Therapeutic interferon alpha 1 proteins

Abstract

To provide chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprising interferon alpha 1, or a variant thereof, and their use as therapeutic agents.SOLUTION: The present invention provides a chimeric protein comprising: (a) an interferon alpha 1 (IFNα1) or a variant thereof, and (b) one or more targeting moieties, comprising recognition domains which specifically bind to an antigen or receptor of interest, wherein the IFNα1 or the variant thereof, and the one or more targeting moieties are optionally connected with one or more linkers.SELECTED DRAWING: Figure 20

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 906,431, filed September 26, 2019, and U.S. Provisional Patent Application No. 62 / 825,569, filed March 28, 2019, the contents of both of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention relates, in part, to chimeric proteins or chimeric protein complexes (including Fc-based chimeric protein complexes) comprising interferon alpha 1 (IFNα1) or variants thereof and their use as therapeutic agents.

[0003] Sequence Listing The contents of the following text file, submitted electronically herewith, are incorporated by reference in their entirety into this specification: Computer-readable copy of the Sequence Listing (File name: ORN-057PC_Sequence_listing_ST25, Recording date: March 26, 2020, File size: 385,024 bytes). [Background technology]

[0004] Type I interferons (IFNs) form a multifunctional cytokine family that plays an important role in immune responses and other biological processes. Human type I IFNs include 13 distinct non-allelic alpha subtypes, one beta subtype, and one omega subtype. All type I interferons appear to bind, to varying degrees, to a common receptor, the type I interferon α / β receptor (IFNAR), composed of the IFNAR1 and IFNAR2 subunits. Upon binding of type I IFN, IFNAR activates the JAK-STAT signaling pathway, inducing various biological effects. Differential activities of IFN subtypes have been reported and, therefore, have been specifically used clinically for the treatment of various diseases and disorders, including viral hepatitis (IFNα2), multiple sclerosis, and cancer therapy (e.g., IFN-β or IFNα2). The formation of IFN-IFNAR ligand-receptor complexes initiates the activation of several signaling pathways that regulate cell differentiation and / or function, depending on the cell type, the IFN subtype that binds to IFNAR, and the type (amplitude, duration, etc.) of receptor-activation-related signals. Although type I interferons share the property of binding to IFNAR, they can bind to different degrees and, as a result, may have nonredundant functions. These differences are, at least in part, related to the type and quality of the signaling responses they induce upon binding to IFNAR. This is related to the various responses of type I IFNs, such as immunostimulatory, antiproliferative, antiviral, and other biological actions. IFNα2 is the most potent IFNAR-binding ligand and IFNAR signaling activator. Various attempts have been reported to generate even more potent IFNAR binders for potential therapeutic applications.

[0005] In contrast to other type I IFNs, interferon alpha 1 (IFNα1) is a member of the type I interferon family characterized by extremely low affinity for the IFNAR2 receptor (20-fold lower binding affinity for IFNAR2 compared to IFNα2; Jaks et al., J. Mol. Biol. 2007;366:525-534). IFNα1 is considered the weakest naturally occurring human IFNAR-binding type I IFN ligand and IFNAR signaling activator within the type I IFN family (Moll et al., Cytokine 2011;53:52-59). Notably, these properties contribute to the long-standing lack of public interest in IFNα1 and its exploration as a potential therapeutic agent. The efficacy of type I IFNs in clinical practice is limited by ineffective administration due to significant systemic toxicity and side effects, including influenza-like syndrome, depression, hepatotoxicity, autoimmune disease, thyroid dysfunction, and weight loss. Therefore, it would be extremely valuable to localize and target IFN activity to only the cell populations that should be treated with IFN (infected organs or tumor masses) or activated by IFN (e.g., immune cell subsets).

[0006] Thus, there remains a need for safe and effective IFNα1-based therapeutics with improved pharmacokinetic and therapeutic properties and minimal toxicity profiles. Summary of the Invention

[0007] Thus, in some aspects, the present invention relates to chimeric protein complexes, including chimeric proteins and Fc-based chimeric protein complexes, comprising wild-type IFNα1 or a variant thereof as a signaling agent. The term "variant" as used herein includes IFNα1 mutants. In some embodiments, the IFNα1 comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof.

[0008] The present disclosure relates, in part, to the discovery that chimeric proteins or chimeric protein complexes comprising wild-type IFNα1 and variants thereof exhibit substantially reduced IFNAR-activating signaling activity compared to wild-type IFNα1. However, this reduced IFNAR-activating signaling activity can be induced and / or restored in such target cells when directed to such target cells via a targeting moiety. Surprisingly, the induced IFNα1 activity in target cells achieved through targeting of a chimeric protein or chimeric protein complex comprising IFNα1 or a variant thereof can be similar to or greater than that of wild-type IFNα1 in the target cells. Furthermore, and equally surprising, the targeted IFNα1 activity of a chimeric protein or chimeric protein complex comprising IFNα1 or a variant thereof can be comparable to or greater than that of the most potent wild-type IFNα2 (e.g., about 10- to 100-fold more potent than wild-type IFNα1, depending on the cell type). Importantly, the IFNα1-containing IFNα1 chimeric proteins and chimeric protein complexes described herein exhibit surprising and valuable selectivity for target cells relative to non-target cells, significantly higher than that achieved, for example, with targeted wild-type IFNα2 chimeric proteins. In summary, a unique combination of high potency and highly cell-target-selective IFNAR signaling activation can be achieved using the IFNα1 compositions and variants thereof described herein. Thus, in various embodiments, the present invention relates to target-selective IFNAR activators with a high therapeutic index and excellent pharmaceutical properties for use in the treatment of various diseases, including cancer, infectious diseases, and autoimmune diseases.

[0009] In some embodiments, incorporating wild-type IFNα1 into a chimeric protein or chimeric protein complex, e.g., by genetic fusion or conjugation (e.g., forming a complex), reduces the biological activity of IFNα1 (sometimes referred to as "fusion-mediated attenuation"). For example, wild-type IFNα1 incorporated into a chimeric protein or chimeric protein complex may have reduced affinity and / or activity for a therapeutic receptor compared to wild-type IFNα1 interferon. In certain embodiments, the therapeutic receptor is the interferon α / β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits. In some embodiments, the reduced affinity and / or activity of wild-type IFNα1 for a therapeutic receptor, e.g., IFNAR, can be induced and restored upon directing or targeting to a target cell via a targeting moiety of a chimeric protein or chimeric protein complex comprising IFNα1. In some embodiments, the induction and restoration of IFNα1-mediated IFNAR activation in target cells can reach levels similar to or greater than those achieved with wild-type (non-chimeric) IFNα1. In some embodiments, the IFNα1 is a variant that contains one or more mutations that maintain or avoid a substantial reduction in IFNAR activation of the modified IFNα1 in a chimeric protein or chimeric protein complex compared to wild-type IFNα1, including reducing undesired disulfide pairing to improve the homogeneity and pharmaceutical properties of the product of the chimeric protein or chimeric protein complex, while maintaining or avoiding a substantial reduction in the restoration and induction of IFNAR activation by the modified IFNα1 when directed or targeted to a target cell via a targeting moiety.

[0010] In some embodiments, the IFNα1 is modified, i.e., variant, and contains one or more mutations in IFNα1. In some embodiments, the one or more mutations reduce the biological activity of IFNα1 (sometimes referred to as "mutational attenuation"). For example, the one or more mutations may reduce the affinity and / or activity of IFNα1 interferon for a therapeutic receptor. In certain embodiments, the therapeutic receptor is the interferon α / β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits. In certain embodiments, the modified IFNα1 contains one or more mutations that reduce its affinity and / or activity for IFNAR1. In other embodiments, the modified IFNα1 contains one or more mutations that reduce its affinity and / or activity for IFNAR2. In certain embodiments, the modified IFNα1 contains one or more mutations that reduce its affinity and / or activity for IFNAR1 and one or more mutations that reduce its affinity and / or activity for IFNAR2. In some embodiments, reduced affinity and / or activity of the modified IFNα1 for therapeutic receptors, e.g., IFNAR1, IFNAR2, and / or IFNAR ("mutationally attenuated") can be induced and restored upon directing or targeting a chimeric protein or chimeric protein complex comprising the modified IFNα1 to a target cell via a targeting moiety. In some embodiments, the modified IFNα1 variants ("mutationally attenuated") comprising one or more mutations that reduce their affinity and / or activity for IFNAR1, IFNAR2, and / or IFNAR further comprise one or more mutations that reduce undesired disulfide pairing to improve the homogeneity and pharmaceutical properties of the chimeric protein or chimeric protein complex product, while maintaining or avoiding a substantial reduction in the induction and / or restoration of IFNAR-activating activity by the modified IFNα1 ("mutationally attenuated") when directed or targeted to a target cell via a targeting moiety.

[0011] In some embodiments, chimeric protein complexes, including chimeric proteins and Fc-based chimeric protein complexes, include one or more additional signal transduction substances, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, which may be modified. In various embodiments, chimeric protein complexes, including chimeric proteins and Fc-based chimeric protein complexes of the present invention, provide improved safety and / or therapeutic activity and / or pharmacokinetic profile (e.g., extended serum half-life) compared to untargeted and / or unmodified IFNα1 or unmodified wild-type IFNα, e.g., IFNα2 or IFNα2b.

[0012] In various embodiments, chimeric protein complexes, including chimeric proteins and Fc-based chimeric protein complexes, comprise one or more targeting moieties having a recognition domain (e.g., an antigen recognition domain, including various antibody formats, including, but not limited to, single-domain antibodies) that specifically binds to a target of interest (e.g., an antigen, a receptor). In various embodiments, the targeting moiety has a recognition domain that specifically binds to a target of interest (e.g., an antigen, a 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, T regulatory cells (Tregs), natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor and tumor macrophages (e.g., M1 and M2 macrophages), B cells, B regulatory (Breg) cells, neutrophils, monocytes, bone marrow-derived cells, and dendritic cells. In various embodiments, the targeting moiety has a recognition domain that specifically binds to a target of interest (e.g., an antigen, receptor), including those found on one or more of tumor cells, endothelial cells, epithelial cells, mesenchymal cells, stromal cells, or other cell types characteristic and / or specific to a particular organ and / or tissue, including those particularly associated with disease. In some embodiments, the recognition domain specifically binds to the target of interest (e.g., an antigen, receptor) and effectively recruits one or more immune cells. In some embodiments, the target of interest (e.g., an antigen, receptor) can be found on one or more tumor cells. In some embodiments, the chimeric protein complexes, including chimeric proteins, including 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 or modulating other immune cells, to a site of action (such as, by way of non-limiting example, the tumor microenvironment). In some embodiments, the chimeric proteins of the present invention, including chimeric protein complexes, including Fc-based chimeric protein complexes, can regulate immune cells at a site of action or recruit immune cells to a site of action (such as, but not limited to, a disease microenvironment) associated with autoimmune disease, inflammatory disease, infectious disease, metabolic and / or cardiovascular disease.In some embodiments, the recognition domain specifically binds to a target of interest (eg, an antigen, a receptor) that is part of a non-cellular structure.

[0013] In various embodiments, the chimeric protein complexes of the present invention, including chimeric proteins and Fc-based chimeric protein complexes, are used to treat a variety of diseases or disorders, such as cancer, infectious diseases, immune disorders, autoimmune diseases, cardiovascular diseases, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and many other diseases and disorders, and the present invention encompasses a variety of treatment methods.

[0014] 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, and 19A-F 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 depression, have knob-in-hole and / or ion-pair (a / k / a 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]

[0015] [Figure 1A-F] 1 shows examples of homodimeric two-chain complexes. These figures show illustrative structures of homodimeric two-chain complexes. [Figures 2A-H] 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-H] 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-D] An example of a heterodimeric two-chain complex with separated 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-F] 1 shows an example of a heterodimeric two-chain complex with separate TM and SA chains, i.e., both TM on the knob chain of the Fc and SA on the hole chain of the 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-J]

[0033] Figure 1 shows an example of a heterodimeric two-chain complex with separate TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and 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-D] An illustrative heterodimeric two-chain complex is shown with separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc. [Figure 8A-F] 1 shows an example of a heterodimeric two-chain complex with separate 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-J] Examples of heterodimeric two-chain complexes are shown, with separate 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-F] 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-L]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-L] 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. [Figures 13A-F] 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-L] 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-L] 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-J]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-J] 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. [Figures 18A-F] 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 separate SA and TM chains: SA on the knob Fc and TM on the hole Fc. [Figure 19A-F] 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 separate SA and TM chains: TM on the knob Fc and SA on the hole Fc. [Figure 20] Figure 1 shows the biological activity of IFNα1 and Clec9A VHH Fc AcTaferon (AFN) on HL116 and HL116-hClec9A cells. Parental HL116 or derivative HL116-hClec9A cells were stimulated with serial dilutions of Fc AFN for 6 hours. The mean luciferase values ​​(±STDEV) of triplicate determinations are plotted. [Figure 21A-D]Figure 1 shows IFNα1 (also referred to herein as IFNα1) and IFN-α2 (also referred to herein as IFNα2) signaling in peripheral blood mononuclear cells (PBMCs) upon targeting. PBMCs from buffy coats of healthy donors were stained for CD20 and then stimulated for 15 minutes with serial dilutions of IFNα2 (i.e., without a targeting moiety), CD20 VHH-IFNα2 (a CD20-specific VHH targeting moiety and a wild-type IFNα2 chimera), IFNα1 (i.e., without a targeting moiety), or CD20 VHH-IFNα1 (a CD20-specific VHH targeting moiety and a wild-type IFNα1 chimera). STAT1 phosphorylation was quantified by FACS and plotted for CD20-positive and CD20-negative PBMCs. The data for IFNα2 is shown in FIG. 21A, the data for CD20 VHH-IFNα2 in FIG. 21B, the data for IFNα1 in FIG. 21C, and the data for CD20 VHH-IFNα1 in FIG. 21D. [Figure 22A-D] Figure 22 shows IFNα1 and IFNα2 signaling in HL116 and HL116-hCD20 cells upon targeting. Parental HL116 or derived HL116-huCD20 cells were stimulated for 6 hours with serial dilutions of IFNα2 (i.e., no targeting moiety), CD20 VHH-IFNα2 (CD20-specific VHH targeting moiety and wild-type IFNα2 chimera), IFNα1 (i.e., no targeting moiety), or CD20 VHH-IFNα1. Mean luciferase values ​​(±STDEV) from triplicate measurements are plotted. IFNα2 data are shown in Figure 22A, CD20 VHH-IFNα2 data in Figure 22B, IFNα1 data in Figure 22C, and CD20 VHH-IFNα1 data in Figure 22D. [Figure 23] 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. [Figure 24A-G]Figure 1 shows the biological activity of IFNα1 AFN on the HL116 reporter. HL116 or HL116-Clec9A cells were stimulated with serial dilutions of wild-type IFNα2 or IFNα1 AFN for 6 hours. The mean luciferase activity (±STDEV) is plotted. [Figure 25A-E] Figure 1 shows pSTAT1 activation in Clec9A- / CD141- and Clec9A+ / CD141+ PBMCs by IFNα2- or IFNα1-based AFN. [Figure 26A-B] Figure 26 shows the biological activity of PD-L1-targeted IFNα1 (Figure 26A) and IFNα2 (Figure 26B) AFNs on the HL116 reporter. HL116 cells were stimulated with serially diluted wild-type IFNα2 or IFNα1 AFNs for 6 hours. Mean luciferase activity (±STDEV) is plotted. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention is based, in part, on the discovery that chimeric protein complexes, such as targeted chimeric proteins and Fc-based chimeric protein complexes, comprising IFNα1 exhibit significantly greater activity and / or target selectivity than unfused, wild-type IFNα1, and exhibit beneficial therapeutic and pharmaceutical properties and reduced side effects. For example, chimeric protein complexes, such as the chimeric proteins or Fc-based chimeric protein complexes of the present invention, are highly target selective, allow for conditional and / or controlled modulation of IFNAR1 / 2 receptor signaling, and are highly active and / or long-acting, while exhibiting minimal off-target effects and inducing minimal side effects.

[0017] The present invention also provides pharmaceutical compositions comprising nucleic acids encoding chimeric proteins, chimeric protein complexes (including Fc-based chimeric protein complexes), and / or chimeric protein complexes, including chimeric proteins and Fc-based chimeric protein complexes. The present invention also includes host cells comprising nucleic acids encoding chimeric proteins and chimeric protein complexes, including Fc-based chimeric protein complexes. The present invention further includes the use of chimeric proteins, chimeric protein complexes (including Fc-based chimeric protein complexes), nucleic acids encoding chimeric proteins and chimeric protein complexes (including Fc-based chimeric protein complexes), pharmaceutical compositions, and / or host cells described herein for the treatment of various diseases.

[0018] Interferon alpha 1 or its variants In one aspect, the present invention provides chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, comprising genetically modified interferons. In one aspect, the present invention provides chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, comprising wild-type IFNα1. In various embodiments, the wild-type IFNα1 comprises the following amino acid sequence: CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO: 1).

[0019] In various embodiments, the present invention provides chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, comprising wild-type IFNα1 fused to one or more targeting moieties. In some embodiments, incorporating wild-type IFNα1 into the chimeric protein or chimeric protein complex, e.g., by gene fusion or conjugation, reduces the biological activity of IFNα1 ("attenuation by fused IFNα1"). For example, wild-type IFNα1 incorporated into the chimeric protein or chimeric protein complex may have reduced affinity and / or activity for a therapeutic receptor compared to wild-type IFNα1 interferon. In certain embodiments, the therapeutic receptor is the interferon α / β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits. In some embodiments, the reduced affinity and / or activity of wild-type IFNα1 for a therapeutic receptor, e.g., IFNAR, can be induced and restored upon directing or targeting the IFNα1-containing chimeric protein or chimeric protein complex to a target cell via the targeting moiety. In some embodiments, the induction and restoration of IFNα1-mediated IFNAR activation in target cells can reach levels similar to or higher than those achieved with wild-type (non-chimeric) IFNα1. In some embodiments, the IFNα1 is a variant containing one or more mutations that reduce undesired disulfide pairing to improve the homogeneity and pharmaceutical properties of the product of the chimeric protein or chimeric protein complex, while maintaining or avoiding a substantial reduction in IFNAR activation of the modified IFNα1 compared to wild-type IFNα1 in the chimeric protein or chimeric protein complex, including maintaining or avoiding a substantial reduction in the restoration and induction of IFNAR activation by the modified IFNα1 when directed or targeted to target cells via a targeting moiety. 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: 1.In some embodiments, the mutation at position C86 can be, for example, C86S, C86A, or C86Y. These C86 mutants of IFNα1 are called reduced-cysteine-based aggregation mutants. In some embodiments, the IFNα1 variant contains mutations at positions C1, C86, and C99 relative to SEQ ID NO: 1. In some embodiments, any of C1, C86, and C99 can be deleted or substituted.

[0020] In some embodiments, the IFNα1 is modified, i.e., variant, and comprises one or more mutations in IFNα1. In some embodiments, the one or more mutations reduce the biological activity of IFNα1 ("mutational attenuation"). For example, the one or more mutations may reduce the affinity and / or activity of IFNα1 interferon for a therapeutic receptor. In certain embodiments, the therapeutic receptor is the interferon α / β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits. In certain embodiments, the modified IFNα1 comprises one or more mutations that reduce its affinity and / or activity for IFNAR1. In other embodiments, the modified IFNα1 comprises one or more mutations that reduce its affinity and / or activity for IFNAR2. In certain embodiments, the modified IFNα1 comprises one or more mutations that reduce its affinity and / or activity for IFNAR1 and one or more mutations that reduce its affinity and / or activity for IFNAR2. In some embodiments, reduced affinity and / or activity of the modified IFNα1 for therapeutic receptors, e.g., IFNAR1, IFNAR2, and / or IFNAR ("mutationally attenuated") can be induced and restored upon delivery or targeting of a chimeric protein or chimeric protein complex comprising the modified IFNα1 to a target cell via a targeting moiety. In some embodiments, the modified IFNα1 variant ("mutationally attenuated") comprising one or more mutations that reduce its affinity and / or activity for IFNAR1, IFNAR2, and / or IFNAR further comprises one or more mutations that reduce undesired disulfide pairing to improve the homogeneity and pharmaceutical properties of the chimeric protein or chimeric protein complex product, while maintaining or avoiding a substantial reduction in the induction and / or restoration of IFNAR-activating activity by the modified IFNα1 ("mutationally attenuated") when delivered or targeted to a target cell via a targeting moiety.In some embodiments, the IFNα1 is a variant comprising one or more mutations that reduce undesired disulfide pairing to improve the homogeneity and pharmaceutical properties of the product of the chimeric protein or chimeric protein complex, while maintaining or avoiding a substantial reduction in IFNAR activation by the modified IFNα1 compared to wild-type IFNα1 in the chimeric protein or chimeric protein complex, including maintaining or avoiding a substantial reduction in restored and induction of IFNAR activation by the modified IFNα1 when directed or targeted to a target cell via a targeting moiety. 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, with reference to SEQ ID NO: 1. In some embodiments, the mutation at position C86 can be, for example, C86S, C86A, or C86Y. These C86 mutants of IFNα1 are called reduced-cysteine ​​based aggregation mutants. In some embodiments, the IFNα1 variant contains mutations at positions C1, C86, and C99 relative to SEQ ID NO:1.

