Chimeric proteins and chimeric protein complexes directed to FMS-like tyrosine kinase 3 (FLT3)

Abstract

To provide chimeric protein complexes used in cancer treatment and the like.SOLUTION: An Fc-based chimeric protein complex is provided, comprising: (i) one or more targeting moieties that specifically bind to an antigen or receptor of interest, the antigen or receptor of interest being FMS-like tyrosine kinase 3 (FLT3); (ii) an Fc domain optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing of the Fc domain, and / or stabilize a hinge region in the Fc domain; and (iii) a signal transduction agent or modified form thereof.SELECTED DRAWING: Figure 28A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 825,580, filed March 28, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION Described herein is FMS-like tyrosine kinase 3 ligand (FLT3L) linked to or complexed with signal transduction agents, including but not limited to, human IFNα2, IFNα1, IFNβ, and IL-1β, for use, e.g., in cancer therapy.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on March 27, 2020, is named ORN-062PC_B_ST25.txt and is 159,744 bytes in size. [Background technology]

[0004] FMS-like tyrosine kinase 3 (FLT3) is expressed on the surface of hematopoietic progenitor cells and some differentiated immune cells. FLT3 signaling is important for the normal development of hematopoietic stem and progenitor cells. The FLT3 gene is one of the most frequently mutated genes in acute myeloid leukemia (AML). Furthermore, FMS-like tyrosine kinase 3 ligand (FLT3L) agents are used to stimulate and activate the immune system, for example, to alter the number of dendritic cells. FLT3 is therefore both a marker for specific immune cells and a functionally important molecule that regulates immune cells and mediates the immunostimulatory function of its ligand, FLT3L.

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

[0006] Clinically, cytokines appear to be applicable to the treatment of various diseases and disorders, including cancer. However, the administration of these soluble substances is not without risks. The therapeutic use of cytokines is often associated with systemic toxicity and adverse side effects, thus limiting the dosage levels at which these agents can be used in various treatment regimens. A possible solution to these problems with cytokine agents is a chimeric protein or chimeric protein complex containing a signal transduction agent (e.g., a cytokine) linked to or complexed with a targeting element, in which the signal transduction agent is wild-type or has been modified (e.g., by mutation) to attenuate the activity of the signal transduction agent (e.g., substantially reduce its ability to interact / bind with its receptor) in such a way that its effector function can be induced, restored, or restored upon binding of the targeting element to its target (e.g., an antigen on a target cell).

[0007] Combining several properties in a drug, including FLT3 targeting, including through its inherent effector function on FLT3L and its cognate receptor FLT3, and additional effector functions, such as cytokine effector function, including inducible cytokine effector function, would be of great interest in creating novel multifunctional protein biologics that function as immune system regulators for the treatment of cancer and other diseases. However, such chimeric proteins or chimeric protein complexes that incorporate an FLT3 targeting moiety, such as FLT3L, and additional other signaling effectors, including cytokines and cytokines such as interferons, interleukins, and tumor necrosis factor family members, can only be applied to therapeutic use if certain conditions are met. This includes, for example: a) important functional properties of such agents, including retention of FLT3 binding in conjunction with delivery of a functional signal transducer signal, e.g., a cytokine signal, including an inducible cytokine signal in FLT3-positive target cells to avoid cytokine cross-reactivity to undesired target cells and associated systemic side effects and toxicity; and b) Important biochemical, biophysical, pharmacological and pharmaceutical properties, such as the ability to be produced on a large scale in a sufficiently homogeneous form (ideally, a single product species), an in vivo half-life that ensures sufficient time for drug exposure to elicit a therapeutically beneficial effect, an appropriate size to avoid rapid elimination or poor tissue penetration and biodistribution, and other properties that ensure adequate solubility, stability and storage without significant loss of function.

[0008] Importantly, all or substantially most of the above properties must be achieved without loss of target selectivity and loss of delivery of intrinsic or conditional effector functions to the intended target, including induction and / or restoration of conditional effector functions for attenuated cytokine activity at the therapeutic target.

[0009] There is a need in the art to be able to obtain biologics with such desirable properties while maintaining the tolerability and therapeutic index of the biologic. Additionally, there is a need for biologics that encode effector functions that are adaptable for manufacturing for use as therapeutics for the treatment or prevention of disease. Summary of the Invention

[0010] Thus, in some aspects, the present invention relates to chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, comprising one or more targeting moieties that specifically bind to an antigen or receptor of interest, such as FMS-like tyrosine kinase 3 (FLT3). In various embodiments, the targeting moiety functionally modulates the 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 FLT3L or the extracellular domain of FLT3L, or a respective portion thereof.

[0011] In some embodiments, FLT3L-ECD, or a variant thereof, is present in two copies on the same polypeptide (single-chain dimeric FLT3L construct). In some embodiments, FLT3L-ECD, or a variant thereof, is present as a single copy on each of two different polypeptides that are otherwise capable of dimerization, such as in an Fc-based chimeric protein complex. In some embodiments, the FLT3L is a FLT3L-ECD, or portion or variant thereof, that contains a mutation that reduces intermolecular FLT3L-ECD homodimerization (i.e., dimerization of FLT3L domains on separate FLT3L-ECD containing molecules that would not otherwise readily dimerize by other mechanisms) and / or favors intramolecular FLT3L-ECD dimerization (i.e., dimerization of two copies of FLT3L-ECD contained within the same single polypeptide, i.e., a single-chain dimeric FLT3L construct), or favors dimerization of a single FLT3-ECD, or variant thereof, on two different polypeptides that can otherwise dimerize within a chimeric protein complex, such as an Fc-based chimeric protein complex. In some embodiments, the mutation in the FLT3L-ECD or variant thereof is L27D (L24D relative to any one of SEQ ID NOS: 2-4, or relative to SEQ ID NO: 5). In some embodiments, the mutation L27D in FLT3-ECD, or variants thereof (relative to any one of SEQ ID NOs: 2-4, or L24D relative to SEQ ID NO: 5), or functionally similar mutations, favors the formation of more homogeneous forms of chimeric proteins and chimeric protein complexes, avoiding higher molecular weight aggregates that may be substantially detrimental to the scale-up of production of FLT3-targeting constructs and the in vivo safety of such constructs (e.g., risk of immunoreactivity, reduced activity, etc.).

[0012] In some embodiments, the present invention provides a FLT3L domain that is a single-chain dimer of the formula ABC, wherein: A is an amino acid sequence having at least 90% identity, or at least 95% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 2 to 5; B is a flexible linker consisting essentially of glycine and serine residues, optionally the flexible linker is (Gly4Ser) n wherein n is from about 1 to about 8, and optionally the flexible linker comprises one or more of SEQ ID NO: 10 to SEQ ID NO: 17; and C is an amino acid sequence having at least 90% identity, or at least 95% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 2 to 5.

[0013] Chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes according to embodiments of the present invention, also include a signal transduction agent or modified version thereof, a signal transduction agent described herein, for example, but not limited to, human IFNα2, IFNα1, IFNβ, and IL-1β. In some embodiments, chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, also include one or more linkers.

[0014] In some embodiments, the signaling agent can be a wild-type signaling agent described herein, such as, but not limited to, human IFNα2, IFNα1, IFNβ, and IL-1β. In some embodiments, incorporation of a wild-type signaling agent into a chimeric protein and / or chimeric protein complex results in attenuation of its activity ("fusion attenuation"). In other embodiments, the signaling agent can be modified to contain one or more mutations. In some embodiments, mutations in the signaling agent attenuate its activity compared to the wild-type signaling agent ("mutation attenuation"). In some embodiments, mutations in the signaling agent can improve its pharmaceutical properties compared to the wild-type signaling agent. One or more mutations introduced into the signaling agent can confer various improved properties to the chimeric protein and chimeric protein complex compared to chimeric proteins and chimeric protein complexes with unmodified (e.g., wild-type) signaling agents. This includes Fc-based chimeric protein complexes compared to Fc-based chimeric protein complexes with unmodified (e.g., wild-type) signaling agents. For example, the signaling agent can be a mutant human signaling agent described herein, such as, but not limited to, human IFNα2, IFNα1, IFNβ, and IL-1β, having one or more mutations that confer improved safety and / or pharmaceutical properties compared to a wild-type signaling agent described herein, such as, but not limited to, human IFNα2, IFNα1, IFNβ, and IL-1β. In various embodiments, the one or more mutations can confer improved safety, reduced affinity of the signaling agent for its receptor, or reduced biological activity of the signaling agent for its receptor compared to the wild-type signaling agent. In some embodiments, the one or more mutations can attenuate activation of the signaling agent, e.g., the agonist or antagonist activity of the signaling agent can be attenuated. In some embodiments, the one or more mutations in the modified signaling agent convert the activity of the signaling agent from agonist activity to antagonist activity.In various embodiments, the mutations confer reduced affinity or activity that is recoverable upon attachment to one or more targeting moieties or upon inclusion in a chimeric protein complex, such as the Fc-based chimeric protein complexes disclosed herein. Further, in various embodiments, the mutations confer reduced or eliminated affinity or activity that is not substantially recoverable upon attachment to a targeting moiety or upon inclusion in an Fc-based chimeric protein complex disclosed herein.

[0015] In various embodiments, the targeting moiety is directed to immune cells, tumor cells, or components of a disease microenvironment, thereby directly or indirectly recruiting immune cells to tumor cells, tumor microenvironments, or other disease microenvironments. Non-limiting examples of immune cells include dendritic cells, T cells, B cells, macrophages, neutrophils, mast cells, myeloid-derived suppressor cells, or NK cells. In some embodiments, the targeting moiety is directed to hematopoietic stem cells (HSCs), early progenitor cells, immature thymocytes, or steady-state dendritic cells (DCs). In some embodiments, targeting is directed to dendritic cells, such as conventional dendritic cells (cDCs) or plasmacytoid dendritic cells (pDCs). In some embodiments, targeting is directed to cDCs, optionally cDC-1, migratory DCs, cDC-2, and Flt3+ DCs. In some embodiments, the targeting moiety can increase the number of dendritic cells. In some embodiments, chimeric protein complexes, including chimeric proteins and Fc-based chimeric protein complexes of the present invention, enhance tumor antigen presentation, optionally by dendritic cells. In some embodiments, the targeting moiety directs distribution of the chimeric protein or chimeric protein complex to diseased tissues or diseased microenvironments.

[0016] In some embodiments, chimeric proteins and chimeric protein complexes comprise a single-chain dimeric FLT3 ligand. In some embodiments, chimeric proteins and chimeric protein complexes comprise a single-chain dimeric FLT3L consisting of tandem repeats of FLT3L monomers (i.e., single-chain dimeric FLT3L), such as FLT3L-ECD (extracellular domain), or portions or variants thereof, wherein individual monomers (e.g., FLT3L-ECD) are linked by a linker. In some embodiments, chimeric proteins and chimeric protein complexes comprise a single-chain dimeric FLT3L-ECD, or portions or variants thereof, wherein two monomers linked via a linker are further linked via one or more linkers to one or more polypeptides, such polypeptides comprising a signal transduction entity or a scaffolding protein, or a scaffolding protein and a signal transduction entity, or, in each case, a modified signal transduction entity. In some embodiments, the targeting moiety in a chimeric protein or chimeric protein complex incorporating the single-chain dimer FLT3L-ECD, or a portion or variant thereof, directs distribution of the chimeric protein or chimeric protein complex to disease tissues or disease microenvironments. In some embodiments, the targeting moiety targets a cellular antigen. In some embodiments, the targeting moiety targets a non-cellular antigen.

[0017] In some embodiments, the targeting moiety comprises the extracellular domain (ECD) of FLT3L, such as the ECD of FLT3L, or a portion or variant thereof, that contains a mutation that reduces intermolecular FLT3L-ECD homodimerization (i.e., dimerization of FLT3L ECD domains present on separate FLT3L-ECD containing molecules that would not otherwise readily dimerize by other mechanisms) and favors intramolecular FLT3L-ECD dimerization (i.e., dimerization of two copies of FLT3L-ECD contained within the same single polypeptide, i.e., a single-chain dimeric FLT3L construct), or dimerization of a single FLT3-ECD on a separate polypeptide that can otherwise dimerize within a particular chimeric protein complex, such as the Fc chain in an Fc-based chimeric protein complex. In some embodiments, the mutation in the FLT3L-ECD is L27D (relative to any one of SEQ ID NOS: 2-4, or L24D relative to SEQ ID NO: 5). In some embodiments, mutations that have a similar functional effect on FLT3L-ECD homodimerization are present in residues 25-30 and / or 63-68 of FLT3L (with reference to SEQ ID NO:2).

[0018] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention are used in patients with various diseases or disorders, such as one or more of cancer, infectious diseases, immune disorders, autoimmune diseases, and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, metabolic diseases, and / or many other diseases and disorders. The present invention encompasses various methods of treating and preventing diseases and disorders, for example, various types of cancer and autoimmune diseases and / or neurodegenerative diseases. In some embodiments, the cancer is acute myeloid leukemia (AML).

[0019] 1A-F, 2A-H, 3A-H, 4A-D, 5A-F, 6A-J, 7A-D, 8A-F, 9A-J, 10A-F, 11A-L, 12A-L, 13A-F, 14A-L, 15A-L, 16A-J, 17A-J, 18A-F, 19A-F, 21A-F, 22A-F, 24A-H, and 25A-L show various non-limiting examples of schematic diagrams of Fc-based chimeric protein complexes of the invention. In some embodiments, each schematic diagram is a composition of the invention. Where applicable in the figures, "TM" means "targeting moiety" as defined herein, "SA" means "signaling agent" as defined herein, [ka] is an optional "linker" as described herein, the two long parallel rectangles optionally have effector knockout and / or stabilizing mutations also as described herein, e.g., are human Fc domains from IgG1-, IgG2-, or IgG4-derived as described herein, and the two long parallel rectangles, one with a protrusion and the other with a recess, have knob-in-hole and / or ion-pair (also known as charge-pair, ion-bond, or charged residue-pair) mutations as described herein, and optionally have effector knockout and / or stabilizing mutations as described herein, e.g., are human Fc domains from IgG1-, IgG2-, or IgG4-derived as also described herein. [Brief explanation of the drawings]

[0020] [Figure 1A] 1 shows examples of homodimeric two-chain complexes. These figures show illustrative structures of homodimeric two-chain complexes. [Figure 1B] 1 shows examples of homodimeric two-chain complexes. These figures show illustrative structures of homodimeric two-chain complexes. [Figure 1C] 1 shows examples of homodimeric two-chain complexes. These figures show illustrative structures of homodimeric two-chain complexes. [Figure 1D]1 shows examples of homodimeric two-chain complexes. These figures show illustrative structures of homodimeric two-chain complexes. [Figure 1E] 1 shows examples of homodimeric two-chain complexes. These figures show illustrative structures of homodimeric two-chain complexes. [Figure 1F] 1 shows examples of homodimeric two-chain complexes. These figures show illustrative structures of homodimeric two-chain complexes. [Figure 2A] 2G and 2H show examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 2G and 2H) have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 2B] 2G and 2H show examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 2G and 2H) have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 2C] 2G and 2H show examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 2G and 2H) have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 2D]2G and 2H show examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 2G and 2H) have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 2E] 2G and 2H show examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 2G and 2H) have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 2F] 2G and 2H show examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 2G and 2H) have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 2G] 2G and 2H show examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 2G and 2H) have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 2H]2G and 2H show examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 2G and 2H) have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 3A] 3A and 3B show examples of homodimeric two-chain complexes with two signaling agents (in some embodiments, more signaling agents may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 3G and 3H) have a TM between SA1 and SA2, or at the N- or C-terminus. [Figure 3B] 3A and 3B show examples of homodimeric two-chain complexes with two signaling agents (in some embodiments, more signaling agents may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 3G and 3H) have a TM between SA1 and SA2, or at the N- or C-terminus. [Figure 3C] 3A and 3B show examples of homodimeric two-chain complexes with two signaling agents (in some embodiments, more signaling agents may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 3G and 3H) have a TM between SA1 and SA2, or at the N- or C-terminus. [Figure 3D]3A and 3B show examples of homodimeric two-chain complexes with two signaling agents (in some embodiments, more signaling agents may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 3G and 3H) have a TM between SA1 and SA2, or at the N- or C-terminus. [Figure 3E] 3A and 3B show examples of homodimeric two-chain complexes with two signaling agents (in some embodiments, more signaling agents may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 3G and 3H) have a TM between SA1 and SA2, or at the N- or C-terminus. [Figure 3F] 3A and 3B show examples of homodimeric two-chain complexes with two signaling agents (in some embodiments, more signaling agents may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 3G and 3H) have a TM between SA1 and SA2, or at the N- or C-terminus. [Figure 3G] 3A and 3B show examples of homodimeric two-chain complexes with two signaling agents (in some embodiments, more signaling agents may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 3G and 3H) have a TM between SA1 and SA2, or at the N- or C-terminus. [Figure 3H]3A and 3B show examples of homodimeric two-chain complexes with two signaling agents (in some embodiments, more signaling agents may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the boxed constructs (i.e., Figures 3G and 3H) have a TM between SA1 and SA2, or at the N- or C-terminus. [Figure 4A] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 4B] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 4C] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 4D] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 5A] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5B] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5C] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5D] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5E] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5F] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 6A]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6B]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6C]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6D]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6E]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6F]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6G]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6H]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6I]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6J]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 7A] An illustrative heterodimeric two-chain complex is shown with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc. [Figure 7B] An illustrative heterodimeric two-chain complex is shown with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc. [Figure 7C] An illustrative heterodimeric two-chain complex is shown with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc. [Figure 7D] An illustrative heterodimeric two-chain complex is shown with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc. [Figure 8A]1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and both TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8B] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and both TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8C] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and both TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8D] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and both TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8E]1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and both TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8F] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and both TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 9A]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9B]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9C]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9D]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9E]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9F]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9G]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9H]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9I]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9J]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling entities (more may be present in some embodiments, as described herein). In these orientations and / or structures, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 10A] An illustrative heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10B] An illustrative heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10C] An illustrative heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10D] An illustrative heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10E] An illustrative heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10F] An illustrative heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 11A] 1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11B] 1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11C]1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11D] 1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11E] 1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11F] 1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11G]1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11H] 1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11I] 1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11J] 1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11K]1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11L] 1 shows an example of a heterodimeric two-chain complex having both the TM and SA chains on the same chain, i.e., both the SA and TM on the knob chain of Fc, and two targeting moieties (as described herein, in some embodiments, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 12A] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12B] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12C] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12D]An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12E] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12F] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12G] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12H] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12I]An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12J] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12K] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12L] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 13A] An illustrative heterodimeric two-chain complex is shown, with TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13B] An illustrative heterodimeric two-chain complex is shown, with TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13C] An illustrative heterodimeric two-chain complex is shown, with TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13D]An illustrative heterodimeric two-chain complex is shown, with TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13E] An illustrative heterodimeric two-chain complex is shown, with TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13F] An illustrative heterodimeric two-chain complex is shown, with TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 14A] 1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14B] 1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14C] 1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14D]1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14E] 1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14F] 1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14G] 1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14H]1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14I] 1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14J] 1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14K] 1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 14L]1 shows an example of a heterodimeric two-chain complex with a TM and SA chain on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two targeting moieties (as described herein, in some embodiments, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical. [Figure 15A] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15B] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15C] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15D] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15E]An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15F] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15G] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15H] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15I] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15J]An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15K] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15L] An example of a heterodimeric two-chain complex is shown, with the TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and with two signaling entities (in some embodiments, more signaling entities may be present, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 16A] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 16B] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 16C]An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 16D] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 16E] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 16F] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 16G] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 16H] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 16I]An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 16J] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17A] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17B] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17C] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17D] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17E]An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17F] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17G] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17H] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17I] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 17J] An example of a heterodimeric two-chain complex is shown, with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein) and an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 can be the same. [Figure 18A]An illustrative heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, there may be more signaling agents, as described herein), and with split SA and TM chains: SA on the knob Fc and TM on the hole Fc. [Figure 18B] An illustrative heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, there may be more signaling agents, as described herein), and with split SA and TM chains: SA on the knob Fc and TM on the hole Fc. [Figure 18C] An illustrative heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, there may be more signaling agents, as described herein), and with split SA and TM chains: SA on the knob Fc and TM on the hole Fc. [Figure 18D] An illustrative heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, there may be more signaling agents, as described herein), and with split SA and TM chains: SA on the knob Fc and TM on the hole Fc. [Figure 18E] An illustrative heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, there may be more signaling agents, as described herein), and with split SA and TM chains: SA on the knob Fc and TM on the hole Fc. [Figure 18F] An illustrative heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, there may be more signaling agents, as described herein), and with split SA and TM chains: SA on the knob Fc and TM on the hole Fc. [Figure 19A] An example of a heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, as described herein, there may be more signaling agents), and with split SA and TM chains: TM on the knob Fc and SA on the hole Fc. [Figure 19B] An example of a heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, as described herein, there may be more signaling agents), and with split SA and TM chains: TM on the knob Fc and SA on the hole Fc. [Figure 19C] An example of a heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, as described herein, there may be more signaling agents), and with split SA and TM chains: TM on the knob Fc and SA on the hole Fc. [Figure 19D] An example of a heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, as described herein, there may be more signaling agents), and with split SA and TM chains: TM on the knob Fc and SA on the hole Fc. [Figure 19E] An example of a heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, as described herein, there may be more signaling agents), and with split SA and TM chains: TM on the knob Fc and SA on the hole Fc. [Figure 19F] An example of a heterodimeric two-chain complex is shown, with two signaling agents (in some embodiments, as described herein, there may be more signaling agents), and with split SA and TM chains: TM on the knob Fc and SA on the hole Fc. [Figure 20] Figure 1 shows pSTAT1 in response to FMS-like tyrosine kinase 3 ligand (FLT3L)-Fc-AFN in transiently transfected Hek293T cells. FLT3- or MOCK (empty vector)-transfected Hek293T cells were stimulated as indicated and stained for pSTAT1. The mean % (±STDEV) of pSTAT1-positive cells from duplicate measurements is plotted. [Figure 21A]Split-dimer FLT3-L(TM): Several embodiments of formats with AFN(SA) on the knob Fc are shown. In some embodiments, the knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 21B] Split-dimer FLT3-L(TM): Several embodiments of formats with AFN(SA) on the knob Fc are shown. In some embodiments, the knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 21C] Split-dimer FLT3-L(TM): Several embodiments of formats with AFN(SA) on the knob Fc are shown. In some embodiments, the knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 21D] Split-dimer FLT3-L(TM): Several embodiments of formats with AFN(SA) on the knob Fc are shown. In some embodiments, the knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 21E] Split-dimer FLT3-L(TM): Several embodiments of formats with AFN(SA) on the knob Fc are shown. In some embodiments, the knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 21F] Split-dimer FLT3-L(TM): Several embodiments of formats with AFN(SA) on the knob Fc are shown. In some embodiments, the knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 22A] Several embodiments of the split-dimer FLT3-L(TM):knobs-into-hole format with AFN(SA) on the hole Fc are shown. In some embodiments, the knobs-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 22B]Several embodiments of the split-dimer FLT3-L(TM):knobs-into-hole format with AFN(SA) on the hole Fc are shown. In some embodiments, the knobs-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 22C] Several embodiments of the split-dimer FLT3-L(TM):knobs-into-hole format with AFN(SA) on the hole Fc are shown. In some embodiments, the knobs-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 22D] Several embodiments of the split-dimer FLT3-L(TM):knobs-into-hole format with AFN(SA) on the hole Fc are shown. In some embodiments, the knobs-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 22E] Several embodiments of the split-dimer FLT3-L(TM):knobs-into-hole format with AFN(SA) on the hole Fc are shown. In some embodiments, the knobs-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 22F] Several embodiments of the split-dimer FLT3-L(TM):knobs-into-hole format with AFN(SA) on the hole Fc are shown. In some embodiments, the knobs-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 23A] The biological activity of FLT3L AFN on the HL116 reporter is shown. HL116 or HL116-hFLT3 cells were stimulated with serial dilutions of wild-type IFNα2 or FLT3L AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 23B] The biological activity of FLT3L AFN on the HL116 reporter is shown. HL116 or HL116-hFLT3 cells were stimulated with serial dilutions of wild-type IFNα2 or FLT3L AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 23C]The biological activity of FLT3L AFN on the HL116 reporter is shown. HL116 or HL116-hFLT3 cells were stimulated with serial dilutions of wild-type IFNα2 or FLT3L AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 23D] The biological activity of FLT3L AFN on the HL116 reporter is shown. HL116 or HL116-hFLT3 cells were stimulated with serial dilutions of wild-type IFNα2 or FLT3L AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 23E] The biological activity of FLT3L AFN on the HL116 reporter is shown. HL116 or HL116-hFLT3 cells were stimulated with serial dilutions of wild-type IFNα2 or FLT3L AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 23F] The biological activity of FLT3L AFN on the HL116 reporter is shown. HL116 or HL116-hFLT3 cells were stimulated with serial dilutions of wild-type IFNα2 or FLT3L AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 23G] The biological activity of FLT3L AFN on the HL116 reporter is shown. HL116 or HL116-hFLT3 cells were stimulated with serial dilutions of wild-type IFNα2 or FLT3L AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 24A] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 24B] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 24C] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 24D] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 24E] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 24F] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 24G] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 24H] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25A] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25B] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25C] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25D] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25E] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25F] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25G] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25H] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25I] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25J] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25K] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 25L] The single-chain dimer FLT3-L(TM):AFN(SA) format is shown. The knob-into-hole format can be substituted or supplemented with ionic-charge pair mutations. [Figure 26A] Figure 1 shows the biological activity of single-chain FLT3L AFN on the HL116 reporter. HL116 or HL116-hFLT3 cells were stimulated with serial dilutions of wild-type IFNα2 or FLT3L AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 26B] Figure 1 shows the biological activity of single-chain FLT3L AFN on the HL116 reporter. HL116 or HL116-hFLT3 cells were stimulated with serial dilutions of wild-type IFNα2 or FLT3L AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 27A]Size exclusion chromatography (SEC) of purified split and single-chain FLT3L AFN is shown. [Figure 27B] Size exclusion chromatography (SEC) of purified split and single-chain FLT3L AFN is shown. [Figure 27C] Size exclusion chromatography (SEC) of purified split and single-chain FLT3L AFN is shown. [Figure 28A] Tumor growth curves in humanized mice after treatment with buffer or scFlt3-Fc (A) and buffer or scFlt3L-Fc-AFN (B) are shown. Mean (+SEM) values ​​(in mm) from 3-5 animals per time point are plotted. [Figure 28B] Tumor growth curves in humanized mice after treatment with buffer or scFlt3-Fc (A) and buffer or scFlt3L-Fc-AFN (B) are shown. Mean (+SEM) values ​​(in mm) from 3-5 animals per time point are plotted. [Figure 29] Changes in body weight of humanized mice at day 12 after treatment with buffer, scFlt3-Fc, or scFlt3L-Fc-AFN are shown. Mean (+SEM) values ​​(in mm) for 3-4 animals per time point are plotted. Animals euthanized before day 26 were excluded. [Figure 30] SEQ ID NO: 73 - Size exclusion chromatography (SEC) profile of purified construct of monomeric Flt3L- linked to interferon via a flexible linker. [Figure 31] Figure 1 shows tumor growth curves in humanized mice after treatment with buffer or Flt3L-IFNα1. Mean values ​​(in mm3) (+SEM) of 5 or 6 animals per time point are plotted. DETAILED DESCRIPTION OF THE INVENTION

