Therapeutic interferon alpha 1 protein

Abstract

The present invention relates in part to chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprising interferon alpha 1 or variants thereof and their use as therapeutic agents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to 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 which are hereby incorporated by reference in their entireties. Technical Field

[0003] 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.

[0004] Sequence Listing

[0005] The contents of the text file submitted electronically with this article are incorporated herein by reference in their entirety. A computer-readable format copy of the sequence listing (file name: ORN-057PC_Sequence_Listing_ST25, record date: March 26, 2020; file size: 385,024 bytes). Background Art

[0006] Type I interferons (IFNs) form a family of multifunctional cytokines that play a major role in immune responses and other biological processes. Human type I IFNs include 13 different non-allelic α subtypes, a β subtype, and an ω subtype. Type I interferons all appear to bind to a common receptor composed of IFNAR1 and IFNAR2 subunits, the type I interferon-α / β receptor (IFNAR), although to varying degrees. After binding to type I IFN, IFNAR activates the JAK-STAT signaling pathway to trigger a variety of biological effects. Different activities of IFN subtypes have been reported, and therefore, they are used clinically to treat various diseases and conditions, including, for example, viral hepatitis (IFN-α2), multiple sclerosis, and cancer therapy (e.g., IFN-β or IFNα2). The assembly of the IFN-IFNAR ligand-receptor complex initiates the activation of several signal transduction pathways, which change cell differentiation and / or function according to the cell type, the IFN subtype engaged with the IFNAR, and the type (amplitude, duration, etc.) of the receptor activation-related signal. The present invention relates to the interferon type I of the present invention.Although type I interferon shares the characteristic of engaging IFNAR, they can play this role to varying degrees, and therefore can also have non-redundant function.These are at least partially relevant with the type of signal reaction that they cause after engaging IFNAR and the difference of quality.This is relevant with various type I IFN reactions, such as immunostimulatory effect, antiproliferative effect, antiviral effect and other biological effects.IFNα2 is one of the most effective IFNAR binding ligands and IFNAR signaling activators.Various efforts in producing even more effective IFNAR binding agents for potential therapeutic use have been reported.

[0007] Compared with other type I IFNs, interferon α1 (IFNα1) is a member of the type I interferon family, characterized by a significantly reduced affinity for the IFNAR2 receptor (compared with IFN-α2, the binding affinity for IFNAR2 is 20 times lower; Jaks et al. J. Mol. Biol. 2007; 366: 525-534). It is considered to be the weakest naturally occurring human type I IFN ligand and IFNAR signaling activator in the type I IFN family that binds to IFNAR (Moll et al., Cytokine 2011; 53: 52-59). These characteristics, as well as other factors, have led to a long-term lack of general interest and pursuit of IFNα1 as a potential therapeutic agent. The efficacy of type I interferons in clinical practice is limited by ineffective administration because there are significant systemic toxicities and side effects, including flu-like syndrome, depression, hepatotoxicity, autoimmune diseases, thyroid dysfunction and weight loss. Therefore, it may be highly desirable to localize and direct IFN activity only to cell populations that are targeted for IFN therapy (e.g., infected organs or tumor masses) or to cell populations that are activated by IFN (e.g., immune cell subsets).

[0008] Therefore, 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

[0009] Thus, in some aspects, the present invention relates to chimeric proteins and chimeric protein complexes comprising wild-type IFNα1 or a variant thereof as a signaling agent, including Fc-based chimeric protein complexes. As used herein, the term variant includes IFNα1 mutants. In one embodiment, the IFNα1 comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof.

[0010] The present disclosure is partly related to the following discovery: compared with wild-type IFN α 1, the chimeric protein or chimeric protein complex comprising wild-type IFN α 1 and its variants show the IFNAR activation signal transduction activity that significantly reduces. However, when directed to target cells via targeting moiety, the IFNAR activation signal transduction activity of this reduction can be induced and / or restored at the target cell. Surprisingly, the IFN α 1 activity induced at the target cell achieved by the targeting of the chimeric protein or chimeric protein complex comprising IFN α 1 or its variants may be similar to or higher than the activity of wild-type IFN α 1 at the target cell. In addition, it is also surprising that the targeting IFN α 1 activity of the chimeric protein or chimeric protein complex comprising IFN α 1 or its variants may be similar to or even higher than the activity of wild-type IFN α 2, and wild-type IFN α 2 is one of the most effective natural type I IFN (for example, efficacy is about 10-100 times stronger than wild-type IFN α 1, for example, depending on cell type). Importantly, the IFNα1 chimeric proteins and chimeric protein complexes comprising IFNα1 described herein exhibit significant and surprising selectivity for target cells relative to non-target cells, and significantly exceed the selectivity achieved, for example, with one or more chimeric proteins targeting wild-type IFNα2. In summary, the use of the IFNα1 compositions and variants thereof described herein can achieve a unique combination of highly efficient and highly cell target-selective IFNAR signaling activation. Therefore, in various embodiments, the present invention relates to target-selective IFNAR activators with a high therapeutic index and excellent drug properties for the treatment of various diseases, including cancer, infectious diseases, and autoimmune diseases.

[0011] In some embodiments, wild-type IFNα1 is incorporated into a chimeric protein or a chimeric protein complex, such as, for example, by gene fusion or connection (for example, formation of a complex), which reduces the biological activity of IFNα1 (sometimes referred to as "fusion-induced attenuation"). For example, compared with wild-type IFNα1 interferon, the wild-type IFNα1 incorporated into a chimeric protein or a chimeric protein complex may have a reduced affinity and / or activity for a therapeutic receptor. In one embodiment, the therapeutic receptor is an interferon-α / β receptor (IFNAR) composed of IFNAR1 and IFNAR2 subunits. In some embodiments, the loss of affinity and / or activity of wild-type IFNα1 for a therapeutic receptor (such as IFNAR) can be induced and restored by directing or targeting the chimeric protein or chimeric protein complex comprising IFNα1 to a target cell via a targeting portion. In some embodiments, the induction and recovery of IFNAR activation mediated by IFNα1 at target cells can reach a level similar to or higher than that achieved with wild-type (non-chimeric) IFNα1. In some embodiments, IFNα1 is a variant comprising one or more mutations that reduce undesirable disulfide bond pairing to improve product homogeneity and pharmaceutical properties of the chimeric protein or chimeric protein complex, while maintaining or avoiding significant loss of IFNAR activation of the modified IFNα1 compared to wild-type IFNα1 in the context of the chimeric protein or chimeric protein complex, including maintaining or avoiding significant loss of restoration and induction of IFNAR activation of the modified IFNα1 when directed or targeted to a target cell via a targeting moiety.

[0012] In some embodiments, the IFNα1 is modified, that is, it is a variant and comprises one or more mutations in IFNα1. In some embodiments, the one or more mutations reduce the biological activity of IFNα1 (sometimes referred to as "mutation-induced attenuation"). For example, the one or more mutations can reduce the affinity and / or activity of IFNα1 interferon to a therapeutic receptor. In one embodiment, the therapeutic receptor is an interferon-α / β receptor (IFNAR) composed of IFNAR1 and IFNAR2 subunits. In one embodiment, the modified IFNα1 comprises one or more mutations that reduce its affinity and / or activity to IFNAR1. In another embodiment, the modified IFNα1 comprises one or more mutations that reduce its affinity and / or activity to IFNAR2. In one embodiment, the modified IFNα1 comprises one or more mutations that reduce its affinity and / or activity to IFNAR1, and comprises one or more mutations that reduce its affinity and / or activity to IFNAR2. In some embodiments, the loss of affinity and / or activity of the modified IFNα1 to therapeutic receptors (e.g., IFNAR1, IFNAR2, and / or IFNAR) ("mutation-induced attenuation") can be induced and restored by directing or targeting the chimeric protein or chimeric protein complex comprising the modified IFNα1 to target cells via a targeting moiety. In some embodiments, the modified IFNα1 variants ("mutation-induced attenuation") comprising one or more mutations that reduce their affinity and / or activity to IFNAR1, IFNAR2, and / or IFNAR further comprise one or more mutations that reduce undesirable disulfide pairing to improve product homogeneity and pharmaceutical properties of the chimeric protein or chimeric protein complex while maintaining or avoiding significant loss ("mutation-induced attenuation") of induction and / or restoration of IFNAR activation of the modified IFNα1 when directed / targeted to target cells via a targeting moiety.

[0013] In some embodiments, the chimeric proteins and chimeric protein complexes (including Fc-based chimeric protein complexes) contain one or more additional signaling agents, such as, but not limited to, interferons, interleukins, and tumor necrosis factors that may be modified. In various embodiments, the chimeric proteins and chimeric protein complexes of the present invention (including Fc-based chimeric protein complexes) provide improved safety and / or therapeutic activity and / or pharmacokinetic characteristics (e.g., increased serum half-life) compared to non-targeted and / or unmodified IFNα1 or unmodified wild-type IFN-α (such as IFN-α2a or IFN-α2b).

[0014] In various embodiments, the chimeric proteins and chimeric protein complexes (including Fc-based chimeric protein complexes) comprise one or more targeting moieties having a recognition domain (e.g., an antigen recognition domain, including but not limited to various antibody formats, including 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), the target including a target found on one or more immune cells, which may include but are not limited to T cells, cytotoxic T lymphocytes, T helper cells, T regulatory cells (Treg), 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, a receptor), including targets found in one or more tumor cells, endothelial cells, epithelial cells, mesenchymal cells, specific organs and / or tissues that are unique and / or unique to the stromal cells or other cell types, including targets on cells particularly associated with the disease. In some embodiments, the recognition domain specifically binds to a target of interest (e.g., an antigen, a receptor) and effectively recruits one or more immune cells. In some embodiments, the target of interest (e.g., an antigen, a receptor) is found on one or more tumor cells. In some embodiments, the chimeric protein, chimeric protein complex (including an Fc-based chimeric protein complex) of the present invention can recruit immune cells, such as immune cells that can kill and / or suppress tumor cells or regulate other immune cells, to a site of action (as a non-limiting example, such as a tumor microenvironment). In some embodiments, the chimeric proteins, chimeric protein complexes of the present invention (including Fc-based chimeric protein complexes) can modulate immune cells at the site of action, or recruit immune cells to sites of action associated with autoimmune diseases, inflammatory diseases, infections, metabolic diseases, and / or cardiovascular diseases (as non-limiting examples, such as disease microenvironments). In some embodiments, the recognition domain specifically binds to a target of interest (e.g., an antigen, a receptor), which is part of a non-cellular structure.

[0015] In various embodiments, the chimeric proteins and chimeric protein complexes of the invention, including Fc-based chimeric protein complexes, can be used to treat a variety of diseases or disorders, such as cancer, infection, immune disorders, autoimmune diseases, cardiovascular disease, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and many other diseases and disorders, and the invention encompasses various treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A-1F 、 Figure 2A-2H 、 Figure 3A-3H 、 Figures 4A-4D 、 Figures 5A-5F 、 Figure 6A-6J 、 Figures 7A-7D 、 Figures 8A-8F 、 Figures 9A-9J 、 Figures 10A-10F 、 Figures 11A-11L 、 Figures 12A-12L 、 Figures 13A-13F 、 Figures 14A-14L 、 Figures 15A-15L 、 Figures 16A-16J 、 Figures 17A-17J 、 Figures 18A-18F and Figures 19A-19F Various non-limiting illustrative schematic diagrams of Fc-based chimeric protein complexes of the present invention are shown. In various embodiments, each schematic diagram is a composition of the present invention. Where applicable in the figures, "TM" refers to a "targeting moiety" as described herein, "SA" refers to a "signaling agent" as described herein, is an optional "linker" as described herein, the two long parallel rectangles are human Fc domains as described herein, e.g., from IgG1, from IgG2 or from IgG4, and optionally have effector knockout and / or stabilizing mutations as also described herein, and the two long parallel rectangles (one of which has a protrusion and the other has a depression) are human Fc domains as described herein, e.g., from IgG1, from IgG2 or from IgG4, with knob-into-hole and / or ion pair (also known as charged pair, ionic bond or charged residue pair) mutations as described herein, and optionally have effector knockout and / or stabilizing mutations as also described herein.

[0017] Figure 1A-1F Illustrative homodimeric 2-chain complexes are shown. These figures show illustrative configurations of homodimeric 2-chain complexes.

[0018] Figure 2A-2H An illustrative homodimer 2-chain complex is shown with two targeting moieties (TM) (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In various embodiments, the construct shown in the box (i.e. Figure 2G and Figure 2H ) has a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc.

[0019] Figures 3A-3HAn illustrative homodimer 2-chain complex is shown with two signaling agents (more signaling agents may be present in some embodiments as described herein). In various embodiments, the positions of SA1 and SA2 are interchangeable. In various embodiments, the construct shown in the box (i.e. Figure 3G and Figure 3H ) has a TM between SA1 and SA2, or has a TM at the N-terminus or C-terminus.

[0020] Figures 4A-4D An illustrative heterodimeric 2-chain complex is shown with a split TM chain and an SA chain, ie, the TM is on the knob chain of Fc and the SA is on the hole chain of Fc.

[0021] Figures 5A-5F An illustrative heterodimer 2-chain complex is shown, which has a split TM chain and an SA chain, i.e., both TMs are on the knob chain of the Fc, and the SA is on the hole chain of the Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical.

[0022] Figure 6A-6J An illustrative heterodimer 2-chain complex is shown, which has a split TM chain and an SA chain, i.e., the TM is on the knob chain of the Fc, and the SA is on the hole chain of the Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In these orientations and / or configurations, one SA is on the knob chain and one SA is on the hole chain. In various embodiments, the positions of SA1 and SA2 are interchangeable.

[0023] Figures 7A-7D An illustrative heterodimeric 2-chain complex is shown with a split TM chain and an SA chain, ie, the SA is on the knob chain of Fc and the TM is on the hole chain of Fc.

[0024] Figures 8A-8F An illustrative heterodimer 2-chain complex is shown, which has a split TM chain and an SA chain, i.e., the SA is on the knob chain of the Fc, and both TMs are on the hole chain of the Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical.

[0025] Figures 9A-9JAn illustrative heterodimer 2-chain complex is shown, which has a split TM chain and an SA chain, i.e., the SA is on the knob chain of the Fc and the TM is on the hole chain of the Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In these orientations and / or configurations, one SA is on the knob chain and one SA is on the hole chain. In various embodiments, the positions of SA1 and SA2 are interchangeable.

[0026] Figures 10A-10F An illustrative heterodimeric 2-chain complex is shown in which the TM and SA are on the same chain, ie, both SA and TM are on the knob chain of Fc.

[0027] Figures 11A-11L An illustrative heterodimer 2-chain complex is shown, wherein the TM and SA are on the same chain, i.e., both SA and TM are on the knob chain of the Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical.

[0028] Figures 12A-12L An illustrative heterodimer 2-chain complex is shown in which the TM and SA are on the same chain, i.e., both SA and TM are on the knob chain of the Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In various embodiments, the positions of SA1 and SA2 are interchangeable.

[0029] Figures 13A-13F An illustrative heterodimeric 2-chain complex is shown, where the TM and SA are on the same chain, ie, both SA and TM are on the hole chain of Fc.

[0030] Figures 14A-14L An illustrative heterodimer 2-chain complex is shown, wherein the TM and SA are on the same chain, i.e., both SA and TM are on the hole chain of the Fc, with two targeting moieties (as described herein, more targeting moieties are present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In various embodiments, TM1 and TM2 can be identical.

[0031] Figures 15A-15L An illustrative heterodimer 2-chain complex is shown, wherein the TM and SA are on the same chain, i.e., both SA and TM are on the hole chain of the Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In various embodiments, the positions of SA1 and SA2 are interchangeable.

[0032] Figures 16A-16JAn illustrative heterodimeric 2-chain complex is shown with two targeting moieties (TMs) (more may be present in some embodiments as described herein), and wherein the SA is on the knob Fc and the TMs are on each chain. In various embodiments, TM1 and TM2 may be the same.

[0033] Figures 17A-17J An illustrative heterodimer 2-chain complex is shown with two targeting moieties (TMs) (more may be present in some embodiments as described herein), wherein the SA is on the hole Fc and the TMs are on each chain. In various embodiments, TM1 and TM2 may be the same.

[0034] Figures 18A-18F An illustrative heterodimeric 2-chain complex is shown with two signaling agents (more signaling agents may be present in some embodiments as described herein) and with split SA and TM chains: SA on the knob and TM on the hole Fc.

[0035] Figures 19A-19F An illustrative heterodimeric 2-chain complex is shown with two signaling agents (more signaling agents may be present in some embodiments as described herein) and with a split SA chain and a TM chain: TM on the knob and SA on the hole Fc.

[0036] Figure 20 The bioactivity of IFNα1 and Clec9A VHH Fc ActaFeron (AFN) on HL116 and HL116-hClec9A cells is shown. Parental HL116 or derived HL116-hClec9A cells were stimulated with serial dilutions of Fc AFN for 6 hours. The average luciferase values ​​(±STDEV) of three measurements are plotted.