[0021] In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises, as a signaling 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.

[0022] Additional IFNα1 variant sequences are known in the art. In various embodiments, the modified IFNα1 is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or less identical to any known amino acid sequence of an IFNα1 interferon variant. or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%). %, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0023] In some embodiments, the IFNα1 variant has an identity of at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, with any of the IFNα1 or IFNα1 variant sequences disclosed herein, e.g., SEQ ID NO:1. %, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 9 0%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0024] In various embodiments, the IFNα1 variant comprises an amino acid sequence having one or more amino acid mutations, which in some embodiments may be independently selected from substitutions, insertions, deletions, and truncations.

[0025] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0026] "Conservative substitutions" can be made, for example, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0027] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid in the same group of the six standard amino acid groups. For example, replacing Asp with Glu maintains one negative charge in the modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0028] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid from a different group of the six standard amino acid groups (1) to (6) above.

[0029] In various embodiments, substitutions also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, C α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general).

[0030] In various embodiments, IFNα1 is modified to have one or more mutations. In some embodiments, the mutations allow the IFNα1 variant to have one or more attenuated activities, such as one or more of reduced binding affinity, reduced intrinsic activity, and reduced specific biological activities, compared to a non-mutated, e.g., wild-type, IFNα1 (e.g., IFNα1 having the amino acid sequence of SEQ ID NO: 1). For example, compared to a non-mutated, e.g., wild-type, IFNα1, the one or more attenuated activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activities, may be for a therapeutic receptor such as IFNAR. As a result, in various embodiments, the mutations allow the IFNα1 variant to have reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to a non-mutated, e.g., wild-type, IFNα1.

[0031] In various embodiments, IFNα1 is modified to have a mutation that reduces its binding affinity or activity to a therapeutic receptor, such as IFNAR. In some embodiments, the activity conferred by IFNα1 is agonism of the therapeutic receptor (e.g., activation of a cellular effect at the site of treatment). For example, IFNα1 can activate the therapeutic receptor. In such embodiments, the mutation results in an IFNα1 variant with reduced activating effect on the therapeutic receptor.

[0032] In some embodiments, the reduced affinity or activity of the modified IFNα1 for a therapeutic receptor can be induced or restored by binding to a targeting moiety or by inclusion of the targeting moiety in a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex disclosed herein. In some embodiments, the activity of IFNα1 is reduced or attenuated, in some cases, by fusing it with another protein, such as by fusion with a targeting moiety described herein. In other embodiments, the activity of IFNα1 is reduced or attenuated by modifying IFNα1, for example, by introducing a mutation described herein. In some embodiments, the attenuation of activity can be restored by binding IFNα1 to a targeting moiety or by the action of the bound targeting moiety. In some embodiments, the targeting moiety induces the activity of IFNα1 by its binding or action.

[0033] In other embodiments, the reduced affinity or activity for a therapeutic receptor is not fully inducible or reversible upon conjugation with a targeting moiety or inclusion of a targeting moiety in a chimeric protein or chimeric protein complex, e.g., an Fc-based chimeric protein complex disclosed herein. In various embodiments, the therapeutic chimeric proteins, chimeric protein complexes, or Fc-based chimeric protein complexes of the present invention reduce off-target effects because wild-type IFNα1 or an IFNα1 variant with one or more mutations exhibits weaker binding affinity or activity for a therapeutic receptor compared to wild-type IFNα1 (unfused). In various embodiments, this reduces side effects observed with, for example, wild-type IFNα1 or other type I interferons. In various embodiments, the IFNα1 constructs and / or IFNα1 variants are substantially inactive en route to the therapeutic site of action and substantially exert their effect specifically on the target cell type, which greatly reduces undesirable cross-reactivity and side effects.

[0034] In various embodiments, the IFNα1 variant is characterized in that the IFNα1 variant has attenuated or reduced affinity, e.g., binding (e.g., K D ) and / or activation (e.g., K A and / or EC 50 In various embodiments, the reduced affinity for the therapeutic receptor allows for attenuated activity and / or signaling from the therapeutic receptor.

[0035] In various embodiments, the IFNα1 variant has one or more mutations that reduce its binding or affinity to the IFNAR1 subunit of IFNAR. In one embodiment, the IFNα1 variant has reduced affinity and / or activity for IFNAR1. In some embodiments, the IFNα1 variant has one or more mutations that reduce its binding or affinity to the IFNAR2 subunit of IFNAR. In some embodiments, the IFNα1 variant has one or more mutations that reduce its binding or affinity to both the IFNAR1 and IFNAR2 subunits.

[0036] In some embodiments, the IFNα1 variant has one or more mutations that reduce its binding or affinity for IFNAR1 and one or more mutations that substantially reduce or eliminate binding or affinity for IFNAR2. In some embodiments, chimeric proteins and chimeric protein complexes, such as Fc-based chimeric protein complexes, having such IFNα1 variants can provide target-selective IFNAR1 activity (e.g., IFNAR1 activity is inducible or restoreable via targeting by a targeting moiety or upon inclusion in an Fc-based chimeric protein complex disclosed herein).

[0037] In some embodiments, the IFNα1 variant has one or more mutations that reduce its binding or affinity for IFNAR2 and one or more mutations that substantially reduce or eliminate its binding or affinity for IFNAR1. In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, having such an IFNα1 variant can provide target-selective IFNAR2 activity (e.g., IFNAR2 activity is inducible or restoreable via targeting by a targeting moiety or upon inclusion in an Fc-based chimeric protein complex disclosed herein).

[0038] In some embodiments, the IFNα1 variant has one or more mutations that reduce its binding or affinity to IFNAR1 and one or more mutations that reduce its binding or affinity to IFNAR2. In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, having such an IFNα1 variant can provide target-selective IFNAR1 and / or IFNAR2 activity (e.g., IFNAR1 and / or IFNAR2 activity is inducible or restoreable via targeting by a targeting moiety or upon inclusion in an Fc-based chimeric protein complex disclosed herein).

[0039] In various embodiments, the IFNα1 variant has about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% to 20%, about 20% to 40%, about 50%, about 40% to 60%, about 60% to 80%, or about 80% to 100% affinity for a therapeutic receptor (e.g., IFNAR or any one of its subunits IFNAR1 and / or IFNAR2) compared to wild-type IFNα1. 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 wild-type IFNα1.

[0040] In some embodiments, the IFNα1 variant contains one or more mutations that reduce the affinity of the IFNα1 variant for a receptor. In some embodiments, the binding affinity of the IFNα1 variant for a receptor is lower than the binding affinity of the targeting moiety for that receptor. In some embodiments, this difference in binding affinity is between the IFNα1 variant / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity allows the IFNα1 variant to have a localized on-target effect and minimize off-target effects that underlie the side effects observed with wild-type IFNα1. In some embodiments, this binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold lower, or at least about 25-fold, or at least about 50-fold lower, or at least about 100-fold, or at least about 150-fold lower.

[0041] 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) or by reflectance interferometry under flow-through conditions, as described by Brecht et al. (1993).The entire contents of these documents are incorporated herein by reference.

[0042] In various embodiments, the chimeric protein complex of the present invention comprises (a) interferon alpha 1 (IFNα1 or a variant thereof) and (b) one or more targeting moieties, wherein the targeting moiety comprises a recognition domain that specifically binds to an antigen or receptor of interest; the IFNα1 or a variant thereof and the one or more targeting moieties are linked to a domain that causes complex formation (e.g., a complexing domain). In some embodiments, the 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, e.g., using electrostatic interactions, hydrogen bonds, and / or hydrophobic effects. In some embodiments, the chimeric protein complex is homomeric (e.g., comprising two or more chimeric proteins described herein, each comprising interferon alpha 1 (IFNα1) or a variant thereof and one or more targeting moieties linked by one or more linkers). In some embodiments, the chimeric protein complex is heteromeric (e.g., comprises a single chimeric protein comprising interferon alpha 1 (IFNα1) or a variant thereof 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 present 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. Patent No. 8,507,222, incorporated herein by reference in its entirety, for methods of generating protein complexes).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.

[0043] In various embodiments, a chimeric protein complex, such as an 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 an 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 an Fc-based chimeric protein complex, allows for conditional activity.

[0044] In various embodiments, a 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 a chimeric protein complex, such as an Fc-based chimeric protein of the present invention, results in 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.

[0045] In some embodiments, the reduced affinity or activity for a receptor is inducible or reversible by binding to one or more targeting moieties described herein or upon inclusion in a chimeric protein complex, such as an Fc-based chimeric protein complex disclosed herein.

[0046] In various embodiments, a 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% affinity for the receptor compared to the wild-type signaling agent, compared to a signaling agent that is not fused to an Fc, or a 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.

[0047] In various embodiments, a chimeric protein complex, such as an 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 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.

[0048] In some embodiments, the attenuated activity for a receptor or reduced affinity for a therapeutic receptor can be induced or restored by binding to a targeting moiety or by inclusion in a chimeric protein complex, such as an Fc-based chimeric protein complex disclosed herein, that has high affinity for an antigen at the site of therapeutic action (e.g., an antibody or antibody format described herein). Targeting is achieved by binding IFNα1 or a variant thereof to the targeting moiety or by inclusion in a chimeric protein complex, such as an Fc-based chimeric protein complex disclosed herein. In one embodiment, IFNα1 or a variant thereof is conjugated to the targeting moiety via its amino terminus. In another embodiment, IFNα1 or a variant thereof is conjugated to the targeting moiety via its carboxy terminus. Thus, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention, in some embodiments, provides a localized, on-target, and controlled therapeutic action at the location of the therapeutic receptor.

[0049] In some embodiments, the IFNα1 interferon is modified to have a mutation at one or more amino acids 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, relative to SEQ ID NO: 1. 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, D115R, L118A, K121A, K121E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E, R145G, R145H, R145I, R145K ... and N157A. In some embodiments, the IFNα1 mutant 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: 1.

[0050] In some embodiments, IFNα1 interferon or its variants are modified to have one or more mutations at amino acid positions C1, C29, C86, C99, or C139 relative to SEQ ID NO: 1. In this regard, Beilharz et al., Journal of interferon research 6.6 (1986):677-685 (incorporated herein by reference in its entirety) describes various mutations of IFNα1 that can be introduced into the modified IFNα1 of the present invention. The mutation at position C86 can be, for example, C86S, C86A, or C86Y. These C86 mutants of IFNα1 are called reduced cysteine ​​aggregation mutants. In some embodiments, the IFNα1 variants contain mutations at positions C1, C86, and C99 relative to SEQ ID NO: 1.

[0051] Therapeutic agents containing interferon or its variants Cellular recruitment of targeting moieties In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention further comprises one or more targeting moieties having a recognition domain that specifically binds to a target of interest (e.g., an antigen, a receptor). In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can comprise two, three, four, five, six, seven, eight, nine, ten, or more targeting moieties. In exemplary embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention can comprise two or more targeting moieties. In such embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can target two different cells (e.g., to create a synapse) or the same cell (e.g., to achieve a higher concentration of a signaling substance effect). In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety that is IFNα1 or a variant thereof, Flt3L, and one targeting moiety that recognizes PD-1 or PD-L1. In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety that is IFNα1 or a variant thereof, Flt3L, and two targeting moieties that recognize PD-1 or PD-L1.

[0052] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety that is IFNα2 or a variant thereof, Flt3L, and one targeting moiety that recognizes PD-1 or PD-L1. In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety that is IFNα2 or a variant thereof, Flt3L, and two targeting moieties that recognize PD-1 or PD-L1.

[0053] In various embodiments, the target of interest (e.g., antigen, receptor) can be 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 or tumor-associated macrophages (e.g., M1 or M2 macrophages), B cells, Breg cells, dendritic cells, or a subset thereof. In some embodiments, the recognition domain specifically binds to the target of interest (e.g., antigen, receptor) and effectively recruits one or more immune cells directly or indirectly. In some embodiments, the target of interest (e.g., antigen, receptor) can be found on one or more tumor cells. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention can recruit immune cells directly or indirectly to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can directly or indirectly recruit immune cells, e.g., immune cells capable of killing and / or suppressing tumor cells, to a site of action (such as, but not limited to, the tumor microenvironment).

[0054] In various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen, a receptor) that is part of a non-cellular structure. In some embodiments, the antigen or receptor is not an integral component of an intact cell or cellular structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-proteinaceous non-cellular marker, which includes, but is not limited to, extracellular deposits such as nucleic acids or cholesterol, including DNA or RNA, such as, for example, DNA released from necrotic tumor cells.

[0055] In some embodiments, the target of interest (e.g., antigen, receptor) is part of the noncellular components of the stroma or extracellular matrix (ECM) or a marker associated therewith. As used herein, stroma refers to the connective and supportive framework of a tissue or organ. The stroma may comprise a collection of cells, such as fibroblasts / myofibroblasts / glial cells, epithelial, adipose, immune, vascular, smooth muscle, and immune cells, along with the extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target of interest (e.g., antigen, receptor) is part of the noncellular components of the stroma, such as the extracellular matrix and extracellular molecules. As used herein, ECM refers to the noncellular components present in all tissues and organs. The ECM consists of a large collection of biochemically distinct components, including, but not limited to, proteins, glycoproteins, proteoglycans, and polysaccharides. These ECM components are typically produced by neighboring cells and secreted into the ECM by exocytosis. Once secreted, ECM components often assemble to form a complex network of macromolecules. In various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein of the present invention, comprises a targeting moiety that recognizes a target (e.g., an antigen or receptor or non-protein molecule) located on any component of the ECM. Examples of components of the ECM include, but are not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers, and other ECM proteins or ECM non-proteins, e.g., polysaccharides and / or lipids, or ECM-associated molecules (e.g., proteins or non-proteins, e.g., polysaccharides, nucleic acids, and / or lipids).

[0056] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on an ECM proteoglycan. Proteoglycans are glycosylated proteins. A basic proteoglycan unit comprises a core protein with one or more covalently attached glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+), which attract water via osmosis and keep the ECM and resident cells hydrated. Proteoglycans can also capture and store growth factors within the ECM. Examples of proteoglycans that can be targeted by the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In certain embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on a non-proteoglycan polysaccharide, such as hyaluronic acid.

[0057] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on ECM fibers. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety recognizes one or more epitopes on collagen or collagen fibers. Collagen is the most abundant protein in the ECM. Collagen exists as a fibrous protein in the ECM and provides structural support for resident cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagen present in the ECM, including, but not limited to, fibrillar collagens (types I, II, III, V, XI), facit collagens (types IX, XII, XIV), short-chain collagens (types VIII, X), basement membrane collagens (type IV), and / or type VI, VII, or XIII collagen. Elastin fibers provide elasticity to tissues, allowing them to stretch as needed and then return to their original state. In some embodiments, the targeting moiety recognizes one or more epitopes on elastin or elastin fibers.

[0058] In some embodiments, the targeting moiety recognizes one or more ECM proteins, including, but not limited to, tenascin, fibronectin, fibrin, laminin, or nidogen / entactin.

[0059] In certain embodiments, the targeting moiety recognizes and binds to tenascin. The tenascin (TN) family of glycoproteins includes at least four members: tenascin-C, tenascin-R, tenascin-X, and tenascin-W. The primary structure of tenascin proteins contains several common motifs ordered in the same contiguous sequence: amino-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain. Each protein member is associated with typical variations in the number and nature of the EGF-like and fibronectin type III repeats. Isoform variants also exist, particularly for tenascin-C. More than 27 splice variants and / or isoforms of tenascin-C are known. In certain embodiments, the targeting moiety recognizes and binds to tenascin-CA1. Similarly, tenascin-R also has various splice variants and isoforms. Tenascin-R typically exists as a dimer or trimer. Tenascin-X is the largest member of the tenascin family and is known to exist as a trimer. Tenascin-W exists as a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on the tenascin protein. In some embodiments, the targeting moiety recognizes the monomeric, dimeric, trimeric, and / or hexameric forms of the tenascin protein.

[0060] In certain embodiments, the targeting moiety recognizes and binds to fibronectin. Fibronectin is a glycoprotein that connects cells to collagen fibers in the ECM, allowing cells to migrate through the ECM. Upon binding to integrins, fibronectin unfolds to form functional dimers. In some embodiments, the targeting moiety recognizes monomeric and / or dimeric forms of fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. In exemplary embodiments, the targeting moiety recognizes fibronectin extracellular domain A (EDA) or fibronectin extracellular domain B (EDB). Elevated EDA levels are associated with various diseases and disorders, including psoriasis, rheumatoid arthritis, diabetes, and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing the EDA isoform and can be used to target chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing the EDB isoform. In various embodiments, such targeting moieties can be used to target chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, to tumor cells, including tumor neovasculature.

[0061] In certain embodiments, the targeting moiety recognizes and binds to fibrin, another protein substance often found in the matrix network of ECM. Fibrin is formed by the action of the protease thrombin on fibrinogen, which polymerizes it. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes both monomeric and polymerized forms of fibrin.

[0062] In certain embodiments, the targeting moiety recognizes and binds to laminin. Laminin is a major component of basement membranes and the protein network foundation for cells and organs. Laminin is a heterotrimeric protein comprising an α chain, a β chain, and a γ chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes monomeric, dimeric, and trimeric forms of laminin.

[0063] In certain embodiments, the targeting moiety recognizes and binds to nidogen or entactin. Nidogen / entactin are a family of highly conserved sulfated glycoproteins. They are major structural components of basement membranes and function to connect laminin with the collagen IV network in the basement membrane. Members of this family include nidogen-1 and nidogen-2. In various embodiments, the targeting moiety recognizes an epitope on nidogen-1 and / or nidogen-2.

[0064] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an epitope present on any of the targets described herein. In certain embodiments, the antigen recognition domain recognizes one or more linear epitopes present on a protein. As used herein, a linear epitope refers to any continuous sequence of amino acids present on a protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on a protein. As used herein, a conformational epitope refers to a portion of one or more amino acids (which may be discontinuous) that forms a three-dimensional surface with characteristics and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0065] In various embodiments, the targeting moiety may bind to the full-length form and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other natural or synthetic analog, variant, or mutant of any of the targets described herein. In various embodiments, the targeting moiety may bind to any form of protein described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers, and associated forms. In various embodiments, the targeting moiety may bind to any post-translationally modified form of protein described herein, such as glycosylated and / or phosphorylated forms.

[0066] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an extracellular molecule such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In certain embodiments, DNA is shed into the extracellular space from necrotic or apoptotic tumor cells or other diseased cells.

[0067] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more noncellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaques are known. Fibro-lipid (fibro-fatty) plaques are characterized by the accumulation of lipid-laden cells beneath the intima of an artery. Beneath the endothelium is a fibrous cap that covers the atherosclerotic plaque core. The core contains lipid-laden cells (macrophages and smooth muscle cells) with increased tissue cholesterol and cholesterol ester content, fibrin, proteoglycans, collagen, elastin, and necrotic cellular debris. In advanced plaques, the central core of the plaque typically contains extracellular cholesterol deposits (released from dead cells), which form areas of cholesterol crystals with empty needle-like spaces. The periphery of the plaque contains younger foam cells and capillaries. Fibrous plaques are also localized subintima within the arterial wall, resulting in wall thickening and proliferation, and sometimes localized narrowing of the lumen, accompanied by slight atrophy of the muscularis. Fibrous plaques contain collagen fibers (eosinophilic), calcium deposits (hematoxylinophilic), and lipid-loaded cells. In some embodiments, the targeting moiety recognizes and binds to one or more noncellular components of these plaques, such as fibrin, proteoglycans, collagen, elastin, necrotic cell debris, and calcium or other mineral deposits or precipitates. In some embodiments, the necrotic cell debris is nucleic acid, e.g., DNA or RNA released from dead cells.