[0021] In some aspects, chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, are provided that include a targeting moiety, wherein the targeting moiety is capable of specifically binding to an antigen or receptor of interest, and the antigen or receptor of interest is FMS-like tyrosine kinase 3 (FLT3). The chimeric protein or chimeric protein complex, such as the Fc-based chimeric protein complex, also includes a wild-type signal transduction agent or a modified version thereof, wherein the signal transduction agent is one of those described herein, such as, but not limited to, human IFNα2, IFNα1, IFNβ, and IL-1β, which, in various embodiments, can be in wild-type human form or a mutant form.

[0022] This technology is based, in part, on the discovery of signal transduction agents that are optionally modified to have reduced affinity or activity for one or more of their receptors, and the discovery, engineering, and integration of targeting moieties that recognize and bind to specific targets, including FLT3. In some embodiments, the chimeric proteins and / or chimeric protein complexes of the present invention comprise FLT3L, such as the ECD (extracellular domain) of FLT3L, or a portion or variant thereof. In some embodiments, the FLT3L-ECD is present in two copies on the same polypeptide (single-chain dimeric FLT3L construct). In some embodiments, the FLT3L-ECD is present in a single copy on each of two different polypeptides that would otherwise dimerize to form a chimeric protein complex, such as mediated by the Fc chain in an Fc-based chimeric protein complex. In some embodiments, the FLT3L is FLT3L-ECD, or a portion or variant thereof, optionally containing a mutation that reduces intermolecular FLT3L-ECD homodimerization (i.e., dimerization of FLT3L domains on separate FLT3L-ECD containing molecules that would not otherwise readily dimerize by other mechanisms) and favors intramolecular FLT3L-ECD dimerization (i.e., dimerization of two copies of FLT3L-ECD contained within the same single polypeptide, i.e., a single-chain dimeric FLT3L construct), or dimerization of a single FLT3-ECD on two different polypeptides that could otherwise dimerize within a chimeric protein complex, such as an Fc-based chimeric protein complex. In some embodiments, the mutation in FLT3L-ECD is L27D (relative to any one of SEQ ID NOS: 2-4, or L24D relative to SEQ ID NO: 5).In some embodiments, the mutation L27D in FLT3-ECD (relative to any one of SEQ ID NOs: 2-4, or L24D relative to SEQ ID NO: 5), or a functionally similar mutation, favors the formation of more homogeneous forms of chimeric proteins and chimeric protein complexes, avoiding undesirable and higher molecular weight complexes and / or aggregates that may be substantially detrimental to the scale-up of production of FLT3-targeting constructs and the in vivo safety of such constructs (e.g., risk of immunoreactivity, reduced activity, etc.).

[0023] Without intending to be bound by theory, the discovery and use of a single-chain dimeric FLT3L (e.g., two copies of FLT3L-ECD in a single polypeptide), optionally with an L27D mutation (relative to any one of SEQ ID NOs: 2-4, or L24D relative to SEQ ID NO: 5), or an ECD domain mutation with a similar or equivalent functional effect, in chimeric proteins and chimeric protein complexes allows for significant simplification of constructs with FLT3 targeting properties, including, but not limited to: a) retention of functionality, specificity, and selectivity of signal transduction activity; b) activation or binding of FLT3; c) generation of more homogeneous preparations of chimeric proteins and chimeric protein complexes by avoiding undesirable FLT3L-driven intermolecular dimerization of chimeric proteins; c) reduction in the size of the final product; d) simplification of product characteristics that affect the purification and scale-up production of such chimeric proteins and chimeric protein complexes; and e) reduced likelihood of aggregation and, therefore, increased safety.

[0024] Similar advantages as described for the single-chain dimeric FLT3L / FLT3L-ECD chimeric proteins and chimeric protein complexes have also been observed for chimeric protein complexes, such as Fc-based chimeric protein complexes. In this case, a single copy of FLT3L-ECD, or a portion thereof, is present in a single polypeptide that can otherwise dimerize with another polypeptide that also contains a single FLT3-ECD. Furthermore, in this case, in each of the two paired polypeptides, the FLT3-ECD optionally has a mutation that reduces undesired intermolecular homodimerization, such as L27D (relative to any one of SEQ ID NOS: 2-4, or L24D relative to SEQ ID NO: 5), or a mutation that has a functionally similar effect in reducing intermolecular ECD homodimerization. This is exemplified by an Fc-based chimeric protein complex, in which each of the two paired / dimerized Fc chains contains a single copy of FLT3L-ECD, or a portion thereof, incorporating such a mutation, such as L27D (relative to any one of SEQ ID NOS: 2-4, or L24D relative to SEQ ID NO: 5). Complex formation through chain pairing promotes FLT3L-ECD mutant (e.g., L27D relative to any one of SEQ ID NOS: 2-4, or L24D relative to SEQ ID NO: 5) dimerization and restoration of FLT3 binding activity to such a "split FLT3L-ECD" construct, while avoiding the high molecular weight and heterogeneous complex formation otherwise observed with FLT3L-ECD that has not been mutated to reduce undesired intermolecular ECD dimerization between the Fc chain and the Fc chimeric complex. Thus, engineering such chimeric protein complexes allows for significant simplification of constructs with FLT3 targeting properties, including, but not limited to: a) retention of functionality, specificity, and selectivity of signal transduction activity, b) activation or binding of FLT3, c) generation of more homogeneous preparations of chimeric protein complexes by avoiding undesirable FLT3L-driven intermolecular dimerization of chimeric proteins or protein complexes, c) reduction in the size of the final product, d) simplification of product characteristics that affect the purification and scale-up production of such chimeric protein complexes, e) reduced likelihood of aggregation and therefore increased safety.

[0025] In some embodiments, one or more targeting moieties are incorporated into chimeric proteins, including chimeric proteins or Fc-based chimeric protein complexes. In some embodiments, one or more signal transduction agents and one or more targeting moieties are conjugated to the chimeric protein, or, in the case of Fc-based chimeric protein complexes, to the Fc domain. Surprisingly, such Fc-based chimeric protein complexes, particularly in the Fc heterodimeric structure described herein, have dramatically improved in vivo half-lives compared to other chimeric proteins and / or targeting signal transduction agent chimeric proteins, and are particularly amenable to production and purification by standard methods. Thus, the Fc-based chimeric protein complex approach of the present invention provides agents that are particularly suitable for use in various therapies, particularly those that benefit from longer-term intermittent administration.

[0026] In some embodiments, the targeting moiety in the chimeric protein or chimeric protein complex comprises FLT3L or a portion thereof. In other embodiments, the targeting moiety comprises the extracellular domain of FLT3L or a portion thereof. In some embodiments, the targeting moiety comprises a functional fragment of the amino acid sequence of SEQ ID NO: 1, for example, a deletion of about 110 residues, or about 100 residues, or about 90 residues, or about 80 residues, or about 70 residues, or about 60 residues, or about 50 residues, or about 40 residues, or about 30 residues, or about 20 residues, or about 10 residues.

[0027] The amino acid sequence of SEQ ID NO: 1 (full length Flt3L) is [ka] where bold = leader sequence, underlined letters: extracellular region not part of the receptor binding domain, italic letters = transmembrane and intracellular domains.

[0028] In some embodiments, the targeting moiety comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2-5, or an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-5.

[0029] The amino acid sequence of SEQ ID NO:2 (mature Flt3L-ec (extracellular domain)) is: TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQP.

[0030] The amino acid sequence of SEQ ID NO:3 (mature Flt3L-ec (extracellular domain) functional, shorter variant, commercially available) is: TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTA.

[0031] 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.

[0032] The amino acid sequence of the mature Flt3L-ec (extracellular domain) minimal functional domain of SEQ ID NO: 5 (Savvides et al., 2000, Nature Structural Biology), truncated by starting with the first cysteine ​​and ending with the last cysteine, is as follows: CSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQC.

[0033] In some embodiments, the chimeric protein or chimeric protein complex has one or more targeting moieties, including a FLT3 targeting moiety. In some embodiments, the targeting moiety can be attached to the FLT3 targeting moiety of the chimeric protein or the signal transduction agent via a linker. In some embodiments, the FLT3 targeting moiety is a single-chain dimer FLT3L, such as FLT3-ECD (extracellular domain), or a portion thereof. In some embodiments, in the chimeric protein complex, the FLT3 targeting moiety, for example, FLT3-ECD, or a variant or fragment thereof, is present on the same or a different polypeptide as the signal transduction agent. In some embodiments, the present invention provides a FLT3L domain that is a single-chain dimer of the formula ABC, wherein: A is an amino acid sequence having at least 90% identity, or at least 95% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 2 to 5, B is a flexible linker consisting essentially of glycine and serine residues, optionally the flexible linker is (Gly4Ser) n wherein n is from about 1 to about 8, and optionally the flexible linker comprises one or more of SEQ ID NO: 10 to SEQ ID NO: 17; and C is an amino acid sequence having at least 90% identity, or at least 95% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 2 to 5.

[0034] Chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes according to embodiments of the present invention, also include a signal transduction agent or modified version thereof, such as a signal transduction agent described herein, for example, but not limited to, human IFNα2, IFNα1, IFNβ, and IL-1β. In some embodiments, chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, also include one or more linkers.

[0035] In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, has one targeting moiety attached to each Fc chain of the Fc domain. In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, comprises two targeting moieties attached to one Fc chain of the Fc domain. In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, has two targeting moieties attached to each other, optionally via a linker. In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, has two targeting moieties attached to the Fc chain, optionally via a linker.

[0036] In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, comprises a polypeptide having an amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 40, 41, and 46-57. In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, comprises an amino acid sequence selected from SEQ ID NOs: 40, 41, and 46-57, and a polypeptide having fewer than 10 mutations relative to that amino acid sequence. In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, comprises an amino acid sequence selected from SEQ ID NOs: 40, 41, and 46-57, and a polypeptide having fewer than 5 mutations relative to that amino acid sequence. In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 40, 41, and 46-66.

[0037] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 40, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 41.

[0038] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 46, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 47.

[0039] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 48, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 49.

[0040] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 50, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 51.

[0041] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 52, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 53.

[0042] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 54, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 55.

[0043] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 56, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 57.

[0044] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 58, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 59.

[0045] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 60-65, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 66.

[0046] In some embodiments, the targeting moiety of the present invention is a single-chain FLT3L AFN with or without an Fc domain. In some embodiments, FLT3L has an L27D mutation (relative to any one of SEQ ID NOS: 2-4, or L24D relative to SEQ ID NOS: 5) in the dimerization interface. In some embodiments, FLT3L has one or more mutations that reduce FLT3L homodimerization. In some embodiments, FLT3L has one or more mutations that reduce FLT3L dimerization in the FLT3L homodimerization domain (amino acid regions 25-30 and 63-68, relative to any one of SEQ ID NOS: 2-4). In some embodiments, the two FLT3L sequences, or modified FLT3L sequences, can be optionally separated by a linker, e.g., a 3′-amino acid residue. * They may be fused together by a GGGGS linker or any linker disclosed herein. In some embodiments, two or more FLT3L sequences or modified FLT3L sequences may be linked to a signal transduction agent described herein. In some embodiments, two FLT3L sequences or modified FLT3L sequences may be linked to a hIFNα sequence (e.g., IFNα1, IFNα2) or a modified version thereof.

[0047] Signal Transduction Agents (SA) In various embodiments, the signal transduction agent is a wild-type signal transduction agent. In various embodiments, the activity of the signal transduction agent is attenuated by attachment, linkage, or fusion of the signal transduction agent in a chimeric protein or chimeric protein complex. In various embodiments, the wild-type signal transduction agent is a type I interferon.

[0048] In some embodiments, the signaling agent comprises an amino acid sequence having at least 95% identity to one of SEQ ID NOs: 6, 7, 38, 39, or 74, or may comprise the amino acid sequence of one of SEQ ID NOs: 6, 7, 38, 39, or 74.

[0049] In various embodiments, the signal transduction agent is a modified (e.g., mutant) signal transduction agent having one or more mutations. In various embodiments, the mutations enable the modified signal transduction agent to have one or more attenuated activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activities, compared to the unmodified or non-mutated, i.e., wild-type, form of the signal transduction agent (e.g., comparing the wild-type and modified (e.g., mutant) forms of the same signal transduction agent). In various embodiments, the mutations enable the modified signal transduction agent to have one or more attenuated activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activities, compared to the unmodified or non-mutated form, e.g., wild-type IFNα2, IFNα1, IFNβ, or IL-1β. In some embodiments, mutations that weaken or reduce binding or affinity include mutations that substantially reduce or eliminate binding or activity. In some embodiments, mutations that weaken or reduce binding or affinity are different from mutations that substantially reduce or eliminate binding or activity. As a result, in various embodiments, the mutations allow the signaling agent to be safer, e.g., have reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the non-mutated, i.e., wild-type, signaling agent (e.g., comparing the wild-type form with an altered (e.g., mutated) form of the same signaling agent). In various embodiments, the mutations allow the signaling agent to be safer, e.g., have reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the non-mutated sequence of a non-mutated interferon, e.g., IFNα2, IFNα1, IFNβ, or IL-1β.

[0050] In various embodiments, the signaling agent is modified to have one or more mutations that reduce binding affinity or activity for one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or eliminate binding affinity or activity for the receptor. In some embodiments, the activity conferred by the wild-type signaling agent is agonism for the receptor (e.g., activation of a cellular effect at the site of treatment). For example, the wild-type signaling agent may activate its receptor. In such embodiments, the mutation results in the signaling agent being modified to reduce or eliminate the activating effect on the receptor. For example, the mutation may result in the signaling agent being modified to send a reduced activation signal to the target cell, or the activation signal may be eliminated. In some embodiments, the effect conferred by the wild-type signaling agent is antagonism for the receptor (e.g., blocking or suppressing a cellular effect at the site of treatment). For example, the wild-type signaling agent may antagonize or inhibit the receptor. In these embodiments, the mutation results in the signaling agent being modified to reduce or eliminate antagonizing activity for the receptor. For example, the mutations can result in the signal transduction agent being altered to send a reduced inhibitory signal to the target cell, or the inhibitory signal can be eliminated. In various embodiments, the signal transduction agent is an antagonist due to one or more mutations, e.g., an agonist signal transduction agent is converted to an antagonist signal transduction agent (e.g., as described in WO 2015 / 007520, the entire contents of which are incorporated herein by reference), and such converted signal transduction agent optionally also has one or more mutations that reduce its binding affinity or activity to one or more of its receptors, or reduce or eliminate its binding affinity or activity to one or more of its receptors.

[0051] In some embodiments, the reduced affinity or activity for a receptor is recoverable by attachment of one or more targeting moieties or upon inclusion in a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex disclosed herein. In other embodiments, the reduced affinity or activity for a receptor is not substantially recoverable by the action of one or more targeting moieties or upon inclusion in a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex disclosed herein.

[0052] In various embodiments, the signaling agent is active on the target cell because the targeting moiety compensates for missing / insufficient binding (e.g., without limitation, and / or avidity) required for substantial activation. In various embodiments, the modified signaling agent is substantially inactive en route to the site of therapeutic action and exerts its effect substantially on the specifically targeted cell type, thereby greatly reducing undesirable side effects.

[0053] In some embodiments, a signaling agent may contain one or more mutations that weaken or reduce binding or affinity for one receptor (i.e., a therapeutic receptor) and one or more mutations that substantially reduce or eliminate binding or activity for a second receptor. In such embodiments, these mutations may be in the same or different positions (i.e., the same mutation or mutations). In some embodiments, the mutation(s) that reduce binding and / or activity for one receptor are different from the mutation(s) that substantially reduce or eliminate binding 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, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex of the present invention, comprises a modified signaling agent that has both mutations that weaken binding and / or activity towards a therapeutic receptor, thus allowing for a more controlled on-target therapeutic effect (e.g., compared to a wild-type signaling agent), and mutations that substantially reduce or eliminate binding and / or activity towards another receptor, thus reducing side effects (e.g., compared to a wild-type signaling agent).

[0054] In some embodiments, the substantial reduction or elimination of binding or activity is not substantially reversible by a targeting moiety or upon inclusion in a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex disclosed herein. In some embodiments, the substantial reduction or elimination of binding or activity is reversible by a targeting moiety or upon inclusion in a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex disclosed herein. In various embodiments, the substantial reduction or elimination of binding or activity to a second receptor may also prevent adverse effects mediated by other receptors. Alternatively, or in addition, the substantial reduction or elimination of binding or activity to other receptors may improve therapeutic efficacy by reducing or eliminating the segregation of the chimeric protein complex, such as a therapeutic chimeric protein or Fc-based chimeric protein complex, away from the therapeutic site of action. For example, in some embodiments, this eliminates the need for high doses of a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex of the present invention, to compensate for losses at other receptors. The ability to reduce such dosages further reduces the potential for side effects.

[0055] In various embodiments, the modified signaling agent may be modified to increase the affinity, e.g., binding (e.g., K D ) and / or activation (e.g., when the altered signal transduction agent is an agonist of that receptor, e.g., K A and / or EC 50 ) and / or inhibition (e.g., if the altered signal transduction agent is an antagonist of that receptor, e.g., K I and / or IC 50The signaling agent may comprise one or more mutations that reduce, substantially reduce, or eliminate affinity (measurable as affinity, agonism, or antagonism) for the receptor. In various embodiments, the reduced affinity of the signaling agent for the receptor allows for attenuated activity (including agonism or antagonism). In such embodiments, the modified signaling agent has about 1%, or about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 10% to 20%, or about 20% to 40%, or about 50%, or about 40% to 60%, or about 60% to 80%, or about 80% to 100% affinity for the receptor compared to the wild-type signaling agent. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower compared to the wild-type signaling agent (including, but not limited to, compared to non-mutated IFNα2, IFNα1, IFNβ, or IL-1β).

[0056] In some embodiments, where a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, has mutations that reduce binding to one receptor and substantially reduce or eliminate binding to a second receptor, the attenuation or reduction in binding affinity of the altered signal transduction agent to one receptor is less than the substantial reduction or elimination of affinity to the other receptor. In some embodiments, the attenuation or reduction in binding affinity of the altered signal transduction agent to one receptor is less than the substantial reduction or elimination of affinity to the other receptor by 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%. In various embodiments, substantial reduction or elimination refers to a reduction in binding affinity and / or activity that is greater than a reduction or reduction.

[0057] In various embodiments, the modified signaling agent comprises one or more mutations that reduce the intrinsic activity of the signaling agent to, for example, about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1% compared to the wild-type signaling agent (including but not limited to, compared to non-mutated IFNα2, IFNα1, IFNβ, or IL-1β).