[0037] Figures 21A-21D Figure 2 shows IFNα1 (also denoted herein as IFNα1) and IFN-α2 (also denoted herein as IFNα2) signaling in peripheral blood mononuclear cells (PBMCs) after targeting. PBMCs from healthy donor buffy coats were stained for CD20 and subsequently stimulated for 15 minutes with serial dilutions of IFNα2 (i.e., no targeting moiety), CD20VHH-IFNα2 (a chimera of a VHH targeting moiety for CD20 and wild-type IFNα2), IFNα1 (i.e., no targeting moiety), or CD20 VHH-IFNα1 (a chimera of a VHH targeting moiety for CD20 and wild-type IFNα1). STAT1 phosphorylation was quantified in FACS and plotted for CD20 positive and CD20 negative PBMCs. Data for IFN-α2 are shown in Table 2. Figure 21A The data of CD20VHH-IFNα2 are shown in Figure 21B The data of IFNα1 are shown in Figure 21C The data for CD20 VHH-IFNα1 are shown in Figure 21D middle.

[0038] Figures 22A-22D Figure 2 shows IFNα1 and IFN-α2 signaling in HL116 and HL116-hCD20 cells after targeting. Parental HL116 or derived HL116-huCD20 cells were stimulated for 6 hours with serial dilutions of IFNα2 (i.e., without a targeting moiety), CD20 VHH-IFNα2 (a chimera of a VHH targeting moiety for CD20 and wild-type IFNα2), IFNα1 (i.e., without a targeting moiety), or CD20 VHH-IFNα1. The average luciferase values ​​(±STDEV) of three measurements are plotted. Data for IFN-α2 are shown in Table 2. Figure 22A The data of CD20VHH-IFNα2 are shown in Figure 22B The data of IFNα1 are shown in Figure 22C The data for CD20 VHH-IFNα1 are shown in Figure 22D middle.

[0039] Figure 23 Tumor growth curves in humanized mice after treatment with buffer or Flt3L-IFNα1 are shown. The mean values ​​(mm) of 5 or 6 animals at each time point are plotted. 3 )(+SEM).

[0040] Figures 24A-24G The biological activity of IFNα1AFN on the HL116 reporter gene is shown. HL116 or HL116-Clec9A cells were stimulated for 6 hours with serial dilutions of wild-type IFNα2 or IFNα1AFN. The mean luciferase activity (±STDEV) is plotted.

[0041] Figures 25A-25E Shown are pSTAT1 activities of AFN in response to IFNα2 or IFNα1 in Clec9A- / CD141- and Clec9A+ / CD141+ PBMCs.

[0042] Figures 26A-26B IFNα1 targeting PD-L1 ( Figure 26A ) and IFNα2( Figure 26B ) AFN bioactivity against HL116 reporter gene. HL116 cells were stimulated with serial dilutions of wild-type IFNα2 or IFNα1AFN for 6 hours. Mean luciferase activity (±STDEV) is plotted. DETAILED DESCRIPTION

[0043] The present invention is based in part on the discovery that targeted chimeric proteins and chimeric protein complexes comprising IFNα1, such as Fc-based chimeric protein complexes, exhibit significantly superior activity and / or target selectivity compared to non-fused, wild-type IFNα1, and exhibit beneficial therapeutic and pharmaceutical properties and reduced side effects. For example, the chimeric proteins or chimeric protein complexes of the present invention, such as Fc-based chimeric protein complexes, have high target selectivity, are capable of conditionally and / or regulatoryally modulating IFNAR1 / 2 receptor signaling, and have high activity and / or long-lasting activity and / or long-lasting effects, while exhibiting minimal off-target effects and causing minimal side effects.

[0044] The present invention also provides pharmaceutical compositions comprising the chimeric proteins, chimeric protein complexes (including Fc-based chimeric protein complexes) and / or nucleic acids encoding the chimeric proteins and chimeric protein complexes (including Fc-based chimeric protein complexes). The present invention also includes host cells comprising the nucleic acids encoding the chimeric proteins and chimeric protein complexes (including Fc-based chimeric protein complexes). The present invention also includes uses of the 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 as described herein for treating various diseases.

[0045] Interferon-α1 or its variants

[0046] In one aspect, the present invention provides a chimeric protein or chimeric protein complex comprising an engineered interferon, such as an Fc-based chimeric protein complex. In one aspect, the present invention provides a chimeric protein or chimeric protein complex comprising wild-type IFNα1, such as an Fc-based chimeric protein complex. In various embodiments, the wild-type IFNα1 comprises the following amino acid sequence:

[0047] CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSSLSLSTNLQERLRRKE (SEQ ID NO: 1).

[0048] In various embodiments, the present invention provides a chimeric protein or chimeric protein complex comprising wild-type IFNα1 fused to one or more targeting moieties, such as a chimeric protein complex based on Fc. In some embodiments, wild-type IFNα1 is incorporated into a chimeric protein or chimeric protein complex, such as, for example, by gene fusion or connection, which reduces the biological activity of IFNα1 ("fusion IFNα1 causes attenuation"). For example, compared to wild-type IFN-α1 interferon, the wild-type IFNα1 incorporated into a chimeric protein or chimeric protein complex may have a reduced affinity and / or activity for a therapeutic receptor. In one embodiment, the therapeutic receptor is an interferon-α / β receptor (IFNAR) composed of IFNAR1 and IFNAR2 subunits. In some embodiments, the loss of affinity and / or activity of wild-type IFNα1 to a therapeutic receptor (e.g., IFNAR) can be induced and restored by directing or targeting a chimeric protein or chimeric protein complex comprising IFNα1 to a target cell via a targeting moiety. In some embodiments, the induction and recovery of IFNAR activation mediated by IFNα1 at target cells can reach a level similar to or higher than that achieved with wild-type (non-chimeric) IFNα1. In some embodiments, IFNα1 is a variant comprising one or more mutations, wherein the one or more mutations reduce undesirable disulfide pairing to improve the product homogeneity and drug properties of the chimeric protein or chimeric protein complex, while maintaining or avoiding the significant loss of IFNAR activation compared to wild-type IFNα1 in the case of chimeric protein or chimeric protein complex, including maintaining or avoiding the significant loss of IFNAR activation of the modified IFNα1 when directed or targeted to the target cell via a targeting portion. In some embodiments, IFNα1 is a mutation comprising one or more reductions in undesirable disulfide pairing, wherein the one or more mutations are, for example, located at amino acid positions C1, C29, C86, C99 or C139 of SEQ ID NO: 1. In some embodiments, the mutation at position C86 can be, for example, C86S or C86A or C86Y. These C86 mutants of IFNα1 are referred to as reduced cysteine-based aggregation mutants. In some embodiments, the IFNα1 variant includes mutations at positions C1, C86, and C99 relative to SEQ ID NO: 1. In various embodiments, any of C1, C86, and C99 is deleted or substituted.

[0049] In some embodiments, IFN-α1 is modified, that is, it is variant and comprises one or more mutations in IFNα1.In some embodiments, the one or more mutations reduce the biological activity of IFN-α1 ("mutation causes attenuation").For example, the one or more mutations can reduce the affinity and / or activity of IFN-α1 interferon to treatment receptors.In one embodiment, the treatment receptor is an interferon-α / β receptor (IFNAR) composed of IFNAR1 and IFNAR2 subunits.In one embodiment, the modified IFN-α1 includes one or more mutations that reduce its affinity and / or activity to IFNAR1.In another embodiment, the modified IFN-α1 includes one or more mutations that reduce its affinity and / or activity to IFNAR2.In one embodiment, the modified IFN-α1 includes one or more mutations that reduce its affinity and / or activity to IFNAR1, and includes one or more mutations that reduce its affinity and / or activity to IFNAR2. In some embodiments, the loss of affinity and / or activity of the modified IFNα1 for a therapeutic receptor (e.g., IFNAR1, IFNAR2 and / or IFNAR) ("mutational attenuation") can be induced and restored by directing or targeting the 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 comprising one or more mutations that reduce its affinity and / or activity for IFNAR1, IFNAR2 and / or IFNAR ("mutational attenuation") further comprises one or more mutations that reduce undesirable disulfide bond pairing to improve the product homogeneity and pharmaceutical properties of the chimeric protein or chimeric protein complex while maintaining or avoiding significant loss of induction and / or restoration of IFNAR activation of the modified IFNα1 when directed or targeted to a target cell via a targeting moiety ("mutational attenuation"). In some embodiments, IFNα1 is a variant comprising one or more mutations that reduce undesirable disulfide pairing to improve product homogeneity and pharmaceutical properties of the chimeric protein or chimeric protein complex, while maintaining or avoiding significant loss of IFNAR activation of the modified IFNα1 compared to wild-type IFNα1 in the context of the chimeric protein or chimeric protein complex, including maintaining or avoiding significant loss of restoration and induction of IFNAR activation of the modified IFNα1 when directed or targeted to a target cell via a targeting moiety. In some embodiments, IFNα1 is a mutation comprising one or more that reduces undesirable disulfide pairing, wherein the one or more mutations are, for example, located at amino acid positions C1, C29, C86, C99, or C139 of SEQ ID NO: 1. In some embodiments, the mutation at position C86 can be, for example, C86S or C86A or C86Y.These C86 mutants of IFNα1 are referred to as reduced cysteine-based aggregation mutants.In some embodiments, the IFNα1 variant comprises mutations at positions C1, C86, and C99 relative to SEQ ID NO: 1.

[0050] In various embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises a modified form of IFNα1, i.e., an IFNα1 variant (including an IFNα1 mutant) as a signaling agent. In various embodiments, the IFNα1 variant encompasses a mutant, functional derivative, analog, precursor, isoform, splice variant, or fragment of the interferon.

[0051] Additional IFNα1 variant sequences are known in the art. In various embodiments, the modified IFNα1 comprises an amino acid sequence that 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% identical to any known amino acid sequence of an IFNα1 interferon variant. , 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 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 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 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or at least about 94%, or at least about 95%, or about 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).

[0052] In some embodiments, the IFNα1 variant comprises an IFNα1 sequence that 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%. 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 96%. 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% 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 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% identity (e.g., about 100%, or about 101%, or about 102%, or about 103%, or about 104%, or about 105%, or about 106%, or about 107%, or about 108%, or about 109%, or about 110%, or about 111%, or about 112%, or about 113%, or about 114%, or about 115%, or about 116%, or about 117%, or about 118%, 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).

[0053] In various embodiments, the IFNα1 variant comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0054] In some embodiments, the amino acid mutation is an amino acid substitution, and can include conservative and / or non-conservative substitutions.

[0055] "Conservative substitutions" can be made, for example, based on similarities 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 grouped into the following 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 affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0056] As used herein, a "conservative substitution" is defined as the exchange of one amino acid listed in the six standard amino acid groups shown above for another amino acid within the same group. For example, exchanging Asp for Glu results in a negative charge being retained in the polypeptide thus modified. In addition, glycine and proline can be substituted for each other based on their ability to disrupt alpha helices.

[0057] As used herein, a "non-conservative substitution" is defined as the exchange of one amino acid listed in different of the six standard amino acid groups (1) to (6) shown above for another amino acid.

[0058] In various embodiments, the substitutions can also include non-classical amino acids (e.g., generally, selenocysteine, pyrrolysine, N-formylmethionine, beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoroamino acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl amino acids, and amino acid analogs).

[0059] In various embodiments, the IFNα1 is modified to have one or more mutations. In some embodiments, the mutation allows the IFNα1 variant to have one or more weakened activities relative to the unmutated (e.g., wild-type) form of the IFNα1 (e.g., IFNα1 with amino acid sequence SEQ ID NO: 1), such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity. For example, relative to the unmutated (e.g., wild-type) form of the IFNα1, the one or more weakened activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity can be at a therapeutic receptor such as IFNAR. As a result, in various embodiments, the mutation allows the IFNα1 variant to have reduced systemic toxicity, reduced side effects, and reduced off-target effects relative to the unmutated (e.g., wild-type) form of the IFNα1.

[0060] In various embodiments, the 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 provided by the IFNα1 is an agonist at the therapeutic receptor (e.g., activating a cellular effect at the therapeutic site). For example, the IFNα1 can activate a therapeutic receptor. In such embodiments, the mutation reduces the activation activity of the IFNα1 variant at the therapeutic receptor.

[0061] In some embodiments, the affinity or activity of the modified IFNα1 to the treatment receptor can be induced or restored by being connected to a targeting moiety or in a chimeric protein or chimeric protein complex as disclosed herein, for example, after including a targeting moiety in an Fc-based complex. In some embodiments, the activity of IFNα1 is reduced or weakened by fusion with another protein, including in some cases by fusion with a targeting moiety as described herein. In other embodiments, the activity of IFNα1 is reduced or weakened by modifying IFNα1, for example, by introducing a mutation as described herein. In some embodiments, the weakening of activity can be restored by connecting IFNα1 to a targeting moiety or by playing a role of the connected targeting moiety. In a number of embodiments, the targeting moiety - by virtue of its connection or its activity - induces the activity of IFNα1.

[0062] In other embodiments, the reduced affinity or activity for the therapeutic receptor is not substantially induced or restored after being connected to a targeting portion or being contained in a chimeric protein or chimeric protein complex as disclosed herein, for example, an Fc-based complex. In various embodiments, the therapeutic chimeric protein, chimeric protein complex, or Fc-based chimeric protein complex of the present invention reduces off-target effects because wild-type IFNα1 or IFNα1 variants with one or more mutations exhibit weak binding affinity or activity to the therapeutic receptor compared to wild-type IFNα1 (non-fusion). In various embodiments, it was observed that side effects were reduced relative to, for example, IFNα1 wild-type forms or other type I interferons. In various embodiments, IFNα1 constructs and / or IFNα1 variants are substantially inactive to the pathway of the therapeutic active site, and their effects are substantially directed against specifically targeted cell types, thereby greatly reducing undesirable cross-reactivity and side effects.

[0063] In various embodiments, the IFNα1 variant has one or more mutations that cause the IFNα1 variant to have a weakened or reduced affinity for one or more therapeutic receptors, e.g., binding (e.g., K D ) and / or activation (which can be measured as, for example, K A and / or EC 50 ). In various embodiments, the decreased affinity at the therapeutic receptor allows for attenuation of activity and / or signaling from the therapeutic receptor.

[0064] In various embodiments, the IFNα1 variant has one or more mutations that reduce the binding or affinity of the IFNα1 variant to the IFNAR1 subunit of IFNAR. In one embodiment, the IFNα1 variant has a reduced affinity and / or activity to IFNAR1. In some embodiments, the IFNα1 variant has one or more mutations that reduce the binding or affinity of the IFNα1 variant to the IFNAR2 subunit of IFNAR. In some embodiments, the IFNα1 variant has one or more mutations that reduce the binding or affinity of the IFNα1 variant to IFNAR1 and IFNAR2 subunits.

[0065] 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 its binding or affinity for IFNAR2. In some embodiments, chimeric proteins and chimeric protein complexes such as or Fc-based chimeric protein complexes having such IFNα1 variants can provide targeted selective IFNAR1 activity (e.g., IFNAR1 activity can be induced or restored by targeting via a targeting moiety or after inclusion in an Fc-based chimeric protein complex disclosed herein).

[0066] 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 having such an IFNα1 variant, such as an Fc-based chimeric protein complex, can provide targeted selective IFNAR2 activity (e.g., IFNAR2 activity can be induced or restored by targeting via a targeting moiety or after inclusion in an Fc-based chimeric protein complex disclosed herein).

[0067] 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 substantially reduce its binding or affinity to IFNAR2. In some embodiments, a chimeric protein or chimeric protein complex having such an IFNα1 variant, such as an Fc-based chimeric protein complex, can provide targeted selective IFNAR1 and / or IFNAR2 activity (e.g., IFNAR1 and / or IFNAR2 activity can be induced or restored by targeting via a targeting moiety or upon inclusion in an Fc-based chimeric protein complex disclosed herein).

[0068] 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%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% of the affinity and / or biological activity for a therapeutic receptor (e.g., IFNAR or any of its subunits IFNAR1 and / or IFNAR2) relative 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 relative to the wild-type IFNα1.

[0069] In some embodiments, the IFNα1 variant comprises one or more mutations, and the one or more mutations cause the IFNα1 variant to have a reduced affinity for receptors. In some embodiments, the binding affinity of the IFNα1 variant to receptors is lower than the binding affinity of the targeting moiety to its receptor. In some embodiments, this binding affinity difference is present between the IFNα1 variant / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this binding affinity difference allows the IFNα1 variant to have a localized on-target effect and minimizes the off-target effect that constitutes the basis of the side effect observed in the case of wild-type IFNα1. In some embodiments, this binding affinity is low at least about 2 times, or at least about 5 times, or at least about 10 times, or at least about 15 times, or at least about 25 times, or at least about 50 times, or at least about 100 times, or at least about 150 times.

[0070] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be assessed by Scatchard plot analysis and computer fitting of the binding data (e.g., Scatchard, 1949) or by reflectance interferometry spectroscopy under flow-through conditions as described in Brecht et al. (1993), all of which are hereby incorporated by reference in their entirety.