[0068] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures found in brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures localized in amyloid plaques found in the brains of patients with Alzheimer's disease. For example, the targeting moiety recognizes and binds to the peptide amyloid beta. The peptide amyloid beta is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in brain plaques found in patients with Huntington's disease. In various embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in plaques associated with other neurodegenerative or musculoskeletal diseases, such as Lewy body dementia and inclusion body myositis.

[0069] In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can have two or more targeting moieties that bind to non-cellular structures. In some embodiments, there are two targeting moieties, one that targets cells and the other that targets non-cellular structures. In various embodiments, the targeting moieties can directly or indirectly recruit cells, such as disease cells and / or effector cells. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes of the present invention, can or find use in methods for engaging immune cells to shift the balance of immune cells in favor of immune attack of tumors. For example, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can shift the ratio of immune cells at clinically important sites in favor of cells capable of killing and / or suppressing tumors (e.g., 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, dendritic cells, or subsets thereof) and against cells that protect tumors (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs); tumor-associated neutrophils (TANs), M2 macrophages, tumor-associated macrophages (TAMs), or subsets thereof). In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes of the present invention, can increase the ratio of effector T cells to regulatory T cells.

[0070] For example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with a T cell. In some embodiments, the recognition domain directly or indirectly recruits a T cell. In certain embodiments, the recognition domain specifically binds to an effector T cell. In some embodiments, the recognition domain directly or indirectly recruits an effector T cell, for example, in some embodiments, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of effector T cells include cytotoxic T cells (e.g., αβTCR, CD3 + , CD8 + , CD45RO + );CD4 + Effector T cells (e.g., αβTCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL7R / CD127 + );CD8 + Effector T cells (e.g., αβTCR, CD3 + , CD8 + , CCR7 + , CD62Lhi, IL7R / CD127 + effector memory T cells (e.g., CD62Llow, CD44 + , TCR, CD3 + , IL7R / CD127 + , IL15R + , CCR7low); central memory T cells (e.g., CCR7 + , CD62L + , CD27 + or CCR7hi, CD44 + , CD62Lhi, TCR, CD3 + , IL7R / CD127 + , IL15R + );CD62L + Effector T cells; early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L -) (TemE and TemL, respectively) containing CD8 + Effector memory T cells (TEM); CD127 ( + )CD25(low / -) effector T cells; CD127( - )CD25( - ) Effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca( + )); TH1 effector T cells (e.g., CXCR3 + , CXCR6 + and CCR5 + ; or αβTCR, CD3 + , CD4 + , IL12R + , IFNγR + , CXCR3 + ), TH2 effector T cells (e.g., CCR3 + , CCR4 + and CCR8 + ; or αβTCR, CD3 + , CD4 + , IL4R + , IL33R + , CCR4 + ,IL17RB + , CRTH2 + ); TH9 effector T cells (e.g., αβTCR, CD3+, CD4+); TH17 effector T cells (e.g., αβTCR, CD3 + , CD4 + , IL23R + , CCR6 + , IL1R + );CD4 + CD45RO + CCR7 + Effector T cells, ICOS + Effector T cells; CD4 + CD45RO + CCR7( - ) effector T cells; and IL2, IL4 and / or IFN-γ secreting effector T cells.

[0071] Examples of T cell antigens of interest include, for example, the following (including the extracellular domain, if applicable): CD8, CD3, SLAMF4, IL2Rα, 4-1BB / TNFRSF9, IL2Rβ, ALCAM, B7-1, IL4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL6R, CCR3, IL7Rα, CCR4, CXCR1 / ILSRA, CCR5, CCR6, IL10Rα, CCR7, IL10Rβ, CCRS, IL12Rβ1, CCR9, IL12Rβ2, CD2, IL13Rα1, IL13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, and lutegrin.α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF 5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common gamma chain / IL2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CR TAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX -1, DNAM-1, thymopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRI II / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAILR1 / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL1R1, and TSLPR. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary T cell antigens.

[0072] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety directed against one or more of checkpoint markers expressed on T cells, e.g., PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, Cd27, CD40L, TIM3, and A2aR.

[0073] For example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with a B cell. In some embodiments, the recognition domain directly or indirectly recruits B cells, e.g., to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of B cell antigens of interest include, e.g., CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, CDw150, CS1, and B-cell maturation antigen (BCMA). In various embodiments, the targeting moiety of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary B cell antigens.

[0074] Further, for example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with natural killer cells. In some embodiments, the recognition domain directly or indirectly recruits natural killer cells, for example, in some embodiments, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of natural killer cell antigens of interest include, for example, TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-epsilon RII, LMIR6 / CD300LE, Fc-γRl / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NK G2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, and ULBP-3. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary NK cell antigens.

[0075] Also, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with macrophages / monocytes. In some embodiments, the recognition domain directly or indirectly recruits macrophages / monocytes, for example, in some embodiments, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of macrophage / monocyte antigens of interest include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin α4 / CD49d, BLAME / SLAMF8, integrin αX / CDllc, CCL6 / C10, integrin β2 / CD18, CD155 / PVR, integrin β3 / CD61, CD31 / PECAM-1, latexin, CD36 / SR-B3, leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, endoglin / CD105, osteoactivin / GPNMB, Fc-γRI / CD64, osteopontin, Fc-γRIIB / CD32b, PD-L2, Fc-γRIIC / CD32c, Siglec-3 / CD33, Fc-γRIIA / CD32a, SIGNR1 / CD209, Fc-γRIII / CD16, SLAM, GM-CSFRα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γRl, TLR4, IFN-γR2, TREM-l, IL-lRII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4, IL10Rα, ALCAM, IL10Rβ, aminopeptida -seN / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, ClqR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, integrin α4 / CD 49d, CCR5, integrin αM / CDllb, CCR8, integrin αX / CDllc, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, L MIR2 / CD300c, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSFR, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD105, NCAM-L1, Fc-γRI / C Examples of macrophage / monocyte antigens include D64, PSGL-1, Fc-γRIIIICD16, RP105, G-CSFR, L-selectin, GM-CSFRα, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL6R, TREM-2, CXCR1 / IL8RA, TREM-3, and TREML1 / TLT-1. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary macrophage / monocyte antigens.

[0076] Additionally, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with a dendritic cell. In some embodiments, the recognition domain directly or indirectly recruits dendritic cells, for example, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of dendritic cell antigens of interest include, for example, CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL12 / IL23p40, 4-Ami no-1, 8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, C lqR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBCCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLl, CD2F-10 / SLAMF9, osteoactivin GPNMB, Chern23, PD-L2 , CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209,These include DEP-1 / CD148, SIGNR4, DLEC / CLEC4C, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, and vanilloid R1. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary DC antigens.

[0077] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) on an immune cell selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, myeloid cells, monocytes, eosinophils, or a subset thereof. In some embodiments, the recognition domain localizes the megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, myeloid cells, monocytes, eosinophils, or a subset thereof, e.g., in some embodiments, directly or indirectly, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). In some embodiments, the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, mast cells, monocytes, erythrocytes, myeloid cells, myeloid-derived suppressor cells, NKT cells, and NK cells, or derivatives thereof.

[0078] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with megakaryocytes and / or platelets. Examples of megakaryocyte and / or platelet antigens of interest include, for example, GPIIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary megakaryocyte and / or platelet antigens.

[0079] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with an erythrocyte. Examples of erythrocyte antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb / IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2 (MHC-II), and Rh antigen. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary erythrocyte antigens.

[0080] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a mast cell. Examples of mast cell antigens of interest include, for example, SCFR / CD117, Fc ε RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMA1, FCERL1A, FCER2, TPSABl. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these mast cell antigens.

[0081] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a basophil. Examples of basophil antigens of interest include, for example, Fc ε RI, CD203c, CD123, CD13, CD107a, CD107b, and CD 164. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these basophil antigens.

[0082] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a neutrophil. Examples of neutrophil antigens of interest include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRIII), CD18 proteins (LFA-1, CR3, and p150, 95), CD45, CD67, and CD177. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these neutrophil antigens.

[0083] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with eosinophils. Examples of eosinophil antigens of interest include, for example, CD35, CD44, and CD69. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these eosinophil antigens.

[0084] In various embodiments, the recognition domain may specifically bind to any suitable target, antigen, receptor, or cell surface marker known to those of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Examples of tissue-specific markers include endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAMl, PROCR, SELE, SELP, TEK, THBD, VCAMl, and VWF; smooth muscle cell surface markers such as ACTA2, MYHlO, MYHl1, MYH9, and MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COLlAl, COL1A2, COL3A1, FAP, and PH-4; epithelial cell surface markers such as CDlD, K6IRS2, KRTlO, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCl, and TACSTDl; and CD13, TFNA, and alpha-v beta-3 (α). Vβ3), angiogenic markers such as E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these antigens. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds cells bearing one or more of these antigens.

[0085] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with a tumor cell. In some embodiments, the recognition domain directly or indirectly recruits tumor cells. For example, in some embodiments, the direct or indirect recruitment of tumor cells is to one or more effector cells (e.g., immune cells described herein) that can kill and / or suppress the tumor cells.

[0086] Tumor cells, or cancer cells, refer to uncontrolled proliferation of cells or tissues and / or abnormally increased cell survival and / or abnormally increased inhibition of apoptosis, which interfere with the normal functioning of bodily organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. Examples of tumor cells include 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, stomach cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney or renal cancer, and tumors of the liver and kidney. 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, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulvar cancer, lymphomas including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, These include, but are not limited to, HL, 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), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (e.g., associated with brain tumors), and cells of Meigs' syndrome.

[0087] Tumor cells or cancer cells also include, but are not limited to, carcinomas, such as various subtypes (including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myeloid, acute lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell), lymphomas and myelomas (including, for example, Hodgkin's and non-Hodgkin's lymphoma, light chain, non-secretory MGUS, and plasmacytoma), and central nervous system cancers (e.g., brain tumors (e.g., gliomas (e.g., astrocytoma, oligodendroglioma, and ependymoma), meningioma, pituitary adenoma, and neuroma), and spinal cord tumors (e.g., meningioma and neurofibroma)).

[0088] Examples of tumor antigens include MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, tumor antigens of the MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, MAGE-A14, MAGE-A15, MAGE-A16, MAGE-A17, MAGE-A18, MAGE-A19, MAGE-A20, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE-A27, MAGE-A28, MAGE-A29, MAGE-A3 ...1, MAGE-A20, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE- MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), tumor antigens of the GAGE ​​family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GA GE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, tumor antigens of the Smad family, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1These tumor antigens include, but are not limited to, CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD47, CS1, CD38, ASGPR, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, and BCMA (TNFRSF17). In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these tumor antigens. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to HER2. In another embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to PD-L2.

[0089] In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises (i) one or more targeting moieties for immune cells selected from T cells, B cells, dendritic cells, macrophages, NK cells, or a subset thereof, and (ii) one or more targeting moieties for tumor cells in combination with any of the signal transduction agents described herein (e.g., IFNα1 or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises (i) a targeting moiety for T cells (including but not limited to, effector T cells), and (ii) the targeting moiety is targeted to tumor cells in combination with any of the signal transduction agents described herein. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises (i) a targeting moiety for B cells, and (ii) the targeting moiety is targeted to tumor cells in combination with any of the signal transduction agents described herein. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for dendritic cells, and (ii) the targeting moiety is targeted to tumor cells in conjunction with any of the signaling agents described herein. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for macrophages, and (ii) the targeting moiety is targeted to tumor cells in conjunction with any of the signaling agents described herein. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for NK cells, and (ii) the targeting moiety is targeted to tumor cells in conjunction with any of the signaling agents described herein.

[0090] By way of non-limiting example, in various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention may comprise any of the following: (i) a chimeric protein or chimeric protein complex comprising, for example, CD8, SLAMF4, IL2Rα, 4-1BB / TNFRSF9, IL2Rβ, ALCAM, B7-1, IL4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL6R, CCR3, IL7Rα, CCR4, CXCR1 / ILSRA, CCR5, CCR6, IL10Rα, CCR7, IL10Rβ, CCRS, IL12Rβ1, CCR9, IL12Rβ2, CD2, IL13Rα1, IL13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrin,α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, LAG-3, CD43, LAIR1, CD45, LAI R2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common gamma chain / IL2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin , CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, thymopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAILRl / TNFRSF10A, ICAM-1 / C and (ii) the targeting moiety is directed to tumor cells in combination with any of the signal transducers described herein (e.g., IFNα1 or a variant thereof).

[0091] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) comprises a targeting moiety directed against one or more of checkpoint markers expressed on T cells, e.g., PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR, and (ii) the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein.

[0092] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties for PD-1. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties that selectively bind a PD-1 polypeptide. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprise one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a PD-1 polypeptide.

[0093] In some embodiments, the targeting moiety comprises the anti-PD-1 antibody pembrolizumab (also known as MK-3475, Keytruda), or a fragment thereof. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, et al. (2013) New England Journal of Medicine 369(2):134-44, US 8,354,509, and International Publication No. WO 2009 / 114335, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, pembrolizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO:7) and / or a light chain comprising the amino acid sequence of (SEQ ID NO:8).

[0094] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody, nivolvam (also known as BMS-936558, MDX-1106, ONO-4538, Opdivo), or a fragment thereof. Nivolvam (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449 and WO 2006 / 121168, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, nivolvam or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO:9) and / or a light chain comprising the amino acid sequence of (SEQ ID NO:10).

[0095] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody pidilizumab (also known as CT-011, hBAT, or hBAT-1), or a fragment thereof. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in U.S. Patent Application Publication No. 2008 / 0025980 and WO 2009 / 101611, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable fragment comprising an amino acid sequence selected from SEQ ID NOS:15-18 of U.S. Patent Application Publication No. 2008 / 0025980: SEQ ID NO:15 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:11); SEQ ID NO:16 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:12); SEQ ID NO:17 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:13); SEQ ID NO:18 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:14). region; and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 20-24 of U.S. Patent Application Publication No. 2008 / 0025980: SEQ ID NO: 20 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 15); SEQ ID NO: 21 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 16); SEQ ID NO: 22 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 17); SEQ ID NO: 23 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 18) and SEQ ID NO: 24 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 19).

[0096] In one embodiment, the targeting moiety comprises a light chain comprising SEQ ID NO: 18 (SEQ ID NO: 14) of U.S. Patent Application Publication No. 2008 / 0025980 and a heavy chain comprising SEQ ID NO: 22 (SEQ ID NO: 17) of U.S. Patent Application Publication No. 2008 / 0025980.

[0097] In certain embodiments, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0098] In some embodiments, the targeting moiety comprises the pd-l2-Fc fusion protein AMP-224, which is disclosed in International Publication Nos. 2010 / 027827 and 2011 / 066342. The entire disclosures of these patents are incorporated herein by reference. In such embodiments, the targeting moiety may comprise a targeting domain comprising SEQ ID NO:4 (SEQ ID NO:20) of International Publication No. 2010 / 027827 and / or a B7-DC fusion protein comprising SEQ ID NO:83 (SEQ ID NO:21) of International Publication No. 2010 / 027827.

[0099] In some embodiments, the targeting moiety comprises the peptide AUNP12 or any other peptide disclosed in U.S. Patent Application Publication No. 2011 / 0318373 or U.S. Patent No. 8,907,053. For example, the targeting moiety can comprise AUNP12 (i.e., Compound 8 or SEQ ID NO: 49 of U.S. Patent Application Publication No. 2011 / 0318373), which has the following sequence: [ka] (SEQ ID NO: 22).

[0100] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E3 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:23); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:24).

[0101] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E8 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:25); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:26).

[0102] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1H3 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1H3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:27); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:28).

[0103] In certain embodiments, the targeting moiety comprises a VHH directed against PD-1, e.g., as disclosed in U.S. Patent No. 8,907,065 and WO 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In exemplary embodiments, the VHH against PD-1 comprises SEQ ID NOS: 347-351 of U.S. Patent No. 8,907,065 (SEQ ID NO: 347 of U.S. Patent No. 8,907,065 (SEQ ID NO: 29); SEQ ID NO: 348 of U.S. Patent No. 8,907,065 (SEQ ID NO: 30); SEQ ID NO: 349 of U.S. Patent No. 8,907,065 (SEQ ID NO: 31); SEQ ID NO: 350 of U.S. Patent No. 8,907,065 (SEQ ID NO: 32); or SEQ ID NO: 351 of U.S. Patent No. 8,907,065 (SEQ ID NO: 33)).

[0104] In certain embodiments, the targeting moiety comprises any one of the anti-PD-1 antibodies or fragments thereof, as disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS: 25-29 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 34); SEQ ID NO: 26 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 35); SEQ ID NO: 27 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 36); SEQ ID NO: 28 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 37); U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 38); a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 29 (SEQ ID NO: 38) of U.S. Patent Application Publication No. 2011 / 0271358; and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 30 to 33 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 30 (SEQ ID NO: 39) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 31 (SEQ ID NO: 40) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 32 (SEQ ID NO: 41) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 33 (SEQ ID NO: 42) of U.S. Patent Application Publication No. 2011 / 0271358).

[0105] In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises one or more antibodies, or antibody fragments thereof, directed against PD-1 selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene Ltd.).

[0106] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties to PD-L1. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties that selectively bind a PD-L1 polypeptide. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprise one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a PD-L1 polypeptide.

[0107] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab), or a fragment thereof. MEDI4736 is selective for PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80 receptors. MEDI4736 and antigen-binding fragments thereof for use in the methods provided herein comprise a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. The sequence of MEDI4736 is disclosed in WO 2016 / 06272, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO:43); and / or a light chain comprising the amino acid sequence of (SEQ ID NO:44).

[0108] In exemplary embodiments, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 (SEQ ID NO:45) of WO 2016 / 06272; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 (SEQ ID NO:46) of WO 2016 / 06272.

[0109] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (also known as MPDL3280A, RG7446), or a fragment thereof. In an exemplary embodiment, atezolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO:47); and / or a light chain comprising the amino acid sequence of (SEQ ID NO:48).

[0110] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody avelumab (also known as MSB0010718C), or a fragment thereof. In an exemplary embodiment, atezolizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO:49); and / or a light chain comprising the amino acid sequence of (SEQ ID NO:50).

[0111] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105), or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, BMS-936559 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:51); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:52).

[0112] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3G10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3G10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:53); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:54).

[0113] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10A5, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 10A5, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:55); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:56).

[0114] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 5F8, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 5F8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:57); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:58).

[0115] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10H10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 10H10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:59); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:60).

[0116] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1B12, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 1B12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:61); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:62).

[0117] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 7H1, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 7H1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:63); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:64).

[0118] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 11E6, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 11E6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:65); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:66).

[0119] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 12B7, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 12B7 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:67); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:68).

[0120] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 13G4, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 13G4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:69); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:70).

[0121] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1E12, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E12, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:71); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:72).

[0122] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1F4, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. 1F4, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:73); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:74).

[0123] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2G11, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 2G11, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:75); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:76).

[0124] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3B6, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 3B6, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:77); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:78).

[0125] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3D10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738 and WO 2012 / 145493. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3D10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:79); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:80).

[0126] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in US Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS: 34-38 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 34 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 81); SEQ ID NO: 35 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 82); SEQ ID NO: 36 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 83); SEQ ID NO: 37 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 84); 358 (SEQ ID NO: 38 (SEQ ID NO: 85)); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 39 to 42 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 39 (SEQ ID NO: 86) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 40 (SEQ ID NO: 87) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 41 (SEQ ID NO: 88) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 42 (SEQ ID NO: 89) of U.S. Patent Application Publication No. 2011 / 0271358).

[0127] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.7A4, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 (SEQ ID NO:90) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:7 (SEQ ID NO:91) of WO 2011 / 066389.

[0128] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.9D10, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.9D10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (SEQ ID NO: 92) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (SEQ ID NO: 93) of WO 2011 / 066389.

[0129] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.14H9, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:22 (SEQ ID NO:94) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 (SEQ ID NO:95) of WO 2011 / 066389.

[0130] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.20A8, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.20A8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:32 (SEQ ID NO:96) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:37 (SEQ ID NO:97) of WO 2011 / 066389.

[0131] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.15G8, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3.15G8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:42 (SEQ ID NO:98) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:47 (SEQ ID NO:99) of WO 2011 / 066389.

[0132] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.18G1, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3.18G1, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:52 (SEQ ID NO:100) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:57 (SEQ ID NO:101) of WO 2011 / 066389.

[0133] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4OPT, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.7A4OPT, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:62 (SEQ ID NO:102) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:67 (SEQ ID NO:103) of WO 2011 / 066389.