[0058] In various embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent described herein to have reduced affinity and / or activity for a receptor for any one of cytokines, growth factors, and hormones, as described herein.

[0059] In some embodiments, the modified signaling agent contains one or more mutations that cause the signaling agent to have a reduced affinity for its receptor that is lower than the binding affinity of the targeting moiety for that receptor. In some embodiments, this difference in binding affinity exists between the signaling agent / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity allows the signaling agent, e.g., the mutant signaling agent, to have a localized on-target effect and minimize off-target effects that underlie side effects observed with wild-type signaling agents. In some embodiments, the binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold lower, or at least about 25-fold, or at least about 50-fold lower, or at least about 100-fold, or at least about 150-fold lower.

[0060] Receptor binding activity can be measured by using known methods in the art.For example, affinity and / or binding activity can be evaluated by Scatchard plot analysis and computer fitting of binding data (for example, Scatchard, 1949 Annals of the New York Academy of Sciences.51(4):660-672) or by reflectance interferometry under flow-through conditions, as described by Brecht et al.(1993), Biosens Bioelectron 1993;8:387-392.The entire contents of these documents are incorporated herein by reference.

[0061] In various embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, comprises a wild-type signaling agent that has improved target selectivity and safety compared to a signaling agent that is not fused to Fc, or a complex, such as, but not limited to, a heterodimeric complex. In various embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, comprises a wild-type signaling agent that has improved target selectivity activity compared to a signaling agent that is not fused to Fc, or a complex, such as, but not limited to, a heterodimeric complex. In various embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, allows for conditional activity.

[0062] In various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a wild-type signaling agent that has improved safety, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to a signaling agent or complex that is not fused to Fc, such as, but not limited to, a signaling agent that is not a heterodimeric complex. In various embodiments, improved safety means that the Fc-based chimeric protein provides lower toxicity (e.g., systemic toxicity and / or tissue / organ-related toxicity) of the wild-type signaling agent, reduced or substantially eliminated side effects, increased tolerability, reduced or substantially eliminated adverse events, reduced or substantially eliminated adverse events, and / or an expanded therapeutic window, compared to a signaling agent or complex that is not fused to Fc, such as, but not limited to, a signaling agent that is not a heterodimeric complex.

[0063] In some embodiments, reduced affinity or activity for a receptor can be restored by attachment of one or more targeting moieties described herein or upon inclusion in an Fc-based chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex disclosed herein.

[0064] In various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has reduced, substantially reduced, or eliminated affinity, e.g., binding (e.g., K D ) and / or activation (e.g., when the altered signal transduction agent is an agonist of that receptor, e.g., K A and / or EC 50 ) and / or inhibition (e.g., if the altered signal transduction agent is an antagonist of that receptor, e.g., K I and / or IC 50In various embodiments, the reduced affinity of the signaling agent for the receptor allows for attenuated activity. In such embodiments, the modified signaling agent has about 1%, or about 3%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 10% to 20%, or about 20% to 40%, or about 50%, or about 40% to 60%, or about 60% to 80%, or about 80% to 100% of the affinity for the receptor compared to the signaling agent that is not fused to Fc, or the signaling agent that is not a complex, such as, but not limited to, a heterodimeric complex. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower, compared to a signaling agent that is not fused to an Fc, or a signaling agent that is not a complex, including but not limited to a heterodimeric complex.

[0065] In various embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, comprises a wild-type signaling agent that has 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% of the intrinsic activity of the signaling agent, compared to, for example, a signaling agent that is not fused to Fc, or a complex, such as, but not limited to, a signaling agent that is not a heterodimeric complex.

[0066] In some embodiments, the wild-type or modified signaling agent is an interferon type I. In some embodiments, the wild-type or modified signaling agent is selected from IFNα2, IFNα1, IFNβ, IFNγ, consensus IFN, IFNε, IFNκ, IFNτ, IFNδ, and IFNν.

[0067] In some embodiments, the wild-type or modified signaling agent is interferon alpha. In such embodiments, the modified IFNα2 agent has reduced affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFNα2 agent has substantially reduced or eliminated affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. Mutant forms of interferon alpha 2 are known to those of skill in the art. In one exemplary embodiment, the modified signal transducer is an allelic IFNα2a having the following amino acid sequence: CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 6).

[0068] In an exemplary embodiment, the altered signal transducer is an allelic IFNα2b having the following amino acid sequence: CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEWRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 7; which differs from IFNα2a at amino acid position 23.

[0069] In some embodiments, the IFNα2 mutant (IFNα2a or IFNα2b) has one or more amino acid mutations introduced at positions 144-154, e.g., amino acid positions 148, 149, and / or 153. In some embodiments, the IFNα2 mutant contains one or more mutations selected from L153A, R149A, and M148A. Such mutants are described, for example, in WO 2013 / 107791 and Piehler et al. (2000) J. Biol. Chem. 275:40425-33, the entire contents of which are incorporated herein by reference.

[0070] In some embodiments, the IFNα2 mutant has reduced affinity and / or activity for IFNAR1. In some embodiments, the IFNα2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A, as described in WO 2010 / 030671, the entire contents of which are incorporated herein by reference.

[0071] In some embodiments, the IFNα2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A, as described in WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0072] In some embodiments, the IFNα2 mutant comprises one or more mutations selected from R120E and R120E / K121E, as described in WO 2015 / 007520 and WO 2010 / 030671, the entire contents of which are incorporated herein by reference. In such embodiments, the IFNα2 mutant antagonizes wild-type IFNα activity 2. In such embodiments, the mutant IFNα2 has reduced affinity and / or activity for IFNAR1 but retains activity for IFNR2.

[0073] In some embodiments, the human IFNα2 mutant comprises one or more mutations selected from (1) R120E and R120E / K121E (which, without wishing to be bound by theory, produce an antagonistic effect), and (2) one or more mutations selected from K133A, R144A, R149A, and L153A (which, without wishing to be bound by theory, enable, for example, an attenuating effect on IFNAR2). In certain embodiments, the human IFNα2 mutant comprises R120E and L153A.

[0074] 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, T106A, T106E, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, as disclosed in WO 2013 / 059885, the entire contents of which are incorporated herein by reference. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L30A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or R33A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or M148A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L153A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations N65A, L80A, Y85A, and / or Y89A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations N65A, L80A, Y85A, Y89A, and / or D114A as disclosed in WO 2013 / 059885.

[0075] In various embodiments, the signal transducer is mutant human IFNα2. In some embodiments, the mutant human IFNα2 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the mutant human IFNα2 has one or more mutations that confer improved safety compared to wild-type IFNα2 having the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, the IFNα2 has one or more mutations at positions 144-154 relative to SEQ ID NO: 6 or 7. In some embodiments, the human IFNα2 has one or more mutations at positions L15, A19, R22, R23, L26, F27, L30, K31, D32, R33, H34, D35, Q40, H57, E58, Q61, F64, N65, T69, L80, Y85, Y89, D114, L117, R120, R125, K133, K134, R144, A145, M148, R149, S152, L153, ​​and N156 relative to SEQ ID NO: 6 or 7. In some embodiments, the mutant IFNα2 has one or more mutations at positions R149, M148, or L153 relative to SEQ ID NO: 6 or 7. In some embodiments, the one or more mutations are one or more of L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57Y, E58N, Q61S, F64A, N65A, T69A, L80A, Y85A, Y89A, D114R, L117A, R120A, R125A, K133A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A relative to SEQ ID NO: 6 or 7. In some embodiments, the mutant human IFNα2 has an R149A mutation relative to SEQ ID NO:6 or 7.

[0076] 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.

[0077] In some embodiments, the mutant human IFNα2 has one or more mutations at positions R33, T106, R144, A145, M148, R149, and L153 relative to SEQ ID NO: 6 or 7. In some embodiments, the mutant human IFNα2 has one or more mutations selected from R33A, T106X3, R120E, R144X1, A145X2, M148A, R149A, and L153A relative to the amino acid sequence of SEQ ID NO: 6 or 7, wherein X1 is selected from A, S, T, Y, L, and I; X2 is selected from G, H, Y, K, and D; and X3 is selected from A and E.

[0078] In some embodiments, the signaling agent is wild-type interferon alpha 1 or modified interferon alpha 1. In some embodiments, the present invention provides a chimeric protein or an Fc-based chimeric protein complex comprising wild-type IFNα1. In various embodiments, the wild-type IFNα1 comprises the following amino acid sequence: CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO: 74).

[0079] In various embodiments, the chimeric protein or Fc-based chimeric protein complex of the present invention comprises, as a signal transduction agent, an IFNα1 variant, including a modified form of IFNα1, i.e., an IFNα1 mutant. In various embodiments, an IFNα1 variant includes a mutant, functional derivative, analog, precursor, isoform, splice variant, or fragment of interferon.

[0080] In some embodiments, the IFNα1 interferon is modified to have one or more amino acid mutations at positions L15, A19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157, relative to SEQ ID NO: 74. The mutations may optionally be hydrophobic mutations, and may be selected from, for example, alanine, valine, leucine, and isoleucine. In some embodiments, the IFNα1 interferon is selected from the group consisting of L15A, A19W, R23A, S25A, L30A, L30V, D32A, R33K, R33A, R33Q, H34A, Q40A, C86S, C86A, D115R, L118A, K121A, K121E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E, R145G, R145H, R145I, R145J, R145K ... and N157A. In some embodiments, the IFNα1 variant comprises one or more mutations selected from L30A / H58Y / E59N_Q62S, R33A / H58Y / E59N / Q62S, M149A / H58Y / E59N / Q62S, L154A / H58Y / E59N / Q62S, R145A / H58Y / E59N / Q62S, D115A / R121A, L118A / R121A, L118A / R121A / K122A, R121A / K122A, and R121E / K122E, relative to SEQ ID NO: 74.

[0081] In some embodiments, the IFNα1 is a variant containing one or more mutations that reduce undesired disulfide pairing, for example, at amino acid positions C1, C29, C86, C99, or C139, relative to SEQ ID NO: 74. In some embodiments, the mutation at position C86 can be, for example, C86S, C86A, or C86Y. These C86 mutants of IFNα1 are referred to as reduced cysteine-aggregating mutants. In some embodiments, the IFNα1 variant contains mutations at positions C1, C86, and C99, relative to SEQ ID NO: 74.

[0082] In some embodiments, the wild-type or modified signal transducer is IFNβ. In some embodiments, the IFNβ is human, having the sequence shown below: MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN (SEQ ID NO: 38).

[0083] 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 complex, such as a chimeric protein or an Fc-based chimeric protein complex, comprises a modified mouse IFNβ. In another embodiment, the chimeric protein complex, such as a chimeric protein or an Fc-based chimeric protein complex, comprises a modified human IFNβ. Human IFNβ is a polypeptide containing 166 amino acid residues and having a molecular weight of approximately 22 kDa. The amino acid sequence of human IFNβ is SEQ ID NO:38.

[0084] In some embodiments, the human IFNβ is IFNβ1a, a glycosylated form of human IFNβ. In some embodiments, the IFNβ is IFNβ1b, a non-glycosylated form of human IFNβ with a Met-1 deletion and a Cys-17 to Ser mutation.

[0085] In various embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity to the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFNβ has reduced affinity and / or activity for IFNAR1. In various embodiments, the modified IFNβ is human IFNβ and has one or more mutations at positions F67, R71, L88, Y92, I95, N96, K123, and R124. In some embodiments, the one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G. In some embodiments, the modified IFNβ comprises an F67G mutation. In some embodiments, the modified IFNβ comprises a K123G mutation. In some embodiments, the modified IFNβ comprises an F67G and an R71A mutation. In some embodiments, the modified IFNβ comprises L88G and Y92G mutations. In some embodiments, the modified IFNβ comprises Y92G, I95A, and N96G mutations. In some embodiments, the modified IFNβ comprises K123G and R124G mutations. In some embodiments, the modified IFNβ comprises F67G, L88G, and Y92G mutations. In some embodiments, the modified IFNβ comprises F67S, L88S, and Y92S mutations.

[0086] In some embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity to the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFNβ has reduced affinity and / or activity for IFNAR2. In various embodiments, the modified IFNβ is human IFNβ and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. In some embodiments, the one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In certain embodiments, the modified IFNβ comprises a W22G mutation. In certain embodiments, the modified IFNβ comprises an L32A mutation. In certain embodiments, the modified IFNβ comprises an L32G mutation. In some embodiments, the modified IFNβ comprises an R35A mutation. In some embodiments, the modified IFNβ comprises an R35G mutation. In some embodiments, the modified IFNβ comprises a V148G mutation. In some embodiments, the modified IFNβ comprises an R152A mutation. In some embodiments, the modified IFNβ comprises an R152G mutation. In some embodiments, the modified IFNβ comprises a Y155G mutation. In some embodiments, the modified IFNβ comprises a W22G and R27G mutation. In some embodiments, the modified IFNβ comprises an L32A and R35A mutation. In some embodiments, the modified IFNβ comprises an L151G and R152A mutation. In some embodiments, the modified IFNβ comprises a V148G and R152A mutation.

[0087] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with C17S or C17A.

[0088] 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.

[0089] The crystal structure of human IFNβ is known and is described in Karpusas et al., (1998) PNAS, 94(22):11813-11818. In particular, the structure of human IFNβ has been shown to contain five α-helices (i.e., A, B, C, D, and E) and four loop regions (i.e., AB, BC, CD, and DE loops) connecting these helices. In various embodiments, the modified IFNβ has one or more mutations in the A, B, C, D, and E helices and / or the AB, BC, CD, and DE loops that reduce its binding affinity or activity to a therapeutic receptor such as IFNAR. Representative mutations are described in International Publication No. 2000 / 023114 and U.S. Patent Application Publication No. 2015 / 0011732, the entire contents of which are incorporated herein by reference. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 28-30, 32, and 33. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 36, 37, 39, and 42. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 71-73. In a representative embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 92, 96, 99, and 100. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 128, 130, 131, and 134. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 149, 153, 156, and 159.

[0090] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at W22, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0091] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R27, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0092] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at W22, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R27, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0093] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L32, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0094] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R35, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0095] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L32, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R35, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0096] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at F67, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0097] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R71, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0098] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at F67, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R71, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0099] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L88, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0100] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at Y92, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0101] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at F67, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at L88, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0102] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at Y92, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0103] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at I95, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and further comprises a mutation at Y92, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0104] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at N96, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0105] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at Y92, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a mutation at I95, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and a mutation at N96, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0106] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at K123, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0107] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R124, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0108] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at K123, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R124, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0109] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L151, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0110] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at R152, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0111] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at L151, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0112] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at V148, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0113] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at V148, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0114] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 38 and comprises a mutation at Y155, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0115] In some embodiments, the wild-type or modified signal transducer is IL-1β. In certain embodiments, wild-type IL-1β has the following amino acid sequence: APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS (SEQ ID NO: 39).

[0116] IL1 is a pro-inflammatory cytokine and an important immune system regulator. It is a potent activator of CD4 T cell responses, promoting the proportion of Th17 cells and increasing the proliferation of IFNγ- and IL4-producing cells. IL1 also activates CD8 + A potent regulator of T cells, antigen-specific CD8 +Enhances T cell proliferation, differentiation, peripheral migration, and memory. IL1 receptors include IL1R1 and IL1R2. Binding to and signaling through IL1R1 constitutes the mechanism by which IL1 mediates many of its biological (and pathological) actions. IL1R2 can function as a decoy receptor, thereby reducing the availability of IL1 for interaction and signaling through IL1R1.

[0117] In some embodiments, the wild-type or modified signal transducer IL1 has reduced affinity and / or activity (e.g., agonist activity) for IL1R1. In some embodiments, the modified IL1 has substantially reduced or eliminated affinity and / or activity for IL1R2. Such embodiments result in the prevention of restoreable IL1 / IL1R1 signaling and loss of the therapeutic chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, for ILR2, resulting in a reduction in the required IL1 dosage (e.g., compared to a wild-type or chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, having only attenuating mutations for ILR1). Such constructs are used, e.g., in methods for treating cancer, including, e.g., stimulating the immune system to mount an anti-cancer response.

[0118] In such embodiments, the modified signal transducer has a deletion of amino acids 52-54, which produces a modified human IL-1β with reduced binding affinity for type I IL1R and reduced biological activity. See, e.g., WO 1994 / 000491, the entire contents of which are incorporated herein by reference. In some embodiments, the modified human IL-1β comprises A117G / P118G, R120X, L122A, T125G / L126G, R127G, Q130X, Q131G, K132A, S137G / Q138Y, L145G, H146X, L145A / L147A, Q148X, Q148G / Q150G, Q150G / D151A, M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209X, K209A / K210A, K219X, E221X, and having one or more substitution mutations selected from E221S / N224A, N224S / K225S, E244K, N245Q (where X can be any amino acid change, e.g., a non-conservative change), which exhibit reduced binding to IL1R, as described, for example, in WO 2015 / 007542 and WO 2015 / 007536, the entire contents of which are incorporated herein by reference (GenBank Accession No. NP_000567, version NP-000567.1, G1:10835145, numbered based on the human IL-1β sequence). In some embodiments, the modified human IL-1β may have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S, and E221K. In certain embodiments, the modified human IL-1β comprises the mutations Q131G and Q148G. In certain embodiments, the modified human IL-1β comprises the mutations Q148G and K208E. In certain embodiments, the modified human IL-1β comprises the mutations R120G and Q131G. In certain embodiments, the modified human IL-1β comprises the mutations R120G and H146A.In some embodiments, the modified human IL-1β comprises the mutations R120G and H146N. In some embodiments, the modified human IL-1β comprises the mutations R120G and H146R. In some embodiments, the modified human IL-1β comprises the mutations R120G and H146E. In some embodiments, the modified human IL-1β comprises the mutations R120G and H146G. In some embodiments, the modified human IL-1β comprises the mutations R120G and K208E. In some embodiments, the modified human IL-1β comprises the mutations R120G, F162A, and Q164E. The modified human IL-1β mutations are relative to SEQ ID NO: 39.

[0119] In various embodiments, one or more mutations in a signal transduction agent may confer improved safety to a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, compared to a wild-type signal transduction agent. The mutations may confer various other beneficial properties, including, but not limited to, reduced affinity of the signal transduction agent for a receptor and / or reduced biological activity of the signal transduction agent for a receptor. In some embodiments, one or more mutations in a signal transduction agent allow the activity of the signal transduction agent to be attenuated. For example, the agonist or antagonist activity of the signal transduction agent may be attenuated. Furthermore, in some embodiments, the modified signal transduction agent contains one or more mutations that convert its activity from agonist activity to antagonist activity.

[0120] In some embodiments, the signaling agent comprises one or more mutations that confer reduced affinity or activity that is recoverable upon attachment to one or more targeting moieties or inclusion in a chimeric protein complex, such as the Fc-based chimeric protein complexes disclosed herein. In other embodiments, the signaling agent has one or more mutations that confer substantially reduced or eliminated affinity or activity that is not substantially recoverable upon attachment to a targeting moiety or inclusion in a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex.

[0121] In some embodiments, the targeting moiety is directed to an immune cell, which can be selected from a dendritic cell, a T cell, a B cell, a macrophage, a neutrophil, a myeloid-derived suppressor cell, or a NK cell. In some embodiments, the targeting moiety is directed to a hematopoietic stem cell (HSC), an early progenitor cell, an immature thymocyte, or a steady-state dendritic cell (DC). The targeting moiety can functionally modulate an antigen or receptor of interest. In some embodiments, the targeting moiety binds but does not functionally modulate an antigen or receptor of interest.

[0122] Fc domain The fragment crystallizable domain (Fc domain) is the tail region of an antibody that interacts with Fc receptors located on the cell surface of cells involved in the immune system, e.g., B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. In IgG, IgA, and IgD antibody isotypes, the Fc domain is composed of two identical protein fragments derived from the second and third constant domains of the antibody's two heavy chains. In IgM and IgE antibody isotypes, the Fc domain is composed of the three heavy chain constant domains (C) in each polypeptide chain. H domains 2-4).

[0123] In some embodiments, the Fc-based chimeric protein complex of the present technology comprises an Fc domain. In some embodiments, the Fc domain is selected from IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from IgG1, IgG2, IgG3, or IgG4.

[0124] In some embodiments, the Fc domain is selected from human IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from human IgG1, IgG2, IgG3, or IgG4.

[0125] In some embodiments, the Fc domain of the chimeric protein or chimeric protein complex, such as the Fc-based chimeric protein complex, comprises the CH2 and CH3 regions of IgG. In some embodiments, the IgG is human IgG. In some embodiments, the human IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0126] In some embodiments, the Fc domain comprises one or more mutations. In some embodiments, the mutations to the Fc domain reduce or eliminate an effector function of the Fc domain. In some embodiments, the mutated Fc domain has reduced affinity or binding to a target receptor. For example, in some embodiments, the mutations to the Fc domain reduce or eliminate binding of the Fc domain to an FcγR. In some embodiments, the FcγR is selected from FcγRI; FcγRIIa, 131R / R; FcγRIIa, 131H / H, FcγRIIb; and FcγRIII. In some embodiments, the mutations to the Fc domain reduce or eliminate binding to a complement protein, such as, for example, C1q. In some embodiments, the mutations to the Fc domain reduce or eliminate binding to both an FcγR and a complement protein, such as, for example, C1q.

[0127] In some embodiments, the Fc domain comprises a LALA mutation to reduce or eliminate effector function of the Fc domain. For example, in some embodiments, the LALA mutation comprises L234A and L235A substitutions in human IgG (e.g., IgG1) (numbering based on the commonly used CH2 residue numbering for human IgG1 according to EU regulations (PNAS, Edelman et al., 1969;63(1)78-85)).

[0128] In some embodiments, the Fc domain of human IgG comprises a mutation at position 46 that reduces or eliminates an effector function of the Fc domain. For example, in some embodiments, the mutation is selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G, and P331S.

[0129] In some embodiments, the Fc domain comprises a FALA mutation that reduces or eliminates the effector function of the Fc domain, for example, in some embodiments, the FALA mutation comprises F234A and L235A substitutions in human IgG4.

[0130] In some embodiments, the Fc domain of human IgG4 comprises mutations at one or more of F234, L235, K322, D265, and P329 to reduce or eliminate effector function of the Fc domain, for example, in some embodiments, the mutations are selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.

[0131] In some embodiments, the mutation in the Fc domain stabilizes the hinge region of the Fc domain. For example, in some embodiments, the Fc domain contains a mutation at the S228 position of human IgG to stabilize the hinge region. In some embodiments, the mutation is S228P.

[0132] In some embodiments, mutations to the Fc domain promote chain pairing of the Fc domain, hi some embodiments, chain pairing is promoted by ion pairing (also known as charge pairing, ionic bonding, or charged residue pairing).

[0133] In some embodiments, the Fc domain contains mutations at the following amino acid residue positions of another IgG to promote ion pairing: D356, E357, L368, K370, K392, D399, and K409.