[0071] In various embodiments, the chimeric protein complex of the present invention includes (a) interferon alpha 1 (IFNα1) or a variant thereof, and (b) one or more targeting moieties, the targeting moiety comprising a recognition domain that specifically binds to a target antigen or receptor; wherein the IFNα1 or a variant thereof, and the one or more targeting moieties are connected to a domain that causes a complex (e.g., a complex domain). In some embodiments, the chimeric protein complex of the present invention further includes one or more proteins or peptides that interact with each other (e.g., complex domains), for example, using electrostatic interactions, hydrogen bonding, and / or hydrophobic effects. In some embodiments, the chimeric protein complex is a homopolymer (e.g., it includes two or more chimeric proteins as described herein, the chimeric protein including, for example, interferon alpha 1 (IFNα1) or a variant thereof and one or more targeting moieties connected to one or more linkers). In some embodiments, the chimeric protein complex is a heteropolymer (e.g., it includes a chimeric protein including interferon alpha 1 (IFNα1) or a variant thereof and one or more targeting moieties connected to one or more linkers and another protein). A variety of protein interaction domains (e.g., composite domains) have been used to generate protein complexes and can be used to prepare the purpose of the chimeric protein complex of the present invention. In some embodiments, the chimeric protein complex can be prepared by using leucine zippers, Jun and Fos protein families, helix-turn-helix self-dimerization peptides, collagen and p53 trimer or tetramer subdomains (see, for example, the method for preparing protein complexes as described in U.S. Patent No. 8507222, which is incorporated herein by reference in its entirety). Other methods for preparing heteropolymeric complexes include charge-based heterodimers as described by, for example, Chang et al. (PNAS 1984; 91: 11408-11412), or heterodimerization leucine zippers as described by Deng et al. (Chemistry & Biology 2008; 15: 908-919), or customized 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 described herein (in the context of Fc-based chimeric protein complexes) to form protein complexes.

[0072] In various embodiments, the chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a wild-type signaling agent that has improved target selectivity and safety relative to a signaling agent that is not fused to an Fc or a signaling agent that is not within the scope of a complex (e.g., but not limited to a heterodimer complex). In various embodiments, the chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a wild-type signaling agent that has improved target selection activity relative to a signaling agent that is not fused to an Fc or a signaling agent that is not within the scope of a complex (e.g., but not limited to a heterodimer complex). In various embodiments, the chimeric protein complex, such as an Fc-based chimeric protein complex, allows for conditional activity.

[0073] In various embodiments, the chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a wild-type signaling agent that has improved safety, such as reduced systemic toxicity, reduced side effects, and reduced off-target effects, relative to a signaling agent that is not fused to an Fc or is not within a complex (e.g., but not limited to a heterodimeric complex). In various embodiments, improved safety means that the chimeric protein complex of the invention, such as an Fc-based chimeric protein, provides lower toxicity (e.g., systemic toxicity and / or tissue / organ-related toxicity) compared to a signaling agent that is not fused to an Fc or is not within a complex (e.g., but not limited to a heterodimeric complex); and / or reduced or substantially eliminated side effects; and / or increased tolerability, reduced or substantially eliminated adverse events; and / or reduced or substantially eliminated off-target effects; and / or an increased therapeutic window.

[0074] In some embodiments, the reduced affinity or activity at the receptor can be induced or restored by attachment to one or more targeting moieties as described herein or upon inclusion in a chimeric protein complex disclosed herein, such as an Fc-based chimeric protein complex.

[0075] In various embodiments, the chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a wild-type signaling agent that has reduced, substantially reduced, or eliminated affinity for one or more of its receptors, e.g., binding (e.g., K D ) and / or activation (e.g., when the modified signaling agent is an agonist of its receptor, which can be measured as, for example, K A and / or EC 50 ) and / or inhibition (e.g., when the modified signaling agent is an antagonist of its receptor, which can be measured as, for example, K I and / or IC 50). In various embodiments, the reduced affinity at the signal transducer receptor allows for reduced activity. In such embodiments, the modified signal transducer 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%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% affinity for the receptor compared to a signal transducer that is not fused to an Fc or a signal transducer that is not within the scope of a complex (such as, but not limited to, a heterodimer 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 is not within a complex (e.g., but not limited to, a heterodimeric complex).

[0076] In various embodiments, the chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a wild-type signaling agent, such as a signaling agent that reduces the intrinsic activity of the signaling agent by 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 a signaling agent that is not fused to an Fc or is not within a complex (e.g., but not limited to, a heterodimeric complex).

[0077] In various embodiments, the activity weakened at the treatment receptor, the affinity weakened at the treatment receptor can be induced or restored by being connected to a targeting portion or being contained in a chimeric protein complex such as an Fc-based chimeric protein complex (e.g., an antibody or antibody form described herein) with high affinity for the antigen at the treatment active site as disclosed herein. The targeting is achieved by being connected to IFNα1 or its variants or being contained in a chimeric protein complex such as an Fc-based chimeric protein complex as disclosed herein. In one embodiment, the IFNα1 or its variants are connected to a targeting portion via its amino terminus. In another embodiment, the IFNα1 or its variants are connected to a targeting portion via its carboxyl terminus. In this way, in some embodiments, the chimeric protein of the present invention or a chimeric protein complex such as an Fc-based chimeric protein complex provides a positioned, targeted and controlled therapeutic effect at the treatment receptor.

[0078] 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 mutation may optionally be a hydrophobic mutation and may, for example, be selected from alanine, valine, leucine, and isoleucine. 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. NO: 1, the IFNα1 interferon is modified to have one or more mutations 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, R145L, R145N, R145Q, R145S, R145T, R145V, R145Y, A146D, A146E, A146G, A146H, A146I, A146K, A146L, A146M, A146N, A146Q, A146R, A146S, A146T, A146V, A146Y, M149A, M149V, R150A, S153A, L154A, and N157A. In some embodiments, relative to SEQ ID NO: 1, the IFNα1 mutant comprises one or more mutations selected from the group consisting of 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.

[0079] In one embodiment, relative to SEQ ID NO:1, the IFNα1 interferon or its variant is modified to have one or more mutations at amino acid positions C1, C29, C86, C99 or C139. In this regard, Beilharz et al. Journal of interferon research 6.6 (1986): 677-685 (hereby incorporated by reference in its entirety) describes various IFNα1 mutations that can be used to introduce the modified IFNα1 of the present invention. The mutation at position C86 can be, for example, C86S or C86A or C86Y. These C86 mutants of IFNα1 are referred to as aggregation mutants based on reduced cysteine. In some embodiments, relative to SEQ ID NO:1, IFNα1 variants include mutations at positions C1, C86 and C99.

[0080] Therapeutic agents comprising interferon or its variants

[0081] Targeted cell recruitment

[0082] In various embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, further comprises one or more targeting moieties, the 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, may comprise two, three, four, five, six, seven, eight, nine, ten or more targeting moieties. In illustrative embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises 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 form a synapse) or the same cell (e.g., to obtain a more concentrated signal transducer effect) targeting moiety. In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises IFNα1 or a variant thereof, a Flt3L targeting moiety and a targeting moiety that recognizes PD-1 or PD-L1. In some embodiments, a chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises IFNα1 or a variant thereof, a Flt3L targeting moiety, and two targeting moieties that recognize PD-1 or PD-L1.

[0083] In some embodiments, a chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises IFNα2 or a variant thereof, a Flt3L targeting moiety, and one targeting moiety that recognizes PD-1 or PD-L1. In some embodiments, a chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises IFNα2 or a variant thereof, a Flt3L targeting moiety, and two targeting moieties that recognize PD-1 or PD-L1.

[0084] In various embodiments, the target of interest (e.g., antigen, receptor) is found on one or more immune cells, which may include, but are not limited to, T cells, cytotoxic T lymphocytes, T helper 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) is found on one or more tumor cells. In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can directly or indirectly recruit immune cells, for example, in some embodiments, recruited to a treatment site (e.g., a location with one or more disease cells or cells regulated for therapeutic effect). In some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, can directly or indirectly recruit immune cells, e.g., immune cells that can kill and / or suppress tumor cells, to a site of action (such as a tumor microenvironment, as a non-limiting example).

[0085] In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has a targeting moiety that specifically binds to a recognition domain of 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 a component of a complete cell or cell structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-protein non-cellular marker, including but not limited to nucleic acids, including DNA or RNA, such as, for example, DNA released from necrotic tumor cells or extracellular deposits such as cholesterol.

[0086] In some embodiments, target (such as antigen, receptor) is a part for the non-cellular component of matrix or extracellular matrix (ECM) or the marker relevant to the non-cellular component. As used herein, matrix refers to the connection and support framework of tissue or organ. Matrix can include the collection of cells such as fibroblasts / myofibroblasts, glial cells, epithelium, fat, immunity, blood vessels, smooth muscle and immune cells and extracellular matrix (ECM) and extracellular molecules. In various embodiments, target (such as antigen, receptor) is a part for the non-cellular component of matrix such as extracellular matrix and extracellular molecules. As used herein, ECM refers to the non-cellular component present in all tissues and organs. ECM is composed of biochemically unique components, including but not limited to the large collection of proteins, glycoproteins, proteoglycans and polysaccharides. These components of ECM are usually produced by adjacent cells and secreted into ECM via exocytosis. Once secreted, ECM components tend to gather and form a complex macromolecular network. In various embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein, comprises a targeting moiety that recognizes a target (e.g., an antigen or receptor or a non-protein molecule) located on any component of the ECM. Illustrative components of the ECM include, but are not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers and other ECM proteins or ECM non-proteins, such as polysaccharides and / or lipids, or ECM-associated molecules (e.g., proteins or non-proteins, such as polysaccharides, nucleic acids and / or lipids).

[0087] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on an ECM proteoglycan. Proteoglycans are glycosylated proteins. The basic proteoglycan unit includes a core protein having one or more covalently linked glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge, thereby attracting positively charged sodium ions (Na+), which attract water molecules via osmotic action, thereby keeping the ECM and resident cells hydrated. Proteoglycans may also contribute to the capture and storage of growth factors within the ECM. Illustrative proteoglycans that the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can target include but are not limited to heparan sulfate, chondroitin sulfate, and keratan sulfate. In one embodiment, the targeting moiety recognizes a target (e.g., antigen, receptor) on a non-proteoglycan polysaccharide such as hyaluronic acid.

[0088] In some embodiments, the targeting moiety identifies a target (e.g., antigen, receptor) on an ECM fiber. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety identifies one or more epitopes on collagen or collagen fibers. Collagen is the most abundant protein in ECM. Collagen is present in ECM in the form of fibrous proteins and provides structural support to resident cells. In one or more embodiments, the targeting moiety identifies and is bound to various types of collagen present in the ECM, including but not limited to fibrous collagen (type I, type II, type III, type V, type XI), interspersed with triple-helical fibril-related collagen (facit collagen) (type IX, type XII, type XIV), short-chain collagen (type VIII, type X), basement membrane collagen (type IV) and / or type VI, type VII or type XIII collagen. Elastin fibers provide elasticity to tissues, allowing them to stretch and then return to their original state when needed. In some embodiments, the targeting moiety identifies one or more epitopes on elastin or elastin fibers.

[0089] In some embodiments, the targeting moiety recognizes one or more ECM proteins, including but not limited to tenascin, fibronectin, fibrin, laminin, or nidogen / entactin.

[0090] In one embodiment, the targeting moiety recognizes and binds to tenascin. The glycoproteins of the tenascin (TN) family include at least four members, namely tenascin C, tenascin R, tenascin X and tenascin W. The primary structure of tenascin includes several common motifs, which are in the same continuous sequence in order: an amino-terminal heptavalent repeat sequence, an epidermal growth factor (EGF)-like repeat sequence, a fibronectin type III domain repeat sequence and a carboxyl-terminal fibrinogen-like globular domain. Each protein member is associated with a typical variation in the number and characteristics of EGF-like repeat sequences and fibronectin type III repeat sequences. Isoform variants also exist in particular with respect to tenascin C. More than 27 splice variants and / or isoforms of tenascin C are known. In a specific embodiment, the targeting moiety recognizes and binds to tenascin CA1. Similarly, tenascin R also has different splice variants and isoforms. Tenascin R usually exists in the form of a dimer or trimer. Tenasin X is the largest member of the tenasin family and is known to exist in trimer form. Tenasin W exists in trimer form. In some embodiments, the targeting moiety recognizes one or more epitopes on tenasin. In some embodiments, the targeting moiety recognizes monomers and / or dimers and / or trimers and / or hexamer forms of tenasin.

[0091] In one embodiment, the targeting moiety recognizes and binds to fibronectin. Fibronectin is a glycoprotein that connects cells to collagen fibers in the ECM, allowing cells to move through the ECM. After binding to integrins, fibronectin unfolds to form a functional dimer. 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 illustrative 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 conditions, including psoriasis, rheumatoid arthritis, diabetes, and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing an EDA isoform and can be used to target the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing an EDB isoform. In various embodiments, such targeting moieties can be used to target the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, to tumor cells, including tumor angiogenesis.

[0092] In one embodiment, the targeting moiety recognizes and binds to fibrin. Fibrin is another proteinaceous substance often found in the matrix network structure of the ECM. Fibrin is formed due to the action of the protease thrombin on fibrinogen that causes fibrin polymerization. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes monomeric and polymeric forms of fibrin.

[0093] In one embodiment, the targeting moiety recognizes and binds to laminin. Laminin is a major component of the basal lamina, which is the basis of the protein network of cells and organs. Laminin is a heterotrimeric protein containing α-chain, β-chain and γ-chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes monomers, dimers and trimers of laminin.

[0094] In one embodiment, the targeting moiety recognizes and binds to nidogen or innexin. Nidogen / innexin are a family of highly conserved sulfated glycoproteins. They constitute the major structural component of the basement membrane and function to connect the laminin and collagen IV networks 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.

[0095] In various embodiments, the targeting moiety comprises an antigen recognition domain that identifies an epitope present on any one of the targets described herein. In one embodiment, the antigen recognition domain identifies one or more linear epitopes present on the protein. As used herein, a linear epitope refers to any continuous amino acid sequence present on the protein. In another embodiment, the antigen recognition domain identifies one or more conformational epitopes present on the protein. As used herein, a conformational epitope refers to one or more amino acid segments (possibly discontinuous) that form a three-dimensional surface with features and / or shapes and / or tertiary structures that can be identified by an antigen recognition domain.

[0096] In various embodiments, the targeting moiety can bind to full-length and / or mature forms and / or isoforms and / or splice variants and / or fragments and / or any other naturally occurring or synthetic analogs, variants or mutants of any of the targets described herein. In various embodiments, the targeting moiety can bind to any form of the proteins described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers and associated forms. In various embodiments, the targeting moiety can bind to any translationally modified form of the proteins described herein, such as glycosylated and / or phosphorylated forms.

[0097] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes extracellular molecules such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In one embodiment, the DNA is shed from necrotic or apoptotic tumor cells or other diseased cells into the extracellular space.

[0098] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaques are known. Fibrolipidic (fibrofatty) plaques are characterized by lipid-laden cells accumulating below the arterial intima. Below the endothelium is a fibrous cap covering the atheromatous core of the plaque. The core includes lipid-laden cells (macrophages and smooth muscle cells) with elevated tissue cholesterol and cholesterol ester content, fibrin, proteoglycans, collagen, elastin, and cellular debris. In advanced plaques, the central core of the plaque typically contains extracellular cholesterol deposits (released from dead cells) that form regions of cholesterol crystals with hollow, needle-like cracks. Outside the plaque are younger foamy cells and capillaries. Fibrous plaques are also localized beneath the intima, within the arterial wall, causing wall thickening and expansion, and sometimes causing multi-spot localized narrowing of the lumen and a certain degree of muscle atrophy. Fibrous plaques contain collagen fibers (eosinophilic), calcium (hematoxylinophilic) and lipid-laden cell deposits. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular components of these plaques, such as fibrin, proteoglycans, collagen, elastin, cellular debris and calcium or other inorganic deposits or precipitates. In some embodiments, the cellular debris is nucleic acids, such as DNA or RNA, released from dead cells.

[0099] 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 located in amyloid plaques found in the brains of patients with Alzheimer's disease. For example, the targeting moiety can recognize and bind to the peptide amyloid beta, which is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures located 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.

[0100] In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can have two or more targeting moieties that bind to non-cellular structures. In some embodiments, there are two targeting moieties, one targeting moiety that targets cells and the other targeting moiety 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 protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can be or can be used in methods involving altering the balance of immune cells that favors immune attack by tumors. For example, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can alter the ratio of immune cells at clinically important sites, thereby favoring cells that can kill and / or suppress tumors (e.g., T cells, cytotoxic T lymphocytes, T helper 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), and opposing 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 a subset thereof). In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can increase the ratio of effector T cells to regulatory T cells.