[0134] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9OPT, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.14H9OPT, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:72 (SEQ ID NO:104) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:77 (SEQ ID NO:105) of WO 2011 / 066389.

[0135] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 061142, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein are selected from the group consisting of SEQ ID NOs: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of WO 2016 / 061142 (SEQ ID NO: 18 of WO 2016 / 061142 (SEQ ID NO: 106); SEQ ID NO: 30 of WO 2016 / 061142 (SEQ ID NO: 107); SEQ ID NO: 38 of WO 2016 / 061142 (SEQ ID NO: 108); SEQ ID NO: 46 of WO 2016 / 061142 (SEQ ID NO: 109); SEQ ID NO: 50 of WO 2016 / 061142 (SEQ ID NO: 110); SEQ ID NO: 54 of WO 2016 / 061142 (SEQ ID NO: 111); SEQ ID NO: 62 of WO 2016 / 061142 (SEQ ID NO: 112); SEQ ID NO: 70 of WO 2016 / 061142 (SEQ ID NO: 113) and SEQ ID NO: 78 of WO 2016 / 061142 (SEQ ID NO: 114)). and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO 2016 / 061142 (SEQ ID NO: 115); SEQ ID NO: 26 of WO 2016 / 061142 (SEQ ID NO: 116); SEQ ID NO: 34 of WO 2016 / 061142 (SEQ ID NO: 117); SEQ ID NO: 4 of WO 2016 / 061142 (SEQ ID NO: 118); 2 (SEQ ID NO:118); SEQ ID NO:58 of WO 2016 / 061142 (SEQ ID NO:119); SEQ ID NO:66 of WO 2016 / 061142 (SEQ ID NO:120); SEQ ID NO:74 of WO 2016 / 061142 (SEQ ID NO:121); SEQ ID NO:82 of WO 2016 / 061142 (SEQ ID NO:122) and SEQ ID NO:86 of WO 2016 / 061142 (SEQ ID NO:123)).

[0136] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 022630, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein are selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WO 2016 / 022630 (SEQ ID NO: 2 of WO 2016 / 022630 (SEQ ID NO: 124); SEQ ID NO: 6 of WO 2016 / 022630 (SEQ ID NO: 125); SEQ ID NO: 10 of WO 2016 / 022630 (SEQ ID NO: 126); SEQ ID NO: 11 of WO 2016 / 022630 (SEQ ID NO: 127); SEQ ID NO: 12 of WO 2016 / 022630 (SEQ ID NO: 128); SEQ ID NO: 13 of WO 2016 / 022630 (SEQ ID NO: 139); SEQ ID NO: 14 of WO 2016 / 022630 (SEQ ID NO: 149); SEQ ID NO: 15 of WO 2016 / 022630 (SEQ ID NO: 159); SEQ ID NO: 16 of WO 2016 / 022630 (SEQ ID NO: 169); SEQ ID NO: 17 of WO 2016 / 022630 (SEQ ID NO: 169)). SEQ ID NO: 14 (SEQ ID NO: 127); SEQ ID NO: 18 of WO 2016 / 022630 (SEQ ID NO: 128); SEQ ID NO: 22 of WO 2016 / 022630 (SEQ ID NO: 129); SEQ ID NO: 26 of WO 2016 / 022630 (SEQ ID NO: 130); SEQ ID NO: 30 of WO 2016 / 022630 (SEQ ID NO: 131); SEQ ID NO: 34 of WO 2016 / 022630 (SEQ ID NO: 132); SEQ ID NO: 38 of WO 2016 / 022630 (SEQ ID NO: 133); a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 42 of WO 2016 / 022630 (SEQ ID NO: 134); and SEQ ID NO: 46 of WO 2016 / 022630 (SEQ ID NO: 135); and / or SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 of WO 2016 / 022630 (SEQ ID NO: 4 of WO 2016 / 022630 (SEQ ID NO: 136); SEQ ID NO: 8 of WO 2016 / 022630 (SEQ ID NO: 137); SEQ ID NO: 12 from WO 2016 / 022630 (SEQ ID NO: 138); SEQ ID NO: 16 from WO 2016 / 022630 (SEQ ID NO: 139); SEQ ID NO: 20 from WO 2016 / 022630 (SEQ ID NO: 140); SEQ ID NO: 24 from WO 2016 / 022630 (SEQ ID NO: 141); SEQ ID NO: 28 from WO 2016 / 022630 (SEQ ID NO: 142); SEQ ID NO: 32 from WO 2016 / 022630 (SEQ ID NO: 143); SEQ ID NO: 36 from WO 2016 / 022630 (SEQ ID NO: 144);and a light chain comprising an amino acid sequence selected from SEQ ID NO: 40 of WO 2016 / 022630 (SEQ ID NO: 145); SEQ ID NO: 44 of WO 2016 / 022630 (SEQ ID NO: 146); and SEQ ID NO: 48 of WO 2016 / 022630 (SEQ ID NO: 147).

[0137] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2015 / 112900, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 38, 50, 82, and 86 of WO 2015 / 112900 (SEQ ID NO: 38 of WO 2015 / 112900 (SEQ ID NO: 148); SEQ ID NO: 50 of WO 2015 / 112900 (SEQ ID NO: 149); SEQ ID NO: 82 of WO 2015 / 112900 (SEQ ID NO: 150); and SEQ ID NO: 86 of WO 2015 / 112900 (SEQ ID NO: 151)); and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of WO 2015 / 112900 (WO 2015 / 112900 SEQ ID NO: 42 of WO 2015 / 112900 (SEQ ID NO: 152); SEQ ID NO: 46 of WO 2015 / 112900 (SEQ ID NO: 153); SEQ ID NO: 54 of WO 2015 / 112900 (SEQ ID NO: 154); SEQ ID NO: 58 of WO 2015 / 112900 (SEQ ID NO: 155); SEQ ID NO: 62 of WO 2015 / 112900 (SEQ ID NO: 156); SEQ ID NO: 66 of WO 2015 / 112900 (SEQ ID NO: 157); SEQ ID NO: 70 of WO 2015 / 112900 (SEQ ID NO: 158); SEQ ID NO: 74 of WO 2015 / 112900 (SEQ ID NO: 159); and SEQ ID NO: 78 of WO 2015 / 112900 (SEQ ID NO: 160).

[0138] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2010 / 077634 and U.S. Patent No. 8,217,149. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, an anti-PD-L1 or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:20 (SEQ ID NO:161) of WO 2010 / 077634; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:21 (SEQ ID NO:162) of WO 2010 / 077634.

[0139] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies obtainable from the hybridomas available under CNCM Accession Nos. CNCM I-4122, CNCM I-4080, and CNCM I-4081, as disclosed in U.S. Patent Application Publication No. 20120039906. The entire disclosures of these patents are incorporated herein by reference.

[0140] In some embodiments, the targeting moiety comprises a VHH directed against a PD-L1 antibody, e.g., as disclosed in U.S. Patent No. 8,907,065 and WO 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the VHH against PD-L1 comprises SEQ ID NOS: 394-399 of U.S. Patent No. 8,907,065 (SEQ ID NO: 394 of U.S. Patent No. 8,907,065 (SEQ ID NO: 163); SEQ ID NO: 395 of U.S. Patent No. 8,907,065 (SEQ ID NO: 164); SEQ ID NO: 396 of U.S. Patent No. 8,907,065 (SEQ ID NO: 165); SEQ ID NO: 397 of U.S. Patent No. 8,907,065 (SEQ ID NO: 166); SEQ ID NO: 398 of U.S. Patent No. 8,907,065 (SEQ ID NO: 167); SEQ ID NO: 399 of U.S. Patent No. 8,907,065 (SEQ ID NO: 168)).

[0141] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties for PD-L2. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties that selectively bind a PD-L2 polypeptide. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprise one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a PD-L2 polypeptide.

[0142] In some embodiments, the targeting moiety comprises a VHH directed against PD-L2, e.g., as disclosed in U.S. Patent No. 8,907,065 and WO 2008 / 071447, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, the VHH against PD-L2 comprises SEQ ID NOs:449-455 of U.S. Patent No. 8,907,065 (SEQ ID NO:449 of U.S. Patent No. 8,907,065 (SEQ ID NO:169); SEQ ID NO:450 of U.S. Patent No. 8,907,065 (SEQ ID NO:170); SEQ ID NO:451 of U.S. Patent No. 8,907,065 (SEQ ID NO:171); SEQ ID NO:452 of U.S. Patent No. 8,907,065 (SEQ ID NO:172); SEQ ID NO:453 of U.S. Patent No. 8,907,065 (SEQ ID NO:173); SEQ ID NO:454 of U.S. Patent No. 8,907,065 (SEQ ID NO:174); and SEQ ID NO:455 of U.S. Patent No. 8,907,065 (SEQ ID NO:175)).

[0143] In some embodiments, the targeting moiety comprises any one of the anti-PD-L2 antibodies disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS: 43-47 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 176); SEQ ID NO: 44 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 177); SEQ ID NO: 45 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 178); SEQ ID NO: 46 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 179); U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 179); No. 8 (SEQ ID NO: 47 (SEQ ID NO: 180)); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 48-51 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 48 (SEQ ID NO: 181) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 49 (SEQ ID NO: 182) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 50 (SEQ ID NO: 183) of U.S. Patent Application Publication No. 2011 / 0271358; and SEQ ID NO: 51 (SEQ ID NO: 184) of U.S. Patent Application Publication No. 2011 / 0271358).

[0144] In various embodiments, the targeting moieties of the present invention align with any of the sequences disclosed herein by at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% , at least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or may comprise sequences targeting PD-1, PD-L1, and / or PD-L2 that are about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% sequence identity).

[0145] In various embodiments, targeting moieties of the invention can include any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences that target PD-1, PD-L1, and / or PD-L2 as disclosed herein.

[0146] Additional antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind or target PD-1, PD-L1, and / or PD-L2 are described in WO 2011 / 066389, U.S. Patent Application Publication No. 2008 / 0025980, U.S. Patent Application Publication No. 2013 / 0034559, U.S. Patent No. 8,779,108, U.S. Patent Application Publication No. No. 2014 / 0356353, U.S. Patent No. 8,609,089, U.S. Patent Application Publication No. 2010 / 028330, U.S. Patent Application Publication No. 2012 / 0114649, WO 2010 / 027827, WO 2011 / 066342, U.S. Patent No. 8,907,065, WO 2016 / 062722, WO 2009 / 101611, WO 2010 / 0278 27, WO 2011 / 066342, WO 2007 / 005874, WO 2001 / 014556, U.S. Patent Application Publication No. 2011 / 0271358, WO 2010 / 036959, WO 2010 / 077634, U.S. Patent No. 8,217,149, U.S. Patent Application Publication No. 2012 / 0039906, WO 2012 / 145493, U.S. and WO 2015 / 112900, the entire disclosures of which are incorporated herein by reference.

[0147] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for T cells, e.g., mediated by targeting to CD8, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for CD8 on T cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0148] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for T cells, e.g., mediated by targeting to CD4, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for CD4 on T cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0149] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for T cells, e.g., mediated by targeting to CD3, CXCR3, CCR4, CCR9, CD70, CD103, or one or more immune checkpoint markers, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 interferon or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for CD3 on T cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0150] In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has one or more targeting moieties for CD3 expressed on T cells. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has one or more targeting moieties that selectively bind a CD3 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a CD3 polypeptide.

[0151] In one embodiment, the targeting moiety comprises the anti-CD3 antibody muromonab-CD3 (also known as Orthoclone OKT3), or a fragment thereof. Muromonab-CD3 is disclosed in U.S. Patent No. 4,361,549 and Wilde et al. (1996) 51:865-894, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, muromonab-CD3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO: 185); and / or a light chain comprising the amino acid sequence of (SEQ ID NO: 186).

[0152] In some embodiments, the targeting moiety comprises the anti-CD3 antibody otelixizumab, or a fragment thereof. Otelixizumab is disclosed in U.S. Patent Application Publication No. 20160000916 and Chatenoud et al. (2012) 9:372-381. The entire disclosures of these documents are incorporated herein by reference. In an exemplary embodiment, otelixizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 187; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 188.

[0153] In some embodiments, the targeting moiety comprises the anti-CD3 antibody teplizumab (also known as MGA031 and hOKT3γ1(Ala-Ala)), or a fragment thereof. Teplizumab is disclosed in Chatenoud et al. (2012) 9:372-381, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, teplizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 189; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 190.

[0154] In some embodiments, the targeting moiety comprises the anti-CD3 antibody visilizumab (also known as Nuvion®; HuM291), or a fragment thereof. Visilizumab is disclosed in U.S. Pat. No. 5,834,597 and WO 2004052397 and Cole et al., Transplantation (1999) 68:563-571, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, visilizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 191; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 192.

[0155] In some embodiments, the targeting moiety comprises the anti-CD3 antibody foralarumab (also known as NI-0401), or a fragment thereof. In various embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent Application Publication No. 2014 / 0193399, U.S. Patent No. 7,728,114, U.S. Patent Application Publication No. 2010 / 0183554, and U.S. Patent No. 8,551,478, the entire disclosures of which are incorporated herein by reference.

[0156] In exemplary embodiments, an anti-CD3 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 2 and 6 of U.S. Patent No. 7,728,114 (SEQ ID NO: 2 of U.S. Patent No. 7,728,114 (SEQ ID NO: 193) and SEQ ID NO: 6 of U.S. Patent No. 7,728,114 (SEQ ID NO: 194)); and / or a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 4 and 8 of U.S. Patent No. 7,728,114 (SEQ ID NO: 4 of U.S. Patent No. 7,728,114 (SEQ ID NO: 195) and SEQ ID NO: 8 of U.S. Patent No. 7,728,114 (SEQ ID NO: 196)).

[0157] In certain embodiments, the targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 of U.S. Patent Application Publication No. 7,728,114; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4 of U.S. Patent Application Publication No. 7,728,114. In certain embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent Application Publication No. 2016 / 0168247, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOs:6-9 of U.S. Patent Application Publication No. 2016 / 0168247 (SEQ ID NO:6 (SEQ ID NO:197) of U.S. Patent Application Publication No. 2016 / 0168247; SEQ ID NO:7 (SEQ ID NO:198) of U.S. Patent Application Publication No. 2016 / 0168247; SEQ ID NO:8 (SEQ ID NO:199) of U.S. Patent Application Publication No. 2016 / 0168247 No. 2016 / 0168247 (SEQ ID NO: 10 (SEQ ID NO: 201); SEQ ID NO: 11 (SEQ ID NO: 202); SEQ ID NO: 12 (SEQ ID NO: 203)).

[0158] In some embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent Application Publication No. 2015 / 0175699, the entire contents of which are incorporated herein by reference. In exemplary embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO:9 (SEQ ID NO:204) of U.S. Patent Application Publication No. 2015 / 0175699; and / or a light chain comprising an amino acid sequence selected from SEQ ID NO:10 (SEQ ID NO:205) of U.S. Patent Application Publication No. 2015 / 0175699.

[0159] In one embodiment, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent No. 8,784,821, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, and 114 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 2 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 206); SEQ ID NO: 18 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 207); SEQ ID NO: 34 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 208); SEQ ID NO: 50 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 209); SEQ ID NO: 66 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 210); SEQ ID NO: 82 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 211); SEQ ID NO: 98 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 212); and SEQ ID NO: 114 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 213)). and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106 and 122 of U.S. Patent No. 8,784,821 (SEQ ID NO: 10 of U.S. Patent No. 8,784,821 (SEQ ID NO: 214); SEQ ID NO: 26 of U.S. Patent No. 8,784,821 (SEQ ID NO: 215); SEQ ID NO: 42 of U.S. Patent No. 8,784,821 (SEQ ID NO: 216); SEQ ID NO: 58 of U.S. Patent No. 8,784,821 (SEQ ID NO: 217); SEQ ID NO: 74 of U.S. Patent No. 8,784,821 (SEQ ID NO: 218); SEQ ID NO: 90 of U.S. Patent No. 8,784,821 (SEQ ID NO: 219); SEQ ID NO: 106 of U.S. Patent No. 8,784,821 (SEQ ID NO: 220); and SEQ ID NO: 122 of U.S. Patent No. 8,784,821 (SEQ ID NO: 221)).

[0160] In certain embodiments, the targeting moiety comprises any one of the anti-CD3 binding constructs disclosed in U.S. Patent Application Publication No. 2015 / 0118252, the entire contents of which are incorporated herein by reference. In exemplary embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 6 and 86 of U.S. Patent Application Publication No. 2015 / 0118252 (SEQ ID NO: 6 of U.S. Patent Application Publication No. 2015 / 0118252 (SEQ ID NO: 222) and SEQ ID NO: 86 of U.S. Patent Application Publication No. 2015 / 0118252 (SEQ ID NO: 223)); and / or a light chain comprising an amino acid sequence selected from SEQ ID NO: 3 of U.S. Patent Application Publication No. 2015 / 0175699 (SEQ ID NO: 3 of U.S. Patent Application Publication No. 2015 / 0118252 (SEQ ID NO: 224)).

[0161] In certain embodiments, the targeting moiety comprises any one of the anti-CD3 binding proteins disclosed in U.S. Patent Application Publication No. 2016 / 0039934, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from SEQ ID NOS: 6-9 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 6 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 225); SEQ ID NO: 7 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 226); SEQ ID NO: 8 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 227); and SEQ ID NO: 9 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 228)). ); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 1-4 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 1 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 229); SEQ ID NO: 2 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 230); SEQ ID NO: 3 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 231); and SEQ ID NO: 4 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 232)).

[0162] In various embodiments, the targeting moieties of the present invention align with any of the sequences disclosed herein by at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% , at least about 97%, at least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or The CD3 targeting sequence may comprise a sequence that is about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% sequence identity).

[0163] In various embodiments, the targeting moieties of the present invention may comprise any combination of CD3-targeting heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences disclosed herein. In various embodiments, targeting moieties of the present invention may comprise any of the heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences of CD3-specific antibodies, including, but not limited to, X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, WT31, and F101.01. These CD3-specific antibodies are well known in the art and are described, inter alia, in Tunnacliffe (1989), Int. Immunol. 1, 546-550, the entire disclosure of which is incorporated herein by reference.

[0164] Additional antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind or target CD3 are disclosed in U.S. Patent Application Publication No. 2016 / 0000916, U.S. Patent Nos. 4,361,549, 5,834,597, 6,491,916, 6,406,696, 6,143,297, 6,750,325, and WO 2004 / 052397, the entire disclosures of which are incorporated herein by reference.

[0165] In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for T cells, e.g., mediated by targeting to PD-1, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 or a variant thereof).

[0166] By way of non-limiting example, in various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention (i) comprise a targeting moiety for B cells mediated by targeting to, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, or CDw150, and (ii) the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof). In certain embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety directed against CD20.

[0167] In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for B cells, e.g., mediated by targeting to CD19, CD20, or CD70, and (ii) the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0168] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for B cells, e.g., mediated by targeting to CD20, and (ii) the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof). In certain embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for CD20 on B cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells. For example, in some embodiments, the CD20 targeting moiety is a recombinant heavy chain-only antibody (VHH) having the following sequence: QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS (SEQ ID NO: 288).