[0134] For example, in some embodiments, a human IgG Fc domain contains one of the combinations of mutations in Table 1 to promote ion pairing. [Table 1]

[0135] In some embodiments, chain pairing is promoted by knob-in-hole mutations. In some embodiments, the Fc domain contains one or more mutations that allow knob-in-hole interactions in the Fc domain. In some embodiments, a first Fc chain is engineered to express a "knob" and a second Fc chain is engineered to express a complementary "hole." For example, in some embodiments, a human IgG Fc domain contains the mutations in Table 2 that allow knob-in-hole interactions. [Table 2]

[0136] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of the present technology comprises any combination of the mutations disclosed above. For example, in some embodiments, the Fc domain comprises mutations that promote ion-pairing and / or knob-in-hole interactions. For example, in some embodiments, the Fc domain comprises mutations that have one or more of the following properties: promote ion-pairing, induce knob-in-hole interactions, reduce or eliminate the effector function of the Fc domain, and provide Fc stabilization (e.g., hinge).

[0137] For example, in some embodiments, a human IgG Fc domain comprises mutations disclosed in Table 3, which promote ion pairing in the Fc domain and / or promote knob-in-hole interactions. [Table 3] TIFF2025169347000007.tif47162

[0138] For example, in some embodiments, a human IgG Fc domain contains mutations disclosed in Table 4 that promote ion pairing of the Fc domain and / or promote knob-in-hole interactions, or a combination thereof. In some embodiments, "chain 1" and "chain 2" in Table 4 are interchangeable (e.g., chain 1 can have Y407T and chain 2 can have T366Y). [Table 4] TIFF2025169347000009.tif239162TIFF2025169347000010.tif239161TIFF2025169347000011.tif216161

[0139] For example, in some embodiments, a human IgG Fc domain comprises mutations disclosed in Table 5 that reduce or eliminate FcγR and / or complement binding in the Fc domain. In some embodiments, the mutations in Table 5 are present in both chains. [Table 5] TIFF2025169347000013.tif239161TIFF2025169347000014.tif147162

[0140] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of the present technology is a homodimer, i.e., the Fc region in the chimeric protein complex comprises two identical protein fragments.

[0141] In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology is a heterodimer, i.e., the Fc domain comprises two non-identical protein fragments.

[0142] In some embodiments, the heterodimeric Fc domain is modified using ion-pairing and / or knobs-in-holes mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation. In the trans orientation, the targeting moiety and the signal transduction agent, in some embodiments, are not found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention.

[0143] In some embodiments, the heterodimeric Fc domain is engineered using ion-pairing and / or knobs-in-holes mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation.

[0144] In the trans orientation, the targeting moiety and the signal transduction agent are, in some embodiments, not found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention. In the trans orientation, the targeting moiety and the signal transduction agent are, in some embodiments, found on separate polypeptide chains in the Fc-based chimeric protein complex of the present invention. In the cis orientation, the targeting moiety and the signal transduction agent are, in some embodiments, found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention.

[0145] In some embodiments where two or more targeting moieties are present in the heterodimeric protein complexes described herein, one targeting moiety may be present in a trans orientation (relative to the signal transduction agent), while another targeting moiety may be present in a cis orientation (relative to the signal transduction agent). In some embodiments, the signal transduction agent and targeting moiety are present on the same end / side (N- or C-terminus) of the Fc domain. In some embodiments, the signal transduction agent and targeting moiety are present on different ends / sides (N- or C-terminus) of the Fc domain.

[0146] In some embodiments in which two or more targeting moieties are present in the heterodimeric protein complex described herein, the targeting moieties can be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties would be in trans relative to each other since they are on different Fc chains). In some embodiments in which two or more targeting moieties are present on the same Fc chain, the targeting moieties can be on the same or different sides / ends of the Fc chain (N-terminus or / C-terminus).

[0147] In some embodiments in which two or more targeting moieties are present in the heterodimeric protein complex described herein, the targeting moieties can be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties would be in trans relative to each other since they are on different Fc chains). In some embodiments in which two or more signaling agents are present on the same Fc chain, the signaling agents can be on the same or different sides / ends of the Fc chain (N- or C-terminus).

[0148] In some embodiments where two or more signal transduction agents are present in the heterodimeric protein complexes described herein, one signal transduction agent may be present in a trans orientation (relative to the targeting moiety) while another signal transduction agent may be present in a cis orientation (relative to the targeting moiety).

[0149] In some embodiments, for "split" targeting moieties, as described for FLT3L-ECD and its variants, such as FLT3L-ECD with the L27D mutation, a portion of the targeting moiety may be present on each of the Fc chains of a homodimeric or heterodimeric protein complex, and the formation of a functional targeting moiety is generated as part of the formation of a chimeric protein complex, as exemplified by FLT3L-ECD monomers dimerizing to form a functional FLT3 targeting moiety for delivery of signaling agents to FLT3-positive target cells.

[0150] In some embodiments, the Fc domain includes or begins with the core hinge region of wild-type human IgG1, which region comprises the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 42). In some embodiments, the Fc domain also includes the upper hinge, or a portion thereof (e.g., DKTHTCPPC (SEQ ID NO: 43); see WO 2009 / 053368), EPKSCDKTHTCPPC (SEQ ID NO: 44), or EPKSSDKTHTCPPC (SEQ ID NO: 45); see Lo et al., Protein Engineering vol. 11 no. 6 pp. 495-500, 1998).

[0151] Fc-based chimeric protein complexes The Fc-based chimeric protein complexes of the present technology comprise at least one Fc domain disclosed herein, at least one signal transduction agent (SA) disclosed herein, and at least one targeting moiety (TM) disclosed herein.

[0152] It is understood that the Fc-based chimeric protein complex of the present invention may comprise two fusion proteins, each containing an Fc domain.

[0153] In some embodiments, the Fc-based chimeric protein complex is a heterodimer. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a cis orientation. In some embodiments, the heterodimeric Fc-based chimeric protein complex does not comprise a signal transduction agent and a targeting moiety on a single polypeptide.

[0154] In some embodiments, the Fc-based chimeric protein has improved in vivo half-life compared to a chimeric protein lacking Fc or a chimeric protein that is not a heterodimeric complex. In some embodiments, the Fc-based chimeric protein has improved solubility, stability, and other pharmacological properties compared to a chimeric protein lacking Fc or a chimeric protein that is not a heterodimeric complex.

[0155] Heterodimeric Fc-based chimeric protein complexes are composed of two different polypeptides. In some embodiments described herein, the targeting domain is located on a different polypeptide from the signal transduction agent, thus allowing for the creation of proteins containing only one copy of the targeting domain and, similarly, only one type of signal transduction agent (which can control for potential interference with desired properties). Furthermore, in some embodiments, the use of only one targeting domain (e.g., VHH) can avoid cross-linking of antigens on the cell surface (which can induce undesirable effects). Furthermore, in some embodiments, the use of only one signal transduction agent can mitigate molecular "crowding" and potential interference with avidity mediated by the recovery of effector function, depending on the targeting domain. Furthermore, in some embodiments, heterodimeric Fc-based chimeric protein complexes can have two targeting moieties, which can be located on two different polypeptides. For example, in some embodiments, the C-termini of both targeting moieties (e.g., VHHs) can be masked to avoid potential or pre-existing autoantibodies (e.g., VHH autoantibodies or pre-existing antibodies). Furthermore, in some embodiments, a heterodimeric Fc-based chimeric protein complex having a targeting domain on a polypeptide different from, for example, a signal transduction agent (e.g., a wild-type signal transduction agent) can preferentially "cross-link" two cell types (e.g., tumor cells and immune cells). Furthermore, in some embodiments, a heterodimeric Fc-based chimeric protein complex has two signal transduction agents, each on a different polypeptide, allowing for more complex effector responses.

[0156] Furthermore, in some embodiments, heterodimeric Fc-based chimeric protein complexes with a variety of targeting moiety and signal transduction agent combinations are provided in a practical manner, e.g., by having a targeting domain on a polypeptide different from the signal transduction agent. For example, in some embodiments, a polypeptide having any of the targeting moieties described herein can be combined "off the shelf" with a polypeptide having any of the signal transduction agents described herein, allowing for the rapid generation of various combinations of targeting moieties and signal transduction agents in a single Fc-based chimeric protein complex.

[0157] In some embodiments, the Fc-based chimeric protein complex comprises one or more linkers. In some embodiments, the Fc-based chimeric protein complex comprises a linker linking the Fc domain, the signal transduction agent, and the targeting moiety. In some embodiments, the Fc-based chimeric protein complex comprises a linker linking each of the signal transduction agent and the targeting moiety (or, in the case of two or more targeting moieties, linking the signal transduction agent to one of the targeting moieties). In some embodiments, the Fc-based chimeric protein complex comprises a linker linking each signal transduction agent to the Fc domain. In some embodiments, the Fc-based chimeric protein complex comprises a linker linking each targeting moiety to the Fc domain. In some embodiments, the Fc-based chimeric protein complex comprises a linker linking a targeting moiety to another targeting moiety. In some embodiments, the Fc-based chimeric protein complex comprises a linker linking a signal transduction agent to another signal transduction agent.

[0158] In some embodiments, the Fc-based chimeric protein complexes of the present invention comprise two or more targeting moieties, which in such embodiments may be the same targeting moiety or different targeting moieties.

[0159] In some embodiments, the Fc-based chimeric protein complex comprises two or more signal transduction agents, which in such embodiments may be the same or different targeting moieties.

[0160] For example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, at least two signal transduction agents (SAs), and at least two targeting moieties (TMs), wherein the Fc domain, signal transduction agents, and targeting moieties are selected from any of the Fc domains, signal transduction agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is a homodimer.

[0161] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 1A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 2A-H. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 3A-H. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 4A-D. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 5A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 6A-J. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 7A-D. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 8A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 9A-J. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 10A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 11A-L. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 12A-L. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 13A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 14A-L. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 15A-L. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 16A-J. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 17A-J. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 18A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 19A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 21A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 22A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 24A-H. In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 25A-L.

[0162] In some embodiments, the signaling agent is linked to a targeting moiety, and the targeting moiety is linked to an Fc domain on the same end (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer. In some embodiments, the signaling agent and targeting moiety are linked to an Fc domain, and the targeting moiety and signaling agent are linked on the same end (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0163] In some embodiments, the targeting moiety is linked to a signaling agent and the signaling agent is linked on the same end to an Fc domain (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0164] In some embodiments, the homodimeric Fc-based chimeric protein complex comprises two or more targeting moieties. In some embodiments, there are four targeting moieties and two signaling agents, the targeting moieties are linked to the Fc domain, and the signaling agents are linked to the targeting moieties on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments, there are four targeting moieties and two signaling agents, two targeting moieties are linked to the Fc domain, and two targeting moieties are linked to the signaling agents, which are linked to the Fc domain on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments, there are four targeting moieties and two signaling agents, the two targeting moieties are linked to each other, one targeting moiety from each pair is linked to the Fc domain on the same end, and the signaling agents are linked to the Fc domain on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four targeting moieties and two signaling agents, two targeting moieties are linked to each other, one targeting moiety from each pair is linked to a signaling agent, and the other targeting moiety of the pair is linked to an Fc domain, with the targeting moieties linked to the Fc domain being linked on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer.

[0165] In some embodiments, the homodimeric Fc-based chimeric protein complex comprises two or more signaling agents. In some embodiments where there are four signaling agents and two signaling agents, the two signaling agents are linked to each other, one signaling agent from a pair is linked to the Fc domain on the same end, and a targeting moiety is linked to the Fc domain on the same end (see Figures 3A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four signaling agents and two signaling agents, the two signaling agents are linked to the Fc domain on the same end, and each of the two signaling agents is linked to a targeting moiety, and the targeting moiety is linked to the Fc domain on the same end (see Figures 3A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four signaling agents and two signaling agents, the two signaling agents are linked to each other, one signaling agent from a pair is linked to a targeting moiety, and the targeting moiety is linked to the Fc domain on the same end (see Figures 3A-H). In some embodiments, the Fc domain is a homodimer.

[0166] For example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, wherein the Fc domain comprises an ion-pairing mutation and / or a knob-in-hole mutation, at least one signal transduction agent, and at least one targeting moiety, wherein the ion-pairing motif and / or knob-in-hole motif, signal transduction agent, and targeting moiety are selected from any of the ion-pairing motifs and / or knob-in-hole motifs, signal transduction agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises mutations that reduce or eliminate its effector function.

[0167] In some embodiments, the signaling agent is linked to a targeting moiety, which is linked to the Fc domain (see Figures 10A-F and 13A-F). In some embodiments, the targeting moiety is linked to a signaling agent, which is linked to the Fc domain (see Figures 10A-F and 13A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0168] In some embodiments, the signaling agent and the targeting moiety are linked to the Fc domain (see Figures 4A-D, 7A-D, 10A-F, and 13A-F). In some embodiments, the targeting moiety and the signaling agent are linked to different Fc chains on the same terminus (see Figures 4A-D and 7A-D). In some embodiments, the targeting moiety and the signaling agent are linked to different Fc chains on different termini (see Figures 4A-D and 7A-D). In some embodiments, the targeting moiety and the signaling agent are linked to the same Fc chain (see Figures 10A-F and 13A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0169] In some embodiments where there is one signaling agent and two targeting moieties, the signaling agent is linked to the Fc domain and the two targeting moieties can be 1) linked to each other with one targeting moiety linked to the Fc domain, or 2) each linked to an Fc domain (see Figures 5A-F, 8A-F, 11A-L, 14A-L, 16A-J, and 17A-J). In some embodiments, the targeting moiety is linked to one Fc chain and the signaling agent is linked to the other Fc chain (see Figures 5A-F and 8A-F). In some embodiments, the targeting moiety and signaling agent of a pair are linked to the same Fc chain (see Figures 11A-L and 14A-L). In some embodiments, a targeting moiety is linked to a signaling agent, another targeting moiety is linked to a signaling agent, and the paired targeting moiety is linked to an Fc domain (see Figures 11A-L and 14A-L, 16A-J, and 17A-J). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked to the same Fc chain (see Figures 11A-L and 14A-L). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked to different Fc chains (see Figures 16A-J and 17A-J). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked on the same terminus (see Figures 16A-J and 17A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0170] In some embodiments where there is one signaling agent and two targeting moieties, the targeting moiety is linked to the signaling agent, which is linked to the Fc domain, and the unpaired targeting moiety is linked to the Fc domain (see Figures 11A-L and 14A-L, 16A-J, and 17A-J). In some embodiments, the paired signaling agent and the unpaired targeting moiety are linked to the same Fc chain (see Figures 11A-L and 14A-L). In some embodiments, the paired signaling agent and the unpaired targeting moiety are linked to different Fc chains (see Figures 16A-J and 17A-J). In some embodiments, the paired signaling agent and the unpaired targeting moiety are linked on the same terminus (see Figures 16A-J and 17A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0171] In some embodiments where there is one signaling agent and two targeting moieties, the targeting moieties are linked together, the signaling agent is linked to one of the paired targeting moieties, and the targeting moiety not linked to the signaling agent is linked to the Fc domain (see Figures 11A-L and 14A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains mutations that reduce or eliminate its effector function.

[0172] In some embodiments where there is one signaling agent and two targeting moieties, the targeting moieties are linked together, the signaling agent is linked to one of the paired targeting moieties, and the signaling agent is linked to the Fc domain (see Figures 11A-L and 14A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains mutations that reduce or eliminate its effector function.

[0173] In some embodiments where there is one signaling agent and two targeting moieties, both targeting moieties are linked to the signaling agent and one of the targeting moieties is linked to the Fc domain (see Figures 11A-L and 14A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0174] In some embodiments where there is one signaling agent and two targeting moieties, the targeting moiety and signaling agent are linked to the Fc domain (see Figures 16A-J and 17A-J). In some embodiments, the targeting moieties are linked terminally (see Figures 16A-J and 17A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains mutations that reduce or eliminate its effector function.

[0175] In some embodiments where there are two signaling agents and one targeting moiety, the signaling agents are linked to the Fc domain on the same end and the targeting moiety is linked to the Fc domain (see Figures 6A-J and 9A-J). In some embodiments, the signaling agents are linked to the Fc domain on the same Fc chain and the targeting moiety is linked on the other Fc chain (see Figures 18A-F and 19A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0176] In some embodiments where there are two signaling agents and one targeting moiety, a signaling agent is linked to a targeting moiety, which is linked to an Fc domain, and another signaling agent is linked to an Fc domain (see Figures 6A-J and 9A-J, 12A-L, and 15A-L). In some embodiments, the targeting moiety and the unpaired signaling agent are linked to different Fc chains (see Figures 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling agent are linked to different Fc chains on the same terminus (see Figures 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling agent are linked to different Fc chains on different termini (see Figures 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling agent are linked to the same Fc chain (see Figures 12A-L and 15A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0177] In some embodiments where there are two signaling agents and one targeting moiety, the targeting moiety is linked to a signaling agent, which is linked to an Fc domain, and the other signaling agent is linked to an Fc domain (see Figures 6A-J and 9A-J). In some embodiments, the paired targeting moiety and the unpaired signaling agent are linked to different Fc chains (see Figures 6A-J and 9A-J). In some embodiments, the paired targeting moiety and the unpaired signaling agent are linked to different Fc chains on the same terminus (see Figures 6A-J and 9A-J). In some embodiments, the paired signaling agent and the unpaired signaling agent are linked to different Fc chains on different termini (see Figures 6A-J and 9A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0178] In some embodiments where there are two signaling agents and one targeting moiety, the signaling agents are linked together, the targeting moiety is linked to one of the paired signaling agents, and the targeting moiety is linked to the Fc domain (see Figures 12A-L and 15A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains mutations that reduce or eliminate its effector function.

[0179] In some embodiments where there are two signaling agents and one targeting moiety, the signaling agents are linked together, one of the signaling agents is linked to the Fc domain, and the targeting moiety is linked to the Fc domain (see Figures 12A-L and 15A-L, 18A-F, and 19A-F). In some embodiments, the paired signaling agent and targeting moiety are linked to the same Fc chain (see Figures 12A-L and 15A-L). In some embodiments, the paired signaling agent and targeting moiety are linked to different Fc chains (see Figures 18A-F and 19A-F). In some embodiments, the paired signaling agent and signaling agent are linked to different Fc chains on the same terminus (see Figures 18A-F and 19A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0180] In some embodiments where there are two signaling agents and one targeting moiety, both signaling agents are linked to a targeting moiety and one of the signaling agents is linked to an Fc domain (see Figures 12A-L and 15A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0181] In some embodiments where there are two signaling agents and one targeting moiety, the signaling agents are linked together, one of the signaling agents is linked to the targeting moiety, and the other signaling agent is linked to the Fc domain (see Figures 12A-L and 15A-L).

[0182] In some embodiments where there are two signaling agents and one targeting moiety, each signaling agent is linked to an Fc domain and the targeting moiety is linked to one of the signaling agents (see Figures 12A-L and 15A-L). In some embodiments, the signaling agents are linked to the same Fc chain (see Figures 12A-L and 15A-L).

[0183] In some embodiments, the targeting moiety or signaling agent is C H 2 and C H The polypeptide is linked to an Fc domain, including one or both of the three domains and optionally a hinge region. For example, such a polypeptide can be produced using a vector encoding a targeting moiety, a signaling agent, or a combination thereof, linked to the Fc domain as a single nucleotide sequence.

[0184] In some embodiments, for "split" targeting moieties, as described for FLT3L-ECD and its variants, such as FLT3L-ECD with the L27D mutation, a portion of the targeting moiety may be present on each of the Fc chains of a homodimeric or heterodimeric protein complex, and the formation of a functional targeting moiety is generated as part of the formation of a chimeric protein complex, as exemplified by FLT3L-ECD monomers dimerizing to form a functional FLT3 targeting moiety for delivery of signaling agents to FLT3-positive target cells.

[0185] Non-Fc-based chimeric protein complexes In various embodiments, a chimeric protein complex of the present invention comprises (a) an FMS-like tyrosine kinase 3 ligand (FLT3L) domain, for example, but not limited to, an extracellular domain of Flt3L described herein, for example, but not limited to, a single-chain or split-chain domain, optionally with the L27D mutation, and (b) one or more targeting moieties, including wild-type or modified signal transducers described herein, wherein (a) and (b) are linked by a domain that results in complex formation (e.g., a complexing domain). In some embodiments, a chimeric protein complex of the present invention further comprises one or more proteins or peptides (e.g., a complexing domain) that interact with each other, for example, using electrostatic interactions, hydrogen bonds, and / or hydrophobic effects. In some embodiments, a chimeric protein complex is homomeric (e.g., comprising two or more chimeric proteins described herein). In some embodiments, the chimeric protein complex is heteromeric (e.g., comprises one chimeric protein comprising a wild-type or modified signal transduction entity and one or more targeting moieties and another protein linked by one or more linkers). Various protein interaction domains (e.g., complexation domains) have been used to generate protein complexes and can be used to generate the chimeric protein complexes of the invention. In some embodiments, chimeric protein complexes can be generated using leucine zippers, the Jun and Fos family of proteins, helix-turn-helix self-dimerizing peptides, collagen, and trimeric and tetrameric subdomains of p53 (see, e.g., U.S. Pat. No. 8,507,222, which is incorporated herein by reference in its entirety).Other methods for generating heteromeric complexes include charge-based heterodimers as described by Chang et al. (PNAS 1984;91:11408-11412) or heterodimerized leucine zippers as described by Deng et al. (Chemistry & Biology 2008;15:908-919) or engineered heterodimers as described by Chen et al. (Nature 2019;565:106-111). In various embodiments, these chimeric protein complexes are not Fc-based. In some embodiments, various protein interaction domains can be used in place of the Fc domain (in the case of Fc-based chimeric protein complexes) described herein to form protein complexes.

[0186] Non-Fc single chain Flt3L construct In some embodiments, the present invention relates to chimeric proteins comprising: (i) one or more targeting moieties comprising a single-chain dimeric FMS-like tyrosine kinase 3 ligand (FLT3L) domain; (ii) one or more flexible linkers connecting (i) and (iii); and (iii) a signal transducer or modified form thereof.

[0187] In some embodiments, the chimeric protein or protein complex has a targeting moiety comprising the extracellular domain of FLT3L, or a portion or variant thereof. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2-5. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-5. In some embodiments, the chimeric protein or protein complex comprises two copies of FLT3L-ECD, or a variant thereof, on the same polypeptide (i.e., a single-chain dimeric FLT3L construct). In some embodiments, FLT3L-ECD, or a portion or variant thereof, comprises a mutation that reduces intermolecular FLT3L-ECD homodimerization (i.e., dimerization of FLT3L domains on separate FLT3L-ECD containing molecules that would not otherwise readily dimerize by other mechanisms) and favors intramolecular FLT3L-ECD dimerization (i.e., dimerization of two copies of FLT3L-ECD contained within the same single polypeptide, i.e., a single-chain dimeric FLT3L construct). In some embodiments, the mutation in FLT3L-ECD or a variant thereof is L27D (L24D relative to any one of SEQ ID NOs: 2-4, or relative to SEQ ID NO: 5). In some embodiments, the mutation L27D in FLT3-ECD, or variants thereof (relative to any one of SEQ ID NOs: 2-4, or L24D relative to SEQ ID NO: 5), or functionally similar mutations, favors the formation of more homogeneous forms of chimeric proteins and protein complexes, avoiding undesirable higher molecular weight complexes and / or aggregates that may be substantially detrimental to the scale-up of production of FLT3-targeting constructs and the in vivo safety of such constructs (e.g., risk of immunoreactivity, reduced activity, etc.).