[0101] For example, in some embodiments, the recognition domain specifically binds to a target associated with a T cell (e.g., an antigen, a receptor). In some embodiments, the recognition domain directly or indirectly recruits T cells. In one embodiment, the recognition domain specifically binds to an effector T cell. In some embodiments, the recognition domain directly or indirectly recruits effector T cells, for example, in some embodiments, to a treatment site (e.g., a location with one or more disease cells or cells that are regulated for a therapeutic effect). Illustrative effector T cells include cytotoxic T cells (e.g., αβ TCR, CD3 + 、CD8 + 、CD45RO + ); CD4 + Effector T cells (e.g., αβTCR, CD3 + 、CD4 + 、CCR7 + 、CD62L high、IL - 7R / CD127 + );CD8 + Effector T cells (e.g., αβTCR, CD3 + 、CD8+ 、CCR7 + 、CD62L high、IL - 7R / CD127 + ); effector memory T cells (e.g., CD62L low, CD44 + , TCR, CD3 + IL - 7R / CD127 + IL-15R + , CCR7 low); central memory T cells (e.g., CCR7 + 、CD62L + 、CD27 + ; or CCR7 high, CD44 + 、CD62L high、TCR、CD3 + IL-7R / CD127 + IL-15R + );CD62L + Effector T cells; CD8 + Effector memory T cells (TEMs), including early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L - ) (TemE and TemL, respectively); 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 + IL-12R + IFNγR + 、CXCR3 + ), TH2 effector T cells (e.g. CCR3 + 、CCR4 + and CCR8 + ; or αβTCR, CD3 + 、CD4 + , IL-4R + IL-33R + 、CCR4+ IL-17RB + , CRTH2 + ); TH9 effector T cells (e.g., αβTCR, CD3 + 、CD4 + ); TH17 effector T cells (e.g., αβTCR, CD3 + 、CD4 + IL-23R + 、CCR6 + , IL-1R + ); CD4 + CD45RO + CCR7 + Effector T cells, ICOS + Effector T cells; CD4 + CD45RO + CCR7( - ) effector T cells; and effector T cells that secrete IL-2, IL-4 and / or IFN-γ.

[0102] Illustrative T cell antigens of interest include, for example (and where appropriate, the extracellular domains): CD8, CD3, SLAMF4, IL-2Rα, 4-1BB / TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL-4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL-6R, CCR3, IL-7Rα, CCR4, CXCR1 / IL-S RA, CCR5, CCR6, IL-10Rα, CCR 7, IL-1 ORβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrin α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD 11b, CDS, integrin αX / CD11c, integrin β2 / CD1S, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, 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 γ chain / IL-2Rγ, 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, TNF RIII / TNFRSF1B, TRAIL R1 / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, and TSLP R. In various embodiments, the targeting moiety of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these illustrative T cell antigens.

[0103] As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has a targeting moiety to one or more of a checkpoint marker expressed on T cells, e.g., PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

[0104] For example, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a B cell. In some embodiments, the recognition domain directly or indirectly raises B cells, for example, in some embodiments, is raised to a treatment site (e.g., a position of cells with one or more disease cells or regulated for therapeutic effect). Illustrative target B cell antigens include, 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, 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 to one or more of these illustrative B cell antigens.

[0105] As another example, in some embodiments, the recognition domain specifically binds to a target associated with natural killer cells (e.g., an antigen, a receptor). In some embodiments, the recognition domain directly or indirectly recruits natural killer cells, for example, in some embodiments, to a treatment site (e.g., a location with one or more diseased cells or cells to be modulated for therapeutic effect). Illustrative natural killer cell antigens of interest include, e.g., TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-εRII, LMIR6 / CD300LE, Fc-γR1 / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Integrin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NKG2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc receptor-like protein 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 the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these illustrative NK cell antigens.

[0106] Moreover, 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 treatment site (e.g., a location with one or more disease cells or cells that are regulated for therapeutic effect). Illustrative target macrophage / monocyte antigens 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, latex, CD36 / SR-B3, leukotriene B4 R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD8 4 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, endothelin / 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-CSF Rα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γR1, TLR4, IFN-γR2, TREM-1, IL-1 RII, 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, 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, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSF In some embodiments, the targeting portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these illustrative macrophage / monocyte antigens.

[0107] Moreover, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a dendritic cell. In some embodiments, the recognition domain directly or indirectly recruits dendritic cells, for example, in some embodiments, recruits to a treatment site (e.g., a location with one or more disease cells or cells regulated for therapeutic effect). Illustrative target dendritic cell antigens include, for example, CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-Pl / COLEC12, SREC-II, LIMPIIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23p40, 4-amino-1,8-naphthalene dicarboxamide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, rutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerhans protein, B7-2 / CD86, leukotriene B4 R1, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, Clq R1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LB CCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAML1, 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, 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 capsaicin R1. In various embodiments, the targeting portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these illustrative DC antigens.

[0108] 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, bone marrow cells, monocytes, eosinophils, or a subset thereof. In some embodiments, the recognition domain directly or indirectly raises megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, bone marrow cells, monocytes, eosinophils, or a subset thereof, for example, in some embodiments, is raised to a treatment site (e.g., with one or more disease cells or the position of cells regulated for therapeutic effect). In some embodiments, the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, mast cells, monocytes, erythrocytes, bone marrow cells, suppressor cells of bone marrow origin, NKT cells, and NK cells, or their derivatives.

[0109] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with megakaryocytes and / or platelets. Illustrative target megakaryocyte and / or platelet antigens include, for example, GP IIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the targeting portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these illustrative megakaryocyte and / or platelet antigens.

[0110] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with an erythrocyte. Illustrative target erythrocyte antigens 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 rhesus monkey antigens. In various embodiments, the targeting portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these illustrative erythrocyte antigens.

[0111] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with mast cells. Illustrative mast cell antigens of interest include, for example, SCFR / CD117, Fc εIn some embodiments, the targeting portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these mast cell antigens.

[0112] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with basophils. Illustrative 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 the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these basophil antigens.

[0113] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with neutrophils. Illustrative target neutrophil antigens include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRIII), CD18 protein (LFA-1, CR3 and p150, 95), CD45, CD67, and CD177. In various embodiments, the targeting portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these neutrophil antigens.

[0114] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with eosinophils. Illustrative target eosinophil antigens include, for example, CD35, CD44, and CD69. In various embodiments, the targeting portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these eosinophil antigens.

[0115] In various embodiments, the recognition domain can bind to any suitable target, antigen, receptor or cell surface marker known to those skilled in the art.In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Illustrative tissue-specific markers include, but are not limited to, endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAM1, PROCR, SELE, SELP, TEK, THBD, VCAM1, VWF; smooth muscle cell surface markers such as ACTA2, MYH10, MYH11, MYH9, MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COL1A1, COL1A2, COL3A1, FAP, PH-4; epithelial cell surface markers such as CD1D, K61RS2, KRT10, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUC1, TACSTD1; neovascularization markers such as CD13, TFNA, α-vβ-3 (α V β3), E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN. In various embodiments, the targeting portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these antigens. In various embodiments, the targeting portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of these antigens in cells bearing these antigens.

[0116] 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, tumor cells are directly or indirectly recruited to one or more effector cells (e.g., immune cells described herein) that can kill and / or suppress tumor cells.

[0117] Tumor cells or cancer cells refer to cells or tissues with uncontrolled growth and / or an abnormal increase in cell survival time and / or inhibition of apoptosis that interferes with the normal function of body organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. Illustrative tumor cells include, but are not limited to, the following: 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 cancer or renal carcinoma; 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, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological 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 NHL; bulky NHL; and disease NHL); mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic 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 nevus hamartomas, edema (e.g., associated with brain tumors), and Meigs' syndrome.

[0118] 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 lymphomas, light chain, non-secretory, MGUS, and plasmacytoma), and central nervous system cancers (including, for example, brain (e.g., gliomas (e.g., astrocytomas, oligodendrogliomas, and ependymomas), meningiomas, pituitary adenomas and neuromas), and spinal cord tumors (e.g., meningiomas and neurofibromas).

[0119] Illustrative tumor antigens include, but are not limited to, MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal-related 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-ζ chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A 11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE ​​family tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-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 idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, Smad family tumor antigens, 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-1CT-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 portion of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to 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.

[0120] In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (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, together with any signal transducers described herein (e.g., IFNα1 or its variants). In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for T cells (including but not limited to effector T cells), and (ii) a targeting moiety for tumor cells, together with any signal transducers described herein. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for B cells, and (ii) a targeting moiety for tumor cells, together with any signal transducers described herein. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for dendritic cells, and (ii) a targeting moiety for tumor cells, together with any signal transducers described herein. In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for macrophages, and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein. In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for NK cells, and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein.

[0121] As non-limiting examples, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety to T cells, for example, mediated by targeting CD8, SLAMF4, IL-2Rα, 4-1BB / TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL-4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL-6R, CCR3, IL-7Rα, CCR4, CXCR1 / IL-S RA, CCR5, CCR6, IL-10Rα, CCR 7, IL-1 ORβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrin α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD 11b, CDS, integrin αX / CD11c, integrin β2 / CD1S, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, 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 / SLAM F6, common γ chain / IL-2Rγ, 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, TRAILR1 / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1 or TSLP R; and (ii) a targeting moiety to tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof).

[0122] As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a checkpoint marker expressed on T cells, e.g., one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR; and (ii) a targeting moiety to tumor cells, together with any of the signaling agents described herein.

[0123] In various embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has one or more targeting moieties against PD-1. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has one or more targeting moieties that selectively bind to a PD-1 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-1 polypeptide.

[0124] In one embodiment, 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 WO 2009 / 114335, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the pembrolizumab or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain comprising the amino acid sequence (SEQ ID NO: 7) and / or a light chain comprising the amino acid sequence (SEQ ID NO: 8).

[0125] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody nivolumab (also known as BMS-936558, MDX-1106, ONO-4538, OPDIVO) or a fragment thereof. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US 8,008,449 and WO 2006 / 121168, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, nivolumab or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence (SEQ ID NO: 9) and / or a light chain comprising the amino acid sequence (SEQ ID NO: 10).

[0126] 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 US 2008 / 0025980 and WO 2009 / 101611, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-18 of US 2008 / 0025980: SEQ ID No: 15 of US 2008 / 0025980 (SEQ ID NO: 11); SEQ ID No: 16 of US 2008 / 0025980 (SEQ ID NO: 12); SEQ ID No: 17 of US 2008 / 0025980 (SEQ ID NO: 13); and SEQ ID No: 18 of US 2008 / 0025980 (SEQ ID NO: 14); and / or a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-24 of US 2008 / 0025980: SEQ ID No: 20 of US 2008 / 0025980 (SEQ ID NO: NO:15); SEQ ID No:21 (SEQ ID NO:16) of US 2008 / 0025980; SEQ ID No:22 (SEQ ID NO:17) of US 2008 / 0025980; SEQ ID No:23 (SEQ ID NO:18) of US 2008 / 0025980; and US SEQ ID No: 24 (SEQ ID NO: 19) of 2008 / 0025980.

[0127] In one embodiment, the targeting moiety comprises a light chain comprising SEQ ID NO: 18 (SEQ ID NO: 14) of US 2008 / 0025980 and a heavy chain comprising SEQ ID NO: 22 (SEQ ID NO: 17) of US 2008 / 0025980.

[0128] In one embodiment, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0129] In one embodiment, the targeting moiety comprises the PD-12-Fc fusion protein AMP-224 disclosed in WO 2010 / 027827 and WO 2011 / 066342, the entire disclosures of which are hereby incorporated by reference. In such an embodiment, the targeting moiety may comprise a targeting domain comprising SEQ ID NO: 4 (SEQ ID NO: 20) of WO 2010 / 027827 and / or a B7-DC fusion protein comprising SEQ ID NO: 83 (SEQ ID NO: 21) of WO 2010 / 027827.

[0130] In one embodiment, the targeting moiety comprises the peptide AUNP 12 or any other peptide disclosed in US 2011 / 0318373 or 8,907,053. For example, the targeting moiety may comprise AUNP 12 having the following sequence (i.e., compound 8 of US 2011 / 0318373 or SEQ ID NO: 49):

[0131]

[0132] SEQ ID NO:22).

[0133] In one embodiment, the targeting moiety comprises an anti-PD-1 antibody 1E3 or a fragment thereof as disclosed in US 2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 23) and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24.

[0134] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E8 or a fragment thereof as disclosed in US 2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 25) and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0135] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1H3 or a fragment thereof as disclosed in US 2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 27) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 28).

[0136] In one embodiment, the targeting moiety comprises a VHH against PD-1 as disclosed, for example, in US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the VHH against PD-1 comprises SEQ ID NOs: 347-351 of US 8,907,065 (SEQ ID No: 347 (SEQ ID NO: 29) of US 8,907,065; SEQ ID No: 348 (SEQ ID NO: 30) of US 8,907,065; SEQ ID No: 349 (SEQ ID NO: 31) of US 8,907,065; SEQ ID No: 350 (SEQ ID NO: 32) of US 8,907,065; and SEQ ID No: 351 (SEQ ID NO: 33) of US 8,907,065).

[0137] In one embodiment, the targeting moiety comprises any one of the anti-PD-1 antibodies or fragments thereof as disclosed in US2011 / 0271358 and WO2010 / 036959, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-29 of US2011 / 0271358 (SEQ ID No: 25 (SEQ ID NO: 34) of US2011 / 0271358; SEQ ID No: 26 (SEQ ID NO: 35) of US2011 / 0271358; SEQ ID No: 27 (SEQ ID NO: 36) of US2011 / 0271358; SEQ ID No: 28 (SEQ ID NO: 37) of US2011 / 0271358; and SEQ ID No: 29 (SEQ ID NO: 38) of US2011 / 0271358); and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-29 of US2011 / 0271358. The amino acid sequences of the peptides A and B are shown in Table 1 (SEQ ID NO: 30-33 of US2011 / 0271358 (SEQ ID NO: 39); SEQ ID NO: 31 (SEQ ID NO: 40); SEQ ID NO: 32 (SEQ ID NO: 41); and SEQ ID NO: 33 (SEQ ID NO: 42) of US2011 / 0271358).

[0138] In various embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more antibodies or antibody fragments thereof against PD-1 selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals) and BGB-A317 (BeiGene Ltd.).

[0139] In various embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has one or more targeting moieties against PD-L1. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has one or more targeting moieties that selectively bind to a PD-L1 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L1 polypeptide.

[0140] 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 PD-1 and CD80 receptors. The MEDI4736 and antigen-binding fragments thereof used 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 hereby incorporated by reference. In illustrative embodiments, the MEDI4736 or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising the amino acid sequence (SEQ ID NO: 43) and / or a light chain comprising the amino acid sequence (SEQ ID NO: 44).

[0141] In illustrative 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.

[0142] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (also known as MPDL3280A, RG7446) or a fragment thereof. In illustrative embodiments, atezolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence (SEQ ID NO: 47) and / or a light chain comprising the amino acid sequence (SEQ ID NO: 48).

[0143] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody avelumab (also known as MSB0010718C) or a fragment thereof. In illustrative embodiments, the avelumab or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain comprising the amino acid sequence (SEQ ID NO: 49) and / or a light chain comprising the amino acid sequence (SEQ ID NO: 50).

[0144] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105) or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 51) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 52).

[0145] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3G10 or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 53) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 54).

[0146] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10A5 or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 55) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 56).

[0147] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 5F8 or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 57) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 58).

[0148] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10H10 or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 59) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 60).

[0149] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 1B12 or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 61) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 62).

[0150] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 7H1 or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 63) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 64).

[0151] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 11E6 or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 65) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 66).

[0152] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 12B7 or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 67) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 68).

[0153] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 13G4 or a fragment thereof as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the 13G4 or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 69) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 70).

[0154] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1E12 or a fragment thereof as disclosed in US 2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 71) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 72).

[0155] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 1F4 or a fragment thereof as disclosed in US 2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 73) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 74).

[0156] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2G11 or a fragment thereof as disclosed in US 2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 75) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 76).

[0157] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3B6 or a fragment thereof as disclosed in US 2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 77) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 78).

[0158] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3D10 or a fragment thereof as disclosed in US 2014 / 0044738 and WO 2012 / 145493, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 (SEQ ID NO: 79) and / or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 80).

[0159] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies as disclosed in US2011 / 0271358 and WO2010 / 036959, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 34-38 of US2011 / 0271358 (SEQ ID No: 34 (SEQ ID NO: 81) of US2011 / 0271358; SEQ ID No: 35 (SEQ ID NO: 82) of US2011 / 0271358; SEQ ID No: 36 (SEQ ID NO: 83) of US2011 / 0271358; SEQ ID No: 37 (SEQ ID NO: 84) of US2011 / 0271358; and SEQ ID No: 38 (SEQ ID NO: 85) of US2011 / 0271358); and / or a light chain comprising an amino acid sequence selected from the group consisting of (SEQ ID No: 34 (SEQ ID NO: 81) of US2011 / 0271358); SEQ ID No: 35 (SEQ ID NO: 82) of US2011 / 0271358; SEQ ID No: 36 (SEQ ID NO: 83) of US2011 / 0271358; SEQ ID No: 37 (SEQ ID NO: 84) of US2011 / 0271358; and SEQ ID No: 38 (SEQ ID NO: 85) of US2011 / 0271358). Amino acid sequences of Nos. 39-42: SEQ ID No. 39 (SEQ ID NO: 86) of US2011 / 0271358; SEQ ID No. 40 (SEQ ID NO: 87) of US2011 / 0271358; SEQ ID No. 41 (SEQ ID NO: 88) of US2011 / 0271358; and SEQ ID No. 42 (SEQ ID NO: 89) of US2011 / 0271358).