[0169] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention may comprise (i) a chimeric protein complex, such as, for example, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-EpsilonRII, LMIR6 / CD300LE, Fc-γRl / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NKG2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IR TA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM and (ii) the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0170] In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for NK cells, e.g., mediated by targeting to Kir1 alpha, DNAM-1, or CD64, and (ii) the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0171] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for NK cells, e.g., mediated by targeting to KIR1, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for KIR1 on NK cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0172] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for NK cells, e.g., mediated by targeting to TIGIT or KIR1, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for TIGIT on NK cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0173] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention may comprise (i) a chimeric protein comprising, for example, CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, SREC-II, LIMPII, ISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL12 / IL23p40, 4-Amino-1, 8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4 Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, ClqR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBCCR7, LMIR6 / CD300LE, CD40 / TNFRS F5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLl, CD2F-10 / SLAMF9, OsteoactivinGPNMB, Chern23, PD-L2, CLEC-1, RP105, CLEC-2, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, DEP-1 / CD148, SIGNR4, DLEC, SLAM, EMMPRIN / and (ii) the targeting moiety is directed to dendritic cells mediated by targeting to CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, or vanilloid R1, and the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0174] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for dendritic cells, e.g., mediated by targeting to CLEC-9A, DC-SIGN, CD64, CLEC-4A, or DEC205, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for Clec9A on dendritic cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0175] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for dendritic cells, e.g., mediated by targeting to Clec9A, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for Clec9A on dendritic cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0176] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for dendritic cells, e.g., mediated by targeting to XCR1, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for XCR1 on dendritic cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0177] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety directed against dendritic cells, e.g., mediated by targeting to RANK, and (ii) the targeting moiety is directed against tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety directed against RANK on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0178] By way of non-limiting example, in various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention may comprise (i) chimeric proteins, such as, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin α4 / CD49d, BLAME / SLAMF8, integrin αX / CDllc, CCL6 / C10, integrin β2 / CD18, CD155 / PVR, integrin β3 / CD61, CD31 / PECAM-1, latexin, CD36 / SR-B3, Leukemia, myeloma ... Cotrien B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, endoglin / CD105, osteoactivin / GPNMB, Fc -γRI / CD64, osteopontin, Fc-γRIIB / CD32b, PD-L2, Fc-γRIIC / CD32c, Siglec-3 / CD33, Fc-γRIIA / CD32a, SIGNR1 / CD209, Fc-γRIII / CD16, SLAM, GM-CSFRα , TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γRl, TLR4, IFN-γR2, TREM-l, IL-lRII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF 4, IL-10Rα, ALCAM, IL-10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, ClqR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / CDllb, CCR8, integrin αX / CDllc, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45Leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSFR, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD105, NCAM-L1, Fc-γRI / CD64, PSGL-1, Fc-γRIIII (ii) the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof), and (iii) the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0179] In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for monocytes / macrophages, e.g., mediated by targeting to B7-H1, CD31 / PECAM-1, CD163, CCR2, or the macrophage mannose receptor CD206, and (ii) the targeting moiety is directed to tumor cells in conjunction with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0180] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention (i) has a targeting moiety for monocytes / macrophages, e.g., mediated by targeting to SIRP1a, and (ii) the targeting moiety is directed to tumor cells in conjunction with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety for SIRP1a on macrophages and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0181] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention comprise one or more targeting moieties directed against one or more of checkpoint markers, e.g., PD-1 / PD-L1 or PD-L2, CD28 / CD80 or CD86, CTLA4 / CD80 or CD86, ICOS / ICOSL or B7RP1, BTLA / HVEM, KIR, LAG3, CD137 / CD137L, OX40 / OX40L, CD27, CD40L, TIM3 / Gal9, CD47, CD70, and A2aR. In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has (i) a targeting moiety directed to a checkpoint marker on T cells, e.g., PD-1, and (ii) a targeting moiety directed to tumor cells, e.g., PD-L1 or PD-L2, in combination with any signal transduction agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety directed to PD-1 on T cells and a second targeting moiety directed to PD-L1 on tumor cells. In another embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention has a targeting moiety directed to PD-1 on T cells and a second targeting moiety directed to PD-L2 on tumor cells.

[0182] In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises two or more targeting moieties directed to the same or different immune cells. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises (i) one or more targeting moieties for immune cells selected from T cells, B cells, dendritic cells, macrophages, NK cells, or a subset thereof, and (ii) one or more targeting moieties for the same or different immune cells selected from T cells, B cells, dendritic cells, macrophages, NK cells, or a subset thereof, together with any signaling substance described herein (e.g., IFNα1 or a variant thereof).

[0183] In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for T cells and one or more targeting moieties for the same or different T cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for T cells and one or more targeting moieties for B cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for T cells and one or more targeting moieties for dendritic cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for T cells and one or more targeting moieties for macrophages. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for T cells and one or more targeting moieties for NK cells. For example, in an exemplary embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can include a targeting moiety for CD8 and a targeting moiety for Clec9A. In another exemplary embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can include a targeting moiety for CD8 and a targeting moiety for CD3. In another exemplary embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can include a targeting moiety for CD8 and a targeting moiety for PD-1.

[0184] In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for B cells and one or more targeting moieties for the same or different B cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for B cells and one or more targeting moieties for T cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for B cells and one or more targeting moieties for dendritic cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for B cells and one or more targeting moieties for macrophages. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for B cells and one or more targeting moieties for NK cells.

[0185] In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for the same or different dendritic cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for T cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for B cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for macrophages. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for NK cells.

[0186] In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for macrophages and one or more targeting moieties for the same or different macrophages. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for macrophages and one or more targeting moieties for T cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for macrophages and one or more targeting moieties for B cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for macrophages and one or more targeting moieties for dendritic cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for macrophages and one or more targeting moieties for NK cells.

[0187] In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for NK cells and one or more targeting moieties for the same or different NK cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for NK cells and one or more targeting moieties for T cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for NK cells and one or more targeting moieties for B cells. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for NK cells and one or more targeting moieties for macrophages. In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for NK cells and one or more targeting moieties for dendritic cells.

[0188] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety directed to a tumor cell and a second targeting moiety directed to the same or a different tumor cell. In such embodiments, the targeting moiety can bind to any of the tumor antigens described herein.

[0189] In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention are expressed in adipocytes (e.g., white adipocytes, brown adipocytes), liver lipid cells, liver cells, kidney cells (e.g., kidney parietal cells, kidney salivary gland, mammary gland, etc.), duct cells (e.g., seminal vesicles, prostate, etc.), intestinal brush border cells (with microvilli), exocrine gland striate duct cells, gallbladder epithelial cells, small efferent tract non-ciliated cells, epididymal principal cells, epididymal basal cells, endothelial cells, ameloblast epithelial cells (tooth enamel secreting cells), ear cells, and the like. Crescentic epithelial cells of the vestibular system (secreting proteoglycans), interdental epithelial cells of the organ of Corti (secreting the tectorial membrane that covers hair cells), loose connective tissue fibroblasts, corneal fibroblasts (keratocytes), tendon fibroblasts, bone marrow reticular fibroblasts, non-epithelial fibroblasts, pericytes, nucleus pulposus cells of the intervertebral disc, cementoblasts / cementocytes (secreting root bone-like Ivan cells), odontoblasts / odontocytes (secreting tooth dentin), hyaline chondrocytes, fibrochondrocytes, elastic cartilage cells Follicles, osteoblasts / osteocytes, bone precursor cells (osteoblast stem cells), vitreous cells of the vitreous body of the eye, stellate cells of the perilymphatic space of the ear, hepatic stellate cells (Ito cells), pancreatic stellate cells, skeletal muscle cells, satellite cells, cardiac muscle cells, smooth muscle cells, myoepithelial cells of the iris, myoepithelial cells of exocrine glands, exocrine secretory epithelial cells (e.g., salivary gland cells, mammary gland cells, lacrimal gland cells, sweat gland cells, sebaceous gland cells, prostate cells, gastric gland cells, pancreatic acinar cells, lung cells), hormone-secreting cells (e.g., pituitary cells, neurosecretory cells, intestinal and airway cells, thyroid cells, parathyroid cells) , adrenal gland cells, Leydig cells of the testes, pancreatic islet cells), keratinizing epithelial cells, moist stratified barrier epithelial cells, neural cells (e.g., sensory transducer cells, autonomic neuron cells, sensory organ and peripheral neural support cells, and central nervous system neurons, and glial cells such as interneurons, principal cells, astrocytes, oligodendrocytes, and ependymal cells), and one or more targeting moieties having a recognition domain that binds to a target of interest (e.g., antigen, receptor), including those found on one or more cells selected from the group consisting of: epithelial cells, cornified epithelial cells, moist stratified barrier epithelial cells, neural cells (e.g., sensory transducer cells, autonomic neuron cells, sensory organ and peripheral neural support cells, and central nervous system neurons, and glial cells such as interneurons, principal cells, astrocytes, oligodendrocytes, and ependymal cells).

[0190] Targeting Part Format In some embodiments, the targeting moiety of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention is a protein-based substance capable of specific binding, such as an antibody or a derivative thereof. In certain embodiments, the targeting moiety comprises an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain contains one variable region (V L ) and one constant region (C L ). The variable regions determine the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity-determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs are termed CDR1, CDR2, and CDR3 and contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0191] In some embodiments, the targeting moiety comprises an antibody derivative or format. In some embodiments, the targeting moiety of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention is a single domain antibody, a recombinant heavy chain antibody (VHH), a single chain antibody (scFv), a shark heavy chain antibody (VNAR), a microprotein (cysteine ​​knot protein, knottin), a DARPin; a tetranectin; an affibody; a transbody; anticalin; an adnectin; an affilin; a microbody; a peptide aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; a shrimpbody; a phylomer; an armadillo repeat protein; a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troibody, a pepbody, a vaccibody, a unibody; an affimer, a duobody, an Fv, a Fab, a Fab', a F(ab')2, a peptidomimetic molecule, or a synthetic molecule. These include U.S. Pat. No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Pat. No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Pat. No. 7,250,297, U.S. Pat. No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Pat. No. 7,838,629, U.S. Pat. No. 7,186,524, U.S. Pat. Nos. 6,004,746, 5,475,096, 2004 / 146938, 2004 / 157209, 6,994,982, 6,794,144, 2010 / 239633, 7,803,907, 2010 / 119446, and / or 7,166,697. See also Storz MAbs. 2011 May-Jun;3(3):310-317.

[0192] In one embodiment, the targeting moiety comprises a VHH derived from an organism that produces VHH antibodies, such as a camelid or shark, or a single-domain antibody, such as a designed VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies derived from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). VHHs are commercially available under the registered trademarks NANOBODY or NANOBODIES.

[0193] In certain embodiments, the targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized or camelized VHH.

[0194] In some embodiments, the VHH is a fully human VHH. H In some embodiments, the fully human VH domain, e.g., a humabody, is monovalent, bivalent, or trivalent. In some embodiments, the fully human VH domain, e.g., a humabody, is monospecific or multispecific, such as monospecific, bispecific, or trispecific. Exemplary fully human VH domains include: H Domains, such as the human body, are described, for example, in WO 2016 / 113555 and WO 2016 / 113557, the entire disclosures of which are incorporated herein by reference.

[0195] In various embodiments, the targeting moiety of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention is a protein-based substance capable of specifically binding to a cellular receptor, such as a natural ligand for the cellular receptor. In various embodiments, the cellular receptor can be found on one or more immune cells, including, but 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, dendritic cells, or a subset thereof. In some embodiments, the cellular receptor is found on megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof.

[0196] In some embodiments, the targeting moiety is a natural ligand, such as a chemokine. Examples of chemokines that can be included in the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention include, but are not limited to, CCL1, CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL29, CCL30, CCL31, CCL32, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39, CCL40, CCL41, CCL42, CCL43, CCL44, CCL45, CCL46, CCL47, CCL48, CCL49, CCL50, CCL51, CCL52, CCL53, CCL54, CCL55, CCL56, CCL57, CCL58, CCL59, CCL60, CCL61, CCL62, CCL63, CCL64, CCL65, CCL66, CCL67, CCL68, CCL69, CCL70, CCL71, CCL72, CCL73, CCL74, CCL75, CCL76, CCL77, CCL78, CCL79, CCL80, CCL81, CCL82, CCL83, CCL84, CCL85, CCL86, CCL87, CCL88, CCL89, CCL90, CCL91, Examples of targeting moieties include L22, CCL23, CCL24, CLL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, HCC-4, and LDGF-PBP. In an exemplary embodiment, the targeting moiety can be XCL1, a chemokine that recognizes and binds to the dendritic cell receptor XCR1. In another exemplary embodiment, the targeting moiety is CCL1, a chemokine that recognizes and binds to CCR8. In another exemplary embodiment, the targeting moiety is CCL2, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL3, a chemokine that recognizes and binds to CCR1, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL4, a chemokine that recognizes and binds to CCR1 or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL5, a chemokine that recognizes and binds to CCR1 or CCR3 or CCR4 or CCR5. In another exemplary embodiment, the targeting moiety is CCL6, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL7, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL8, a chemokine that recognizes and binds to CCR1 or CCR2 or CCR2B or CCR5 or CCR9.In another exemplary embodiment, the targeting moiety is CCL9, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL10, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL11, a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL13, a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL14, a chemokine that recognizes and binds to CCR1 or CCR9. In another exemplary embodiment, the targeting moiety is CCL15, a chemokine that recognizes and binds to CCR1 or CCR3. In another exemplary embodiment, the targeting moiety is CCL16, a chemokine that recognizes and binds to CCR1, CCR2, CCR5, or CCR8. In another exemplary embodiment, the targeting moiety is CCL17, a chemokine that recognizes and binds to CCR4. In another exemplary embodiment, the targeting moiety is CCL19, a chemokine that recognizes and binds to CCR7. In another exemplary embodiment, the targeting moiety is CCL20, a chemokine that recognizes and binds to CCR6. In another exemplary embodiment, the targeting moiety is CCL21, a chemokine that recognizes and binds to CCR7. In another exemplary embodiment, the targeting moiety is CCL22, a chemokine that recognizes and binds to CCR4. In another exemplary embodiment, the targeting moiety is CCL23, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL24, a chemokine that recognizes and binds to CCR3. In another exemplary embodiment, the targeting moiety is CCL25, a chemokine that recognizes and binds to CCR9. In another exemplary embodiment, the targeting moiety is CCL26, a chemokine that recognizes and binds to CCR3. In another exemplary embodiment, the targeting moiety is CCL27, a chemokine that recognizes and binds to CCR10.In another exemplary embodiment, the targeting moiety is CCL28, a chemokine that recognizes and binds to CCR3 or CCR10. In another exemplary embodiment, the targeting moiety is CXCL1, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL2, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL3, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL4, a chemokine that recognizes and binds to CXCR3B. In another exemplary embodiment, the targeting moiety is CXCL5, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL6, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL8, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL9, a chemokine that recognizes and binds to CXCR3. In another exemplary embodiment, the targeting moiety is CXCL10, a chemokine that recognizes and binds to CXCR3. In another exemplary embodiment, the targeting moiety is CXCL11, a chemokine that recognizes and binds to CXCR3 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL12, a chemokine that recognizes and binds to CXCR4 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL13, a chemokine that recognizes and binds to CXCR5. In another exemplary embodiment, the targeting moiety is CXCL16, a chemokine that recognizes and binds to CXCR6. In another exemplary embodiment, the targeting moiety is LDGF-PBP, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is XCL2, a chemokine that recognizes and binds to XCR1. In another exemplary embodiment, the targeting moiety is CX3CL1, a chemokine that recognizes and binds to CX3CR1.

[0197] In some embodiments, the targeting moiety is a natural ligand, such as FMS-like tyrosine kinase 3 ligand (Flt3L) or a truncated region thereof (e.g., a region capable of binding Flt3). In some embodiments, the targeting moiety is the extracellular domain of Flt3L. In some embodiments, the targeting moiety comprises a Flt3L domain, wherein the Flt3L domain is a single-chain dimer, optionally wherein one Flt3L domain is linked to another Flt3L domain via one or more linkers, wherein the linkers are flexible linkers. In some embodiments, the targeting moiety of the present invention comprises a Flt3L domain, wherein the Flt3L domain is a single-chain dimer and an Fc domain, optionally comprising 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. In some embodiments, the targeting moiety recognizes CD20. In some embodiments, the targeting moiety recognizes PD-L1. In some embodiments, the targeting moiety recognizes Clec9A.

[0198] In various embodiments, the chimeric protein complexes, such as the chimeric protein or Fc-based chimeric protein complexes, of the present invention contain various combinations of targeting moieties. In an exemplary embodiment, the chimeric protein complexes, such as the chimeric protein or Fc-based chimeric protein complexes of the present invention, can contain two targeting moieties, both of which are antibodies or derivatives thereof. In another exemplary embodiment, the chimeric protein complexes, such as the chimeric protein or Fc-based chimeric protein complexes of the present invention, can contain two targeting moieties, both of which are natural ligands for cell receptors. In a further exemplary embodiment, the chimeric protein complexes, such as the chimeric protein or Fc-based chimeric protein complexes of the present invention, can contain two targeting moieties, one of which is an antibody or a derivative thereof, and the other of which is a natural ligand for cell receptors.

[0199] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention functionally modulates (including, but not limited to, partially or completely neutralizes) a target of interest (e.g., an antigen, receptor), e.g., substantially inhibits, reduces, or neutralizes the biological effect of the antigen. For example, various recognition domains can be directed to one or more tumor antigens that actively suppress or have the ability to suppress the immune system of, for example, a tumor-bearing patient. For example, in some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention functionally modulates one or more immunosuppressive signals (e.g., checkpoint inhibitors), such as TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2. For example, in some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention is genetically modified to disrupt, block, reduce, and / or inhibit the transmission of immunosuppressive signals, including, but not limited to, the binding of PD-1 to PD-L1 or PD-L2, and / or the binding of CTLA-4 to one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A.

[0200] In various embodiments, the recognition domain of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention binds but does not functionally modulate a target of interest (e.g., an antigen, a receptor); e.g., the recognition domain is or resembles a binding antibody. For example, in various embodiments, the recognition domain only targets an antigen or receptor without substantially inhibiting, reducing, or functionally modulating the biological action of the antigen or receptor. For example, some of the above-described antibody formats (e.g., compared to whole antibodies) have the ability to target difficult-to-access epitopes, providing a broader range of specific binding surfaces. In various embodiments, the recognition domain binds to an epitope that is physically distant from the antigen or receptor site (e.g., the active site of the antigen) important for its biological activity.

[0201] Such non-neutralizing binding is used in various embodiments of the present invention, including methods in which chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention are used to directly or indirectly recruit active immune cells to a required site via an effector antigen. For example, in various embodiments, chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can be used to directly or indirectly recruit cytotoxic T cells to tumor cells via CD8 in methods of shrinking or eliminating tumors (e.g., chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, can include an anti-CD8 recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit CD8-expressing cytotoxic T cells but not functionally modulate CD8 activity. In contrast, in these embodiments, CD8 signaling is an important part of the tumor reduction or elimination process. As a further example, in various embodiments, chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention are used to directly or indirectly recruit dendritic cells (DCs) via Clec9A (e.g., chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, can include an anti-Clec9A recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit Clec9A-expressing DCs but not functionally modulate Clec9A activity. In contrast, in these embodiments, Clec9A signaling is an important part of tumor reduction or elimination.

[0202] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention binds to XCR1 on dendritic cells, for example. For example, the recognition domain, in some embodiments, comprises all or a portion of XCL1 or a non-neutralizing anti-XCR1 agent.

[0203] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention binds to an immunomodulatory antigen (e.g., an immunostimulatory antigen or an immunosuppressive antigen). In various embodiments, the immunomodulatory antigen is one or more of 4-1BB, OX-40, HVEM, GITR, CD27, CD28, CD30, CD40, ICOS ligand; OX-40 ligand, LIGHT (CD258), GITR ligand, CD70, B7-1, B7-2, CD30 ligand, CD40 ligand, ICOS, ICOS ligand, CD137 ligand, and TL1A. In various embodiments, the immunostimulatory antigen is expressed on tumor cells. In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention binds but does not functionally regulate such immunostimulatory antigens, thus allowing the recruitment of cells expressing these antigens without reducing or losing potential tumor reduction or elimination capabilities.

[0204] In various embodiments, the recognition domains of the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can be associated with chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, that include two recognition domains with neutralizing activity, or that include two recognition domains with non-neutralizing (e.g., binding) activity, or that include one recognition domain with neutralizing activity and one recognition domain with non-neutralizing (e.g., binding) activity.

[0205] 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).

[0206] 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.

[0207] 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.

[0208] In some embodiments, the Fc domain of 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.

[0209] 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.

[0210] 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)).

[0211] 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.

[0212] In some embodiments, the Fc domain comprises a FALA mutation that reduces or eliminates an effector function of the Fc domain, for example, in some embodiments, the FALA mutation comprises F234A and L235A substitutions in human IgG4.

[0213] 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.

[0214] 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.

[0215] In some embodiments, mutations to the Fc domain promote chain pairing of the Fc domain, hi some embodiments, chain pairing is promoted by ionic pairing (a / k / a charge pairs, ionic bonds, or charged residue pairs).

[0216] 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.

[0217] 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]

[0218] 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]

[0219] 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).

[0220] 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] TIFF2025072580000007.tif94162

[0221] 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] TIFF2025072580000009.tif242160TIFF2025072580000010.tif242162TIFF2025072580000011.tif242160

[0222] 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] TIFF2025072580000013.tif242161TIFF2025072580000014.tif163162

[0223] 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.

[0224] 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.

[0225] 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 / structure, in which the targeting moiety and the signal transduction agent, e.g., IFNα1, are not found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention.

