein required to achieve a therapeutic effect may be determined empirically according to conventional procedures for a particular purpose. Generally, when administering a therapeutic agent for a therapeutic purpose, the therapeutic agent is administered in a pharmacologically effective amount. "Pharmacologically effective amount", "pharmacologically effective dosage", "therapeutically effective amount", or "effective amount" means an amount sufficient to produce the desired physiological effect or an amount capable of achieving the 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 the symptoms of a disorder or disease, alter the course of the symptoms of a disorder or disease (e.g., slow the progression of the symptoms of a disease), reduce or eliminate one or more symptoms or manifestations of a disorder or disease, and restore the symptoms of a disorder or disease. A therapeutic effect also includes arresting or slowing the progression of the underlying disease or disorder, whether or not an improvement is realized.

[0171] The effective amount, toxicity, and therapeutic effect can be determined by standard pharmaceutical procedures, for example, by measuring LD50 (the lethal dose for about 50% of a population) and ED50 (the therapeutically effective amount for about 50% of a population) in cell culture or experimental animals. The dosage can vary depending on the dosage form used and the route of administration utilized. The dosage ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. In some embodiments, compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective amount can initially be inferred from in vitro assays, including, for example, cell culture assays. Also, the dosage 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. The plasma levels of the described composition can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by appropriate bioassays. The dosage may be determined by a physician and adjusted as needed to conform to the observed therapeutic effect.

[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. The therapeutic effect also includes halting or delaying the progression of the underlying disease or disorder, whether or not an improvement is achieved.

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

[0174] Examples "AFN" or "AcTaferon" is used as needed 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-targeted 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 irradiated with a sublethal dose of 100 cGy and then 5 10 5Individual human RL follicular lymphoma cells (ATCC CRL-2261; not sensitive to the direct anti-proliferative effect of IFN) were subcutaneously inoculated. Mice were intraperitoneally treated daily with 30 μg of human Flt3L protein from 8 days to 18 days after tumor inoculation. When palpable tumors were observed, daily intravenous injection with buffer or Flt3L-AFN (30 μg) was started on day 10 after tumor inoculation (n = 5 or 6 mice per group). Tumor size (vernier caliper measurement), body weight, and body temperature were evaluated daily. The data in Figure 1 show tumor growth until 2 days after the last treatment, demonstrating that Flt3-targeted AcTaferon potently inhibits tumor growth. The data on body weight and body temperature showed no significant difference between buffer treatment and AFN treatment, confirming that AFN treatment had good tolerance. The sequence of the mature Flt3L (bold)_linker (italic)_human IFNα2 (R149A) (italic and bold) GGS_his9 used in this example is as follows:

Chemical formula

Chemical formula

[0175] Example 2: FLT3L-AFN fusion forms a dimer The Flt3L-linker-human IFNα2 (R149A)_GGS_his9 fusion protein of Example 1 was recloned into the pcDNA3.4 vector for expression in CHO cells to obtain plasmid P-2373. Production was carried out 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 using a 1 ml HisTrap Excel column (GE Healthcare) with an AKTA pure instrument (GE Healthcare). The eluted protein was desalted against PBS-H8.0 buffer using Sephadex G25 (5 ml column). Finally, the sample was further analyzed using size exclusion chromatography with an AKTA on a Superdex 75 Increase 10 / 30 column (GE Healthcare) in 10 mM NH4-acetate pH 5.0 buffer containing 123.5 mM NaCl. Analysis by SEC profile and subsequent SDS-PAGE of the peak fractions showed that the protein behaved as a protein of approximately 150 kD in SEC (i.e., eluting at the position of the 158 kD marker) (Figure 2), while it behaved as a protein of approximately 55 kD in SDS-PAGE (Figure 3). These data support a non-covalently linked dimer configuration. Such dimers are not observed with VHH-based AFN and are thus the result of Flt3L dimerization 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 to 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 95% identity to SEQ ID NO: 6 or 7, and having a mutation at position R33, M148, R149, or L153 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 chimeric protein described in claim 1, wherein the mutation is R33A, M148A, R149A, or L153A based on sequence number 6 or 7.

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

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

5. The chimeric protein described in claim 1, wherein the targeting portion increases the number of dendritic cells.

6. The chimeric protein described in claim 1, wherein the targeting portion enhances tumor antigen presentation by dendritic cells.

7. A chimeric protein as described in claim 1, comprising two targeting moieties, which may or may not be identical.

8. A chimeric protein as described in claim 1, comprising an additional signal transduction substance.

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

10. A chimeric protein as described in claim 1, wherein the protein is a dimer and / or the protein is a non-covalently linked dimer.

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

12. A host cell containing the nucleic acid described in claim 11.

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

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

15. Use of a chimeric protein described in 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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