[0160] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4 or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US Pat. No. 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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.

[0161] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 2.9D10 or a fragment thereof as disclosed in WO 2011 / 066389, US 8,779,108, and US 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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.

[0162] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9 or a fragment thereof as disclosed in WO 2011 / 066389, US 8,779,108, and US 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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.

[0163] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.20A8 or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US Pat. No. 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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.

[0164] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3.15G8 or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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.

[0165] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3.18G1 or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US Pat. No. 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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.

[0166] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A40PT or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US 2014 / 0356353, and US 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.7A40PT 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.

[0167] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H90PT or a fragment thereof as disclosed in WO 2011 / 066389, US 8,779,108, and US 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.14H90PT 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.

[0168] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2016 / 061142, the entire contents of which are hereby incorporated by reference. In illustrative 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 the group consisting of SEQ ID No: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of WO2016 / 061142 (SEQ ID No: 18 (SEQ ID NO: 106) of WO2016 / 061142; SEQ ID No: 30 (SEQ ID NO: 107) of WO2016 / 061142; SEQ ID No: 38 (SEQ ID NO: 108) of WO2016 / 061142; SEQ ID No: 46 (SEQ ID NO: 109) of WO2016 / 061142; SEQ ID No: 50 (SEQ ID NO: 110) of WO2016 / 061142; SEQ ID No: 54 (SEQ ID NO: 55) of WO2016 / 061142). NO: 111); SEQ ID No: 62 (SEQ ID NO: 112) of WO2016 / 061142; SEQ ID No: 70 (SEQ ID NO: 113) of WO2016 / 061142; and SEQ ID No: 78 (SEQ ID NO: 114) of WO2016 / 061142); and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 22, 26, 34, 42, 58, 66, 74, 82 and 86 of WO2016 / 061142 (SEQ ID No: 22 (SEQ ID NO: 115) of WO2016 / 061142; SEQ ID No: 26 (SEQ ID NO: 116) of WO2016 / 061142; and SEQ ID No: 34 (SEQ ID NO: 117) of WO2016 / 061142. NO: 117 of WO2016 / 061142); SEQ ID No: 42 (SEQ ID NO: 118) of WO2016 / 061142; SEQ ID No: 58 (SEQ ID NO: 119) of WO2016 / 061142; SEQ ID No: 66 (SEQ ID NO: 120) of WO2016 / 061142; SEQ ID No: 74 (SEQ ID NO: 121) of WO2016 / 061142; SEQ ID No: 82 (SEQ ID NO: 122) of WO2016 / 061142; and SEQ ID No: 86 (SEQ ID NO: 123) of WO2016 / 061142).

[0169] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2016 / 022630, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID No: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WO2016 / 022630 (SEQ ID No: 2 (SEQ ID NO: 124) of WO2016 / 022630; SEQ ID No: 6 (SEQ ID NO: 125) of WO2016 / 022630; SEQ ID No: 10 (SEQ ID NO: 126) of WO2016 / 022630; SEQ ID No: 14 (SEQ ID NO: 127) of WO2016 / 022630; SEQ ID No: 18 (SEQ ID NO: 128) of WO2016 / 022630; SEQ ID No: 22 (SEQ ID NO: 229) of WO2016 / 022630). ID NO: 129); SEQ ID No: 26 (SEQ ID NO: 130) of WO2016 / 022630; SEQ ID No: 30 (SEQ ID NO: 131) of WO2016 / 022630; SEQ ID No: 34 (SEQ ID NO: 132) of WO2016 / 022630; SEQ ID No: 38 (SEQ ID NO: 133) of WO2016 / 022630; SEQ ID No: 42 (SEQ ID NO: 134) of WO2016 / 022630; and SEQ ID No: 46 (SEQ ID NO: 135) of WO2016 / 022630); and / or a light chain comprising a SEQ ID NO selected from the group consisting of SEQ ID NOs. No.: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 amino acid sequences (SEQ ID No: 4 (SEQ ID NO: 136) of WO2016 / 022630; SEQ ID No: 8 (SEQ ID NO: 137) of WO2016 / 022630; SEQ ID No: 12 (SEQ ID NO: 138) of WO2016 / 022630; SEQ ID No: 16 (SEQ ID NO: 139) of WO2016 / 022630; SEQ ID No: 20 (SEQ ID NO: 140) of WO2016 / 022630);SEQ ID No: 24 (SEQ ID NO: 141) of WO2016 / 022630; SEQ ID No: 28 (SEQ ID NO: 142) of WO2016 / 022630; SEQ ID No: 32 (SEQ ID NO: 143) of WO2016 / 022630; SEQ ID No: 36 (SEQ ID NO: 144) of WO2016 / 022630; SEQ ID No: 40 (SEQ ID NO: 145) of WO2016 / 022630; SEQ ID No: 44 (SEQ IDNO: 146) of WO2016 / 022630; and SEQ ID No: 48 (SEQ ID NO: 147) of WO2016 / 022630).;

[0170] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2015 / 112900, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 38, 50, 82, and 86 of WO 2015 / 112900 (SEQ ID No: 38 (SEQ ID NO: 148) of WO 2015 / 112900; SEQ ID No: 50 (SEQ ID NO: 149) of WO 2015 / 112900; SEQ ID No: 82 (SEQ ID NO: 150) of WO 2015 / 112900; and SEQ ID No: 86 (SEQ ID NO: 151) of WO 2015 / 112900); and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 38, 50, 82, and 86 of WO 2015 / 112900. No.: 42, 46, 54, 58, 62, 66, 70, 74 and 78 of WO 2015 / 112900 (SEQ ID No: 42 (SEQ ID NO: 152) of WO 2015 / 112900; SEQ ID No: 46 (SEQ ID NO: 153) of WO 2015 / 112900; SEQ ID No: 54 (SEQ ID NO: 154) of WO 2015 / 112900; SEQ ID No: 58 (SEQ ID NO: 155) of WO 2015 / 112900; SEQ ID No: 62 (SEQ ID NO: 156) of WO 2015 / 112900; SEQ ID No: 66 (SEQ ID NO: 157) of WO 2015 / 112900; SEQ ID No: 70 (SEQ ID NO: 158) of WO 2015 / 112900; SEQ ID No: 71 (SEQ ID NO: 72) of WO 2015 / 112900; SEQ ID NO: 74 (SEQ ID NO: 159) of WO 2015 / 112900; and SEQ ID No: 78 (SEQ ID NO: 160) of WO 2015 / 112900).

[0171] In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO 2010 / 077634 and US 8,217,149, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof used 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.

[0172] In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies obtainable from hybridomas obtainable under CNCM deposit numbers CNCM 1-4122, CNCM 1-4080, and CNCM 1-4081 as disclosed in US 20120039906, the entire disclosure of which is hereby incorporated by reference.

[0173] In one embodiment, the targeting moiety comprises a VHH directed against PD-L1 as disclosed in, for example, US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the VHHs against PD-L1 comprise SEQ ID Nos: 394-399 of US 8,907,065 (SEQ ID No: 394 of US 8,907,065 (SEQ ID NO: 163); SEQ ID No: 395 of US 8,907,065 (SEQ ID NO: 164); SEQ ID No: 396 of US 8,907,065 (SEQ ID NO: 165); SEQ ID No: 397 of US 8,907,065 (SEQ ID NO: 166); SEQ ID No: 398 of US 8,907,065 (SEQ ID NO: 167); and SEQ ID No: 399 of US 8,907,065 (SEQ ID NO: 168)).

[0174] In various embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has one or more targeting moieties against PD-L2. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has one or more targeting moieties that selectively bind to a PD-L2 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L2 polypeptide.

[0175] In one embodiment, the targeting moiety comprises a VHH directed against PD-L2 as disclosed in, for example, US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the VHHs against PD-L2 comprise SEQ ID Nos: 449-455 of US 8,907,065 (SEQ ID No: 449 of US 8,907,065 (SEQ ID NO: 169); SEQ ID No: 450 of US 8,907,065 (SEQ ID NO: 170); SEQ ID No: 451 of US 8,907,065 (SEQ ID NO: 171); SEQ ID No: 452 of US 8,907,065 (SEQ ID NO: 172); SEQ ID No: 453 of US 8,907,065 (SEQ ID NO: 173); SEQ ID No: 454 of US 8,907,065 (SEQ ID NO: 174); and SEQ ID No: 455 of US 8,907,065 (SEQ ID NO: 175)).

[0176] In one embodiment, the targeting moiety comprises any one of the anti-PD-L2 antibodies as disclosed in US 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 43-47 of US2011 / 0271358 (SEQ ID No: 43 (SEQ ID NO: 176) of US2011 / 0271358; SEQ ID No: 44 (SEQ ID NO: 177) of US2011 / 0271358; SEQ ID No: 45 (SEQ ID NO: 178) of US2011 / 0271358; SEQ ID No: 46 (SEQ ID NO: 179) of US2011 / 0271358; and SEQ ID No: 47 (SEQ ID NO: 180) of US2011 / 0271358); and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 43-47 of US2011 / 0271358. The amino acid sequences of Nos. 48-51 are as follows (SEQ ID No: 48 (SEQ ID NO: 181) of US2011 / 0271358; SEQ ID No: 49 (SEQ ID NO: 182) of US2011 / 0271358; SEQ ID No: 50 (SEQ ID NO: 183) of US2011 / 0271358; and SEQ ID No: 51 (SEQ ID NO: 184) of US2011 / 0271358).

[0177] In various embodiments, the targeting moiety of the present invention can comprise a sequence that is 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%, or at least about 77% identical to any of the sequences disclosed herein. , 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 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%, or about 100% identical to any of the sequences disclosed herein 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) of a sequence targeting PD-1, PD-L1 and / or PD-L2.

[0178] In various embodiments, the targeting moieties of the present invention may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences as disclosed herein that target PD-1, PD-L1, and / or PD-L2.

[0179] Other antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to or target PD-1, PD-L1, and / or PD-L2 are disclosed in WO 2011 / 066389, US 2008 / 0025980, US 2013 / 0034559, US 8,779,108, US 2014 / 0356353, US 8,609,089, US 2010 / 028330, US 2012 / 0114649, WO 2010 / 027827, WO 2011 / 066342, US 8,907,065, WO 2016 / 062722, WO 2009 / 101611, WO 2010 / 027827, WO 2011 / 066342, WO 2007 / 005874, WO 2001 / 014556, US2011 / 0271358, WO 2010 / 036959, WO 2010 / 077634, US 8,217,149, US 2012 / 0039906, WO 2012 / 145493, US 2011 / 0318373, US Patent No. 8,779,108, US 20140044738, WO 2009 / 089149, WO2007 / 00587, WO 2016061142, WO 2016,02263, WO 2010 / 077634 and WO 2015 / 112900, the entire disclosure of which is hereby incorporated by reference.

[0180] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for T cells, for example, mediated by targeting CD8; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a targeting moiety for CD8 on T cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0181] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for T cells, for example, mediated by targeting CD4; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a targeting moiety for CD4 on T cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0182] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting portion for T cells, for example, mediated by targeting CD3, CXCR3, CCR4, CCR9, CD70, CD103, or one or more immune checkpoint markers; and (ii) a targeting portion for tumor cells, together with any signaling agent described herein (e.g., IFNα1 interferon or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a targeting portion for CD3 on T cells and a second targeting portion for PD-L1 or PD-L2 on tumor cells.

[0183] In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has one or more targeting moieties directed against CD3 expressed on T cells. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has one or more targeting moieties that selectively bind to 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 antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a CD3 polypeptide.

[0184] 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 hereby incorporated by reference. In illustrative embodiments, muromonab-CD3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence (SEQ ID NO: 185) and / or a light chain comprising the amino acid sequence (SEQ ID NO: 186).

[0185] In one embodiment, the targeting moiety comprises the anti-CD3 antibody otelixizumab or a fragment thereof. Otelixizumab is disclosed in U.S. Patent No. 20160000916 and Chatenoud et al. (2012) 9:372-381, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the otelixizumab or an antigen-binding fragment thereof used 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.

[0186] In one embodiment, the targeting moiety comprises the anti-CD3 antibody teplizumab (also known as AKA 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 hereby incorporated by reference. In illustrative embodiments, 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.

[0187] In one embodiment, the targeting moiety comprises the anti-CD3 antibody visilizumab (also known as HuM291) or a fragment thereof. Visiluzumab is disclosed in US Pat. No. 5,834,597 and WO2004052397, as well as in Cole et al., Transplantation (1999) 68:563-571, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, Visiluzumab 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.

[0188] In one embodiment, the targeting moiety comprises the anti-CD3 antibody foralumab (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 US20140193399, US 7,728,114, US20100183554 and US 8,551,478, the entire disclosures of which are hereby incorporated by reference.

[0189] In illustrative embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 2 and 6 of US 7,728,114 (SEQ ID NO: 2 (SEQ ID NO: 193) of US 7,728,114 and SEQ ID NO: 6 (SEQ ID NO: 194) of US 7,728,114) and / or a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 4 and 8 of US 7,728,114 (SEQ ID NO: 195) of US 7,728,114 and SEQ ID NO: 196 of US 7,728,114).

[0190] In some embodiments, the targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 of US 7,728,114 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 of US 7,728,114. In one embodiment, the targeting moiety comprises any of the anti-CD3 antibodies disclosed in US2016 / 0168247, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 6-9 of US2016 / 0168247 (SEQ ID No: 6 (SEQ ID NO: 197) of US2016 / 0168247; SEQ ID No: 7 (SEQ ID NO: 198) of US2016 / 0168247; SEQ ID No: 8 (SEQ ID NO: 199) of US2016 / 0168247; and SEQ ID No: 9 (SEQ ID NO: 200) of US2016 / 0168247); and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 10-12 of US2016 / 0168247 (SEQ ID No: 10 (SEQ ID NO: 100) of US2016 / 0168247). NO:201); SEQ ID No: 11 (SEQ ID NO:202) of US2016 / 0168247; and SEQ ID No: 12 (SEQ ID NO:203) of US2016 / 0168247).

[0191] In one embodiment, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in US2015 / 0175699, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or antigen-binding fragment thereof used 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 US2015 / 0175699; and / or a light chain comprising an amino acid sequence selected from SEQ ID No: 10 (SEQ ID NO: 205) of US2015 / 0175699.

[0192] In one embodiment, the targeting moiety comprises any of the anti-CD3 antibodies disclosed in US 8,784,821, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 2, 18, 34, 50, 66, 82, 98, and 114 of US 8,784,821 (SEQ ID No: 2 of US 8,784,821 (SEQ ID NO: 206); SEQ ID No: 18 of US 8,784,821 (SEQ ID NO: 207); SEQ ID No: 34 of US 8,784,821 (SEQ ID NO: 208); SEQ ID No: 50 of US 8,784,821 (SEQ ID NO: 209); SEQ ID No: 66 of US 8,784,821 (SEQ ID NO: 210); SEQ ID No: 82 of US 8,784,821 (SEQ ID NO: 211); 8,784,821 (SEQ ID NO: 214); SEQ ID No: 26 of US 8,784,821 (SEQ ID NO: 215); SEQ ID No: 42 of US 8,784,821 (SEQ ID NO: 216); SEQ ID No: 58 of US 8,784,821 (SEQ ID NO: 217); SEQ ID No: 74 of US 8,784,821 (SEQ ID NO: 218); SEQ ID No: 86 of US 8,784,821 (SEQ ID NO: 219); SEQ ID No: 90 of US 8,784,821 (SEQ ID NO: 220); SEQ ID No: 91 of US 8,784,821 (SEQ ID NO: 221); SEQ ID No: 92 of US 8,784,821 (SEQ ID NO: 222); SEQ ID No: 93 of US 8,784,821 (SEQ ID NO: 223); SEQ ID No: 94 of US 8,784,821 (SEQ ID NO: 224); SEQ ID No: 95 of US 8,784,821 (SEQ ID NO: 225); ID NO:218); SEQ ID No:90 (SEQ ID NO:219) of US 8,784,821; SEQ ID No:106 (SEQ ID NO:220) of US 8,784,821; and SEQ ID No:122 (SEQ ID NO:221) of US 8,784,821).

[0193] In one embodiment, the targeting moiety comprises any one of the anti-CD3 binding constructs disclosed in US20150118252, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID No: 6 and 86 of US20150118252 (SEQ ID No: 6 (SEQ ID NO: 222) of US20150118252 and SEQ ID No: 86 (SEQ ID NO: 223) of US20150118252); and / or a light chain comprising an amino acid sequence selected from SEQ ID No: 3 of US2015 / 0175699 (SEQ ID No: 3 (SEQ ID NO: 224) of US20150118252).