[0226] In some embodiments, the Fc domain includes or begins with the core hinge region of wild-type human IgG1, which region includes the sequence Cys-Pro-Pro-Cys. In some embodiments, the Fc domain also includes the upper hinge, or a portion thereof (e.g., DKTHTCPPC (see WO2009053368), EPKSCDKTHTCPPC, or EPKSSDKTHTCPPC (see Lo et al., Protein Engineering vol. 11 no. 6 pp. 495-500, 1998)).

[0227] 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 transducer (SA) disclosed herein, e.g., IFNα1, and at least one targeting moiety (TM) disclosed herein.

[0228] It is understood that an Fc-based chimeric protein complex of the present invention may comprise two fusion proteins, each containing an Fc domain.

[0229] 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 / structure. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a cis orientation / structure.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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).

[0234] In some embodiments in which two or more targeting moieties are present in the heterodimeric protein complex described herein, the targeting moieties are found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties must 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).

[0235] 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).

[0236] In some embodiments, the heterodimeric Fc-based chimeric protein complex does not comprise a signaling agent, eg, IFNα1, and a targeting moiety on a single polypeptide.

[0237] 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.

[0238] 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, e.g., IFNα1, thus allowing for the creation of proteins containing only one copy of the targeting domain and, similarly, only one type of signal transduction agent, e.g., IFNα1 (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 a single signal transduction agent, e.g., IFNα1, can mitigate molecular "crowding" and potential interference with avidity-mediated induction or restoration 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., IFNα1 (e.g., a wild-type signal transduction agent, e.g., wild-type IFNα1) 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.

[0239] Furthermore, in some embodiments, a heterodimeric Fc-based chimeric protein complex having a variety of combinations of targeting moieties and signal transduction agents, e.g., IFNα1, is provided in a practical manner, e.g., having a targeting domain on a polypeptide different from the signal transduction agent, e.g., IFNα1. 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.

[0240] 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, a signal transduction agent, e.g., IFNα1, and a targeting moiety. In some embodiments, the Fc-based chimeric protein complex comprises a linker linking each signal transduction agent, e.g., IFNα1, and a targeting moiety (or, in the case of two or more targeting moieties, linking the signal transduction agent, e.g., IFNα1, to one of the targeting moieties). In some embodiments, the Fc-based chimeric protein complex comprises a linker linking each signal transduction agent, e.g., IFNα1, 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, e.g., IFNα1, to another signal transduction agent.

[0241] In some embodiments, the Fc-based chimeric protein complex comprises two or more targeting moieties, which in such embodiments may be the same targeting moiety or different targeting moieties.

[0242] In some embodiments, the Fc-based chimeric protein complex comprises two or more signaling agents, which in such embodiments may be the same or different targeting moieties.

[0243] 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.

[0244] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 1A-F.

[0245] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 2A-H.

[0246] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 3A-H.

[0247] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 4A-D.

[0248] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 5A-F.

[0249] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 6A-J.

[0250] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 7A-D.

[0251] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 8A-F.

[0252] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 9A-J.

[0253] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 10A-F.

[0254] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 11A-L.

[0255] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 12A-L.

[0256] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 13A-F.

[0257] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 14A-L.

[0258] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 15A-L.

[0259] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 16A-J.

[0260] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 17A-J.

[0261] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 18A-F.

[0262] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 19A-F.

[0263] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 20A-E.

[0264] In some embodiments, the signaling agent is linked to a targeting moiety and the targeting moiety is linked on the same end to the Fc domain (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0265] In some embodiments, the signaling agent and targeting moiety are linked to the Fc domain, and the targeting moiety and signaling agent are linked on the same terminus (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0266] 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.

[0267] 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, and 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, and two targeting moieties are linked to the Fc domain and two targeting moieties are linked to the signaling agents 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, and two targeting moieties are linked to each other, and 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, e.g., IFNα1, 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.

[0268] 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 the pair is linked to the Fc domain on the same end, 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 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 the 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.

[0269] 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, e.g., IFNα1, and at least one targeting moiety, wherein the ion-pairing motif and / or knob-in-hole motif, signal transduction agent, e.g., IFNα1, 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.

[0270] In some embodiments, a signaling agent, e.g., IFNα1, is linked to a targeting moiety, which is linked to an Fc domain (see Figures 10A-F and 13A-F). In some embodiments, a targeting moiety is linked to a signaling agent, e.g., IFNα1, which is linked to an 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.

[0271] In some embodiments, the signaling agent, e.g., IFNα1, 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, e.g., IFNα1, 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, e.g., IFNα1, 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, e.g., IFNα1, 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.

[0272] In some embodiments where there is one signaling agent, e.g., IFNα1, and two targeting moieties, the signaling agent, e.g., IFNα1, is linked to the Fc domain, and the two targeting moieties can be 1) linked to each other using 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, e.g., IFNα1, is linked to the other Fc chain (see Figures 5A-F and 8A-F). In some embodiments, the paired targeting moiety and signaling agent, e.g., IFNα1, are linked to the same Fc chain (see Figures 11A-L and 14A-L). In some embodiments, a targeting moiety is linked to the Fc domain, another targeting moiety is linked to a signaling agent, e.g., IFNα1, and a paired targeting moiety is linked to the 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.

[0273] In some embodiments where there is one signaling agent, e.g., IFNα1, and two targeting moieties, the targeting moiety is linked to the signaling agent, e.g., IFNα1, which is Fc domain-linked, 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, e.g., IFNα1, 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, e.g., IFNα1, and the paired targeting moiety are linked to different Fc chains (see Figures 16A-J and 17A-J). In some embodiments, the paired signaling agent, e.g., IFNα1, 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.

[0274] In some embodiments where there is one signaling agent, e.g., IFNα1, and two targeting moieties, the targeting moieties are linked together, the signaling agent, e.g., IFNα1, is linked to one of the paired targeting moieties, and the targeting moiety not linked to the signaling agent, e.g., IFNα1, 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 comprises a mutation that reduces or eliminates its effector function.

[0275] In some embodiments where there is one signaling agent, e.g., IFNα1, and two targeting moieties, the targeting moieties are linked together, the signaling agent, e.g., IFNα1, is linked to one of the paired targeting moieties, and the signaling agent, e.g., IFNα1, 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.

[0276] In some embodiments where there is one signaling agent, e.g., IFNα1, and two targeting moieties, both targeting moieties are linked to the signaling agent, e.g., IFNα1, 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 comprises a mutation that reduces or eliminates its effector function.

[0277] In some embodiments where there is one signaling agent, e.g., IFNα1, and two targeting moieties, the targeting moiety and signaling agent, e.g., IFNα1, 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 comprises a mutation that reduces or eliminates its effector function.

[0278] In some embodiments where there are two signaling agents and one targeting moiety, the signaling agents are linked to the Fc domain and the targeting moiety is linked to the Fc domain on the same end (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.

[0279] In some embodiments where there are two signaling agents and one targeting moiety, the signaling agent, e.g., IFNα1, is linked to a targeting moiety, which is linked to an Fc domain, and the other signaling agent, e.g., IFNα1, 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, e.g., IFNα1, are linked to different Fc chains (see Figures 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling agent, e.g., IFNα1, 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, e.g., IFNα1, 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, e.g., IFNα1, 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.

[0280] In some embodiments where there are two signaling agents and one targeting moiety, the targeting moiety is linked to a signaling agent, e.g., IFNα1, which is linked to the Fc domain, and the other signaling agent, e.g., IFNα1, is linked to the Fc domain (see Figures 6A-J and 9A-J). In some embodiments, the paired signaling agent, e.g., IFNα1, and the unpaired signaling agent, e.g., IFNα1, are linked to different Fc chains (see Figures 6A-J and 9A-J). In some embodiments, the paired signaling agent, e.g., IFNα1, and the unpaired signaling agent, e.g., IFNα1, are linked to different Fc chains on the same end (see Figures 6A-J and 9A-J). In some embodiments, the paired signaling agent, e.g., IFNα1, and the unpaired signaling agent, e.g., IFNα1, are linked to different Fc chains on different ends (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.

[0281] 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.

[0282] 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.

[0283] In some embodiments where there are two signaling agents and one targeting moiety, both signaling agents are linked to targeting moieties 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 comprises a mutation that reduces or eliminates its effector function.

[0284] 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, e.g., IFNα1, is linked to the Fc domain (see Figures 12A-L and 15A-L).

[0285] In some embodiments where there are two signaling agents and one targeting moiety, each signaling agent, e.g., IFNα1, 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).

[0286] In some embodiments, a targeting moiety or signal transduction agent, e.g., IFNα1, is linked to an Fc domain, which includes one or both of the CH2 and CH3 domains, and optionally a hinge region. For example, such polypeptides can be produced using a vector encoding a targeting moiety, a signal transduction agent, e.g., IFNα1, or a combination thereof linked to an Fc domain as a single nucleotide sequence.

[0287] In some embodiments, 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: 290, 291, 293-303. In some embodiments, the Fc-based chimeric protein complex comprises an amino acid sequence selected from SEQ ID NOs: 290, 291, 293-303 and a polypeptide having fewer than 10 mutations relative to that amino acid sequence. In some embodiments, the Fc-based chimeric protein complex comprises an amino acid sequence selected from SEQ ID NOs: 290, 291, 293-303 and a polypeptide having fewer than 5 mutations relative to that amino acid sequence. In some embodiments, the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 290, 291, 293-303.

[0288] In some embodiments, 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: 290, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 291. In some embodiments, 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: 293, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 294, 295, 296, 297, 298, or 299. In some embodiments, 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 SEQ ID NOs: 300, 301, 302, 303, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 294.

[0289] Additional signaling substances In one aspect, the present invention provides a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, that includes one or more signal transduction agents (e.g., immunomodulatory agents) in addition to IFNα1 or a variant thereof described herein. In exemplary embodiments, the chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, can include two, three, four, five, six, seven, eight, nine, ten, or more signal transduction agents in addition to IFNα1 or a variant thereof described herein. In various embodiments, the additional signal transduction agents are modified to have reduced affinity or activity for their one or more receptors, which allows for attenuation of the activity (including agonism or antagonism) of the chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, and / or prevents nonspecific signal transduction or undesired sequestration.

[0290] In various embodiments, the additional signaling substance is an antagonist in its wild-type form and has one or more mutations that attenuate its antagonist activity. In various embodiments, the additional signaling substance is an antagonist due to one or more mutations, e.g., an agonist signaling substance is converted to an antagonist signaling substance, and such a converted signaling substance optionally also has one or more mutations that attenuate its antagonist activity (e.g., as described in WO 2015 / 007520, the entire contents of which are incorporated herein by reference).

[0291] In various embodiments, the additional signaling substance is selected from modified forms of cytokines, growth factors, and hormones. Examples of such cytokines, growth factors, and hormones include, but are not limited to, lymphokines, monokines, traditional polypeptide hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; protein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factors; fibroblast growth factors; prolactin; placental lactogen; tumor necrosis factor α and tumor necrosis factor β; Müllerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors such as NGFα; platelet growth factors; and transforming growth factors (TG). F) For example, TGFα and TGFβ; insulin-like growth factor-I and II; osteoinductive factors; interferons such as interferon α, interferon β, and interferon γ (and interferons type I, II, and III); colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL1β, IL1α, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, and IL18; tumor necrosis factors such as TNF-α or TNFβ; and other polypeptide factors such as LIF and Kit ligand (KL). As used herein, cytokines, growth factors, and hormones include products from natural sources or recombinant bacterial, eukaryotic, or mammalian cell culture systems and biologically active equivalents of the native sequence cytokines.

[0292] In some embodiments, the additional signaling agent is a modified form of a growth factor selected from, but not limited to, transforming growth factors (TGFs), such as TGF-α and TGFβ, epidermal growth factor (EGF), insulin-like growth factors, such as insulin-like growth factor-I and II, fibroblast growth factor (FGF), heregulin, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF).

[0293] In some embodiments, the growth factor is a modified fibroblast growth factor (FGF). Examples of FGFs include, but are not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, mouse FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0294] In one embodiment, the growth factor is a modified vascular endothelial growth factor (VEGF). Examples of VEGF include, but are not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF and their isoforms. 121 , VEGF 121 b, VEGF 145 , VEGF 165 , VEGF 165 b, VEGF 189 , and VEGF 206 It includes various VEGF-A isoforms such as:

[0295] In one embodiment, the growth factor is a modified transforming growth factor (TGF). Examples of TGF include, but are not limited to, TGF-α and TGF-β and their subtypes, including the various subtypes of TGF-β, including TGFβ1, TGFβ2, and TGFβ3.

[0296] In some embodiments, the additional signaling substance may be, but is not limited to, human chorionic gonadotropin, gonadotropin-releasing hormone, androgen, estrogen, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growth hormone-releasing hormone, corticotropin-releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, steroid hormone, steroid hormone-releasing ... and modified forms of hormones selected from glandular hormones, glucocorticoids, mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin, glucagon, amylin, calcitriol, calciferol, atrial natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor (IGF), leptin, thrombopoietin, erythropoietin (EPO), and angiotensinogen.

[0297] In some embodiments, the additional signaling agent is an immunomodulatory agent, for example, one or more of an interleukin, an interferon, and a tumor necrosis factor.

[0298] In some embodiments, the additional signaling substance is an interleukin, including, for example, IL1β, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, or a fragment, variant, analog, or family member thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulating the proliferation of immune cells (e.g., helper T cells, B cells, eosinophils, and lymphocytes), chemotactic activity of neutrophils and T lymphocytes, and / or inhibiting interferon. Interleukin activity can be measured using assays known in the art (Matthews et al., in Lymphokines and Interferons: A Practical Approach, Clemens et al., eds., IRL Press, Washington, DC 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20).

[0299] In some embodiments, the signaling agent is a modified interferon, such as interferon types I, II, and III. Examples of interferons include, for example, interferon alpha-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21, interferon beta and interferon gamma, interferon kappa, interferon epsilon, and interferon tau.

[0300] In some embodiments, the additional signaling agent is a type I interferon, hi some embodiments, the type I interferon is selected from IFNα2, IFNα1, IFN-β, IFNγ, consensus IFN, IFN-ε, IFN-κ, IFN-τ, IFN-δ, and IFN-ν.

[0301] In some embodiments, the additional signal transduction agent is a modified form of tumor necrosis factor (TNF) or a protein of the TNF family, including, but not limited to, TNFα, TNFβ, LTβ, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TRAIL.

[0302] In various embodiments, the additional 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 one or more of 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 one or more of reduced binding affinity, reduced intrinsic activity, and reduced specific biological activities, compared to the unmodified or non-mutated, i.e., non-mutated, IFNα1. 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 a 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 a non-mutated sequence of a non-mutated interferon, e.g., IFNα1.

[0303] In various embodiments, the additional 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.

[0304] In some embodiments, the reduced affinity or activity for the receptor is inducible or reversible by the binding of one or more targeting moieties or upon inclusion in an Fc-based chimeric protein complex disclosed herein, hi other embodiments, the reduced affinity or activity for the receptor is not fully inducible or reversible by the action of one or more targeting moieties or upon inclusion in an Fc-based chimeric protein complex disclosed herein.

[0305] In various embodiments, the additional signaling agent is active on the target cell because the targeting moiety(s) compensate 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.

[0306] In some embodiments, the additional signaling agent may contain one or more mutations that weaken or reduce binding or affinity for one receptor (i.e., the 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, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention have modified signaling agents that have 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).

[0307] In some embodiments, substantial reduction or elimination of binding or activity cannot be fully induced or restored by a targeting moiety or upon inclusion in an Fc-based chimeric protein complex disclosed herein. In some embodiments, substantial reduction or elimination of binding or activity can be induced or restored by a targeting moiety or upon inclusion in an Fc-based chimeric protein complex disclosed herein. In various embodiments, 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, substantial reduction or elimination of binding or activity to other receptors may improve therapeutic efficacy by reducing or eliminating the segregation of a 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, to compensate for losses at other receptors. The ability to reduce such dosages further reduces the potential for side effects.

[0308] In various embodiments, the additional altered signaling agent comprises one or more mutations that reduce, substantially reduce, or eliminate the affinity, e.g., binding (e.g., KD) and / or activation (e.g., if the altered signaling agent is an agonist of the receptor, measurable, e.g., as K and / or EC50) and / or inhibition (e.g., if the altered signaling agent is an antagonist of the receptor, measurable, e.g., as K and / or IC50) of the signaling agent for one or more of its receptors. 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%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% to 20%, about 20% to 40%, about 50%, about 40% to 60%, about 60% to 80%, or about 80% to 100% of the 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 signal transduction agent (including but not limited to, compared to non-mutated IFNα1).

[0309] In some embodiments, where a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has mutations that reduce binding to one receptor and substantially reduce or eliminate binding to a second receptor, the binding affinity of the altered signal transduction agent for one receptor is reduced or eliminated to a lesser extent than the affinity for the other receptor. In some embodiments, the binding affinity of the altered signal transduction agent for one receptor is reduced or eliminated by about 1%, or by about 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% less than the affinity for the other receptor. In various embodiments, a substantial reduction or elimination refers to a greater reduction in binding affinity and / or activity than a reduction or elimination.

[0310] In various embodiments, the additional 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 IFNα1).

[0311] In various embodiments, the additional modified signal transduction agent comprises one or more mutations that cause the signal transduction agent to have reduced affinity and / or activity for a receptor for any one of cytokines, growth factors, and hormones.

[0312] In some embodiments, the additional 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.

[0313] 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) or by reflectance interferometry under flow-through conditions, as described by Brecht et al. (1993).The entire contents of these documents are incorporated herein by reference.

[0314] The amino acid sequences of the wild-type signal transduction factors described herein are well known in the art. Thus, in various embodiments, the additional modified signaling agent is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0315] In various embodiments, the additional modified signaling agent is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%. %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity to the amino acid sequence.

[0316] In various embodiments, the additional modified signaling agent comprises an amino acid sequence having one or more amino acid mutations, which in some embodiments may be independently selected from substitutions, insertions, deletions, and truncations.

[0317] In some embodiments, the amino acid mutations are amino acid substitutions, which may include conservative and / or non-conservative substitutions, as described herein.

[0318] As described herein, additional modified signal transduction agents have mutations that affect affinity and / or activity for one or more receptors. In various embodiments, there is reduced affinity and / or activity for a therapeutic receptor, e.g., a receptor through which a desired therapeutic effect is mediated (e.g., agonism or antagonism). In various embodiments, the modified signal transduction agent has mutations that substantially reduce or eliminate affinity and / or activity for a receptor, e.g., a receptor through which a desired therapeutic effect is not mediated (e.g., as a result of disrupted binding). Receptors for modified signal transduction agents, e.g., receptors for one of the cytokines, growth factors, and hormones described herein, are known in the art.

[0319] Examples of mutations that result in reduced affinity and / or activity (e.g., agonist activity) for a receptor can be found in WO 2013 / 107791 (e.g., for interferons), WO 2015 / 007542 (e.g., for interleukins), and WO 2015 / 007903 (e.g., for TNF), the entire contents of each of which are incorporated herein by reference. Examples of mutations that reduce affinity and / or activity (e.g., antagonist activity) for a therapeutic receptor can be found in WO 2015 / 007520, the entire contents of which are incorporated herein by reference.

[0320] In some embodiments, the additional modified signal transducer comprises one or more mutations that reduce the affinity and / or activity of the signal transducer for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a receptor of the tumor necrosis factor receptor (TNFR) superfamily, a TGF beta receptor, a receptor of the immunoglobulin (Ig) superfamily, and / or a receptor of the tyrosine kinase superfamily.

[0321] In various embodiments, the receptor for the additional signal transduction substance is a type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to, receptors for IL2 (beta subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and also receptors for thrombopoietin (TPO), prolactin, and growth hormone. Exemplary type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptor.

[0322] In various embodiments, the receptor for the additional signal transduction substance is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterogeneous subunits and are primarily receptors for interferons. This receptor family includes, but is not limited to, receptors for interferon-α, interferon-β, and interferon-γ, IL10, IL22, and tissue factor. Exemplary type II cytokine receptors include, but are not limited to, IFN-α receptors (e.g., IFNAR1 and IFNAR2), IFN-β receptors, IFN-γ receptors (e.g., IFNGR1 and IFNGR2), and type II IL receptors.

[0323] In various embodiments, the receptor for the additional signal transduction substance is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors that have a seven-transmembrane structure and are coupled to G proteins for signal transduction. Chemokine receptors include, but are not limited to, CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors (XCR1). Examples of chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.