[0188] In some embodiments, a single copy of FLT3L-ECD with an L27D mutation or a functionally equivalent mutation in the ECD can be present in a polypeptide that can dimerize with the same or a different polypeptide that also has a single copy of FLT3L-ECD with an L27D mutation or a functionally equivalent mutation in the ECD. Upon protein complex formation, dimerization of the FLT3L-ECD-L27D domain is induced, thereby generating a functional FLT3 targeting moiety. This type of chimeric protein complex is considered to have a "split FLT3L-ECD" because a functional targeting moiety derived from the split FLT3-ECD (i.e., on a different polypeptide molecule) is formed during assembly of the chimeric protein complex. A signaling agent can be attached or fused to any one of the polypeptides forming the chimeric protein complex.

[0189] Several types of signal transduction substances may be incorporated into chimeric proteins with single-chain dimeric FLT3L, or portions or variants thereof, and chimeric protein complexes formed by the "split FLT3L-ECD" approach.

[0190] In some embodiments, the chimeric protein has a targeting moiety comprising the extracellular domain of FLT3L, or a portion thereof. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2-5. In some embodiments, the targeting moiety comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-5. In some embodiments, the signal transduction agent is wild-type human IFNα2, IFNα1, IFNβ, or IL-1β. In some embodiments, the signal transduction agent comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 6, 7, 38, 39, or 74. In some embodiments, the signal transduction agent comprises the amino acid sequence of any one of SEQ ID NOs: 6, 7, 38, 39, or 74.

[0191] In some embodiments, a signaling agent is modified to contain one or more mutations. In some embodiments, the one or more mutations confer improved safety, or reduced affinity for the signaling agent's receptor, or reduced biological activity for the signaling agent's receptor, or allow for attenuation of the signaling agent's activity compared to the wild-type signaling agent. In some embodiments, the one or more mutations confer reduced affinity or activity that can be restored by attachment to one or more targeting moieties.

[0192] In some embodiments, the chimeric protein comprises a signal transducer and / or activator comprising a mutant human IFNα2 comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 6 or 7, wherein 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, and optionally the human IFNα2 has one or more mutations at positions 144-154 relative to SEQ ID NO: 6 or 7, and optionally the human IFNα2 has one or more mutations at positions 144-154 relative to SEQ ID NO: 6 or 7. and optionally the mutant human IFNα2 has one or more mutations at positions L15, A19, R22, R23, L26, F27, L30, 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; and one or more mutations at R148, R149, and L153, ​​and optionally the mutations are L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57Y, E58N, Q61S, F64A, N65A, T69A, L80A, Y85A, Y89A, D114R, L117A, R120A, R125A, K133A, K134A, R144A, A145G, A145M, or the like, relative to SEQ ID NO: 6 or 7. , M148A, R149A, S152A, L153A, and N156A, and optionally 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, and is a chimeric protein in which 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.

[0193] In some embodiments, the signal transduction agent is wild-type interferon α1 or modified interferon α1. In some embodiments, the present invention provides a chimeric protein or Fc-based chimeric protein complex comprising wild-type IFNα1. In various embodiments, the wild-type IFNα1 comprises the amino acid sequence of SEQ ID NO:74.

[0194] In various embodiments, the chimeric protein or Fc-based chimeric protein complex of the present invention comprises, as a signal transduction agent, an IFNα1 variant, including a modified form of IFNα1, i.e., an IFNα1 mutant. In various embodiments, an IFNα1 variant includes a mutant, functional derivative, analog, precursor, isoform, splice variant, or fragment of interferon.

[0195] In some embodiments, the IFNα1 interferon is modified to have one or more amino acid mutations at positions L15, A19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157 relative to SEQ ID NO: 74. The mutations may optionally be hydrophobic mutations, and may be selected from, for example, alanine, valine, leucine, and isoleucine. In some embodiments, the IFNα1 interferon is selected from the group consisting of L15A, A19W, R23A, S25A, L30A, L30V, D32A, R33K, R33A, R33Q, H34A, Q40A, C86S, C86A, D115R, L118A, K121A, K121E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E, R145G, R145H, R145I, R145J, R145K ... and N157A. In some embodiments, the IFNα1 variant comprises one or more mutations selected from L30A / H58Y / E59N_Q62S, R33A / H58Y / E59N / Q62S, M149A / H58Y / E59N / Q62S, L154A / H58Y / E59N / Q62S, R145A / H58Y / E59N / Q62S, D115A / R121A, L118A / R121A, L118A / R121A / K122A, R121A / K122A, and R121E / K122E relative to SEQ ID NO: 74.

[0196] In some embodiments, the IFNα1 is a variant comprising one or more mutations that reduce undesired disulfide pairing, for example, at amino acid positions C1, C29, C86, C99, or C139 relative to SEQ ID NO: 74. In some embodiments, the mutation at position C86 can be, for example, C86S, C86A, or C86Y. These C86 mutants of IFNα1 are referred to as reduced cysteine-aggregating mutants. In some embodiments, the IFNα1 variant comprises mutations at positions C1, C86, and C99 relative to SEQ ID NO: 74.

[0197] In some embodiments, the chimeric protein is a chimeric protein in which the signal transducer is a mutant human IFNβ comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 38, and the mutant human IFNβ has one or more mutations that confer improved safety compared to wild-type IFNβ having the amino acid sequence of SEQ ID NO: 38, and the mutations are one or more of W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, relative to the amino acid sequence of SEQ ID NO: 38.

[0198] In some embodiments, the chimeric protein comprises a signal transducer that is a mutant human IL-1β comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 39, wherein the mutant human IL-1β has one or more mutations that confer improved safety compared to wild-type IL-1β having the amino acid sequence of SEQ ID NO: 39, the mutations being A117G / P118G, R120G, R120A, L122A, T125G / L126G, R127G, Q130A, Q130W, Q131G, K132A, S137G / Q138Y, L14 5G, H146A, H146G, H146E, H146N, H146R, L145A / L147A, Q148E, Q148G, Q148L, Q 148G / Q150G, Q150G / D151A, M152G, F162A, F162A / Q164E, F166A, Q164E / E167K , N169G / D170G, I172A, V174A, K208E, K209A, K209D, K209A / K210A, K219S, K219Q, E221S, E221K, E221S / N224A, N224S / K225S, E244K and N245Q.

[0199] In some embodiments, the chimeric protein comprises a flexible linker, wherein the flexible linker consists essentially of glycine and serine residues, and optionally the flexible linker consists essentially of (Gly4Ser) n wherein n is from about 1 to about 8, and optionally the flexible linker comprises one or more of SEQ ID NO:10 to SEQ ID NO:17.

[0200] In some embodiments, the invention relates to recombinant nucleic acid compositions encoding one or more chimeric proteins described herein. In some embodiments, the invention relates to host cells comprising the recombinant nucleic acids.

[0201] In some embodiments, the chimeric proteins are suitable for use in patients with one or more of cancer, infectious diseases, immune disorders, autoimmune and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases. Some aspects of the present invention relate to methods for treating or preventing cancer, infectious diseases, immune disorders, autoimmune and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases, the methods comprising administering to a patient in need thereof an effective amount of a chimeric protein.

[0202] Single-chain Fc-based Flt3L constructs In some aspects, the present invention relates to an Fc-based chimeric protein complex comprising: (i) one or more targeting moieties comprising a single-chain dimeric FMS-like tyrosine kinase 3 ligand (FLT3L) domain; and (ii) an Fc domain, optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain.

[0203] In some embodiments, the Fc-based chimeric protein complex is an Fc-based chimeric protein complex in which a single-chain dimeric FLT3L is attached to one Fc chain of an Fc domain. In some embodiments, the Fc-based chimeric protein complex is an Fc-based chimeric protein complex in which the single-chain FLT3L comprises the extracellular domain of FLT3L, or a portion thereof. In some embodiments, the Fc-based chimeric protein complex is an Fc-based chimeric protein complex in which the single-chain dimeric FLT3L comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2-5. In some embodiments, the single-chain dimeric FLT3L comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-5.

[0204] In some embodiments, the Fc-based chimeric protein complex comprises two copies of FLT3L-ECD (FLT3L extracellular domain) or a variant thereof attached to one Fc chain of the Fc domain (i.e., a single-chain dimeric FLT3L-ECD construct). In some embodiments, the FLT3L-ECD, or a portion or variant thereof, comprises a mutation that reduces intermolecular FLT3L-ECD homodimerization (i.e., dimerization of FLT3L domains present on separate FLT3L-ECD containing molecules) and favors intramolecular FLT3L-ECD dimerization (i.e., dimerization of two copies of FLT3L-ECD contained within the same single polypeptide, i.e., a single-chain dimeric FLT3L construct). In some embodiments, the mutation in FLT3L-ECD or a variant thereof is L27D (relative to any one of SEQ ID NOS: 2-4, or L24D relative to SEQ ID NO: 5). In some embodiments, the mutation L27D in FLT3-ECD, or variants thereof (relative to any one of SEQ ID NOs: 2-4, or L24D relative to SEQ ID NO: 5), or functionally similar mutations, favors the formation of more homogeneous forms of chimeric proteins and protein complexes, avoiding undesirable higher molecular weight complexes and / or aggregates that may be substantially detrimental to the scale-up of production of FLT3-targeting constructs and the in vivo safety of such constructs (e.g., risk of immunoreactivity, reduced activity, etc.).

[0205] In some aspects, the invention relates to recombinant nucleic acid compositions encoding one or more chimeric proteins described herein. In some embodiments, the invention relates to host cells comprising the recombinant nucleic acids.

[0206] In some embodiments, the Fc-based chimeric protein complexes are suitable for use in patients with one or more of cancer, infectious diseases, immune disorders, autoimmune and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases. In some embodiments, the present invention relates to methods for treating or preventing cancer, infectious diseases, immune disorders, autoimmune and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases, comprising administering to a patient in need thereof an effective amount of an Fc-based chimeric protein complex.

[0207] How to use In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, recruits one or more immune cells directly or indirectly to diseased cells, for example, via a targeting moiety, among other features. Thus, in some embodiments, the targeting moiety directly or indirectly recruits immune cells to tumor cells or the tumor microenvironment. In this manner, the targeting moiety can increase the number of dendritic cells. In some embodiments, the targeting moiety optionally enhances tumor antigen presentation by dendritic cells.

[0208] In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is suitable for use in patients with one or more of cancer, infectious disease, immune disorder, autoimmune disease and / or neurodegenerative disease, cardiovascular disease, wound, ischemia-related disease, and / or metabolic disease. In some aspects, methods of treating or preventing cancer are provided, comprising administering to a patient in need thereof an effective amount of a chimeric protein complex, such as a chimeric protein or an Fc-based chimeric protein complex, according to various embodiments of the present disclosure.

[0209] In various embodiments, the cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney or renal cancer cancer); laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, tonsil, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland carcinoma; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; Hodgkin's lymphoma and non-Hodgkin's lymphoma; and lymphomas, including B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; The cancer is selected from one or more of the following: intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD); and abnormal vascular proliferation associated with phacomatosis; edema (for example, associated with brain tumor); and Meigs syndrome. In certain embodiments, the cancer is acute myeloid leukemia (AML).

[0210] Furthermore, in some aspects, the present invention includes methods for treating or preventing autoimmune and / or neurodegenerative diseases, the methods comprising administering to a patient in need thereof an effective amount of a chimeric protein complex, such as a chimeric protein or an Fc-based chimeric protein complex, according to various embodiments of the present disclosure. The autoimmune and / or neurodegenerative disease can be selected from multiple sclerosis, diabetes, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barré syndrome, scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, primary sclerosing cholangitis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Menier's syndrome, transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, myasthenia gravis, Reiter's syndrome, and Graves' disease.

[0211] In some embodiments, chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention optionally comprise one or more linkers. In some embodiments, chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes of the present invention, comprise a linker linking one or more of the Fc domain, targeting moiety, and signal transduction agent (e.g., IFNα2, IFNα1, IFNβ, or IL-1β, or variants thereof). In some embodiments, chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes of the present invention, comprise a linker within the signal transduction agent (e.g., IFNα2, IFNα1, IFNβ, or IL-1β, or variants thereof). In some embodiments, linkers may be utilized to link various functional groups, residues, or moieties described herein to chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes. In some embodiments, the linker is a single amino acid or multiple amino acids that do not affect or reduce the stability, orientation, binding, neutralization, and / or excretion properties of the binding region and binding protein. In various embodiments, the linker is selected from a peptide, a protein, a sugar, or a nucleic acid.

[0212] In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more additional signal transduction agents, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, which may be wild-type or modified as described herein. In various embodiments, the chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex of the present invention, comprises a modified signal transduction agent, providing improved safety compared to the unmodified wild-type. Note that, in some embodiments, the present invention includes chimeric protein complexes, such as an Fc-based chimeric protein complex, comprising one, two, or three signal transduction agents.

[0213] In various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties (e.g., various antibody formats, including, but not limited to, single domain antibodies, including VHHs) that specifically bind to a target of interest (e.g., antigen, receptor). In various embodiments, the targeting moiety specifically binds to a target of interest (e.g., antigen, receptor), including those found on one or more immune cells, which may include, but are not limited to, T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendritic cells. In some embodiments, the targeting moiety specifically binds to a target of interest (e.g., antigen, receptor) and effectively recruits one or more immune cells. In some embodiments, the target of interest (e.g., antigen, receptor) may be found on one or more tumor cells. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can recruit immune cells, e.g., immune cells capable of killing and / or suppressing tumor cells, to a site of action (such as, by way of non-limiting example, the tumor microenvironment). In some embodiments, the targeting moiety specifically binds to a target of interest (e.g., an antigen, a receptor) that is part of a non-cellular structure. Note that the present invention includes, in some embodiments, chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, having one, two, or three targeting moieties.

[0214] In some embodiments, vectors encoding chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention linked as a single nucleotide sequence to any of the linkers described herein are provided and can be used to prepare such chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes.

[0215] In some embodiments, the length of the linker allows for effective binding of one targeting moiety and one signaling agent (e.g., IFNα2, IFNα1, IFNβ, or IL-1β or a variant thereof) to their receptors. For example, in some embodiments, the length of the linker allows for effective binding of one of the targeting moieties and one of the signaling agents to their receptors on the same cell.

[0216] In some embodiments, the length of the linker is at least equal to the shortest distance between the binding sites of a targeting moiety and a signaling agent to a receptor on the same cell, hi some embodiments, the length of the linker is at least 2, 3, 4, 5, 10, 20, 25, 50, 100, or more times the shortest distance between the binding sites of a targeting moiety and a signaling agent to a receptor on the same cell.

[0217] As described herein, the length of the linker allows for effective binding of a single targeting moiety and signaling agent to a receptor on the same cell, and binding is sequential, e.g., targeting moiety / receptor binding precedes signaling agent / receptor binding.

[0218] In some embodiments, there are two linkers in a single chimera, each linking a signaling agent to a targeting moiety. In various embodiments, the linkers have a length that allows for the formation of a moiety that holds disease cells and effector cells without steric hindrance that could interfere with the regulation of either cell.

[0219] The present invention contemplates the use of various linker sequences. In various embodiments, the linker can be derived from natural multidomain proteins or can be an empirical linker, for example, as described in Chichili et al., (2013), Protein Sci. 22(2):153-167; Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369. The entire contents of these documents are incorporated herein by reference. In some embodiments, the linker can be designed using a linker design database and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369 and Crasto et al., (2000), Protein Eng. 13(5):309-312. In various embodiments, the linker can be functional. For example, but not limited to, a linker may function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve the biological activity of a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex of the present invention.

[0220] In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids in length. For example, the 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 linker is a polypeptide. In some embodiments, the linker is more than about 100 amino acids in length. For example, the 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. In some embodiments, the linker is flexible. In other embodiments, the linker is rigid.

[0221] In various embodiments, the 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 linker is (Gly4Ser) n wherein n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NOs: 10 to 17, respectively). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 18). Examples of additional linkers include, but are not limited to, the sequences: LE, GGGGS (SEQ ID NO: 10), (GGGGS) n (n=1 to 4) (SEQ ID NOs: 10 to 13), (Gly) (SEQ ID NO: 19), (Gly) (SEQ ID NO: 20), (EAAAK) n (n = 1 to 3) (SEQ ID NOs: 21 to 23), A(EAAAK) nA (n = 2 to 5) (SEQ ID NOs: 24 to 27), AEAAAKEAAAKA (SEQ ID NO: 24), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 28), PAPAP (SEQ ID NO: 29), KESGSVSSEQLAQFRSLD (SEQ ID NO: 30), EGKSSGSGSESKST (SEQ ID NO: 31), GSAGSAAGSGEF (SEQ ID NO: 32), and (XP) n (wherein X represents any amino acid, for example, Ala, Lys, or Glu). In various embodiments, the linker is GGS.

[0222] In some embodiments, the 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 linker of G, S, and E randomly spaced every four amino acids.

[0223] In some embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region found in IgG, IgA, IgD, and IgE class antibodies functions as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to constant regions, hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies within IgG subclasses. The hinge region of IgG1 encompasses amino acids 216–231 and is freely flexible, allowing the Fab fragments to rotate around their axis of symmetry and move within a sphere centered on the first two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks glycine residues and is relatively short, containing a rigid polyproline double helix stabilized by additional inter-heavy chain disulfide bridges. These characteristics limit the flexibility of the IgG2 molecule. IgG3, unlike other subclasses, has a unique extended hinge region (approximately four times longer than the IgG1 hinge) containing 62 amino acids (including 21 prolines and 11 cysteines) that forms a rigid polyproline double helix. In IgG3, the Fab fragments are relatively far from the Fc fragment, giving the molecule greater flexibility. The extended hinge of IgG3 is also responsible for its higher molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge region has been reported to decrease in the following order: IgG3 > IgG1 > IgG4 > IgG2.

[0224] Crystallographic studies have shown that the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge, the core, and the lower hinge. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region is comprised of C H1 The length of the upper hinge region correlates with the flexibility of the antibody segment. The core hinge region contains the inter-heavy chain disulfide bridges, while the lower hinge region contains the C H 2 domain, and C H The core hinge region of wild-type human IgG1 comprises the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 37), which upon dimerization by disulfide bond formation generates a cyclic octapeptide that is believed to function as a pivot point, thereby conferring flexibility. In various embodiments, the linkers of the invention comprise one, two, or three upper hinge, core, and lower hinge regions of any antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region may also contain one or more glycosylation sites, which contain several structurally distinct types of carbohydrate attachment sites. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of the hinge region, which confers resistance of the hinge region polypeptide to intestinal proteases, a property believed to be advantageous for secretory immunoglobulins. In various embodiments, the linkers of the invention contain one or more glycosylation sites.

[0225] In some embodiments, the linker is a synthetic linker, such as PEG. In various embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve the biological activity of a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex of the present invention. In another example, the linker may function to target a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, to a specific cell type or site.

[0226] In various embodiments, chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention can contain one or more functional groups, residues, or moieties. In various embodiments, the one or more functional groups, residues, or moieties are attached to or genetically fused to any of the signal transduction or targeting moieties described herein. In some embodiments, such functional groups, residues, or moieties impart one or more desirable properties or functionalities to chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes of the present invention. Examples of such functional groups and techniques for introducing them into chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, are known in the art; see, for example, Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

[0227] In various embodiments, each of the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, may be conjugated and / or fused with another agent to extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, may be fused or conjugated with one or more of PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, etc.

[0228] In various embodiments, each individual chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is fused to one or more substances described in BioDrugs (2015) 29:215-239, the entire contents of which are incorporated herein by reference.

[0229] In some embodiments, the functional group, residue, or moiety comprises a suitable pharmaceutically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly(ethylene glycol) or mPEG). In some embodiments, the attachment of a PEG moiety extends the half-life and / or reduces the immunogenicity of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex. Any suitable form of PEGylation is commonly used, such as PEGylation used in the art for antibodies and antibody fragments (including, but not limited to, single-domain antibodies such as VHHs); see, e.g., Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev., 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003); and WO 04 / 060965. The entire contents of these documents are incorporated herein by reference. Various reagents for protein PEGylation are also commercially available, for example, from Nektar Therapeutics, USA. In some embodiments, site-specific PEGylation, particularly via cysteine ​​residues, is used (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003)), the entire contents of which are incorporated herein by reference). In some embodiments, the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex of the present invention, is modified to appropriately introduce one or more cysteine ​​residues for PEG attachment, or an amino acid sequence containing one or more cysteine ​​residues for PEG attachment can be fused to the amino and / or carboxy terminus of the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex, using techniques known in the art.

[0230] In some embodiments, the functional group, residue, or moiety comprises N-linked or O-linked glycosylation, hi some embodiments, the N-linked or O-linked glycosylation is introduced as part of a co-translational and / or post-translational modification.

[0231] 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 suitable 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. The VHHs and polypeptides of the invention so labelled may, depending on the choice of the particular label, be used, for example, for in vitro, in vivo or in situ assays (immunoassays known per se such as ELISAs, RIAs and EIAs and other "sandwich" methods) and for in vivo diagnostic and imaging purposes.

[0232] In some embodiments, the functional group, residue, or moiety comprises a tag attached or genetically fused to a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex. In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, can comprise a single tag or multiple tags. For example, the tag is a peptide, sugar, or DNA molecule that does not inhibit or interfere with binding of the chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, to its target or any other antigen of interest, such as a tumor antigen. In various embodiments, the tag is at least about: 3-5 amino acids in length, 5-8 amino acids in length, 8-12 amino acids in length, 12-15 amino acids in length, or 15-20 amino acids in length. Examples of tags are described, for example, in U.S. Patent Application Publication No. 2013 / 0058962. In some embodiments, the tag is an affinity tag, such as a glutathione-S-transferase (GST) or histidine (His) tag. In certain embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a histidine tag.

[0233] In some embodiments, the functional group, residue, or moiety comprises a chelating group, for example, for chelating a metal or metal cation. Suitable chelating groups include, for example, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0234] In some embodiments, the functional group, residue, or moiety comprises a functional group that is one half of a specific binding pair, such as a biotin-(streptavidin) binding pair. Such a functional group can be used to link a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex of the present invention, to another protein, polypeptide, or chemical compound that is bound to the other half of the binding pair, i.e., through the formation of a binding pair. For example, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex of the present invention, can be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier that is conjugated to avidin or streptavidin. For example, in diagnostic systems in which a detectable signal-generating substance is conjugated to avidin or streptavidin, such a chimeric protein complex, such as a binding chimeric protein or Fc-based chimeric protein complex, can be used, for example, as a reporter. For example, such a binding pair can be used to bind a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, 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). Such binding pairs can also be used to link therapeutically active agents to chimeric protein complexes, such as the chimeric proteins or Fc-based chimeric protein complexes of the present invention.