[0194] In one embodiment, the targeting moiety comprises any of the anti-CD3 binding proteins disclosed in US 2016 / 0039934, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 6-9 of US2016 / 0039934 (SEQ ID No: 6 (SEQ ID NO: 225) of US2016 / 0039934; SEQ ID No: 7 (SEQ ID NO: 226) of US2016 / 0039934; SEQ ID No: 8 (SEQ ID NO: 227) of US2016 / 0039934; and SEQ ID No: 9 (SEQ ID NO: 228) of US2016 / 0039934); and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 1-4 of US2016 / 0039934 (SEQ ID No: 1 (SEQ ID NO: 229) of US2016 / 0039934). ID NO: 229); SEQ ID No: 2 of US2016 / 0039934 (SEQ ID NO: 230); SEQ ID No: 3 of US2016 / 0039934 (SEQ ID NO: 231); and SEQ ID No: 4 of US2016 / 0039934 (SEQ ID NO: 232).

[0195] In various embodiments, the targeting moiety of the present invention can comprise a sequence that is 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 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% to any of the sequences disclosed herein). 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%, about 99%, or about 100% sequence identity) of a sequence targeting CD3.

[0196] In various embodiments, the targeting moieties of the invention may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences as disclosed herein that target CD3. In various embodiments, the targeting moiety of the present invention can comprise any heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequence of a CD3-specific antibody, 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 hereby incorporated by reference.

[0197] Other antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to or target CD3 are disclosed in U.S. Patent 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 International Publication No. WO 2004 / 052397, the entire disclosures of which are hereby incorporated by reference.

[0198] In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety that is directed to T cells, for example, mediated by targeting PD-1; and (ii) a targeting moiety that is directed to tumor cells, together with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0199] As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety that is directed to B cells, e.g., mediated by targeting 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, CDwl30, CD138, or CDwl50; and (ii) a targeting moiety that is directed to tumor cells, together 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 of the invention, such as an Fc-based chimeric protein complex, has a targeting moiety to CD20.

[0200] In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety that is directed to B cells, e.g., mediated by targeting CD19, CD20, or CD70; and (ii) a targeting moiety that is directed to tumor cells, together with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0201] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for B cells, for example, mediated by targeting CD20; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a targeting moiety for CD20 on B cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells. As an example, in some embodiments, the CD20 targeting moiety is a recombinant heavy chain-only antibody (VHH) having the following sequence:

[0202] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISSRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS (SEQ ID NO: 288).

[0203] As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety to NK cells, for example, mediated by targeting 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-εRII, LMIR6 / CD300LE, Fc-γRl / CD64, MICA, Fc-γRIIB / CD32b, MI CB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Bindin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NK G2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc receptor-like protein 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 or ULBP-3; and (ii) a targeting moiety to tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof).

[0204] In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting portion that is directed to NK cells, for example, by targeting Kir1α, DNAM-1, or CD64; and (ii) a targeting portion that is directed to tumor cells, together with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0205] In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for NK cells, for example, mediated by targeting KIR1; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has a targeting moiety for KIR1 on NK cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0206] In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for NK cells, for example, mediated by targeting TIGIT or KIR1; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has a targeting moiety for TIGIT on NK cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0207] As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety to dendritic cells, for example, mediated by targeting CLEC-9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, SREC-II, LIMPIIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB Ligand / TNFSF9, IL-12 / IL-23p40, 4-Amino-1,8-naphthalene dicarboxamide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, rutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerhans protein, B7-2 / CD86, leukotriene B4 R1, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, Clq R1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LB CCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAML1, CD2F-10 / SLAMF9, osteoactivin GPNMB, Chern 23. 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 / 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 capsaicin R1; and (ii) a targeting moiety to tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof).

[0208] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for dendritic cells, for example, mediated by targeting CLEC-9A, DC-SIGN, CD64, CLEC4A, or DEC205; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a targeting moiety for CLEC9A on dendritic cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0209] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for dendritic cells, for example, mediated by targeting CLEC9A; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a targeting moiety for CLEC9A on dendritic cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0210] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for dendritic cells, for example, mediated by targeting XCR1; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a targeting moiety for XCR1 on dendritic cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0211] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for dendritic cells, for example, mediated by targeting RANK; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a targeting moiety for RANK on dendritic cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0212] As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety to monocytes / macrophages, for example, mediated by targeting SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin α4 / CD49d, BLAME / SLAMF8, integrin αX / CD11c, CCL6 / C10, integrin β2 / CD18, CD155 / PVR, integrin β3 / CD61, CD31 / PECAM-1, latex, CD36 / SR-B3, leukotriene B4, leukocytes / macrophages ... R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD8 4 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, endothelin / 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-CSF Rα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γR1, TLR4, IFN-γR2, TREM-1, IL-1 RII, 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, C1q R1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / CD11b, CCR8, integrin αX / CD11c, 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, LMIR2 / CD300c, CD163, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endothelin / CD105, NCAM-L1, Fc-γRI / CD64, PSGL-1, Fc-γRIIIICD16, RP105, G-CSF R, L-selectin, GM-CSF Rα, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL-6R, TREM-2, CXCR1 / IL-8RA, TREM-3, or TREML1 / TLT-1; and (ii) a targeting moiety to tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof).

[0213] In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety that is directed to monocytes / macrophages, e.g., mediated by targeting B7-H1, CD31 / PECAM-1, CD163, CCR2, or the macrophage mannose receptor CD206; and (ii) a targeting moiety that is directed to tumor cells, together with any of the signaling agents described herein (e.g., IFNα1 or a variant thereof).

[0214] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for monocytes / macrophages, for example, mediated by targeting SIRP1a; and (ii) a targeting moiety for tumor cells, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a targeting moiety for SIRP1a on macrophages and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0215] In various embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has one or more targeting moieties for checkpoint markers, such as 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, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (i) a targeting moiety for a checkpoint marker on T cells, such as PD-1, and (ii) a targeting moiety for tumor cells, such as PD-L1 or PD-L2, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has a targeting moiety that is directed against PD-1 on T cells and a second targeting moiety that is directed against PD-L1 on tumor cells. In another embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has a targeting moiety that is directed against PD-1 on T cells and a second targeting moiety that is directed against PD-L2 on tumor cells.

[0216] In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises two or more targeting moieties for the same or different immune cells. In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has (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 another immune cell selected from T cells, B cells, dendritic cells, macrophages, NK cells, or a subset thereof, together with any signaling agent described herein (e.g., IFNα1 or a variant thereof).

[0217] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for T cells and one or more targeting moieties for the same or another T cell. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for T cells and one or more targeting moieties for B cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for T cells and one or more targeting moieties for dendritic cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for T cells and one or more targeting moieties for macrophages. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for T cells and one or more targeting moieties for NK cells. For example, in illustrative embodiments, the 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 illustrative embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may include a targeting moiety for CD8 and a targeting moiety for CD3. In another illustrative embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may include a targeting moiety for CD8 and a targeting moiety for PD-1.

[0218] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for B cells and one or more targeting moieties for the same or another B cell. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for B cells and one or more targeting moieties for T cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for B cells and one or more targeting moieties for dendritic cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for B cells and one or more targeting moieties for macrophages. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for B cells and one or more targeting moieties for NK cells.

[0219] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for the same or another dendritic cell. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for T cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for B cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for macrophages. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for dendritic cells and one or more targeting moieties for NK cells.

[0220] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for macrophages and one or more targeting moieties for the same or another macrophage. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for macrophages and one or more targeting moieties for T cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for macrophages and one or more targeting moieties for B cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for macrophages and one or more targeting moieties for dendritic cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties for macrophages and one or more targeting moieties for NK cells.

[0221] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as a chimeric protein complex based on Fc, comprises one or more targeting moieties for NK cells and one or more targeting moieties for the same or another NK cell. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as a chimeric protein complex based on Fc, comprises one or more targeting moieties for NK cells and one or more targeting moieties for T cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as a chimeric protein complex based on Fc, comprises one or more targeting moieties for NK cells and one or more targeting moieties for B cells. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as a chimeric protein complex based on Fc, comprises one or more targeting moieties for NK cells and one or more targeting moieties for macrophages. In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as a chimeric protein complex based on Fc, comprises one or more targeting moieties for NK cells and one or more targeting moieties for dendritic cells.

[0222] In one embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises a targeting moiety directed to a tumor cell and a second targeting moiety directed to the same or different tumor cell. In such embodiments, the targeting moiety can bind to any tumor antigen described herein.

[0223] In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties having a recognition domain that binds to a target of interest (e.g., an antigen, a receptor), including targets found on one or more cells selected from the group consisting of: adipocytes (e.g., white adipocytes, brown adipocytes), hepatic adipocytes, hepatocytes, renal cells (e.g., renal parietal cells, renal salivary glands, renal mammary glands, etc.), ductal cells (ductal cells of seminal vesicles, prostate, etc.), intestinal brush border cells (with microvilli), exocrine gland striated ductal cells, gallbladder epithelial cells, efferent ductules, etc. Ciliated cells, epididymal chief cells, epididymal basal cells, endothelial cells, ameloblast epithelial cells (enamel secretion), semilunar squamous epithelial cells of the vestibular system (proteoglycan secretion), organ of Corti interdental epithelial cells (secretion of the membrane covering the hair cells), loose connective tissue fibroblasts, corneal fibroblasts (keratocytes), tendon fibroblasts, bone marrow reticular tissue fibroblasts, non-epithelial fibroblasts, pericytes, nucleus pulposus cells of the intervertebral disc, cementoblasts / cementocytes (root bone-like ewan cells secretion), odontoblast / odontocyte (dentin secretion), hyaline chondrocytes, fibrochondrocytes, elastic chondrocytes, osteoblast / osteocytes, osteoprogenitor cells (osteoblast stem cells), clear cells of the vitreous humor, 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, secretory epithelial cells of the exocrine glands (e.g. salivary glands), cells, mammary gland cells, lacrimal gland cells, sweat gland cells, sebaceous gland cells, prostate cells, gastric gland cells, pancreatic acinar cells, alveolar cells), hormone-secreting cells (e.g., pituitary cells, neurosecretory cells, intestinal and respiratory tract cells, thyroid cells, parathyroid cells, adrenal gland cells, testicular interstitial cells, pancreatic islet cells), keratinized epithelial cells, wet stratified barrier epithelial cells, neuronal cells (e.g., sensory transducing cells, autonomic neuronal cells, sensory organ and peripheral nerve supporting cells, and central nervous system neurons and glial cells such as interneurons, chief cells, astrocytes, oligodendrocytes, and ependymal cells).

[0224] Targeting moiety form

[0225] In various embodiments, the targeting moiety of the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, is a protein-based agent capable of specific binding, such as an antibody or derivative thereof. In one embodiment, 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 a variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain includes a variable region (V L ) and a constant region (C L ). The variable region determines the specificity of the antibody. Each variable region includes three hypervariable regions flanked by four relatively conserved framework regions (FR), also known as complementarity determining regions (CDRs). The three CDRs (referred to as CDR1, CDR2, and CDR3) contribute to the antibody's binding specificity. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0226] In some embodiments, the targeting moiety comprises an antibody derivative or antibody form. In some embodiments, the targeting moiety of the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, is a single domain antibody, a heavy chain only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain only shark antibody (VNAR), a microprotein (cysteine ​​knot protein, knottin), a DARPin; a tetranectin; an Affibody; a transbody; an anticalin; an AdNectin; an Affilin; a microbody; a peptide aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, or a mitochondrial domain. domain), avimer, atrimer, probody, immunobody, triomab, troybody; pepbody; vaccibody, unibody; affimer, duobody, Fv, Fab, Fab', F(ab')2, peptide mimetic molecule or synthetic molecule as described in the following U.S. Patent Nos. or Patent Publication Nos.: US 7,417,130, US 2004 / 132094, US 5,831,012, US 2004 / 023334, US 7,250,297, US 6,818,418, US 2004 / 209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004 / 146938, US 2004 / 157209, US 6,994,982, US 6,794,144, US 2010 / 239633, US 7,803,907, US 2010 / 119446 and / or US 7,166,697, the contents of which are hereby incorporated by reference in their entirety. See also Storz MAbs. 2011 May-June; 3(3):310-317.

[0227] In one embodiment, the targeting moiety comprises a single domain antibody, such as a VHH from an organism (such as a camel, shark) that produces a VHH antibody, or a designed VHH. VHH is a therapeutic protein derived from an antibody that contains the unique structure and functional properties of naturally occurring heavy chain antibodies. VHH technology is a fully functional antibody based on the lack of light chains from camels. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). VHH can be purchased under the trademarks of NANOBODY or NANOBODIES.

[0228] In one embodiment, the targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

[0229] In some embodiments, the VHH comprises a fully human VH domain, such as HUMABODY (Crescendo Biologics, Cambridge, UK). In some embodiments, the fully human VH domain (e.g., HUMABODY) is monovalent, bivalent, or trivalent. In some embodiments, the fully human VH domain (e.g., HUMABODY) is monospecific or multispecific, such as monospecific, bispecific, or trispecific. Illustrative fully human VH domains (e.g., HUMABODIES) are described in, for example, WO 2016 / 113555 and WO 2016 / 113557, the entire contents of which are incorporated by reference.

[0230] In various embodiments, the targeting portion of a chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, is a protein-based agent that is capable of specifically binding to a cell receptor, such as a natural ligand of a cell receptor. In various embodiments, the cell receptor is found on one or more immune cells, which may include, but are not limited to, T cells, cytotoxic T lymphocytes, T helper 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 cell receptor is found on megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof.

[0231] In some embodiments, the targeting moiety is a natural ligand, such as a chemokine. Illustrative chemokines that can be included in the chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, include, but are not limited to, CCL1, CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CL17, 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 In one embodiment, the targeting moiety is a chemokine that recognizes and binds to the dendritic cell receptor XCR1. In another embodiment, the targeting moiety is a chemokine that recognizes and binds to CCR8. In another embodiment, the targeting moiety is a chemokine that recognizes and binds to CCR2 or CCR9. In another illustrative embodiment, the targeting moiety is CCL3, which is a chemokine that identifies and is bound to CCR1, CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL4, which is a chemokine that identifies and is bound to CCR1 or CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL5, which is a chemokine that identifies and is bound to CCR1 or CCR3 or CCR4 or CCR5. In another illustrative embodiment, the targeting moiety is CCL6, which is a chemokine that identifies and is bound to CCR1. In another illustrative embodiment, the targeting moiety is CCL7, which is a chemokine that identifies and is bound to CCR2 or CCR9. In another illustrative embodiment, the targeting moiety is CCL8, which is a chemokine that identifies and is bound to CCR1 or CCR2 or CCR2B or CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL9, which is a chemokine that identifies and is bound to CCR1. In another illustrative embodiment, the targeting moiety is CCL10, which is a chemokine that recognizes and binds to CCR1. In another illustrative embodiment, the targeting moiety is CCL11, which is a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR9.In another illustrative embodiment, the targeting moiety is CCL13, which is a chemokine that identifies and is bound to CCR2 or CCR3 or CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL14, which is a chemokine that identifies and is bound to CCR1 or CCR9. In another illustrative embodiment, the targeting moiety is CCL15, which is a chemokine that identifies and is bound to CCR1 or CCR3. In another illustrative embodiment, the targeting moiety is CCL16, which is a chemokine that identifies and is bound to CCR1, CCR2, CCR5 or CCR8. In another illustrative embodiment, the targeting moiety is CCL17, which is a chemokine that identifies and is bound to CCR4. In another illustrative embodiment, the targeting moiety is CCL19, which is a chemokine that identifies and is bound to CCR7. In another illustrative embodiment, the targeting moiety is CCL20, which is a chemokine that identifies and is bound to CCR6. In another illustrative embodiment, the targeting moiety is CCL21, which is a chemokine that recognizes and is bound to CCR7. In another illustrative embodiment, the targeting moiety is CCL22, which is a chemokine that recognizes and is bound to CCR4. In another illustrative embodiment, the targeting moiety is CCL23, which is a chemokine that recognizes and is bound to CCR1. In another illustrative embodiment, the targeting moiety is CCL24, which is a chemokine that recognizes and is bound to CCR3. In another illustrative embodiment, the targeting moiety is CCL25, which is a chemokine that recognizes and is bound to CCR9. In another illustrative embodiment, the targeting moiety is CCL26, which is a chemokine that recognizes and is bound to CCR3. In another illustrative embodiment, the targeting moiety is CCL27, which is a chemokine that recognizes and is bound to CCR10. In another illustrative embodiment, the targeting moiety is CCL28, which is a chemokine that recognizes and is bound to CCR3 or CCR10. In another illustrative embodiment, the targeting moiety is CXCL1, which is a chemokine that recognizes and binds to CXCR1 or CXCR2. In another illustrative embodiment, the targeting moiety is CXCL2, which is a chemokine that recognizes and binds to CXCR2. In another illustrative embodiment, the targeting moiety is CXCL3, which is a chemokine that recognizes and binds to CXCR2. In another illustrative embodiment, the targeting moiety is CXCL4, which is a chemokine that recognizes and binds to CXCR3B. In another illustrative embodiment, the targeting moiety is CXCL5, which is a chemokine that recognizes and binds to CXCR2. In another illustrative embodiment, the targeting moiety is CXCL6, which is a chemokine that recognizes and binds to CXCR1 or CXCR2.In another illustrative embodiment, the targeting moiety is CXCL8, which is a chemokine that recognizes and binds to CXCR1 or CXCR2. In another illustrative embodiment, the targeting moiety is CXCL9, which is a chemokine that recognizes and binds to CXCR3. In another illustrative embodiment, the targeting moiety is CXCL10, which is a chemokine that recognizes and binds to CXCR3. In another illustrative embodiment, the targeting moiety is CXCL11, which is a chemokine that recognizes and binds to CXCR3 or CXCR7. In another illustrative embodiment, the targeting moiety is CXCL12, which is a chemokine that recognizes and binds to CXCR4 or CXCR7. In another illustrative embodiment, the targeting moiety is CXCL13, which is a chemokine that recognizes and binds to CXCR5. In another illustrative embodiment, the targeting moiety is CXCL16, which is a chemokine that recognizes and binds to CXCR6. In another illustrative embodiment, the targeting moiety is LDGF-PBP, which is a chemokine that recognizes and binds to CXCR2. In another illustrative embodiment, the targeting moiety is XCL2, a chemokine that recognizes and binds to XCR1. In another illustrative embodiment, the targeting moiety is CX3CL1, a chemokine that recognizes and binds to CX3CR1.