[0324] In various embodiments, the receptor for the additional signal transduction agent is a TNFR family member. Tumor necrosis factor receptor (TNFR) family members share a cysteine-rich domain (CRD) formed from three disulfide bonds surrounding a CXXCXXC core motif that creates an elongated molecule. Examples of tumor necrosis factor receptor family members include: CD120a (TNFRSF1A), CD120b (TNFRSF1B), lymphotoxin beta receptor (LTBR, ​​TNFRSF3), CD134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSF10A (TNFRSF10A), TNFRSF10B (TNFRSF10B), TNFRSF10C (TNFRSF10C), and TNFRSF10D. 10C), TNFRSF10D (TNFRSF10D), RANK (TNFRSF11A), osteoclast differentiation inhibitor (TNFRSF11B), TNFRSF12A (TNFRSF12A), TNFRSF13B (TNFRSF13B), TNFRSF13C (TNFRSF13C), TNFRSF14 (TNFRSF14), nerve growth factor receptor (NGFR, TNFRSF16), TNFRSF17 (TNFRSF17), TNFRSF18 (TNFRSF18), TNFRSF19 (TNFRSF19), TNFRSF21 (TNFRSF21), and TNFRSF25 (TNFRSF25).

[0325] In various embodiments, the receptor for the additional signal transduction substance is a TGF-beta receptor.TGF-beta receptor is a single transmembrane serine / threonine kinase receptor.TGF-beta receptors include but are not limited to TGFBR1, TGFBR2 and TGFBR3.

[0326] In various embodiments, the receptor for the additional signal transduction substance is an Ig superfamily receptor. Receptors of the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Receptors of the Ig superfamily include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

[0327] In various embodiments, the receptor for the additional signal transduction substance is a tyrosine kinase superfamily receptor. Receptors of the tyrosine kinase tyrosine kinase superfamily are well known in the art. There are approximately 58 receptor tyrosine kinases (RTKs) classified into 20 subfamilies. Receptors of the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0328] In certain embodiments, the additional modified signal transduction agent is interferon alpha. In such embodiments, the modified IFN alpha agent has reduced affinity and / or activity for the IFN alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFN alpha agent has substantially reduced or eliminated affinity and / or activity for the IFN alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0329] Mutant forms of interferon alpha are known to those of skill in the art. In an exemplary embodiment, the modified signal transducer is an allelic IFNα2a having the amino acid sequence of SEQ ID NO:233.

[0330] In an exemplary embodiment, the altered signal transducer is the allelic IFNα2b having the amino acid sequence of SEQ ID NO: 234 (which differs from IFNα2a at amino acid position 23):

[0331] 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.

[0332] 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.

[0333] 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.

[0334] 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.

[0335] 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.

[0336] In some embodiments, the human IFNα2 variant comprises one or more mutations selected from L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, as disclosed in WO 2013 / 059885, the entire contents of which are incorporated herein by reference. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L30A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or R33A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or M148A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L153A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations N65A, L80A, Y85A, and / or Y89A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations N65A, L80A, Y85A, Y89A, and / or D114A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises one or more mutations selected from R144X1, A145X2, and R33A, where X1 is selected from A, S, T, Y, L, and I, and X2 is selected from G, H, Y, K, and D.In some embodiments, the human IFNα2 comprises 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: 233 or 234, 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.

[0337] In certain embodiments, the additional modified signal transduction agent is interferon beta. In such embodiments, the modified interferon beta agent also has reduced affinity and / or activity for the IFN alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFN beta agent has substantially reduced or eliminated affinity and / or activity for the IFN alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0338] In an exemplary embodiment, the additional modified signal transduction substance is IFNβ. 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 or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a modified mouse IFNβ. In another embodiment, the chimeric protein or chimeric protein complex, such as 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: 277.

[0339] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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. Exemplary 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 an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 28-30, 32, and 33. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 36, 37, 39, and 42. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 71-73. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising 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. In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0345] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0346] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0347] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0348] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0349] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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), and further 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).

[0350] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0351] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0352] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0353] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0354] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0355] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0356] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and comprises 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 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).

[0357] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0358] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0359] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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); and a mutation at I95, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V); and 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).

[0360] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0361] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0362] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0363] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0364] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0365] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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), and further 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).

[0366] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0367] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and comprises a mutation at V148, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0368] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 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).

[0369] In some embodiments, the invention relates to a chimeric protein complex, such as an Fc-based chimeric protein complex, comprising: (a) an amino acid sequence of SEQ ID NO: 277 and a mutation at position W22, where the mutation is an aliphatic hydrophobic residue and a modified IL2 or modified IL2 variant disclosed herein; and (b) one or more targeting moieties, where the targeting moiety comprises a recognition domain that specifically binds to an antigen or receptor of interest, and the modified IFNβ and the one or more targeting moieties may optionally be linked with one or more linkers. In various embodiments, the mutation at position W22 is an aliphatic hydrophobic residue selected from G, A, L, I, M, and V. In various embodiments, the mutation at position W22 is G.

[0370] Additional examples of IFNβ variants are provided in International Application No. PCT / EP2017 / 061544, the entire disclosure of which is incorporated herein by reference.

[0371] In some embodiments, the additional modified signal transduction agent is interferon gamma. In such embodiments, the modified interferon gamma agent has reduced affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains. In some embodiments, the modified interferon gamma agent has substantially reduced or eliminated affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains.

[0372] In some embodiments, the additional modified signal transduction substance is a consensus interferon. Consensus interferon is generated by scanning the sequences of several human non-allelic IFNα subtypes and assigning the most frequently observed amino acid at each corresponding position. Consensus interferon differs from IFNα2b at 20 of 166 amino acids (88% homology), and comparison with IFNβ shows identity at more than 30% of amino acid positions. In various embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO: 278:

[0373] In some embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO:279, which differs from the amino acid sequence of SEQ ID NO:278 by one amino acid, i.e., SEQ ID NO:279 lacks the first methionine residue of SEQ ID NO:278:

[0374] In various embodiments, the consensus interferon comprises a modified consensus interferon, i.e., a consensus interferon variant, as a signaling agent. In various embodiments, the consensus interferon variant includes a functional derivative, analog, precursor, isoform, splice variant, or fragment of consensus interferon.

[0375] In some embodiments, the consensus interferon variant is selected from the consensus interferon variants disclosed in U.S. Patent Nos. 4,695,623, 4,897,471, 5,541,293 and 8,496,921.The entire contents of these documents are incorporated herein by reference.For example, the consensus interferon variant can comprise the amino acid sequence of IFN-CON2 or IFN-CON3 as disclosed in U.S. Patent Nos. 4,695,623, 4,897,471 and 5,541,293.In some embodiments, the consensus interferon variant comprises the amino acid sequence of IFN-CON2, SEQ ID NO:280.

[0376] In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON3, SEQ ID NO:281.

[0377] In some embodiments, the consensus interferon variant comprises the amino acid sequence of any one of the variants disclosed in U.S. Patent No. 8,496,921. For example, the consensus variant may comprise the amino acid sequence of SEQ ID NO:282.

[0378] In another embodiment, the consensus interferon variant may comprise the amino acid sequence of SEQ ID NO:283.

[0379] In some embodiments, the consensus interferon variant may be pegylated, i.e., include a PEG moiety. In certain embodiments, the consensus interferon variant may include a PEG moiety attached at position S156C of SEQ ID NO: 283.

[0380] In some embodiments, the modified interferon is a variant of human IFNα2a, with an Asp insertion near position 41 of the sequence Glu-Glu-Phe-Gly-Asn-Gln (SEQ ID NO: 284) resulting in Glu-Glu-Phe-Asp-Gly-Asn-Gln (SEQ ID NO: 285) (which results in a renumbering of the sequence relative to the IFNα2a sequence), and the following mutations: Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser, Asp86Glu, Ile104Thr, Gly106Glu, Thr110Glu, Lys117Asn, Arg125Lys, and Lys136Thr. All embodiments herein describing consensus interferon similarly apply to this genetically modified interferon.

[0381] In some embodiments, the additional signal modifier is vascular endothelial growth factor (VEGF). VEGF is a potent growth factor that plays an important role in both physiological and pathological angiogenesis, regulating vascular permeability and acting as a growth factor for cells expressing VEGF receptors. Additional functions include stimulating cell migration, particularly of macrophage lineages and endothelial cells. In addition to at least three receptors (VEGFR1, VEGFR2, and VEGFR3), several members of the VEGF growth factor family exist. VEGF family members can bind to and activate two or more VEGFR types. For example, VEGF-A binds VEGFR1 and VEGFR2, while VEGF-C can bind VEGFR2 and VEGFR3. VEGFR1 and VEGFR2 activation regulates angiogenesis, and VEGFR3 activation is involved in lymphangiogenesis. Most pro-angiogenic signals are generated from VEGFR2 activation. It has been reported that VEGFR1 activation may be associated with a negative role in angiogenesis. VEGFR1 signaling has also been reported to be important for in vivo tumor progression via bone marrow-derived VEGFR1-positive cells (contributing to the formation of a premetastatic microenvironment in bone). Several VEGF-A-based therapeutic therapies, primarily directed against or neutralizing therapeutic antibodies, have been developed for use in the treatment of various human tumors that depend primarily on angiogenesis. However, these are not without side effects. This is not surprising given that these agents act as general, non-cell / tissue-specific inhibitors of VEGF / VEGFR interactions. Therefore, it would be desirable to restrict VEGF (e.g., VEGF-A) / VEGFR2 inhibition to specific target cells (e.g., tumor vasculature endothelial cells).

[0382] In some embodiments, the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E and VEGF 121 , VEGF 121 b, VEGF 145 , VEGF 165 , VEGF 165 b, VEGF189 , and VEGF 206 These isoforms include various VEGF-A isoforms such as those described above. In some embodiments, the altered signaling agent has reduced affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In some embodiments, the altered signaling agent has reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In certain embodiments, the altered signaling agent has reduced affinity and / or activity for VEGFR-2 (KDR / Flk-1) and / or has reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1). Such embodiments are used, for example, in wound healing methods or in the treatment of ischemia-related diseases (mediated, without intending to be bound by theory, by the effects of VEGFR2 on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1 (Flt-1), which is associated with cancer and pro-inflammatory activity, is avoided. In various embodiments, VEGFR-1 (Flt-1) functions as a decoy receptor, thereby substantially reducing or eliminating affinity for this receptor and avoiding sequestration of the therapeutic agent. In certain embodiments, the modified signal transduction agent has reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or reduced or eliminated affinity and / or activity for VEGFR-2 (KDR / Flk-1). In some embodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signal transduction agent has reduced affinity and / or activity for VEGFR3. Alternatively, the modified signal transduction agent has substantially reduced or eliminated affinity and / or activity for VEGFR3.

[0383] Pro-angiogenic therapies are also important in various diseases (e.g., ischemic heart disease, hemorrhage, etc.), and include VEGF-based therapeutics. Activation of VEGFR2 is pro-angiogenic (acting on endothelial cells). VEGFR1 can stimulate the migration of inflammatory cells (including, e.g., macrophages), leading to inflammation associated with vascular hyperpermeability. Activation of VEGFR1 can also activate myeloid cells associated with tumor microenvironment formation. Therefore, VEGF-based therapeutics selective for VEGFR2 activation would be desirable in this case. Furthermore, cells that specifically target, for example, endothelial cells would be desirable.

[0384] In some embodiments, the additional modified signal transduction agent has reduced affinity and / or activity (e.g., antagonist activity) for VEGFR2 and / or substantially reduced or eliminated affinity and / or activity for VEGFR1. When targeted to tumor vasculature endothelial cells via a targeting moiety that binds to a tumor endothelial cell marker (e.g., PSMA), such a construct will inhibit VEGFR2 activation specifically on such marker-positive cells but will not activate VEGFR1 en route to or on the target cells (when activity is eliminated), thus, for example, eliminating the induction of an inflammatory response. This would provide a more selective and safer antiangiogenic therapy for many tumor types than VEGF-A neutralizing therapy.

[0385] In some embodiments, the additional modified signal transduction agent has reduced affinity and / or activity (e.g., agonist activity) for VEGFR2 and / or has substantially reduced or eliminated affinity and / or activity for VEGFR1. By targeting vascular endothelial cells, in some embodiments, such constructs promote angiogenesis without inducing the inflammatory response associated with VEGFR1. Thus, such constructs will have targeted pro-angiogenic effects with substantially reduced risk of side effects resulting from systemic activation of VEGFR2 and VEGFR1.

[0386] In an exemplary embodiment, the altered signal transduction agent is a VEGF having the amino acid sequence of SEQ ID NO: 235. 165 is.

[0387] In another exemplary embodiment, the additional modified signal transduction agent is a VEGF having the amino acid sequence of SEQ ID NO: 236. 165 b.

[0388] In these embodiments, the modified signal transducer has a mutation at amino acid 183 (e.g., a substitution mutation at 183, e.g., 183K, 183R, or 183H). Without intending to be bound by theory, it is believed that such mutations may result in reduced receptor binding affinity. See, e.g., U.S. Patent No. 9,078,860, the entire contents of which are incorporated herein by reference.

[0389] In one embodiment, the additional signal-modifying substance is interferon-α. TNF is a pleiotropic cytokine with many diverse functions, including regulating cell proliferation, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and trafficking, inflammation, and septic shock. It binds to two distinct membrane receptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits a very broad expression pattern, while TNFR2 is selectively expressed on specific populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. In response to receptor activation, distinct biological pathways are activated, although some overlap exists. As a general rule, and without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), while TNFR2 signaling is associated with the activation of cell survival signals (e.g., activation of the NFκB pathway). Administration of TNF results in systemic toxicity, primarily due to the involvement of TNFR1. However, like TNFR1, activation of TNFR2 is also associated with a variety of actions, and it should be noted that control of TNF targeting and activity is important in the development of TNF-based therapeutic agents.

[0390] In some embodiments, the additional modified signal transduction substance has reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signal transduction substance has substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2. TNFR1 is expressed in most tissues and is involved in cell death signaling; in contrast, TNFR2 is involved in cell survival signaling. Thus, in embodiments relating to cancer therapy, the modified signal transduction substance has reduced affinity and / or activity for TNFR1 and / or substantially reduced or eliminated affinity and / or activity for TNFR2. In these embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can target cells in which apoptosis is desired, e.g., tumor cells or tumor vascular endothelial cells. For example, in embodiments relating to methods of promoting cell survival in neurogenesis for the treatment of neurodegenerative disorders, the modified signal transduction substance has reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1. In other words, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention, in some embodiments, comprise modified TNFα agents that can favor either death or survival signals.

[0391] In some embodiments, chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, have modified TNF with reduced affinity and / or activity for TNFR1 and / or substantially reduced or eliminated affinity and / or activity for TNFR2. Such chimeras are, in some embodiments, more potent inducers of apoptosis than chimeras having only wild-type TNF and / or mutations that result in reduced affinity and / or activity for TNFR1. Such chimeras are, in some embodiments, used to induce tumor cell death or tumor vascular endothelial cell death (e.g., in the treatment of cancer). Also, in some embodiments, these chimeras inhibit TNF via, for example, TNFR2. reg This avoids or reduces cellular activation, thus further supporting TNFR1-mediated anti-tumor activity in vivo.

[0392] In some embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has a modified TNF with reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1. Such chimeras, in some embodiments, are more potent activators of cell survival in some cell types, which may be of particular therapeutic interest in various diseases, including, but not limited to, stimulation of neurogenesis. Furthermore, such TNFR2-selected chimeras are also useful in the treatment of autoimmune diseases (e.g., Crohn's disease, diabetes, MS, colitis, etc., and many other diseases described herein). In some embodiments, the chimeras target autoreactive T cells. In some embodiments, the chimeras target T cells. reg Promotes cell activation and indirect suppression of cytotoxic T cells.

[0393] In some embodiments, the chimeric protein results in the death of autoreactive T cells, e.g., by activating TNFR2 and / or circumventing TNFR1 (e.g., by modified TNF with reduced affinity and / or activity for TNFR2 and / or with substantially reduced or eliminated affinity and / or activity for TNFR1). Without wishing to be bound by theory, these autoreactive T cells have altered apoptosis / survival signals, e.g., due to changes in NFκB pathway activity / signaling.

[0394] In some embodiments, TNFR2-based chimeras have additional therapeutic applications for a variety of autoimmune diseases, particularly diseases including cardiac disease, demyelinating and neurodegenerative disorders, and infectious diseases.

[0395] In one embodiment, the wild-type TNFα has the amino acid sequence of SEQ ID NO:237.

[0396] In such embodiments, ...

Claims

1. A chimeric protein comprising: (a) interferon alpha 1 (IFNα1) or a variant thereof, and (b) one or more targeting moieties that contain a recognition domain that specifically binds to an antigen or receptor of interest; Including, A chimeric protein, wherein said IFNα1 or variant thereof and said one or more targeting moieties are optionally linked with one or more linkers.

2. The chimeric protein of claim 1, wherein the IFNα1 comprises an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to SEQ ID NO:

1.

3. The chimeric protein of claim 1 or 2, wherein the IFNα1 comprises the amino acid sequence of SEQ ID NO:1, and optionally the IFNα1 is pegylated, one or more targeting moieties are pegylated, or the linker is pegylated.

4. The chimeric protein of claim 1 or 2, wherein the variant IFNα1 comprises one or more mutations.

5. The chimeric protein of claim 4 , wherein the one or more mutations of the variant IFNα1 confer reduced affinity for the interferon α / β receptor (IFNAR).

6. The chimeric protein of claim 5 , wherein the IFNα1 exhibits reduced affinity for IFNAR1.

7. The chimeric protein of claim 5 , wherein the IFNα1 exhibits reduced affinity for IFNAR2.

8. The amino acid positions of the one or more mutations in said variant IFNα1 are selected from the group consisting of 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, and said positions are based on SEQ ID NO:

1. Based on the above, 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, R145L, R145N, R145Q , R145S, R145T, R145V, R145Y, A146D, A146E, A146G, A146H, A146I, A146K, A146L, A146M, A146N, A146Q, A14 6R, A146S, A146T, A146V, A146Y, M149A, M149V, R150A, S153A, L154A, N157A, L30A-H58Y-E59N-Q62S, R33A-H 6. The chimeric protein of claim 5, wherein the chimeric protein is arbitrarily selected from the group consisting of 58Y-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.

9. The chimeric protein of any one of claims 1 to 8, further comprising a mutation at positions C1, C29, C86, C99, C139 relative to SEQ ID NO:1, selected from the group consisting of C86S, C86A, and C86Y.

10. The chimeric protein of any one of claims 5 to 9, wherein the one or more mutations confer a reduced affinity that is recoverable by binding to one or more targeting moieties or upon inclusion in the Fc-based chimeric protein complex.

11. The chimeric protein of any one of claims 1 to 10, wherein the targeting moiety is directed to a tumor cell.

12. 12. The chimeric protein of any one of claims 1 to 11, wherein the targeting moiety comprises a recognition domain that recognizes and / or binds to an antigen or receptor on a tumor cell, an endothelial cell, an epithelial cell, a mesenchymal cell, a tumor stroma or stromal cell, an ECM and / or an immune cell, an organ cell, and / or a tissue cell.

13. The chimeric protein of claim 12, wherein the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, mast cells, monocytes, erythrocytes, myeloid cells, myeloid-derived suppressor cells, NKT cells, and NK cells, or derivatives thereof.

14. The targeting moiety may be a full-length antibody or a fragment thereof, a single domain antibody, a recombinant heavy chain only antibody (VHH), a single chain antibody (scFv), a humabody, a shark heavy chain only antibody (VNAR), a microprotein (e.g., cysteine ​​knot proteins, knottins), a darpin, an anticalin, an adnectin, an aptamer, an Fv, an Fab, an Fab', an F(ab') ２ The chimeric protein of any one of claims 1 to 13, comprising a recognition domain which is a peptide mimetic molecule, a natural ligand for a receptor, or a synthetic molecule.

15. 15. The chimeric protein of any one of claims 1 to 14, wherein the recognition domain i) is a natural ligand and optionally comprises Flt3L or a truncated form thereof, optionally the extracellular domain of Flt3L, optionally a single chain Flt3L; ii) recognizes CD20; iii) recognizes PD-1 or PD-L1; or iv) recognizes Clec9A.

16. The recognition domain is a single domain antibody (V ＨＨ ), optionally V ＨＨ , humanized V ＨＨ , or camelized V ＨＨ The chimeric protein according to any one of claims 1 to 15,

17. The chimeric protein of any one of claims 1 to 16, wherein the recognition domain functionally modulates the antigen or receptor of interest.

18. The chimeric protein of any one of claims 1 to 17, wherein the recognition domain binds but does not functionally modulate the antigen or receptor of interest.