[0235] Methods for producing chimeric protein complexes, such as the chimeric proteins or Fc-based chimeric protein complexes of the present invention, are described herein. For example, DNA sequences encoding chimeric protein complexes, such as the chimeric proteins or Fc-based chimeric protein complexes of the present invention (e.g., DNA sequences encoding a signal transduction agent (e.g., IFNα2, IFNα1, IFNβ, or IL-1β or a variant thereof) and a targeting moiety and linker, or polypeptide components of a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex) can be chemically synthesized using methods known in the art. The synthetic DNA sequence can be linked to other appropriate nucleotide sequences, including, for example, expression control sequences, to produce a gene expression construct encoding a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex of interest. Thus, in various embodiments, the present invention provides isolated nucleic acids comprising a nucleotide sequence encoding a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex of the present invention, or a polypeptide subunit thereof.

[0236] Nucleic acids encoding chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention can be incorporated (linked) into expression vectors, which can be introduced into host cells by gene transfer, transformation, or transduction techniques. For example, nucleic acids encoding chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes of the present invention, can be introduced into host cells by retroviral transduction. Examples of host cells include E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, cultured monkey kidney (COS) cells, or human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. Transformed host cells can be grown under conditions that allow the host cells to express genes encoding chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes of the present invention. Thus, in various embodiments, the present invention provides expression vectors containing nucleic acids encoding chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes of the present invention. In various embodiments, the invention further provides a host cell comprising such an expression vector.

[0237] Specific expression and purification conditions vary depending on the expression system used.For example, if a gene is expressed in E. coli, the gene is first inserted into an expression vector by placing the engineered gene downstream of a bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence.In another example, if a engineered gene is expressed in a eukaryotic host cell, such as CHO cell, the gene is first inserted into an expression vector that includes, for example, a suitable eukaryotic promoter, a secretion signal, a transcription factor, and various introns.The gene construct can be introduced into host cells using gene transfer, transformation, or transduction techniques.

[0238] Chimeric protein complexes, such as the chimeric proteins or Fc-based chimeric protein complexes of the present invention, can be produced by growing host cells transfected with an expression vector encoding the chimeric protein or chimeric protein complex, such as the Fc-based chimeric protein complex, under conditions that allow expression of the protein. After expression, the protein can be collected and purified using techniques well known in the art, for example, by affinity tags, such as glutathione-S-transferase (GST) and histidine tags, or by chromatography.

[0239] Thus, in various embodiments, the present invention provides nucleic acids encoding chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention. In various embodiments, the present invention provides host cells comprising nucleic acids encoding chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention. In various embodiments, the present invention provides nucleic acids encoding chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention, that are suitable for production in a cell-free system (e.g., in vitro transcription and / or in vitro translation).

[0240] In various embodiments, chimeric protein complexes, such as IFNα2, IFNα1, IFNβ, or IL-1β, variants thereof, or chimeric proteins or Fc-based chimeric protein complexes comprising IFNα2, IFNα1, IFNβ, or IL-1β, or variants thereof, can be expressed in vivo, for example, in a patient. For example, in various embodiments, chimeric protein complexes, such as IFNα2, IFNα1, IFNβ, or IL-1β, variants thereof, or chimeric proteins or Fc-based chimeric protein complexes comprising IFNα2, IFNα1, IFNβ, or IL-1β, or variants thereof, can be administered in the form of nucleic acids encoding IFNα2, IFNα1, IFNβ, or IL-1β, or variants thereof, or chimeric proteins or Fc-based chimeric protein complexes comprising IFNα2, IFNα1, IFNβ, or IL-1β, or variants thereof. In various embodiments, the nucleic acid is DNA or RNA. In some embodiments, chimeric protein complexes, such as IFNα2, IFNα1, IFNβ, or IL-1β, variants thereof, or chimeric proteins or Fc-based chimeric protein complexes comprising IFNα2, IFNα1, IFNβ, or IL-1β, or variants thereof, are encoded by modified mRNA, i.e., mRNA comprising one or more modified nucleotides. In some embodiments, the modified mRNA comprises one or more modifications found in U.S. Pat. No. 8,278,036, the entire contents of which are incorporated herein by reference. In some embodiments, the modified mRNA comprises one or more of m5C, m5U, m6A, s2U, Ψ, and 2'-O-methyl-U. In some embodiments, the invention relates to the administration of modified mRNA encoding one or more chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention. In some embodiments, the invention relates to gene therapy vectors comprising the modified mRNA. In some embodiments, the invention relates to gene therapy methods comprising the modified mRNA.In various embodiments, the nucleic acid is in the form of an oncolytic virus, such as an adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus, or vaccinia.

[0241] Chimeric protein complexes, such as the chimeric proteins or Fc-based chimeric protein complexes described herein, may have sufficiently basic functional groups that can react with inorganic or organic acids or carboxyl groups, or with inorganic or organic bases, to form pharmaceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids as is well known in the art. Such salts include, for example, the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich (Switzerland), 2002. These references are incorporated herein by reference in their entireties.

[0242] Pharmaceutically acceptable salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate. Examples of suitable salts include benzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, alpha-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate.

[0243] The term "pharmaceutically acceptable salt" also refers to a salt of a composition of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, and unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, organic amines such as dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines) such as mono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

[0244] In some embodiments, the compositions described herein are in the form of pharmaceutically acceptable salts.

[0245] In various embodiments, the present invention relates to pharmaceutical compositions comprising a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex described herein, and a pharmaceutically acceptable carrier or excipient. Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally contain an appropriate amount of a pharmaceutically acceptable excipient to provide a suitable dosage form.

[0246] In various embodiments, pharmaceutical excipients can be liquids such as water and oils, including those of petroleum, animal, vegetable, or artificial origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Pharmaceutical excipients can be, for example, saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, and urea. Additionally, auxiliary agents, stabilizers, thickeners, lubricants, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Saline and aqueous dextrose and glycerin solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, etc. Any of the formulations described herein may contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Further examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0247] The present invention includes the described pharmaceutical compositions (and / or additional therapeutic agents) in a variety of formulations. Any of the pharmaceutical compositions (and / or additional therapeutic agents) of the present invention described herein can be in the form of a solution, suspension, emulsion, infusion, tablet, pill, pellet, capsule, liquid-containing capsule, gelatin capsule, powder, sustained-release formulation, suppository, emulsion, aerosol, spray, suspension, lyophilized powder, frozen suspension, dry powder, or any other form suitable for use. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In a further embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a liquid.

[0248] If desired, the pharmaceutical compositions of the present invention (and / or additional agents) may also include a solubilizing agent. Additionally, the agents may be delivered using suitable vehicles or delivery devices known in the art. The combination therapeutic agents outlined herein may be co-delivered in a single delivery vehicle or carrier.

[0249] Formulations containing the pharmaceutical composition of the present invention (and / or additional agents) can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. Such methods typically include the step of combining the therapeutic agent with a carrier, which may constitute one or more accessory ingredients. Typically, formulations are prepared by uniformly and intimately admixing the therapeutic agent with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation (e.g., wet or dry granulation, powder blending, etc., followed by tabletting using conventional methods known in the art).

[0250] In various embodiments, any pharmaceutical composition (and / or additional agent) described herein is formulated in accordance with routine procedures as a composition adapted for the methods of administration described herein.

[0251] Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical. Administration can be local or systemic. In some embodiments, administration is oral. In other embodiments, administration is by parenteral injection. The method of administration is at the discretion of the practitioner and depends, in part, on the site of the medical condition. In most cases, administration results in the release of any agent described herein into the bloodstream.

[0252] In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex described herein, is formulated according to conventional methods as a composition adapted for oral administration. Compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more agents, such as sweeteners such as lactose, aspartame, or saccharin, flavorings such as peppermint, wintergreen oil, or cherry oil, coloring agents, and preservatives, to provide a pharmaceutically palatable formulation. Furthermore, in tablet or pill form, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby enabling a sustained action over an extended period of time. A selectively permeable membrane surrounding a chimeric protein complex, such as any of the osmotically activated chimeric proteins or Fc-based chimeric protein complexes described herein, is also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is absorbed by the carrier compound, which swells and expels the drug or drug composition through an opening. These delivery platforms can provide an essentially zero-order delivery profile, as opposed to the spiked profile of immediate-release formulations. Time-delay agents 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 pharmaceutical grade. In addition to the active compound, the suspension may contain a suspending agent, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and the like, and mixtures thereof.

[0253] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may be prepared in the form of sterile solid compositions (e.g., lyophilized compositions), which can be dissolved or suspended in a sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art. Suitable formulation components for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetate, citrate, or phosphate; and agents for regulating osmolality such as sodium chloride or dextrose.

[0254] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier must be stable under the conditions of manufacture and storage and must be protected against microorganisms. The carrier may be a solvent or dispersion medium, for example, containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0255] The compositions provided herein, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., "nebulized" formulations) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0256] Any pharmaceutical composition of the present invention (and / or additional agent) described herein can be administered by controlled release or sustained release means or delivery device known to those skilled in the art.Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556.Each of these patents is incorporated herein by reference in its entirety. Such dosage forms are useful for enabling controlled or sustained release of one or more active ingredients using, for example, hydropropyl cellulose, hydropropyl methyl cellulose, polyvinylpyrrolidone, other polymer matrices, gels, osmotic membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, and can provide desired release profiles at various rates. Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the active ingredients of the pharmaceutical compositions described herein. The present invention thus provides unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel capsules, and caplets, adapted for controlled or sustained release.

[0257] Controlled or sustained release of an active ingredient can be stimulated by various conditions including, but not limited to, changes in pH, temperature, stimulation with light of a suitable wavelength, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0258] In another embodiment, a sustained-release system can be placed in the vicinity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review in Langer, 1990, Science 249:1527-1533, can be used.

[0259] Pharmaceutical preparations are preferably sterile. Sterilization can be achieved, for example, by filtration through sterile filtration membranes. If the composition is lyophilized, filter sterilization can be performed before or after lyophilization and reconstitution.

[0260] It will be understood that the actual dose of a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, administered according to the present invention will vary depending on the specific dosage form and administration method. Those skilled in the art can take into account many factors that alter the action of a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex (e.g., body weight, sex, diet, administration time, administration route, excretion rate, subject condition, drug combinations, genetic predisposition, and reaction sensitivity). Administration can be carried out continuously or in one or more separate doses within the maximum tolerated dose range. The optimal administration rate for a given set of conditions can be ascertained by those skilled in the art using conventional dose administration tests.

[0261] In some embodiments, a suitable dosage of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is in the range of about 0.01 μg / kg to about 100 mg / kg of the subject's body weight, about 0.01 μg / kg to about 10 mg / kg of the subject's body weight, or about 0.01 μg / kg to about 1 mg / kg of the subject's body weight, e.g., about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.06 μg / kg, about 0.07 μg / kg, about 0.08 μg / kg, about 0.09 μg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0. 3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, 1.9 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg body weight, or about 100 mg / kg body weight (including all values ​​and ranges therebetween).

[0262] Individual doses of chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can be administered as a unit dosage form (e.g., tablet, capsule, or liquid formulation) containing, for example, about 1 μg to about 100 mg, about 1 μg to about 90 mg, about 1 μg to about 80 mg, about 1 μg to about 70 mg, about 1 μg to about 60 mg, about 1 μg to about 50 mg, about 1 μg to about 40 mg, about 1 μg to about 30 mg, about 1 μg to about 20 mg, about 1 μg to about 10 mg, about 1 μg to about 5 mg, about 1 μg to about 3 mg, about 1 μg to about 1 mg, or about 1 μg to about 50 μg. For example, the unit dosage form may contain about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 0.1 mg, about 0.2 mg g, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg (including all values ​​and ranges therebetween).

[0263] In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is administered in an amount of about 1 μg to about 100 mg daily, about 1 μg to about 90 mg daily, about 1 μg to about 80 mg daily, about 1 μg to about 70 mg daily, about 1 μg to about 60 mg daily, about 1 μg to about 50 mg daily, about 1 μg to about 40 mg daily, about 1 μg to about 30 mg daily, about 1 μg to about 20 mg daily, about 1 μg to about 10 mg daily, about 1 μg to about 5 mg daily, about 1 μg to about 3 mg daily, or about 1 μg to about 1 mg daily. In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, 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 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg g, 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).

[0264] In certain embodiments of the present invention, a pharmaceutical composition comprising a chimeric protein or a chimeric protein complex, such as an Fc-based chimeric protein complex, can be administered, for example, twice or more times daily (e.g., about twice, about three times, about four times, about five times, about six times, about seven times, about eight times, about nine times, or about ten times daily), about once daily, about every other day, about every third day, about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every six months, or about once a year. In certain embodiments, a pharmaceutical composition comprising a chimeric protein or a chimeric protein complex, such as an Fc-based chimeric protein complex, is administered about three times a week.

[0265] In various embodiments, the chimeric protein or chimeric protein complex, such as the Fc-based chimeric protein complex, of the present invention can be administered for a long period of time. For example, the chimeric protein or chimeric protein complex, such as the Fc-based chimeric protein complex, 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 or chimeric protein complex, such as the Fc-based chimeric protein complex, can be administered for 12 weeks, 24 weeks, 36 weeks, or 48 weeks. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, 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 or chimeric protein complex, such as an Fc-based chimeric protein complex, 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.

[0266] In various embodiments, the pharmaceutical compositions of the present invention are co-administered with an additional therapeutic agent. The co-administration can be simultaneous or sequential.

[0267] In one embodiment, the additional therapeutic agent and the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex, of the present invention are administered to a subject simultaneously. As used herein, the term "simultaneously" means that the additional therapeutic agent and the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex, are administered at a time interval of about 60 minutes or less, for example, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, about 5 minutes or less, or about 1 minute or less. The administration of the additional therapeutic agent and the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex, can be performed by simultaneous administration of a single formulation (e.g., a formulation containing the additional therapeutic agent and the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex) or separate formulations (e.g., a first formulation containing the additional therapeutic agent and a second formulation containing the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex).

[0268] Co-administration does not require that the therapeutic agents be administered simultaneously, provided that the timing of their administration is such that the pharmacological activities of the additional therapeutic agent and the chimeric protein overlap over time, thereby achieving a combined therapeutic effect. For example, the additional therapeutic agent and the chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, can be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and the chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, are administered at a time interval of more than about 60 minutes. For example, the time between the sequential administration of the additional therapeutic agent and the chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, can be more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, more than about 1 week, more than about 2 weeks, or more than about 1 month. The optimal administration time will depend on the metabolism, excretion rate, and / or pharmacodynamic activity of the additional therapeutic agent and the chimeric protein or chimeric protein conjugate, such as an Fc-based chimeric protein conjugate, being administered. The additional therapeutic agent or chimeric protein or chimeric protein conjugate, such as an Fc-based chimeric protein conjugate, may be administered first.

[0269] Co-administration also does not require that the therapeutic agents be administered to a subject by the same route of administration. Rather, each therapeutic agent can be administered by any suitable route, e.g., parenterally or non-parenterally.

[0270] In some embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex described herein, acts synergistically when co-administered with another therapeutic agent. In such embodiments, the chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, and the additional therapeutic agent may be administered at a lower dose than would be employed if the therapeutic agent were used in monotherapy.

[0271] In some embodiments, the present invention relates to a chemotherapeutic agent as an additional therapeutic agent. For example, but not limited to, such combinations of chimeric protein complexes of the present invention with chemotherapeutic agents, including chimeric proteins or Fc-based chimeric protein complexes, are used to treat cancer, as described elsewhere herein. Examples of chemotherapeutic agents include alkylating agents such as thiotepa, CYTOXAN (cyclophosphamide), alkyl sulfonates such as busulfan, improsulfan, and piposulfan, aziridines such as benzodopa, carboquone, mesuredopa, and uredopa, ethylenimines, methylmelamines such as altretamine, triethylenemelamine, triethylenenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine, acetogenins (e.g., bullatacin, bullatatinone), canthotecins (including the synthetic analog topotecan), bryostatin, kallistatin (callystatin), and the like. statin), CC-1065 (including synthetic analogs of adozelesin, carzelesin, and bizelesin), cryptophycins (e.g., cryptophycin 1, cryptophycin 8, etc.), dolastatins, duocarmycins (including synthetic analogs KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictine, spongistatin, nitrogen mustards, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine, antibiotics such as enediyne antibiotics (calicheamicin, especially calicheamicin gamma II and calicheamicin omega II (see Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodol Bicine, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine , azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal substances such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as florinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate, demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine;Maytansinoids, e.g., maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (ara-C); cyclophosphamide; thiotepa; taxoids, e.g., taxol, paclitaxel (Bristol-Myers Squibb Company, Squibb Oncology, Princeton, NJ), Abraxane Cremophor-free, albumin-treated nanoparticle-formed paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), Taxotere doxetaxel (Rhone-Poulenc Rorer, Antony, France), chlorambucil, Gemzar (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine, platinum, etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (cancer ptsar, CPT-11) (including irinotecan plus 5-FU and leucovorin therapy); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin therapy (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A, which reduce cell proliferation;and pharmaceutically acceptable salts, acids, or derivatives of any of the above-mentioned agents. Furthermore, the method of treatment may further include the use of radiation. Furthermore, the method of treatment may further include the use of photodynamic therapy.

[0272] In some embodiments, chimeric protein complexes, such as the chimeric proteins or Fc-based chimeric protein complexes described herein, include modified derivatives, i.e., derivatives modified by the covalent attachment of any type of molecule to the composition, provided that the covalent attachment does not interfere with the activity of the composition. For example, but not limited to, derivatives include compositions modified by glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, conjugation to cellular ligands or other proteins, among others. Any of a number of chemical modifications can be performed using known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

[0273] In still other embodiments, the chimeric protein complexes, such as the chimeric proteins or Fc-based chimeric protein complexes described herein, further comprise cytotoxic agents, which in exemplary embodiments include toxins, chemotherapeutic agents, radioisotopes, and agents that cause apoptosis or cell death. Such agents can be conjugated to the compositions described herein.

[0274] Chimeric proteins or chimeric protein complexes, such as the Fc-based chimeric protein complexes described herein, can be post-translationally modified in this manner to add effector moieties such as chemical linkers, detectable moieties such as fluorescent dyes, enzymes, substrates, bioluminescent, radioactive, and chemiluminescent moieties, or functional moieties such as streptavidin, avidin, biotin, cytotoxins, cytotoxic drugs, and radioactive moieties.

[0275] Examples of cytotoxic agents include methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, These include, but are not limited to, cis-dichlorodiamineplatinum(II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines (including daunorubicin (formerly daunomycin) and doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone, and bisantrene); antibiotics (including dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC)); antimytotic agents (e.g., vinca alkaloids, vincristine, and vinblastine). Other cytotoxic agents include paclitaxel (Taxol), ricin, Pseudomonas aeruginosa exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicine, dihydroxyanthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (O,P'-(DDD)), interferon, and mixtures of these cytotoxic agents.

[0276] Additional cytotoxic agents include chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platins, taxol, irinotecan, 5-fluorouracil, gemcytabine, leucovorin, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine). ), mustine, tyrosine kinase inhibitor, radiation therapy, sex hormone antagonist, selective androgen receptor modulator, selective estrogen receptor modulator, PDGF antagonist, TNF antagonist, IL1 antagonist, interleukin (e.g., IL12 or IL2), IL12R antagonist, toxin-conjugated monoclonal antibody, tumor antigen-specific monoclonal antibody, erbitux, avastin, pertuzumab, anti-CD20 antibody, rituxan, ocrelizumab, ofatumumab, DXL625, Herceptin®, or any combination thereof. Toxic enzymes from plants and bacteria, such as ricin, diphtheria toxin, and Pseudomonas toxin, can be conjugated with therapeutic agents (e.g., antibodies) to produce cell-type-specific killing agents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).

[0277] Other cytotoxic agents include cytotoxic ribonucleases, as described in U.S. Patent No. 6,653,104 to Goldenberg.

[0014] Embodiments of the present invention also relate to radioimmunoconjugates in which alpha- or beta-particle-emitting radionuclides are stably attached to chimeric proteins or chimeric protein conjugates, such as Fc-based chimeric protein conjugates, with or without the use of complexing agents. 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.

[0278] 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.

[0279] In some embodiments, including but not limited to autoimmune applications, the additional therapeutic agent is an immunosuppressant that is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a nonsteroidal anti-inflammatory drug (NSAID). Steroids, particularly corticosteroids, and their synthetic analogs are well known in the art. Examples of corticosteroids useful in the present invention include hydroxyltriamcinolone, α-methyldexamethasone, β-methyl β-methasone, beclomethasone dipropionate, β-methasone benzoate, β-methasone dipropionate, β-methasone valerate, clobetasol valerate, desonide, desoximetasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluchlorolone acetonide, flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortine butyl ester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortolone, hydroxypropyltriamcin ... These include, but are not limited to, flumethasone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolon acetonide, medrysone, amcinafide, amcinafide, betamethasone and its remaining esters, chloroprednisone, clocortelone, clesinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, and beclomethasone dipropionate. NSAIDS that may be used in the present invention include, but are not limited to, salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate, salicylamide, benzyl-2,5-diacetoxybenzoate, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin.In some embodiments, the immunosuppressant may be a cytostatic agent such as an alkylating agent, antimetabolite (e.g., azathioprine, methotrexate), cytotoxic antibiotic, antibody (basiliximab, daclizumab, and muromonab), anti-immunophilin (e.g., cyclosporine, tacrolimus, sirolimus), interferon, opioid, TNF-binding protein, mycophenolate, and small molecule biologic (e.g., fingolimod, myriocin). Additional anti-inflammatory agents are described, for example, in U.S. Pat. No. 4,537,776, the entire contents of which are incorporated herein by reference.

[0280] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is used in combination with one or more disease-modifying therapeutics (DMTs) described herein (e.g., agents in Table A) in methods for treating multiple sclerosis. In some embodiments, the present invention provides improved therapeutic effects compared to the use of one or more DMTs described herein (e.g., agents listed in Table A below) without one or more disclosed binding agents. In certain embodiments, the combination of a chimeric protein complex, such as a chimeric protein or an Fc-based chimeric protein complex, with one or more DMTs results in a synergistic therapeutic effect. Examples of disease-modifying therapeutic agents include, but are not limited to: [Table 6] TIFF2025169347000016.tif240161TIFF2025169347000017.tif238162TIFF2025169347000018.tif223162

[0281] The present invention also provides kits for the administration of any of the agents described herein (e.g., chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, with or without various additional therapeutic agents). A kit is a collection of materials or components, including at least one pharmaceutical composition of the invention described herein. Thus, in some embodiments, the kit comprises at least one pharmaceutical composition described herein.