[0232] 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., it is capable of binding to 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 connected to another Flt3L domain via one or more linkers, wherein the linker is a flexible linker. 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, wherein the Fc domain optionally has one or more mutations, wherein the one or more mutations 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.

[0233] In various embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, comprises various combinations of targeting moieties. In one illustrative embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can comprise two targeting moieties, wherein both targeting moieties are antibodies or derivatives thereof. In another illustrative embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can comprise two targeting moieties, wherein both targeting moieties are natural ligands of cell receptors. In another illustrative embodiment, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can comprise two targeting moieties, wherein one targeting moiety is an antibody or derivative thereof, and the other targeting moiety is a natural ligand of a cell receptor.

[0234] In various embodiments, the chimeric protein of the present invention or chimeric protein complex such as the recognition domain of the chimeric protein complex based on Fc functionally regulates (in a non-limiting manner, partially or completely neutralizes) target (e.g., antigen, receptor), for example, substantially suppresses, reduces or neutralizes the biological effect of the antigen. For example, each recognition domain can be directed to one or more active suppressions or has the ability to suppress, for example, tumor antigens of the immune system of a patient with a tumor. For example, in some embodiments, the chimeric protein of the present invention or chimeric protein complex such as the chimeric protein complex based on Fc functionally regulates immunosuppressive signals (e.g., checkpoint inhibitors), for example, 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. One or more. For example, in some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, is engineered to disrupt, block, reduce and / or inhibit the transmission of immunosuppressive signals, as non-limiting examples, 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.

[0235] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, binds to but does not functionally modulate a target of interest (e.g., an antigen, a receptor), e.g., the recognition domain is or is similar to a binding antibody. For example, in various embodiments, the recognition domain only targets an antigen or receptor, but does not substantially inhibit, reduce, or functionally modulate the biological effect of the antigen or receptor. For example, some of the smaller antibody forms described above (e.g., compared to, for example, complete antibodies) have the ability to target inaccessible epitopes and provide a larger range of specific binding sites. In various embodiments, the recognition domain binds to an epitope that is physically separated from an antigenic site (e.g., an active site of an antigen) that is important for the biological activity of the antigen or receptor.

[0236] Such non-neutralizing binding can be used in various embodiments of the present invention, including methods of recruiting active immune cells to desired sites using chimeric proteins or chimeric protein complexes of the present invention, such as Fc-based chimeric protein complexes, directly or indirectly via effector antigens such as any effector antigens described herein. For example, in various embodiments, chimeric proteins or chimeric protein complexes of the present invention, such as Fc-based chimeric protein complexes, can be used in methods of reducing or eliminating tumors to recruit cytotoxic T cells directly or indirectly to tumor cells via CD8 (e.g., the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can comprise an anti-CD8 recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit cytotoxic T cells expressing CD8 but not functionally modulate the activity of the CD8. On the contrary, in these embodiments, CD8 signaling is an important part of the tumor reduction or elimination effect. As another example, in various methods of reducing or eliminating tumors, chimeric proteins or chimeric protein complexes of the present invention, such as Fc-based chimeric protein complexes, are used to directly or indirectly recruit dendritic cells (DCs) via CLEC9A (e.g., the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can comprise an anti-CLEC9A recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit DCs that express CLEC9A without functionally modulating the activity of CLEC9A. Quite the contrary, in these embodiments, CLEC9A signaling is an important part of the tumor reduction or elimination effect.

[0237] In various embodiments, the recognition domain of a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, binds to XCR1 on, for example, dendritic cells. For example, in some embodiments, the recognition domain comprises all or part of XCL1 or a non-neutralizing anti-XCR1 agent.

[0238] In various embodiments, the chimeric protein of the present invention or chimeric protein complex such as the recognition domain of the chimeric protein complex based on Fc is bound to immunomodulatory antigens (such as immunostimulatory or immunosuppressive antigens). In various embodiments, the immunomodulatory antigen is one or more of the following: 4-1BB, OX-40, HVEM, GITR, CD27, CD28, CD30, CD40, ICOS ligands; OX-40 ligands, LIGHT (CD258), GITR ligands, CD70, B7-1, B7-2, CD30 ligands, CD40 ligands, ICOS, ICOS ligands, CD137 ligands and TL1A. In various embodiments, such immunostimulatory antigens are expressed on tumor cells. In various embodiments, the chimeric protein of the present invention or chimeric protein complex such as the recognition domain of the chimeric protein complex based on Fc binds to such immunostimulatory antigens but does not functionally regulate such immunostimulatory antigens, therefore allowing the cells expressing these antigens to be raised without reducing or losing their potential tumor reduction or elimination ability.

[0239] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, can be within the range of comprising two recognition domains with neutralizing activity, or comprising two recognition domains with non-neutralizing (e.g., binding) activity, or comprising one recognition domain with neutralizing activity and one recognition domain with non-neutralizing (e.g., binding) activity.

[0240] Fc domain

[0241] The fragment crystallizable domain (Fc domain) is the tail region of an antibody that interacts with Fc receptors 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 two heavy chains of the antibody. In IgM and IgE antibody isotypes, the Fc domain contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0242] In some embodiments, the Fc-based chimeric protein complexes of the present technology comprise 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.

[0243] 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.

[0244] 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.

[0245] In some embodiments, the Fc domain comprises one or more mutations. In some embodiments, the one or more mutations of the Fc domain reduce or eliminate the effector function of the Fc domain. In some embodiments, the mutated Fc domain has a reduced affinity or binding to the target receptor. As an example, in some embodiments, the mutation of the Fc domain reduces or eliminates the binding of the Fc domain to FcγR. In some embodiments, FcγR is selected from FcγRI; FcγRIIa, 131R / R; FcγRIIa, 131H / H, FcγRIIb; and FcγRIII. In some embodiments, the mutation of the Fc domain reduces or eliminates binding to complement proteins (such as, for example, C1q). In some embodiments, the mutation of the Fc domain reduces or eliminates binding to both FcγR and complement proteins (such as, for example, C1q).

[0246] In some embodiments, the Fc domain comprises a LALA mutation to reduce or eliminate the effector function of the Fc domain. As an example, in some embodiments, the LALA mutation comprises L234A and L235A substitutions in human IgG (e.g., IgG1) (wherein the numbering is based on the conventional numbering of CH2 residues of human IgG1 according to EU convention (PNAS, Edelman et al., 1969; 63(1):78-85).

[0247] In some embodiments, the Fc domain of the human IgG comprises a mutation at position 46 to reduce or eliminate the effector function of the Fc domain. As an example, in some embodiments, the mutation is selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G and P331S.

[0248] In some embodiments, the Fc domain comprises a FALA mutation to reduce or eliminate the effector function of the Fc domain. As an example, in some embodiments, the FALA mutation comprises F234A and L235A substitutions in human IgG4.

[0249] 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 the effector function of the Fc domain. As an example, in some embodiments, the mutations are selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.

[0250] In some embodiments, the one or more mutations in the Fc domain stabilize the hinge region in the Fc domain. As an example, in some embodiments, the Fc domain comprises a mutation at S228 of human IgG to stabilize the hinge region. In some embodiments, the mutation is S228P.

[0251] In some embodiments, the one or more mutations in the Fc domain promote chain pairing in the Fc domain.In some embodiments, chain pairing is promoted by ionic pairing (also known as charged pairs, ionic bonds, or pairs of charged residues).

[0252] In some embodiments, the Fc domain comprises a mutation at one or more of the following amino acid residues of IgG to promote ion pairing: D356, E357, L368, K370, K392, D399, and K409.

[0253] As an example, in some embodiments, the human IgG Fc domain comprises one of the combinations of mutations in Table 1 to promote ion pairing.

[0254]

[0255]

[0256] In some embodiments, chain pairing is facilitated via knob-into-hole mutations. In some embodiments, the Fc domain comprises one or more mutations to enable knob-into-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." As an example, in some embodiments, a human IgG Fc domain comprises the mutations of Table 2 to enable knob-into-hole interactions.

[0257]

[0258] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of the present technology comprises any combination of mutations disclosed above. As an example, in some embodiments, the Fc domain comprises mutations that promote ion pairing and / or knob-into-hole interactions. As an example, in some embodiments, the Fc domain comprises mutations having one or more of the following properties: promoting ion pairing, inducing knob-into-hole interactions, reducing or eliminating the effector function of the Fc domain, and causing Fc stabilization (e.g., at the hinge).

[0259] As an example, in some embodiments, the human IgG Fc domain comprises mutations disclosed in Table 3 that promote ion pairing and / or promote knob-into-hole interactions in the Fc domain.

[0260]

[0261]

[0262] As an example, in some embodiments, the human IgG Fc domain comprises mutations disclosed in Table 4 that promote ion pairing in the Fc domain, promote knob-into-hole interactions, or a combination thereof. In various embodiments, "Chain 1" and "Chain 2" of Table 4 can be interchanged (e.g., Chain 1 can have Y407T and Chain 2 can have T366Y).

[0263]

[0264]

[0265]

[0266]

[0267] As an example, in some embodiments, the human IgG Fc domain comprises mutations disclosed in Table 5 that reduce or eliminate FcγR and / or complement binding in the Fc domain. In various embodiments, the mutations of Table 5 are present in both chains.

[0268]

[0269]

[0270]

[0271]

[0272] In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology is a homodimer, ie, the Fc region in the chimeric protein complex comprises two identical protein fragments.

[0273] In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology is a heterodimer, ie, the Fc domain comprises two different protein fragments.

[0274] In some embodiments, the heterodimeric Fc domain is engineered using ion pairing and / or knob-into-hole mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation / configuration. In the trans orientation / configuration, in various embodiments, the targeting moiety and the signaling agent (e.g., IFNα1) are not present on the same polypeptide chain of the Fc-based chimeric protein complex of the invention.

[0275] In some embodiments, the Fc domain comprises or begins with the core hinge region of wild-type human IgG1, wherein the core hinge region comprises the sequence Cys-Pro-Pro-Cys. In some embodiments, the Fc domain further comprises an upper hinge or portion thereof (e.g., DKTHTCPPC; see WO 2009053368), EPKSCDKTHTCPPC, or EPKSSDKTHTCPPC; see Lo et al., Protein Engineering, Vol. 11, No. 6, pp. 495-500, 1998).

[0276] Fc-based chimeric protein complexes

[0277] The Fc-based chimeric protein complexes of the present technology comprise at least one Fc domain disclosed herein, at least one signaling agent such as IFNα1 (SA) disclosed herein (eg, IFNα1), and at least one targeting moiety (TM) disclosed herein.

[0278] It will be understood that the Fc-based chimeric protein complexes of the present invention may encompass a complex of two fusion proteins, each fusion protein comprising an Fc domain.

[0279] In some embodiments, the Fc-based chimeric protein complex is a heterodimer. In some embodiments, the heterodimer Fc-based chimeric protein complex has a trans orientation / configuration. In some embodiments, the heterodimer Fc-based chimeric protein complex has a cis orientation / configuration.

[0280] In some embodiments, the heterodimeric Fc domain is engineered using the ion pairing and / or knob-into-hole mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation.

[0281] In the trans orientation, in various embodiments, the targeting moiety and the signaling agent are not found on the same polypeptide chain of the Fc-based chimeric protein complex of the present invention. In the cis orientation, in various embodiments, the targeting moiety and the signaling agent are found on separate polypeptide chains of the Fc-based chimeric protein complex. In the cis orientation, in various embodiments, the targeting moiety and the signaling agent are found on the same polypeptide chain of the Fc-based chimeric protein complex.

[0282] In some embodiments, when more than one targeting moiety is present in the heterodimeric protein complex described herein, one targeting moiety can be in a trans orientation (relative to the signaling agent) while another targeting moiety can be in a cis orientation (relative to the signaling agent). In some embodiments, the signaling agent and the targeting moiety are on the same end / side (N-terminus or C-terminus) of the Fc domain. In some embodiments, the signaling agent and the targeting moiety are on different sides / ends (N-terminus and C-terminus) of the Fc domain.

[0283] In some embodiments, when more than one targeting moiety is present in a heterodimeric protein complex described herein, the targeting moieties may 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 are trans relative to each other because they are on different Fc chains). In some embodiments, when more than one targeting moiety is present on the same Fc chain, the targeting moieties may be on the same or different sides / termini (N-terminus or / and C-terminus) of the Fc chain.

[0284] In some embodiments, when more than one signaling agent is present in a heterodimeric protein complex described herein, the signaling agent may be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the signaling agents are trans relative to each other because they are on different Fc chains). In some embodiments, when more than one signaling agent is present on the same Fc chain, the signaling agents may be on the same or different sides / termini (N-terminus or / and C-terminus) of the Fc chain.

[0285] In some embodiments, when more than one signaling agent is present in a heterodimeric protein complex described herein, one signaling agent can be in a trans orientation (e.g., relative to a targeting moiety) while another signaling agent can be in a cis orientation (e.g., relative to a targeting moiety).

[0286] 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.

[0287] In some embodiments, the Fc-based chimeric protein has an improved in vivo half-life relative 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 relative to a chimeric protein lacking Fc or a chimeric protein that is not a heterodimeric complex.

[0288] The Fc-based chimeric protein complex of the heterodimer is composed of two different polypeptides. In the embodiments described herein, the targeting domain and the signal transducer (e.g., IFNα1) are located on different polypeptides. Therefore, the protein contains only one copy of the targeting domain and can only prepare one copy of the signal transducer (e.g., IFNα1) (this provides a configuration in which potential interference with the desired properties can be controlled). In addition, in multiple embodiments, only one targeting domain (e.g., VHH) can avoid cross-linking of antigens on the cell surface (which may cause adverse effects in some cases). In addition, in multiple embodiments, a signal transducer (e.g., IFNα1) can alleviate molecular "crowding" and potential interference with the recovery of effector functions mediated by the affinity of the targeting domain. In addition, in multiple embodiments, the Fc-based chimeric protein complex of the heterodimer can have two targeting moieties, and these targeting moieties can be placed on two different polypeptides. For example, in multiple embodiments, the C-termini of the two targeting moieties (e.g., VHH) can be masked to avoid potential autoantibodies or pre-existing antibodies (e.g., VHH autoantibodies or pre-existing antibodies). In addition, in various embodiments, for example, heterodimeric Fc-based chimeric protein complexes having a targeting domain other than a signaling agent (e.g., IFNα1) on a different polypeptide (e.g., a wild-type signaling agent, such as IFNα1) may facilitate "cross-linking" of two cell types (e.g., tumor cells and immune cells). In addition, in various embodiments, heterodimeric Fc-based chimeric protein complexes may have two signaling agents, each located on a different polypeptide to allow for more complex effector responses.

[0289] In addition, in various embodiments, for example, heterodimers of Fc-based chimeric protein complexes having targeting domains on different polypeptides other than signaling agents (e.g., IFNα1) provide for combinatorial diversity of targeting moieties and signaling agents (e.g., IFNα1) in a practical manner. For example, in various embodiments, polypeptides having any targeting moiety described herein can be "ready-to-use" combined with polypeptides having any signaling agents described herein to allow rapid generation of various combinations of targeting moieties and signaling agents in a single Fc-based chimeric protein complex.

[0290] 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 connecting an Fc domain, one or more signaling agents (e.g., IFNα1) and one or more targeting moieties. In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting each signaling agent (e.g., IFNα1) and a targeting moiety (or connecting a signaling agent (e.g., IFNα1) to one of the targeting moieties when there is more than one targeting moiety). In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting each signaling agent (e.g., IFNα1) to an Fc domain. In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting each targeting moiety to an Fc domain. In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting a targeting moiety to another targeting moiety. In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting a signaling agent (e.g., IFNα1) to another signaling agent.

[0291] In some embodiments, the Fc-based chimeric protein complex comprises two or more targeting moieties. In such embodiments, the targeting moieties can be the same targeting moiety or they can be different targeting moieties.

[0292] In some embodiments, the Fc-based chimeric protein complex comprises two or more signaling agents. In such embodiments, the signaling agents can be the same targeting moiety or they can be different targeting moieties.