19. The chimeric protein of any one of claims 1 to 18, comprising two or more targeting moieties.

20. The chimeric protein of any one of claims 1 to 19, further comprising one or more additional modified signal transduction entities.

21. The chimeric protein of any one of claims 1 to 20, wherein the chimeric protein comprises two signal transduction entities or two targeting moieties or two of both.

22. The chimeric protein of any one of claims 1 to 21, wherein the chimeric protein comprises three signal transduction entities or three targeting moieties or three of both.

23. 21. The chimeric protein of claim 20, wherein the modified signal transduction agent contains one or more mutations that confer reduced affinity or activity for the receptor compared to the non-mutated signal transduction agent.

24. The chimeric protein of claim 23, wherein the one or more mutations allow for attenuation of activity.

25. The chimeric protein of claim 24, wherein the agonist or antagonist activity is attenuated.

26. 26. The chimeric protein of claim 24 or 25, wherein the modified signal transduction substance contains one or more mutations that convert its activity from an agonist activity to an antagonist activity.

27. The chimeric protein of claim 23, wherein the one or more mutations confer reduced affinity or activity that is recoverable by binding to one or more targeting moieties or upon inclusion in the Fc-based chimeric protein complex.

28. 28. The chimeric protein of any one of claims 1 to 27, wherein the chimeric protein is suitable for use in patients with one or more of cancer, infectious diseases, immune disorders, autoimmune diseases, cardiovascular diseases, wounds, ischemia-related diseases, neurodegenerative diseases, and / or metabolic diseases.

29. A recombinant nucleic acid composition encoding one or more chimeric proteins according to any one of claims 1 to 28.

30. 30. A host cell comprising the nucleic acid of claim 29.

31. A method for treating cancer comprising administering to a patient in need thereof an effective amount of i) a chimeric protein of any one of claims 1 to 28; ii) a recombinant nucleic acid of claim 29 to a patient in need thereof; or iii) a host cell of claim 30 to a patient in need thereof.

32. Preclinical cancer, basal cell carcinoma, bile duct 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, lingual, intraoral, and pharyngeal); 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; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; and 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 The method of claim 31, wherein the cancer is selected from one or more of: hematopoietic NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; post-transplant lymphoproliferative disorder (PTLD); and abnormal blood vessel proliferation associated with phacomatosis; edema (e.g. associated with brain tumors); and Meigs syndrome.

33. 33. The method of claim 32, wherein the cancer is hairy cell leukemia.

34. 33. The method of claim 32, wherein the cancer is melanoma.

35. 33. The method of claim 32, wherein the cancer is Kaposi's sarcoma.

36. A chimeric protein according to any one of claims 1 to 28 for use as a drug.

37. The chimeric protein of any one of claims 1 to 28 for use in the treatment of cancer, autoimmune diseases, inflammatory diseases, metabolic diseases, cardiovascular diseases, infectious diseases, degenerative diseases and neurodegenerative diseases.

38. Use of a chimeric protein according to any one of claims 1 to 28 in the manufacture of a medicament.

39. 1. An Fc-based chimeric protein complex, comprising: (a) interferon alpha 1 (IFNα1) or a variant thereof, and (b) one or more targeting moieties that comprise a recognition domain that specifically binds to an antigen or receptor of interest, and (c) an Fc domain, optionally with one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in said Fc domain, and / or stabilize the hinge region in said Fc domain; An Fc-based chimeric protein complex comprising:

40. The Fc-based chimeric protein complex of claim 39, wherein the IFNα1 comprises an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to SEQ ID NO:

1.

41. The Fc-based chimeric protein complex of claim 39 or 40, wherein the IFNα1 comprises the amino acid sequence of SEQ ID NO:1, and optionally the IFNα1 is pegylated, one or more targeting moieties are pegylated, and / or the Fc domain is pegylated.

42. The Fc-based chimeric protein complex of claim 39, wherein the variant IFNα1 comprises one or more mutations.

43. The Fc-based chimeric protein complex of claim 42, wherein one or more mutations of the variant IFNα1 confer reduced affinity for the interferon α / β receptor (IFNAR).

44. The Fc-based chimeric protein complex of claim 43, wherein the IFNα1 exhibits reduced affinity for IFNAR1.

45. The Fc-based chimeric protein complex of claim 43, wherein the IFNα1 exhibits reduced affinity for IFNAR2.

46. the amino acid positions of the one or more mutations in said variant IFNα1 are selected from the group consisting of 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, and said positions are selected from the group consisting of 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, with respect to SEQ ID NO:1 Then, 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, R145L, R145N, R145Q, R14 5S, R145T, R145V, R145Y, A146D, A146E, A146G, A146H, A146I, A146K, A146L, A146M, A146N, A146Q, A146R, A14 6S, A146T, A146V, A146Y, M149A, M149V, R150A, S153A, L154A, N157A, L30A-H58Y-E59N-Q62S, R33A-H58Y-E59 43. The Fc-based chimeric protein complex of claim 42, optionally selected from the group consisting of N-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.

47. The chimeric protein of any one of claims 39 to 46, further comprising a mutation at positions C1, C29, C86, C99, C139 relative to SEQ ID NO:1, selected from the group consisting of C86S, C86A, and C86Y.

48. The Fc-based chimeric protein complex of any one of claims 39 to 47, wherein the one or more mutations confer reduced affinity that is recoverable upon binding to one or more targeting moieties or inclusion in the Fc-based chimeric protein complex.

49. The Fc-based chimeric protein complex of any one of claims 39 to 48, wherein the targeting moiety is directed to a tumor cell.

50. The Fc-based chimeric protein complex of any one of claims 39 to 49, wherein the targeting moiety is directed to an immune cell.

51. The Fc-based chimeric protein complex of claim 50, wherein the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, mast cells, monocytes, erythrocytes, myeloid cells, myeloid-derived suppressor cells, NKT cells, and NK cells, or derivatives thereof.

52. 52. The Fc-based chimeric protein complex of any one of claims 39 to 51, wherein the targeting moiety comprises a recognition domain that is a single domain antibody, a recombinant heavy chain only antibody (VHH), a single chain antibody (scFv), a shark heavy chain only antibody (VNAR), a microprotein (e.g., cysteine ​​knot proteins, knottins), a darpin, anticalin, an adnectin, an aptamer, Fv, Fab, Fab', F(ab')2, a peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule.

53. The Fc-based chimeric protein complex of any one of claims 39 to 52, wherein the recognition domain i) is a natural ligand and optionally comprises Flt3L or a truncated form thereof, optionally the extracellular domain of Flt3L; ii) recognizes CD20; iii) recognizes PD-1 or PD-L1; or iv) recognizes Clec9A.

54. The recognition domain is a single domain antibody (V ＨＨ ), optionally V ＨＨ , humanized V ＨＨ , or camelized V ＨＨ The Fc-based chimeric protein complex according to any one of claims 39 to 53,

55. The Fc-based chimeric protein complex of any one of claims 39 to 54, wherein the recognition domain functionally modulates the antigen or receptor of interest.

56. The Fc-based chimeric protein complex of any one of claims 39 to 55, wherein the recognition domain binds but does not functionally modulate the antigen or receptor of interest.

57. 57. The Fc-based chimeric protein complex of any one of claims 39 to 56, comprising two or more targeting moieties.

58. The Fc-based chimeric protein complex of any one of claims 39 to 57, further comprising one or more additional modified signal transduction entities.

59. The Fc-based chimeric protein complex of any one of claims 39 to 58, wherein the Fc-based chimeric protein complex comprises two signal transduction entities or two targeting moieties or two of both.

60. The Fc-based chimeric protein complex of any one of claims 39 to 59, wherein the Fc-based chimeric protein complex comprises three signal transduction entities or three targeting moieties or three of both.

61. The Fc-based chimeric protein complex of claim 58, wherein the modified signal transduction agent contains one or more mutations that confer reduced affinity or activity for a receptor compared to the non-mutated signal transduction agent.

62. The Fc-based chimeric protein complex of claim 61, wherein the one or more mutations allow for attenuation of activity.

63. The Fc-based chimeric protein complex of claim 62, wherein the agonist or antagonist activity is attenuated.

64. The Fc-based chimeric protein complex of claim 61 or 62, wherein the modified signal transduction substance contains one or more mutations that convert its activity from an agonist activity to an antagonist activity.

65. The Fc-based chimeric protein complex of claim 39, wherein the one or more mutations confer reduced affinity or activity that is recoverable by binding to one or more targeting moieties or upon inclusion in the Fc-based chimeric protein complex.

66. The Fc-based chimeric protein complex according to any one of claims 39 to 65, wherein the Fc-based chimeric protein complex is suitable for use in patients with one or more of cancer, infectious diseases, immune disorders, autoimmune diseases, cardiovascular diseases, wounds, ischemia-related diseases, neurodegenerative diseases, and / or metabolic diseases.

67. 67. A recombinant nucleic acid composition encoding one or more Fc-based chimeric protein complexes or component polypeptides thereof according to any one of claims 39 to 66.

68. 68. A host cell comprising the nucleic acid of claim 67.

69. A method for treating cancer comprising administering to a patient in need thereof an effective amount of: i) an Fc-based chimeric protein according to any one of claims 39 to 66; ii) a recombinant nucleic acid according to claim 67 to a patient in need thereof; or iii) a host cell according to claim 68 to a patient in need thereof.

70. Preclinical cancer, basal cell carcinoma, bile duct 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, lingual, intraoral, and pharyngeal); 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; lymphomas, including Hodgkin's and non-Hodgkin's lymphomas; and 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 70. The method of claim 69, wherein the cancer 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 disorders (PTLDs); and abnormal blood vessel proliferation associated with phacomatosis; edema (e.g., associated with brain tumors); and Meigs syndrome.

71. 71. The method of claim 70, wherein the cancer is hairy cell leukemia.

72. 71. The method of claim 70, wherein the cancer is melanoma.

73. 71. The method of claim 70, wherein the cancer is Kaposi's sarcoma.

74. An Fc-based chimeric protein complex according to any one of claims 39 to 66 for use as a drug.

75. The Fc-based chimeric protein complex according to any one of claims 39 to 66 for use in the treatment of cancer, autoimmune diseases, inflammatory diseases, metabolic diseases, cardiovascular diseases, infectious diseases, degenerative diseases and neurodegenerative diseases.

76. Use of an Fc-based chimeric protein complex according to any one of claims 39 to 66 in the manufacture of a medicament.

77. The Fc-based chimeric protein complex of any one of claims 39 to 66, wherein the Fc domain is derived from IgG, IgA, IgD, IgM, or IgE.

78. The Fc-based chimeric protein complex of claim 77, wherein the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

79. The Fc-based chimeric protein complex according to any one of claims 39 to 66, wherein the Fc domain is derived from human IgG, IgA, IgD, IgM or IgE.

80. The Fc-based chimeric protein complex of claim 79, wherein the human IgG is selected from human IgG1, IgG2, IgG3, or IgG4.

81. The Fc-based chimeric protein complex of any one of claims 39 to 66 and 74 to 80, wherein the Fc chain pairing is facilitated by ion pairing and / or knob-in-hole pairing.

82. The Fc-based chimeric protein complex of any one of claims 39 to 66 and 74 to 81, wherein the one or more mutations to the Fc domain result in ionic pairing between Fc chains in the Fc domain.

83. The Fc-based chimeric protein complex of any one of claims 39 to 66 and 74 to 82, wherein the one or more mutations to the Fc domain result in knob-in-hole pairing in the Fc domain.

84. The Fc-based chimeric protein complex of any one of claims 39 to 66 and 74 to 83, wherein the one or more mutations to the Fc domain result in a reduced or eliminated effector function of the Fc domain.

85. The Fc-based chimeric protein complex of any one of claims 39 to 66 or 74 to 84, wherein the Fc-based chimeric protein complex is a heterodimer and has a trans orientation / structure with respect to any targeting moiety and signal transduction agent relative to each other, or any targeting moiety relative to each other, or any signal transduction agent relative to each other.

86. The Fc-based chimeric protein complex of any one of claims 39 to 66 or 74 to 85, wherein the Fc-based chimeric protein complex is a heterodimer and has a cis orientation with respect to any targeting moiety and signal transduction agent relative to each other, or with respect to any targeting moiety relative to each other, or with respect to any signal transduction agent relative to each other.

87. The Fc-based chimeric protein complex according to any one of claims 39 to 66 and 74 to 86, wherein the Fc comprises L234A, L235A, and K322Q substitutions (according to EU numbering) in human IgG1.

88. The Fc-based chimeric protein complex according to any one of claims 39 to 66 and 74 to 87, wherein the Fc is human IgG1 and optionally comprises one or more mutations at L234, L235, K322, D265, P329, and P331 (according to EU numbering).

89. The Fc-based chimeric protein complex of any one of claims 39 to 66 and 74 to 88, wherein the Fc-based chimeric protein complex has an orientation and / or 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, and 19A-F.

90. The Fc-based chimeric protein complex of any one of claims 39 to 66 and 74 to 89, 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: 290, 291, 293 to 303.

91. The Fc-based chimeric protein complex according to any one of claims 39 to 66 and 74 to 89, wherein the Fc-based chimeric protein complex comprises an amino acid sequence selected from SEQ ID NOs: 290, 291, 293 to 303 and a polypeptide having less than 10 mutations relative to the amino acid sequence.

92. The Fc-based chimeric protein complex of claim 91, comprising an amino acid sequence selected from SEQ ID NOs: 290, 291, 293-303, and a polypeptide having less than 5 mutations relative to said amino acid sequence.

93. The Fc-based chimeric protein complex of claim 91, wherein the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 290, 291, 293-303.

94. The Fc-based chimeric protein complex of claim 90, comprising a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 290 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

291.

95. The Fc-based chimeric protein complex of claim 90, comprising a first amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 293 and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 294, 295, 296, 297, 298, or 299.

96. The Fc-based chimeric protein complex of claim 90, comprising 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: 300, 301, 302, 303, and a second amino acid sequence having at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO:

294.

97. 1. A chimeric protein complex comprising: (a) interferon alpha 1 (IFNα1) or a variant thereof; (b) one or more targeting moieties that comprise a recognition domain that specifically binds to an antigen or receptor of interest, and (c) a complexing domain; A chimeric protein complex comprising:

98. The chimeric protein complex of claim 97, wherein the complex formation domain comprises a leucine zipper.

99. The chimeric protein complex of claim 97 or 98, wherein the IFNα1 comprises an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to SEQ ID NO:1, and optionally the IFNα1 is pegylated, one or more targeting moieties are pegylated, or the linker is pegylated.

100. The chimeric protein complex of any one of claims 97 to 99, wherein the variant IFNα1 comprises one or more mutations.

101. The chimeric protein complex of claim 100, wherein one or more mutations of the variant IFNα1 confer reduced affinity for the interferon α / β receptor (IFNAR).

102. The chimeric protein complex of claim 101, wherein the IFNα1 exhibits reduced affinity for IFNAR1.

103. The chimeric protein complex of claim 101, wherein the IFNα1 exhibits reduced affinity for IFNAR2.

104. The amino acid positions of the one or more mutations in said variant IFNα1 are selected from the group consisting of 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, and said positions are selected from the group consisting of 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, Then, 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, R145L, R145N, R145Q, R 145S, 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, R33A-H58Y- The chimeric protein complex of claim 100, optionally selected from the group consisting of 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.

105. The chimeric protein of any one of claims 97 to 104, further comprising a mutation at positions C1, C29, C86, C99, C139 relative to SEQ ID NO:1, selected from the group consisting of C86S, C86A, and C86Y.

106. The chimeric protein complex of any one of claims 97 to 105, wherein the one or more mutations confer reduced affinity that is recoverable upon binding to one or more targeting moieties or inclusion in the chimeric protein complex.

107. The chimeric protein complex of any one of claims 97 to 106, wherein the targeting moiety is directed to tumor cells, endothelial cells, epithelial cells, mesenchymal cells, tumor stroma or stromal cells, ECM and / or immune cells, organ cells, and / or tissue cells.

108. The chimeric protein complex of any one of claims 97 to 107, wherein the targeting moiety is directed to an immune cell.

109. The chimeric protein complex of claim 108, wherein the immune cell is selected from a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, a mast cell, a monocyte, an erythrocyte, a myeloid cell, a myeloid-derived suppressor cell, a NKT cell, and a NK cell, or a derivative thereof.

110. 110. The chimeric protein complex of any one of claims 97 to 109, wherein the targeting moiety comprises a recognition domain that is a single domain antibody, a recombinant heavy chain only antibody (VHH), a single chain antibody (scFv), a shark heavy chain only antibody (VNAR), a microprotein (e.g., cysteine ​​knot proteins, knottins), a darpin, anticalin, an adnectin, an aptamer, Fv, Fab, Fab', F(ab')2, a peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule.

111. 111. The chimeric protein complex of any one of claims 97-110, wherein the recognition domain i) is a natural ligand and optionally comprises Flt3L or a truncated form thereof, optionally the extracellular domain of Flt3L, optionally a single chain Flt3L; ii) recognizes CD20; iii) recognizes PD-1 or PD-L1; or iv) recognizes Clec9A.

112. The recognition domain is a single domain antibody (V ＨＨ ), optionally V ＨＨ , humanized V ＨＨ , or camelized V ＨＨ The chimeric protein complex according to any one of claims 97 to 111,

113. The chimeric protein complex of any one of claims 97 to 112, wherein the recognition domain functionally modulates the antigen or receptor of interest.

114. The chimeric protein complex of any one of claims 97 to 113, wherein the recognition domain binds but does not functionally modulate the antigen or receptor of interest.

115. The chimeric protein complex of any one of claims 97 to 114, comprising two or more targeting moieties.

116. The chimeric protein complex of any one of claims 97 to 115, further comprising one or more additional modified signal transduction entities.

117. 117. The chimeric protein complex of any one of claims 97 to 116, wherein the chimeric protein complex comprises two signal transduction entities or two targeting moieties or two of both.

118. 118. The chimeric protein complex of any one of claims 97 to 117, wherein the chimeric protein complex comprises three signal transduction agents or three targeting moieties or three of both.

119. The chimeric protein complex of claim 116, wherein the modified signal transduction substance contains one or more mutations that confer reduced affinity or activity for the receptor compared to the non-mutated signal transduction substance.

120. The chimeric protein complex of claim 119, wherein the one or more mutations enable attenuation of activity.

121. The chimeric protein complex of claim 120, wherein the agonist or antagonist activity is attenuated.

122. The chimeric protein complex of claim 119 or 120, wherein the modified signal transduction substance contains one or more mutations that convert its activity from an agonist activity to an antagonist activity.

123. The chimeric protein complex of claim 97, wherein the one or more mutations confer reduced affinity or activity that is recoverable by binding to one or more targeting moieties or upon inclusion in the chimeric protein complex.

124. The chimeric protein complex of any one of claims 97 to 123, wherein the chimeric protein complex is suitable for use in patients with one or more of cancer, infectious diseases, immune disorders, autoimmune diseases, cardiovascular diseases, wounds, ischemia-related diseases, neurodegenerative diseases, and / or metabolic diseases.

125. A recombinant nucleic acid composition encoding one or more chimeric protein complexes according to any one of claims 97 to 124, or component polypeptides thereof.

126. A host cell comprising the nucleic acid of claim 125.

127. A method for treating cancer comprising administering to a patient in need thereof an effective amount of: i) a chimeric protein of any one of claims 97-124; ii) a recombinant nucleic acid of claim 125 to a patient in need thereof; or iii) a host cell of claim 126 to a patient in need thereof.

128. Preclinical cancer, basal cell carcinoma, bile duct 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, lingual, intraoral, and pharyngeal); 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; lymphomas, including Hodgkin's and non-Hodgkin's lymphomas; and 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 noncleaved cell The method of claim 127, wherein the cancer is selected from one or more of: 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 disorders (PTLDs); and abnormal blood vessel proliferation associated with phacomatosis; edema (e.g. associated with brain tumors); and Meigs syndrome.

129. 129. The method of claim 128, wherein the cancer is hairy cell leukemia.

130. 129. The method of claim 128, wherein the cancer is melanoma.

131. 129. The method of claim 128, wherein the cancer is Kaposi's sarcoma.

132. A chimeric protein complex according to any one of claims 97 to 124 for use as a drug.

133. The chimeric protein complex of any one of claims 97 to 124 for use in the treatment of cancer, autoimmune diseases, inflammatory diseases, metabolic diseases, cardiovascular diseases, infectious diseases, degenerative diseases and neurodegenerative diseases.

134. Use of the chimeric protein complex according to any one of claims 97 to 124 in the manufacture of a medicament.

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