[0282] The precise nature of the components comprising the kit will depend on its intended purpose, hi one embodiment, the kit is configured for the purpose of treating a human subject.

[0283] Instructions for use may be included in the kit. The instructions typically include clear language describing techniques to be employed in using the components of the kit to achieve a desired result, such as treating cancer. Optionally, the kit also includes other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings, or other useful accessories as would be readily apparent to one of ordinary skill in the art.

[0284] The materials and components incorporated into the kit can be stored and provided to the practitioner in any convenient and appropriate manner that maintains their operability and usefulness. For example, the components can be provided at room temperature, refrigerated, or frozen. The components are typically contained in suitable packaging. In various embodiments, the packaging is constructed in a well-known manner, preferably one that provides a sterile, contaminant-free environment. The packaging may have an exterior label indicating the contents and / or the purpose of the kit and / or its components.

[0285] definition As used herein, "a," "an," or "the" may mean one (one) or more than one (one).

[0286] Unless otherwise stated or clear from context, as used herein, the term "or" includes and covers both "or" and "and."

[0287] Additionally, the term "about" when used in connection with a reference numerical designation means the reference numerical designation plus or minus up to 10% of the reference numerical designation, e.g., a value within (±) 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the indicated value. For example, the term "about 50" covers a range of 45 to 55.

[0288] The term "effective amount," when used in relation to medical use, is an amount that is effective to produce a measurable treatment, prevention, or reduction in the rate of onset of the disease of interest.

[0289] As used herein, something is "reduced" when, in the presence of a substance or stimulus, the activity and / or effect output value is reduced by a significant amount, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to at least about 100% (including about 100%), compared to the absence of such modulation. As will be understood by one of skill in the art, in some embodiments, activity is decreased and some downstream output values ​​are decreased, while others may increase.

[0290] Conversely, activity is "higher" if the activity and / or effect output value increases by a significant amount, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to at least about 100% (including about 100%) or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, or at least about 100-fold, in the presence of a substance or stimulus compared to the absence of such substance or stimulus.

[0291] When referenced herein, percentages for all compositions are by weight of the total composition unless otherwise specified. As used herein, the word "include" and variations thereof are intended to be non-limiting, such that the description of items in a list is not intended to exclude other similar items that may also be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and variations thereof are intended to be non-limiting, such that a description that an embodiment "can" or "may" include certain elements or features does not exclude other embodiments of the present technology that do not include these elements or features.

[0292] Although the invention is described and claimed herein using the open-ended term "comprising" as a synonym for terms such as including, containing, or having, the invention, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

[0293] As used herein, the terms "preferred" and "preferably" refer to embodiments of the present technology that offer certain advantages, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present technology.

[0294] The amount of the compositions described herein required to achieve a therapeutic effect may be empirically determined according to conventional procedures for a particular purpose. Generally, when a therapeutic agent is administered for therapeutic purposes, the therapeutic agent is administered in a pharmacologically effective amount. A "pharmacologically effective amount," "pharmacologically effective dose," "therapeutically effective amount," or "effective amount" refers to an amount sufficient to produce a desired physiological effect or to achieve a desired result, particularly for treating a disorder or disease. As used herein, an effective amount may include, for example, an amount sufficient to slow the progression of symptoms of a disorder or disease, alter the course of symptoms of a disorder or disease (e.g., slow the progression of symptoms of a disease), reduce or eliminate one or more symptoms or onset of a disorder or disease, and ameliorate symptoms of a disorder or disease. A therapeutic effect also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is achieved.

[0295] Effective doses, toxicity, and therapeutic effects can be determined by standard pharmaceutical procedures, for example, in cell cultures or experimental animals, to determine the LD50 (the dose lethal to approximately 50% of the population) and ED50 (the dose therapeutically effective in approximately 50% of the population). Dosages can vary depending on the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. In some embodiments, compositions and methods exhibiting large therapeutic indices are preferred. Therapeutically effective doses can be initially estimated from in vitro assays, including, for example, cell culture assays. Alternatively, doses can be formulated in animal models to achieve a circulating plasma concentration range, such as the IC50, determined in cell culture or an appropriate animal model. Plasma levels of the described compositions can be measured, for example, by high-performance liquid chromatography. The effects of any particular dosage can be monitored by an appropriate bioassay. Dosages can be determined by a physician and, if necessary, adjusted to accommodate observed therapeutic effects.

[0296] In certain embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. A therapeutic effect also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether an improvement is realized.

[0297] As used herein, "method of treatment" is equally applicable to compositions for the treatment of a disease or disorder described herein and / or compositions for use in the manufacture of a medicament and / or multiple uses for the treatment of a disease or disorder described herein. The present invention is further illustrated by the following non-limiting examples.

[0298] Example "AFN" is used where appropriate (as indicated herein) to refer to the interferon-based chimeric proteins or chimeric protein complexes described herein. Example 1: FLT3L Fc AFN In this example, we designed and evaluated an FMS-like tyrosine kinase 3 ligand (FLT3L)-based AFN for targeting FLT3L-positive cells. construct [ka]

[0299] Production and purification of FLT3L-based AFN Construct FLT3L-20 * GGS-Fc3 and Fc4-20 * GGS-IFNa2_R149A was conjugated to the AFN variant with the structure outlined in Figure 7B and transiently expressed in the ExpiCHO expression system (Thermo Fisher Scientific) according to the manufacturer's instructions. One week after transfection, the supernatant was collected and the cells were removed by centrifugation. The recombinant protein was purified from the supernatant using a Pierce Protein A spin plate (Thermo Fisher Scientific).

[0300] Biological activity: STAT1 phosphorylation in transiently transfected Hek293T cells Hek293T cells were transiently transfected with a FLT3 expression plasmid or an empty vector (MOCK). Two days after transfection, cells were stimulated with serial dilutions (as indicated) of wild-type IFNa2 or FLT3L-Fc-AFN for 15 minutes at 37°C. After fixation (10 minutes at 37°C in Fix Buffer I; BD Biosciences), permeabilization (30 minutes on ice in Perm III Buffer I; BD Biosciences), and washing, cells were stained with anti-STAT1 pY701 Ab (BD Biosciences). Samples were acquired on a MACSQuant X instrument (Miltenyi Biotec) and analyzed using FlowLogic software (Miltenyi Biotec). The data in Figure 20 clearly demonstrate that FLT3L Fc AFN can induce STAT1 phosphorylation only in FLT3 but not in MOCK-transfected cells, demonstrating the feasibility of targeting with this ligand. Of note, FLT3 or MOCK transfected cells are equally sensitive to wild-type IFNa2.

[0301] Example 2: Split FLT3L Fc AFN variant In this example, we evaluated the potential of FLT3L-targeted Fc-format IFN-based AFN to selectively activate FLT3-positive cells using constructs in which one copy of FLT3L is displayed on each Fc chain. The potential of split-FLT3L Fc constructs in such a "knobs-in-holes" Fc context is shown in Figures 21A-F and 22A-F. For the exemplified heterodimer (see structure Figure 21A), FLT3L sequences were cloned N-terminal to both Fc arms, resulting in the so-called "split" configuration. The first FLT3L sequence was fused to a human IgG1 Fc sequence containing the L234A_L235A_K322Q effector mutation and the "hole" modification Y349C_T366S_L368A_Y407V in the pcDNA3.4 expression vector. The second AFN partner, also cloned in the pcDNA3.4 vector, consists of a fusion between a human IgG1 Fc sequence containing the FLT3L sequence, L234A_L235A_K322Q effector mutations and the "knob" modification S354C_T366W, and hIFNa2 with the AFN mutation R149A and deletion of the O-glycosylation site by mutation of T106 to E. Three variants were tested: in the first variant, the sequence of FLT3L amino acids (aa) 27 to 184 is substituted for the 5 * In the second variant, the same FLT3L sequence was fused directly to the Fc arm via a GGS linker. In the third variant, FLT3L amino acids 27-160 and 5 * A combination of GGS linkers was used. In each variant, residue L27 in the FLT3-L dimerization interface was optionally further mutated to D. This resulted in 12 different sequences (see below).

[0302] To produce these "knobs-in-hole" AFNs, a combination of both the "hole" and "knobs" plasmids was transfected into ExpiCHO cells (ThermoFisher) according to the manufacturer's instructions. Seven days after transfection, the recombinant proteins were purified using Protein A spin plates (ThermoFisher), quantified, and tested for purity using SDS-PAGE. SDS-PAGE under non-reducing conditions showed a more homogenous profile for constructs containing the L27D mutation.

[0303] The biological activity of the resulting proteins was measured in parental HL116 cells (an IFN-responsive cell line stably transfected with the p6-16 luciferase reporter) and the resulting stably transfected HL116-FLT3 cells. Cells were seeded overnight and stimulated with serial dilutions of FLT3L AFN for 6 hours. Luciferase activity was measured using an EnSight Multimode Plate Reader (Perkin Elmer). The data in Figures 23A–G clearly demonstrate that (i) both parental HL116 and HL116-FLT3 cells are equally responsive to wild-type IFNa2, (ii) all FLT3L AFNs are significantly more active in targeted (FLT3-expressing) cells than in non-targeted cells, (iii) there are no clear differences in IFN-like signaling between FLT3L variants, and (iv) the L27D mutation has no clear effect on signaling in either targeted or non-targeted cells. array: [ka] TIFF2025169347000021.tif226161TIFF2025169347000022.tif226161

[0304] Example 3: Comparison of split and single chain formats for FLT3L Fc AFN variants In this example, we evaluated the potential of FLT3L-targeted Fc-format IFN-based AFNs to selectively activate FLT3-positive cells using a construct in which two copies of FLT3L are displayed on one single Fc chain. The potential of single-chain FLT3L Fc constructs in such a "knobs-in-holes" Fc context is shown in Figures 24A-H and 25A-L. For the exemplified heterodimer (structure see Figure 24A), the single-chain FLT3L sequence (from Lu et al. 2002 Acta Biochimica Et Biophysica Sinica 2002, 34(6):697-702) allows for flexible 10-fold activation. * It was fused via a GGS linker and in the pcDNA3.4 expression vector to a human IgG1 Fc sequence containing the L234A_L235A_K322Q effector mutation and the "hole" modification Y349C_T366S_L368A_Y407V. The second AFN partner, also cloned in the pcDNA3.4 vector, consisted of a fusion between a human IgG1 Fc sequence containing the L234A_L235A_K322Q effector mutation and the "knob" modification S354C_T366W and hIFNa2 with the AFN mutation R149A and deletion of the O-glycosylation site by mutation of T106 to E (see sequence below).

[0305] Single-chain FLT3L AFN was produced, purified, and bioactivity was measured as described in Example 2. The data in Figures 26A-B clearly demonstrate that single-chain FLT3L-targeted AFN is active only against FLT3-expressing cells. Even at the highest concentration, no luciferase induction was observed. Notably, both cell lines were equally sensitive to wild-type IFN. array: [ka]

[0306] Example 4: Manufacturability of split and single-chain FLT3L AFN In this example, we compared the manufacturability of FLT3L AFN in split or single-chain Fc format. For the split format, we used FLT3L aa27-184 sequence. * The variants fused to the Fc arm via a GGS linker (P-2168 + P-2169) and its L27D mutant (P-2174 + P-2175) were selected. As a single-chain FLT3L AFN, (P-2180 + P-1414; see Example 3 for the sequence) was selected. FLT3L AFN was produced in ExpiCHO cells (ThermoFisher) according to the manufacturer's guidelines. Seven days after transfection, the supernatant was collected and the cells were removed by centrifugation. The protein was purified on a 5 ml Protein A column (GE Healthcare) using an AKTA pure instrument (GE Healthcare). The purified protein was further analyzed by size exclusion chromatography on a Superdex 200 Increase 10 / 300 column (GE Healthcare) using the AKTA. The SEC profiles in Figures 27A-C and subsequent SDS-PAGE analysis of each peak show that (i) the peak eluting at the 158 kD marker is primarily the dimeric P-1414 form (observed only for the p-2180 + P-1414 combination); (ii) the elution peak to the left of the 158 kD marker is identified as the desired product, found in highest abundance for P-2180 + P-1414 (78%, compared to 57% for P-2168 + P-2169 and 25% for P-2174 + P-2175); and (iii) in the split-strand product with the L27D mutation (P-2174 + P-2175), a predominant single high molecular weight species is observed, while the other split-strand construct has a high propensity to form multiple higher molecular weight species (P-2168 + P-2169). HL116 reporter assays in parental HL116 versus HL116-FLT3 cells confirmed that for P-2180+P-1414, the peak to the left of the 158 kD marker was indeed the correct substance, as it was approximately 25-fold more active in HL116-FLT3 cells compared to the peak at 158 ​​kD.

[0307] Example 5: Single-chain FLT3L Fc AFN variants In this example, the expression and manufacturability of all constructs listed in the "Sequence" section below were tested. These single-chain FLT3L Fc AFN variants differ fundamentally in the length of the FLT3L sequence used (aa 27-182, aa 27-177, and aa 27-160) and the presence or absence of the L27D mutation in the FLT3L dimerization interface. Two such FLT3L sequences are shown in Table 1. * The resulting FLT3L sequence is a flexible 10 * The AFN partner was fused to a human IgG1 Fc sequence containing the L234A_L235A_K322Q effector mutation and the "hole" modification Y349C_T366S_L368A_Y407V via a GGS linker and in the pcDNA3.4 expression vector. The second AFN partner, also cloned in the pcDNA3.4 vector, consisted of a fusion between a human IgG1 Fc sequence containing the L234A_L235A_K322Q effector mutation and the "knob" modification S354C_T366W and hIFNa2 with the AFN mutation R149A and a deletion of the O-glycosylation site by mutation of T106 to E. The construct was expressed in ExpiCHO cells (ThermoFisher) according to the manufacturer's guidelines. Seven days after transfection, recombinant proteins were purified using Protein A spin plates (ThermoFisher), quantified, and purity assessed using SDS-PAGE.

[0308] The biological activity of the resulting proteins was measured in two independent experiments in parental HL116 and HL116-FLT3 cells as described in Example 2. The data in Table 6 show that (i) all tested FLT3L AFN variants induce IFNAR-dependent signaling in HL116-FLT3 cells, (ii) such signaling was not detectable in the parental HL116 cell line, even at the highest concentrations tested, thus all these variants have an extremely high selectivity window that is completely lacking in wild-type IFNa2, (iii) FLT3L variants aa27-182 and aa27-177 have comparable activity, while even aa27-160-based FLT3L is still significantly more potent, and (iv) the L27D mutation tends to somewhat reduce signaling ability in all three length variants, paving the way for optimizing manufacturability. [Table 7] array: [ka] TIFF2025169347000026.tif232162

[0309] Example 6: Single-chain FLT3L AFN variants without an Fc domain In this example, we evaluate the expression, manufacturability, and biological activity of different single-chain FLT3L AFNs that do not contain an Fc domain and compare them with a single-copy FLT3L AFN (P-2373). The different variants differ primarily in the length of the FLT3L sequence used (aa 27-182, aa 27-177, and aa 27-160) and the presence or absence of the L27D mutation in the FLT3L dimerization interface. The two FLT3L sequences are 3 * The resulting FLT3L single chain was fused with a GGGGS linker, and the resulting FLT3L single chain was flexible 10 *It is fused via a GGS linker to an AFN partner consisting of hIFNa2 with the AFN mutation R149A and a deletion of an O-glycosylation site by mutating T106 to E. The construct is cloned into pcDNA3.4 with a C-terminal histidine tag, expressed in ExpiCHO, and purified by metal affinity. array: [ka] TIFF2025169347000028.tif209162

[0310] The construct of SEQ ID NO: 73 was expressed and purified. Figure 30 shows the size-exclusion chromatography (SEC) profile of the purified construct of SEQ ID NO: 73, a monomeric form of Flt3L linked to interferon via a flexible linker. The purified FLT3L-AFN is shown as a dark line, and the protein marker is shown as a gray line. The SEC profile of the peak fraction shows that the protein behaves as an approximately 150 kD protein on SEC (i.e., elutes at the 158 kD marker). These data support the formation of a non-covalently linked dimer. Such a dimer is not observed with VHH-based AFN and is therefore the result of FLT3L dimerization, which is required for FLT3L receptor binding and signal transduction. Compare with the size-exclusion chromatography (SEC) profiles in Figures 27A-C.

[0311] Example 7: Use of FLT3-targeted AFN for therapy To evaluate the antitumor activity of FLT3-targeted AFN, the following two single-chain Flt3L chimeric protein constructs were expressed in ExpiCHO and purified by Protein A followed by size-exclusion chromatography. scFLT3-Fc-AFN: P-2180+P-1414 scFLT3-Fc: P-2180+P-2038 (P-2038 encodes the Fc4 protein without the AFN moiety).

[0312] The purified protein was then evaluated in a humanized mouse tumor model. Briefly, neonatal NSG mice (1-2 days old) were sublethally irradiated with 100 cGy and then immunized with 1x10 5 CD34+ human stem cells (HLA-A2 positive umbilical cord blood derived) were delivered intrahepatically. Thirteen weeks after stem cell transfer, mice received 25x10 5 Human RL follicular lymphoma cells (ATCC CRL-2261; not susceptible to the direct antiproliferative effects of IFN) were inoculated subcutaneously. Intravenous treatment with equimolar doses of scFlt3-Fc fusion (19.8 μg) and scFlt3L-Fc-AFN (25 μg) or buffer was performed on days 12 and 20 after tumor inoculation (n = 3–5 mice / group). Tumor size (caliper measurements) and body weight were assessed every 2 or 3 days.

[0313] The data in Figure 28A show tumor growth up to 6 days after the second treatment, demonstrating that the scFlt3L-Fc fusion is functional, as tumor growth is reduced compared to buffer treatment. The data in Figure 28B demonstrate the efficacy of the scFlt3L-Fc-AFN protein in even more potently suppressing tumor growth. The weight changes in Figure 29 show that both groups of mice treated with the Flt3L construct gained weight equally, in contrast to the buffer-treated animals, which lost weight.

[0314] Example 8: Generation, production, purification and characterization of further Flt3L / IFNα1 AFN In this example, we evaluated the activity of IFNα1 fused to FLT3L. The purified protein was evaluated in a humanized mouse tumor model. Briefly, neonatal NSG mice (1-2 days old) were sublethally irradiated with 100 cGy and then immunized with 1x10 5 CD34+ human stem cells (HLA-A2 positive umbilical cord blood derived) were delivered intrahepatically. Thirteen weeks after stem cell transfer, mice received 25x10 5Human RL follicular lymphoma cells (ATCC CRL-2261; insensitive to the direct antiproliferative effects of IFN) were inoculated subcutaneously. Mice were treated intraperitoneally with 30 μg of human Flt3L protein daily from days 8 to 18 after tumor inoculation. Daily intravenous injections of buffer or Flt3L-IFNα1 (30 μg) were initiated on day 10 after tumor inoculation, when palpable tumors were observed (n = 5 or 6 mice per group). Tumor size (caliper measurements), body weight, and body temperature were assessed daily.

[0315] The data in Figure 31 show tumor growth up to 2 days after the last treatment, demonstrating that targeted Flt3L / IFNα1 potently suppresses tumor growth. Body weight and temperature data showed no significant differences between buffer and IFNα1 treatments, confirming that the treatments were well tolerated.

Claims

1. (i) one or more targeting moieties that specifically bind to an antigen or receptor of interest, wherein the antigen or receptor of interest is FMS-like tyrosine kinase 3 (FLT3); (ii) an Fc domain, optionally with one or more mutations that reduce or eliminate one or more effector functions of said Fc domain, promote Fc chain pairing of said Fc domain, and / or stabilize the hinge region in said Fc domain; and (iii) a signal transducer or modified form thereof; 1. An Fc-based chimeric protein complex comprising:

2. The Fc-based chimeric protein complex of claim 1, wherein one targeting moiety is attached to each Fc chain of the Fc domain.

3. The Fc-based chimeric protein complex of claim 1, wherein two targeting moieties are attached to one Fc chain of the Fc domain.

4. The Fc-based chimeric protein complex of claim 3, wherein the two targeting moieties are attached to each other, optionally via a linker.

5. The Fc-based chimeric protein complex of claim 4, wherein the two targeting moieties are attached to the Fc chain, optionally via a linker.

6. The Fc-based chimeric protein complex of any one of claims 1 to 5, wherein the targeting moiety comprises FLT3L, or a portion thereof.

7. The Fc-based chimeric protein complex of any one of claims 1 to 6, wherein the targeting moiety comprises the extracellular domain of FLT3L, or a portion thereof.

8. The Fc-based chimeric protein complex of claim 7, wherein the targeting moiety comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 to 5.

9. The Fc-based chimeric protein complex of claim 8, wherein the targeting moiety comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2 to 5.

10. 10. The Fc-based chimeric protein complex of any one of claims 1 to 9, wherein the targeting moiety comprises the extracellular domain of FLT3L, or a portion thereof, and optionally a mutation that reduces intermolecular extracellular domain dimerization, which is a substitution at position L27 of any one of SEQ ID NOs: 2 to 4, optionally L27D.

11. The Fc-based chimeric protein complex of any one of claims 1 to 9, wherein the targeting moiety comprises the extracellular domain of FLT3L, or a portion thereof, and the extracellular domain is a single-chain dimer.

12. The Fc-based chimeric protein complex according to any one of claims 1 to 11, wherein the signal transduction substance is wild-type human IFNα2, IFNα1, IFNβ, or IL-1β.

13. The Fc-based chimeric protein complex of any one of claims 1 to 12, wherein the signal transduction agent comprises an amino acid sequence having at least 95%, or at least 97%, or at least 98% identity to any one of SEQ ID NOs: 6, 7, 38, 39, or 74.

14. The Fc-based chimeric protein complex according to any one of claims 1 to 13, wherein the signal transduction agent is modified to contain one or more mutations.

15. The Fc-based chimeric protein complex of claim 14, wherein the one or more mutations confer improved safety compared to the wild-type signal transduction agent.

16. The Fc-based chimeric protein complex of claim 14, wherein the one or more mutations confer a reduced affinity of the signal transduction substance to a receptor.

17. The Fc-based chimeric protein complex of claim 14, wherein the one or more mutations confer reduced biological activity to a signal transduction substance receptor.

18. The Fc-based chimeric protein complex according to any one of claims 14 to 17, wherein the one or more mutations allow for attenuation of the activity of the signal transduction substance.

19. The Fc-based chimeric protein complex of claim 18, wherein the agonist or antagonist activity of the signal transduction substance is attenuated.

20. The Fc-based chimeric protein complex according to any one of claims 15 to 19, wherein the modified signal transduction agent comprises one or more mutations that convert its activity from agonist activity to antagonist activity.

21. The Fc-based chimeric protein complex of any one of claims 15 to 20, wherein the one or more mutations confer reduced affinity or activity that is recoverable by attachment to one or more targeting moieties or upon inclusion in an Fc-based chimeric protein complex.

22. 22. The Fc-based chimeric protein complex of any one of claims 15 to 21, wherein the one or more mutations confer substantially reduced or eliminated affinity or activity that is not substantially recoverable upon attachment to a targeting moiety or inclusion in an Fc-based chimeric protein complex.

23. The Fc-based chimeric protein complex according to any one of claims 1 to 22, wherein the signal transduction substance is a mutant human IFNα2 comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the mutant human IFNα2 has one or more mutations that confer improved safety compared to wild-type IFNα2 having the amino acid sequence of SEQ ID NO: 6 or 7.

24. The Fc-based chimeric protein complex of claim 23, wherein the human IFNα2 has one or more mutations at positions 144-154 relative to SEQ ID NO: 6 or 7.

25. The Fc-based chimeric protein complex of claim 23, wherein the human IFNα2 has one or more mutations at positions L15, A19, R22, R23, L26, F27, L30, K31, D32, R33, H34, D35, Q40, H57, E58, Q61, F64, N65, T69, L80, Y85, Y89, D114, L117, R120, R125, K133, K134, R144, A145, M148, R149, S152, L153, ​​and N156 relative to SEQ ID NO: 6 or 7.

26. 26. The Fc-based chimeric protein complex of claim 25, wherein the mutations are one or more of L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57Y, E58N, Q61S, F64A, N65A, T69A, L80A, Y85A, Y89A, D114R, L117A, R120A, R125A, K133A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, relative to SEQ ID NO: 6 or 7.

27. The mutant human IFNα2 has one or more mutations at positions R33, R144, A145, M148, R149, and L153 relative to the amino acid sequence of SEQ ID NO: 6 or 7, or the mutant human IFNα2 has one or more mutations at positions R33A, R120E, R144X, A145, M148, R149, and L153 relative to the amino acid sequence of SEQ ID NO: 6 or 7. １ , A145X ２ , M148A, R149A, and L153A; and １ is selected from A, S, T, Y, L, and I, and X ２ The Fc-based chimeric protein complex of claim 23, wherein is selected from G, H, Y, K, and D.

28. The Fc-based chimeric protein complex according to any one of claims 13 to 27, wherein the human IFNα2 has a mutation selected from T106A or T106E.

29. wherein said signal transducer is wild-type or variant human IFNα1 comprising an amino acid sequence having at least 90%, or at least 93%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or 100% identity to SEQ ID NO: 74, and optionally said IFNα1 is: (i) one or more amino acids that confer reduced affinity for the interferon alpha / beta receptor (IFNAR), optionally selected from substitutions at positions L15, A19, R23, S25, L30, D32, R33, H34, Q40, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157, or a combination thereof; is a plurality of mutations, the positions of which are, based on SEQ ID NO: 74, L15A, A19W, R23A, S25A, L30A, L30V, D32A, R33K, R33A, R33Q, H34A, Q40A, D115R, L118A, K121A, K121E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E, R145G, R145H, R145I, R145K ... 5L, R145N, R145Q, R145S, R145T, R145V, R145Y, A146D, A146E, A146G, A146H, A146I, A146K, A146L, A146M, A146N, A146Q, A146R, A146S, A146T, A146V, A146Y, M149A, M149V, R150A, S153A, L154A, N157A, L30A-H58Y-E59N-Q62S, R one or more mutations optionally selected from the group consisting of 33A-H58Y-E59N-Q62S, M149A-H58Y-E59N-Q62S, L154A-H58Y-E59N-Q62S, R145A-H58Y-E59N-Q62S, D115A-R121A, L118A-R121A, L118A-R121A-K122A, R121A-K122A, and R121E-K122E; and / or (ii) one or more mutations that affect aggregation, optionally selected from one or more substitutions or deletions at positions C1, C29, C86, C99, C139 relative to SEQ ID NO: 74, and optionally selected from C86S, C86A, and C86Y; The Fc-based chimeric protein complex according to any one of claims 1 to 22, comprising:

30. 23. The Fc-based chimeric protein complex of any one of claims 1 to 22, wherein the signal transduction agent is a mutant human IFNβ comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 38, and wherein the mutant human IFNβ has one or more mutations that confer improved safety compared to wild-type IFNβ having the amino acid sequence of SEQ ID NO:

38.

31. The Fc-based chimeric protein complex of claim 30, wherein the mutations are one or more of W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, and R152G based on the amino acid sequence of SEQ ID NO:

38.

32. 23. The Fc-based chimeric protein complex according to any one of claims 1 to 22, wherein the signal transduction agent is a mutant human IL-1β comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 39, and the mutant human IL-1β has one or more mutations that confer improved safety compared to wild-type IL-1β having the amino acid sequence of SEQ ID NO:

39.

33. The mutations are A117G / P118G, R120G, R120A, L122A, T125G / L126G, R127G, Q130A, Q130W, Q131G, K132A, S137G / Q138Y, L145G, H146A, H146G, H146E, H146N, H146R, L145A / L147A, Q148E, Q148G, Q148L, Q148G / Q150G, Q150G / D151G, and Q151G / D151G, based on the amino acid sequence of SEQ ID NO:

39.

33. The Fc-based chimeric protein complex of claim 32, wherein the amino acid sequence is one or more of: A, M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209A, K209D, K209A / K210A, K219S, K219Q, E221S, E221K, E221S / N224A, N224S / K225S, E244K, and N245Q.

34. The Fc-based chimeric protein complex of any one of claims 1 to 33, wherein the targeting moiety is directed against an immune cell, optionally a dendritic cell.

35. 35. The Fc-based chimeric protein complex of claim 34, wherein the dendritic cells are conventional dendritic cells (cDCs), optionally cDC-1, migratory DC, CDC-2, and Flt3+ DC.

36. 36. The Fc-based chimeric protein complex of claim 35, wherein the targeting moiety is directed to hematopoietic stem cells (HSCs), early progenitor cells, immature thymocytes, or steady-state dendritic cells (DCs).

37. 37. The Fc-based chimeric protein complex of any one of claims 1 to 36, wherein the targeting moiety functionally modulates the antigen or receptor of interest.

38. 38. The Fc-based chimeric protein complex of any one of claims 1 to 37, wherein the targeting moiety binds but does not functionally modulate the antigen or receptor of interest.

39. The Fc-based chimeric protein complex of any one of claims 1 to 38, wherein the targeting moiety directly or indirectly recruits immune cells to tumor cells or to the tumor microenvironment.

40. The Fc-based chimeric protein complex of any one of claims 1 to 39, wherein the targeting moiety increases the number of dendritic cells.

41. 41. The Fc-based chimeric protein complex of any one of claims 1 to 40, wherein the targeting moiety optionally enhances tumor antigen presentation by dendritic cells.

42. 42. The Fc-based chimeric protein complex of any one of claims 1 to 41, further comprising an additional targeting moiety, which may be identical or non-identical, optionally two targeting moieties.

43. 43. The Fc-based chimeric protein complex of any one of claims 1 to 42, comprising an additional signal transduction agent.

44. 44. The Fc-based chimeric protein complex according to any one of claims 1 to 43, comprising two signal transduction substances.

45. The Fc-based chimeric protein complex according to any one of claims 1 to 44, wherein the Fc-based chimeric protein complex is suitable for use in patients with one or more of cancer, infectious diseases, immune disorders, autoimmune and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases.

46. The Fc-based chimeric protein complex of any one of claims 1 to 45, wherein the Fc domain is derived from IgG, IgA, IgD, IgM, or IgE.

47. The Fc-based chimeric protein complex of claim 46, wherein the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

48. 46. ​​The Fc-based chimeric protein complex of any one of claims 1 to 45, wherein the Fc domain is derived from human IgG, IgA, IgD, IgM, or IgE.

49. The Fc-based chimeric protein complex of claim 48, wherein the human IgG is selected from human IgG1, IgG2, IgG3, or IgG4.

50. 50. The Fc-based chimeric protein complex of any one of claims 1 to 49, wherein the Fc chain pairing is facilitated by ionic pairing and / or knob-in-hole pairing.

51. 51. The Fc-based chimeric protein complex of any one of claims 1 to 50, wherein the one or more mutations to the Fc domain result in ionic pairing between Fc chains in the Fc domain.

52. 52. The Fc-based chimeric protein complex of any one of claims 1 to 51, wherein the one or more mutations to the Fc domain result in knob-in-hole pairing in the Fc domain.

53. 53. The Fc-based chimeric protein complex of any one of claims 1 to 52, wherein the one or more mutations to the Fc domain result in a reduction or elimination of an effector function of the Fc domain.

54. The Fc-based chimeric protein complex according to any one of claims 1 to 53, wherein the Fc-based chimeric protein complex is a heterodimer and has a trans orientation.

55. The Fc-based chimeric protein complex according to any one of claims 1 to 53, wherein the Fc-based chimeric protein complex is a heterodimer and has a cis orientation.

56. 56. The Fc-based chimeric protein complex according to any one of claims 1 to 55, wherein the Fc comprises L234A, L235A, and K322Q substitutions (according to EU numbering) in human IgG1.

57. The Fc-based chimeric protein complex according to any one of claims 1 to 52, wherein the Fc is human IgG1 and optionally comprises one or more of L234, L235, K322, D265, P329 and P331 (according to EU numbering).

58. 58. The Fc-based chimeric protein complex of any one of claims 1 to 57, wherein the Fc-based chimeric protein complex has an orientation / structure according to any one of Figures 1A-F, 2A-H, 3A-H, 4A-D, 5A-F, 6A-J, 7A-D, 8A-F, 9A-J, 10A-F, 11A-L, 12A-L, 13A-F, 14A-L, 15A-L, 16A-J, 17A-J, 18A-F, 19A-F, 21A-F, 22A-F, 24A-H, and 25A-L.

59. The Fc-based chimeric protein complex of any one of claims 1 to 58, wherein the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 40, 41, and 46 to 66.

60. The Fc-based chimeric protein complex according to any one of claims 1 to 58, wherein the Fc-based chimeric protein complex comprises an amino acid sequence selected from SEQ ID NOs: 40, 41, and 46 to 66 and a polypeptide having fewer than 10 mutations relative to the amino acid sequence.

61. The Fc-based chimeric protein complex of claim 60, wherein the Fc-based chimeric protein complex comprises an amino acid sequence selected from SEQ ID NOs: 40, 41, and 46-66 and a polypeptide having fewer than five mutations relative to the amino acid sequence.

62. The Fc-based chimeric protein complex of claim 60, wherein the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 40, 41, and 46-66.

63. The Fc-based chimeric protein complex of claim 59, wherein the Fc-based chimeric protein complex comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 40 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

41.

64. The Fc-based chimeric protein complex of claim 59, wherein the Fc-based chimeric protein complex comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 46 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

47.

65. The Fc-based chimeric protein complex of claim 59, wherein the Fc-based chimeric protein complex comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 48 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

49.

66. The Fc-based chimeric protein complex of claim 59, wherein the Fc-based chimeric protein complex comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 50 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

51.

67. The Fc-based chimeric protein complex of claim 59, wherein the Fc-based chimeric protein complex comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 52 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

53.

68. The Fc-based chimeric protein complex of claim 59, wherein the Fc-based chimeric protein complex comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 54 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

55.

69. The Fc-based chimeric protein complex of claim 59, wherein the Fc-based chimeric protein complex comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 56 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

57.

70. The Fc-based chimeric protein complex of claim 59, wherein the Fc-based chimeric protein complex comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 58 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

59.

71. 60. The Fc-based chimeric protein complex of claim 59, wherein the Fc-based chimeric protein complex comprises a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to any one of the sequences selected from SEQ ID NOs: 60-65, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

66.

72. The FLT3L domain is a single chain dimer of the formula ABC, wherein: A is an amino acid sequence having at least 90% identity, or at least 95% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 2 to 5; B is a flexible linker consisting essentially of glycine and serine residues, optionally the flexible linker is ４ Ser) ｎ wherein n is from about 1 to about 8, and optionally the flexible linker comprises one or more of SEQ ID NOs: 10 to 17; and C is an amino acid sequence having at least 90% identity, or having at least 95% identity, or having at least 97% identity, or having at least 98% identity, or having at least 99% identity to any one of SEQ ID NOs: 2 to 5; An Fc-based chimeric protein complex according to any one of claims 1 to 71.

73. 73. The Fc-based chimeric protein complex of claim 72, wherein the FLT3L domain comprises an L27D mutation.

74. A method for treating or preventing cancer, comprising administering to a patient in need thereof an effective amount of an Fc-based chimeric protein complex of any one of claims 1 to 73.

75. Preface: Basal cell carcinoma, biliary tract cancer; bladder cancer; Bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; 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 lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, tonsil, mouth, 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; cancer of the urinary system; vulvar cancer; Hodgkin's lymphoma and non-Hodgkin's lymphoma; and lymphomas, including B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell 75. The method of claim 74, wherein the lymphoma is selected from one or more of: chronic NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD); and abnormal blood vessel proliferation associated with phacomatosis; edema (e.g., associated with brain tumors); and Meigs' syndrome.

76. 76. The method of claim 75, wherein the cancer is acute myeloid leukemia (AML).

77. A method for treating or preventing an autoimmune disease and / or a neurodegenerative disease, comprising administering to a patient in need thereof an effective amount of an Fc-based chimeric protein complex described in any one of claims 1 to 73.

78. 78. The method of claim 77, wherein the autoimmune and / or neurodegenerative disease is selected from multiple sclerosis, diabetes, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barré syndrome, scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, primary sclerosing cholangitis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Menier's syndrome, transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, myasthenia gravis, Reiter's syndrome, and Graves' disease.

79. 74. A recombinant nucleic acid composition encoding one or more Fc-based chimeric protein complexes or polypeptide components thereof according to any one of claims 1 to 73.

80. 80. A host cell comprising the nucleic acid of claim 79.

81. (i) one or more targeting moieties comprising an FMS-like tyrosine kinase 3 ligand (FLT3L) domain, wherein FLT3L is a single-chain dimer; (ii) one or more flexible linkers connecting elements (i) and (iii); and (iii) a signal transducer or modified form thereof; A chimeric protein comprising:

82. 82. The chimeric protein of claim 81, wherein the targeting moiety comprises the extracellular domain of FLT3L, or a portion thereof.

83. The chimeric protein of claim 81 or 82, wherein the targeting moiety comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2-5.

84. The chimeric protein of claim 83, wherein the targeting moiety comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2-5.

85. The chimeric protein of any one of claims 81 to 84, wherein the signal transduction substance is wild-type human IFNα2, IFNα1, IFNβ, or IL-1β.

86. 86. The chimeric protein of claim 85, wherein the signal transduction agent comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 6, 7, 38, 39 or 74.

87. 87. The chimeric protein of claim 86, wherein the signal transduction substance comprises the amino acid sequence of any one of SEQ ID NOs: 6, 7, 38, 39 or 74.

88. 88. The chimeric protein of any one of claims 81 to 87, wherein the signal transduction substance is modified to contain one or more mutations.

89. 89. The chimeric protein of claim 88, wherein the one or more mutations confer improved safety, or reduced affinity of the signal transduction agent for its receptor, or reduced biological activity of the signal transduction agent for its receptor, compared to a wild-type signal transduction agent, or allow attenuation of the activity of the signal transduction agent.

90. 89. The chimeric protein of claim 88, wherein the one or more mutations confer reduced affinity or activity that is reversible by attachment to one or more targeting moieties.

91. (a) the signal transduction agent is a mutant human IFNα2 comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 6 or 7, and the mutant human IFNα2 has one or more mutations that confer improved safety compared to wild-type IFNα2 having the amino acid sequence of SEQ ID NO: 6 or 7, optionally the human IFNα2 has one or more mutations at positions 144-154 relative to SEQ ID NO: 6 or 7, and optionally Alternatively, the human IFNα2 may contain one or more amino acids at positions L15, A19, R22, R23, L26, F27, L30, 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. and optionally said 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, and optionally said mutations are L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q4 0A, H57Y, E58N, Q61S, F64A, N65A, T69A, L80A, Y85A, Y89A, D114R, L117A, R120A, R125A, K133A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, and optionally said mutant human IFNα2 is one or more of R33A, T106X ... ３ , R120E, R144X １ , A145X ２ , M148A, R149A, and L153A; and １ is selected from A, S, T, Y, L, and I; X ２ is selected from G, H, Y, K, and D, and X ３ is selected from A and E; (b) the signal transducer is a variant human IFNβ comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 38, and the variant human IFNβ has one or more mutations that confer improved safety compared to wild-type IFNβ having the amino acid sequence of SEQ ID NO: 38, optionally the mutations are one or more of W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G relative to the amino acid sequence of SEQ ID NO: 38; (c) the signal transducer is a mutant human IL-1β comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 39, and the mutant human IL-1β has one or more mutations that confer improved safety compared to wild-type IL-1β having the amino acid sequence of SEQ ID NO: 39, and optionally the mutations are A117G / P118G, R120G, R120A, L122A, T125G / L126G, R127G, Q130A, Q130W, Q131G, K132A, S137G / Q138G, S139G / Q140G, S141G / Q142G, S142G / Q143G, S144G / Q145G, S145G / Q146G, S146G / Q147G, S147G / Q148G, S148G / Q149G, S150G / Q151G, S152G / Q153G, S154G / Q155G, S156G / Q157G, S158G / Q159G, S159G / Q160G, S161G / Q162G, S163G / Q164G, S165G / Q166G, S167G / Q168G, S169G / Q170G, S171G / Q172G, S172G / Q173G, S173G / Q174G, S175G / Q176G, S176G / Q177G, S177G / Q178G, S178G / Q179G, S179G / Q180G, S179G / Q181G, S1 38Y, L145G, H146A, H146G, H146E, H146N, H146R, L145A / L147A, Q148E, Q1 48G, Q148L, Q148G / Q150G, Q150G / D151A, M152G, F162A, F162A / Q164E, F16 6A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209A, K209D, K209 A / K210A, K219S, K219Q, E221S, E221K, E221S / N224A, N224S / K225S, E244K and one or more of N245Q, or (d) said signal transducer is wild-type or variant human IFNα1 comprising an amino acid sequence having at least 90%, or at least 93%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or 100% identity to SEQ ID NO: 74, optionally said IFNα1 being: (i) one or more amino acids that confer reduced affinity for the interferon alpha / beta receptor (IFNAR), optionally selected from substitutions at positions L15, A19, R23, S25, L30, D32, R33, H34, Q40, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157, or a combination thereof; is a plurality of mutations, the positions of which are, based on SEQ ID NO: 74, L15A, A19W, R23A, S25A, L30A, L30V, D32A, R33K, R33A, R33Q, H34A, Q40A, D115R, L118A, K121A, K121E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E, R145G, R145H, R145I, R145K ... 5L, R145N, R145Q, R145S, R145T, R145V, R145Y, A146D, A146E, A146G, A146H, A146I, A146K, A146L, A146M, A146N , A146Q, A146R, A146S, A146T, A146V, A146Y, M149A, M149V, R150A, S153A, L154A, N157A, L30A-H58Y-E59N-Q62S, one or more mutations optionally selected from the group consisting of R33A-H58Y-E59N-Q62S, M149A-H58Y-E59N-Q62S, L154A-H58Y-E59N-Q62S, R145A-H58Y-E59N-Q62S, D115A-R121A, L118A-R121A, L118A-R121A-K122A, R121A-K122A, and R121E-K122E; and / or (ii) one or more mutations that affect aggregation, optionally selected from one or more substitutions or deletions at positions C1, C29, C86, C99, C139 relative to SEQ ID NO: 74, and optionally selected from C86S, C86A, and C86Y; Including, A chimeric protein according to any one of claims 81 to 90.

92. The flexible linker consists essentially of glycine and serine residues, and optionally the ... serine and serine residues, and optionally the flexible linker consists essentially of serine and serine residues, and optionally the flexible link ４ Ser) ｎ wherein n is from about 1 to about 8, and optionally the flexible linker comprises one or more of SEQ ID NOs: 10 to 17.

93. 93. The chimeric protein of any one of claims 81 to 92, wherein the targeting moiety comprises the extracellular domain of FLT3L, or a portion thereof, and optionally a mutation that reduces intermolecular extracellular domain dimerization, which is a substitution at position L27 of any one of SEQ ID NOs: 2 to 4, optionally L27D.

94. 94. The chimeric protein of any one of claims 81 to 93, further comprising an additional targeting moiety and / or an additional signal transduction agent or modified form thereof.

95. A recombinant nucleic acid composition encoding one or more chimeric proteins of any one of claims 81 to 94.

96. 96. A host cell comprising the recombinant nucleic acid of claim 95.

97. 95. The chimeric protein of any one of claims 81 to 94, wherein the chimeric protein is suitable for use in patients with one or more of cancer, infectious diseases, immune disorders, autoimmune and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases.

98. 95. A method for treating or preventing cancer, infectious diseases, immune disorders, autoimmune diseases and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases, comprising administering to a patient in need thereof an effective amount of a chimeric protein of any of claims 81-94.

99. (i) one or more targeting moieties comprising an FMS-like tyrosine kinase 3 ligand (FLT3L) domain, wherein FLT3L is a single-chain dimer; and (ii) an Fc domain, optionally with one or more mutations that reduce or eliminate one or more effector functions of said Fc domain, promote Fc chain pairing of said Fc domain, and / or stabilize the hinge region in said Fc domain; An Fc-based chimeric protein complex comprising:

100. 100. The Fc-based chimeric protein complex of claim 99, wherein the single-chain dimer FLT3L is attached to one Fc chain of the Fc domain.

101. The Fc-based chimeric protein complex of claim 99 or claim 100, wherein the single-chain dimer FLT3L comprises the extracellular domain of FLT3L, or a portion thereof.

102. The Fc-based chimeric protein complex according to any one of claims 99 to 101, wherein the single-chain dimer FLT3L comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 to 5, or at least 95% identity to any one of SEQ ID NOs: 2 to 5, or at least 98% identity to any one of SEQ ID NOs: 2 to 5.

103. 103. The Fc-based chimeric protein complex of any one of claims 98-102, wherein the targeting moiety comprises the extracellular domain of FLT3L, or a portion thereof, and optionally a mutation that reduces intermolecular extracellular domain dimerization, which is a substitution at position L27 of any one of SEQ ID NOs: 2-4, optionally L27D.

104. The Fc-based chimeric protein complex of any one of claims 98 to 103, further comprising an additional targeting moiety.

105. A recombinant nucleic acid composition encoding one or more chimeric proteins of any one of claims 99-104.

106. A host cell comprising the recombinant nucleic acid of claim 105.

107. 105. The Fc-based chimeric protein complex of any one of claims 99 to 104, wherein the chimeric protein is suitable for use in patients with one or more of cancer, infectious diseases, immune disorders, autoimmune and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases.

108. A method for treating or preventing cancer, infectious diseases, immune disorders, autoimmune diseases and / or neurodegenerative diseases, cardiovascular diseases, wounds, ischemia-related diseases, and / or metabolic diseases, comprising administering to a patient in need thereof an effective amount of an Fc-based chimeric protein complex according to any one of claims 99 to 104.

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