[0293] As an example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, at least two signaling agents (SAs), and at least two targeting moieties (TMs), wherein the Fc domain, signaling agent, and targeting moiety are selected from any one of the Fc domains, signaling agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is a homodimer.

[0294] In various embodiments, the Fc-based chimeric protein complex takes the form Figure 1A-1F in the form of any of the schematic diagrams of .

[0295] In various embodiments, the Fc-based chimeric protein complex takes the form Figure 2A-2H in the form of any of the schematic diagrams of .

[0296] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 3A-3H in the form of any of the schematic diagrams of .

[0297] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 4A-4D in the form of any of the schematic diagrams of .

[0298] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 5A-5F in the form of any of the schematic diagrams of .

[0299] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 6A-6J in the form of any of the schematic diagrams of .

[0300] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 7A-7D in the form of any of the schematic diagrams of .

[0301] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 8A-8F in the form of any of the schematic diagrams of .

[0302] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 9A-9J in the form of any of the schematic diagrams of .

[0303] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 10A-10F in the form of any of the schematic diagrams of .

[0304] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 11A-11L in the form of any of the schematic diagrams of .

[0305] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 12A-12L in the form of any of the schematic diagrams of .

[0306] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 13A-13F in the form of any of the schematic diagrams of .

[0307] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 14A-14L in the form of any of the schematic diagrams of .

[0308] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 15A-15L in the form of any of the schematic diagrams of .

[0309] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 16A-16J in the form of any of the schematic diagrams of .

[0310] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 17A-17J in the form of any of the schematic diagrams.

[0311] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 18A-18F in the form of any of the schematic diagrams.

[0312] In various embodiments, the Fc-based chimeric protein complex takes the form Figures 19A-19F in the form of any of the schematic diagrams of .

[0313] In various embodiments, the Fc-based chimeric protein complex takes the form Figure 20 A- Figure 20 Any form of the schematic diagram of E.

[0314] In some embodiments, the signaling agent is attached to the targeting moiety and the targeting moiety is attached to the same end of the Fc domain (see Figure 1A-1F In some embodiments, the Fc domain is a homodimer.

[0315] In some embodiments, the signaling agent and the targeting moiety are linked to an Fc domain, wherein the targeting moiety and the signaling agent are linked at the same terminus (see Figure 1A-1F In some embodiments, the Fc domain is a homodimer.

[0316] In some embodiments, the targeting moiety is attached to the signaling agent and the signaling agent is attached to the same end of the Fc domain (see Figure 1A-1F In some embodiments, the Fc domain is a homodimer.

[0317] In some embodiments, the homodimeric Fc-based chimeric protein complex has two or more targeting moieties. In some embodiments, there are four targeting moieties and two signaling agents, the targeting moieties being linked to the Fc domain, and the signaling agents being linked to the same end of the targeting moiety (see Figure 2A-2H 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 attached to the Fc domain and two targeting moieties are attached to the signaling agents, which are attached to the same end of the Fc domain (see Figure 2A-2H In some embodiments, the Fc domain is a homodimer. In some embodiments, where there are four targeting moieties and two signaling agents, the two targeting moieties are linked to each other, and one targeting moiety in each pair is linked to the same end of the Fc domain, and the signaling agent is linked to the same end of the Fc domain (see Figure 2A-2H In some embodiments, the Fc domain is a homodimer. In some embodiments, where there are four targeting moieties and two signaling agents, the two targeting moieties are linked to each other, wherein one targeting moiety in each pair is linked to the signaling agent (e.g., IFNα1) and the other targeting moiety in the pair is linked to the Fc domain, wherein the targeting moieties linked to the Fc domain are linked to the same end (see Figure 2A-2H In some embodiments, the Fc domain is a homodimer.

[0318] In some embodiments, the homodimeric Fc-based chimeric protein complex has two or more signaling agents. In some embodiments, where there are four signaling agents and two targeting moieties, the two signaling agents are linked to each other, and one signaling agent in the pair is linked to the same end of the Fc domain, and the targeting moiety is linked to the same end of the Fc domain (see Figure 3A-3H In some embodiments, the Fc domain is a homodimer. In some embodiments, where there are four signaling agents and two targeting moieties, the two signaling agents are linked to the same end of the Fc domain, and two of the signaling agents are each linked to a targeting moiety, wherein the targeting moieties are linked to the same end of the Fc domain (see Figure 3A-3H In some embodiments, the Fc domain is a homodimer. In some embodiments, where there are four signaling agents and two targeting moieties, the two signaling agents are linked to each other and one of the signaling agents in the pair is linked to the targeting moiety, and the targeting moieties are linked to the same end of the Fc domain (see Figure 3A-3HIn some embodiments, the Fc domain is a homodimer.

[0319] As an example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, wherein the Fc domain comprises one or more ion-pairing mutations and / or one or more knob-into-hole mutations; at least one signaling agent (e.g., IFNα1); and at least one targeting moiety, wherein the ion-pairing motif and / or knob-into-hole motif, signaling agent (e.g., IFNα1), and targeting group are selected from any one of the ion-pairing groups and / or knob-into-hole motifs, signaling agents, and targeting moieties disclosed herein. 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.

[0320] In some embodiments, the signaling agent (eg, IFNα1) is linked to the targeting moiety, which is linked to the Fc domain (see Figures 10A-10F and Figures 13A-13F In some embodiments, the targeting moiety is linked to the signaling agent (eg, IFNα1), which is linked to the Fc domain (see Figures 10A-10F and Figures 13A-13F 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.

[0321] In some embodiments, the signaling agent (eg, IFNα1) and the targeting moiety are linked to the Fc domain (see Figures 4A-4D 、 Figures 7A-7D 、 Figures 10A-10F and Figures 13A-13F In some embodiments, the targeting moiety and the signaling agent (eg, IFNα1) are linked to the same terminus of different Fc chains (see Figures 4A-4D and Figures 7A-7D In some embodiments, the targeting moiety and the signaling agent (eg, IFNα1) are attached to different ends of different Fc chains (see Figures 4A-4D and Figures 7A-7D In some embodiments, the targeting moiety and the signaling agent (eg, IFNα1) are linked to the same Fc chain (see Figures 10A-10F and Figures 13A-13F 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.

[0322] 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 an Fc domain, and the two targeting moieties can be: 1) linked to each other, with one targeting moiety linked to the Fc domain; or 2) each linked to an Fc domain (see Figures 5A-5F 、 Figures 8A-8F 、 Figures 11A-11L 、 Figures 14A-14L 、 Figures 16A-16J and Figures 17A-17J In some embodiments, the targeting moiety is attached to one Fc chain and the signaling agent (eg, IFNα1) is on the other Fc chain (see Figures 5A-5F and Figures 8A-8F In some embodiments, a paired targeting moiety and signaling agent (eg, IFNα1) are linked to the same Fc chain (see Figures 11A-11L and Figures 14A-14L In some embodiments, one targeting moiety is linked to the Fc domain, the other targeting moiety is linked to the signaling agent (e.g., IFNα1), and the paired targeting moieties are linked to the Fc domain (see Figures 11A-11L 、 Figures 14A-14L 、 Figures 16A-16J and Figures 17A-17J In some embodiments, the unpaired targeting moiety and the paired targeting moiety are attached to the same Fc chain (see Figures 11A-11L and Figures 14A-14L In some embodiments, the unpaired targeting moiety and the paired targeting moiety are attached to different Fc chains (see Figures 16A-16J and Figures 17A-17J In some embodiments, the unpaired targeting moiety and the paired targeting moiety are attached to the same end (see Figures 16A-16J and Figures 17A-17J 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.

[0323] In some embodiments, where there is one signaling agent (e.g., IFNα1) and two targeting moieties, the targeting moiety is attached to the signaling agent (e.g., IFNα1), the signaling agent is attached to the Fc domain, and the unpaired targeting moiety is attached to the Fc domain (see Figures 11A-11L 、 Figures 14A-14L 、 Figures 16A-16J and Figures 17A-17J In some embodiments, the paired signaling agent (eg, IFNα1) and the unpaired targeting moiety are attached to the same Fc chain (see Figures 11A-11L and Figures 14A-14LIn some embodiments, the paired signaling agent (e.g., IFNα1) and the unpaired targeting moiety are linked to different Fc chains (see Figures 16A-16J and Figures 17A-17J In some embodiments, the paired signaling agent (eg, IFNα1) and the unpaired targeting moiety are linked to the same terminus (see Figures 16A-16J and Figures 17A-17J 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.

[0324] In some embodiments, where there is one signaling agent (e.g., IFNα1) and two targeting moieties, the targeting moieties are linked together and the signaling agent (e.g., IFNα1) is linked to one of the pair of targeting moieties, wherein the targeting moiety not linked to the signaling agent (e.g., IFNα1) is linked to an Fc domain (see Figures 11A-11L and Figures 14A-14L 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.

[0325] In some embodiments, where there is one signaling agent (e.g., IFNα1) and two targeting moieties, the targeting moieties are linked together and the signaling agent (e.g., IFNα1) is linked to one of the pair of targeting moieties, wherein the signaling agent (e.g., IFNα1) is linked to an Fc domain (see Figures 11A-11L and Figures 14A-14L 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.

[0326] 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), wherein one of the targeting moieties is linked to an Fc domain (see Figures 11A-11L and Figures 14A-14L 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.

[0327] In some embodiments, where there is one signaling agent (e.g., IFNα1) and two targeting moieties, the targeting moiety and the signaling agent (e.g., IFNα1) are linked to an Fc domain (see Figures 16A-16J and Figures 17A-17JIn some embodiments, the targeting moiety is attached to the terminus (see Figures 16A-16J and Figures 17A-17J 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.

[0328] In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are attached to the same terminus of the Fc domain and the targeting moiety is attached to the Fc domain (see Figures 6A-6J and Figures 9A-9J In some embodiments, the signaling agent is attached to an Fc domain on the same Fc chain, while the targeting moiety is attached to another Fc chain (see Figures 18A-18F and Figures 19A-19F 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.

[0329] In some embodiments, where there are two signaling agents and a targeting moiety, the signaling agent (e.g., IFNα1) is linked to the targeting moiety, which is linked to the Fc domain, and the other signaling agent (e.g., IFNα1) is linked to the Fc domain (see Figures 6A-6J 、 Figures 9A-9J 、 Figures 12A-12L and Figures 15A-15L In some embodiments, the targeting moiety and the unpaired signaling agent (eg, IFNα1) are linked to different Fc chains (see Figure 6A-6J and Figures 9A-9J In some embodiments, the targeting moiety and the unpaired signaling agent (eg, IFNα1) are linked to the same terminus of different Fc chains (see Figure 6A-6J and Figures 9A-9J In some embodiments, the targeting moiety and the unpaired signaling agent (eg, IFNα1) are attached to different termini of different Fc chains (see Figures 6A-6J and Figures 9A-9J In some embodiments, the targeting moiety and the unpaired signaling agent (eg, IFNα1) are linked to the same Fc chain (see Figures 12A-12L and Figures 15A-15L 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.

[0330] In some embodiments, where there are two signaling agents and a targeting moiety, the targeting moiety is linked to the signaling agent (e.g., IFNα1), the signaling agent is linked to the Fc domain, and the other signaling agent (e.g., IFNα1) is linked to the Fc domain (see Figure 6A-6J and Figures 9A-9J In some embodiments, the paired signaling agent (eg, IFNα1) and the unpaired signaling agent (eg, IFNα1) are linked to different Fc chains (see Figures 6A-6J and Figures 9A-9J In some embodiments, a paired signaling agent (eg, IFNα1) and an unpaired signaling agent (eg, IFNα1) are linked to the same terminus of different Fc chains (see Figures 6A-6J and Figures 9A-9J In some embodiments, the paired signaling agent (eg, IFNα1) and the unpaired signaling agent (eg, IFNα1) are linked to different ends of different Fc chains (see Figure 6A-6J and Figures 9A-9J 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.

[0331] In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are linked together and the targeting moiety is linked to one of the signaling agents in the pair, wherein the targeting moiety is linked to an Fc domain (see Figures 12A-12L and Figures 15A-15L 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.

[0332] In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are linked together, and one of the signaling agents is linked to an Fc domain, and the targeting moiety is linked to the Fc domain (see Figures 12A-12L 、 Figures 15A-15L 、 Figures 18A-18F and Figures 19A-19F In some embodiments, the paired signaling agent and targeting moiety are linked to the same Fc chain (see Figures 12A-12L and Figures 15A-15L In some embodiments, the paired signaling agent and targeting moiety are linked to different Fc chains (see Figures 18A-18F and Figures 19A-19F In some embodiments, the paired signaling agent and targeting moiety are attached to the same terminus of different Fc chains (see Figures 18A-18F and Figures 19A-19FIn some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0333] In some embodiments, where there are two signaling agents and one targeting moiety, both signaling agents are linked to the targeting moiety, wherein one of the signaling agents is linked to an Fc domain (see Figures 12A-12L and Figures 15A-15L 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.

[0334] In some embodiments, where two signaling agents and a targeting moiety are present, the signaling agents are linked together, with one of the signaling agents being linked to the targeting moiety and the other signaling agent (e.g., IFNα1) being linked to the Fc domain (see Figures 12A-12L and Figures 15A-15L ).

[0335] 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-12L and Figures 15A-15L In some embodiments, the signaling agents are linked to the same Fc chain (see Figures 12A-12L and Figures 15A-15L ).

[0336] In some embodiments, a targeting moiety or signaling agent (eg, IFNα1) is attached to a C H 2 and C H For example, vectors encoding a targeting moiety, a signaling agent (eg, IFNα1), or a combination thereof linked as a single nucleotide sequence to an Fc domain can be used to prepare such polypeptides.

[0337] In some embodiments, the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence that is at least 95%, or at least 98%, or at least 99% identical to any one of SEQ ID NOs: 290, 291, and 293-303. In various embodiments, the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 290, 291, 293-303 and having fewer than 10 mutations relative to the amino acid sequence. In various embodiments, the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 290, 291, 293-303 and having fewer than 5 mutations relative to the amino acid sequence. In some embodiments, the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 290, 291, 293-303.

[0338] In some embodiments, the Fc-based chimeric protein complex comprises a first amino acid sequence that is at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 290 and a second amino acid sequence that is at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 291. In some embodiments, the Fc-based chimeric protein complex comprises a first amino acid sequence that is at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 293 and a second amino acid sequence that is at least 95%, or at least 98%, or at least 99% identical to any one of SEQ ID NOs: 294, 295, 296, 297, 298, or 299. In various embodiments, the Fc-based chimeric protein complex comprises a first amino acid sequence that is at least 95%, or at least 98%, or at least 99% identical to any one of SEQ ID NOs: 300, 301, 302, 303 and a second amino acid sequence that is at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 294.

[0339] Additional signaling agents

[0340] On the one hand, the present invention provides chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes, which include one or more signal transducers (e.g., immunomodulators) in addition to IFNα1 or variants thereof described herein. In illustrative embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may also include two, three, four, five, six, seven, eight, nine, ten or more signal transducers in addition to IFNα1 or variants described herein. In various embodiments, the additional signal transducer is modified so as to have a reduced affinity or activity for one or more of its receptors, thereby allowing for attenuation of activity (including excitement or antagonism) and / or prevention of nonspecific signaling or undesirable chelation of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex.

[0341] In various embodiments, the additional signaling agent is antagonistic in its wild-type form and carries one or more mutations that reduce its antagonistic activity. In various embodiments, the additional signaling agent is antagonistic due to one or more mutations, such as an agonist signaling agent is converted into an antagonist signaling agent, and optionally, this converted signaling agent also carries one or more mutations that reduce its antagonistic activity (e.g., as described in WO2015 / 007520, the entire contents of which are hereby incorporated by reference).

[0342] In various embodiments, the additional signaling agent is selected from modified forms of cytokines, growth factors, and hormones. Illustrative 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; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and tumor necrosis factor-β; Müllerian inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor such as NGF-α; platelet growth factor; transforming growth factor (TGF) such as TGF-α and TGF-β ; insulin-like growth factor-I and insulin-like growth factor-II; osteoinductive factors; interferons such as, for example, interferon-α, interferon-β and interferon-γ (as well as type I interferons, type II interferons and type III interferons), colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as, for example, IL-1β, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13 and IL-18; tumor necrosis factor such as, for example, TNF-α or TNF-β; and other polypeptide factors, including, for example, LIF and kit ligand (KL). As used herein, cytokines, growth factors, and hormones include proteins obtained from natural sources or produced by recombinant bacterial, eukaryotic, or mammalian cell culture systems, as well as biologically active equivalents of the native sequence cytokines.

[0343] In some embodiments, the additional signaling agent is a modified version of a growth factor selected from, but not limited to, transforming growth factor (TGF) such as (TGF-α and TGF-β), epidermal growth factor (EGF), insulin-like growth factor (such as insulin-like growth factor-I and insulin-like growth factor-II), fibroblast growth factor (FGF), heregulin, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF).

[0344] In one embodiment, the growth factor is a modified version of fibroblast growth factor (FGF). Illustrative FGFs include, but are not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, murine FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0345] In one embodiment, the growth factor is a modified form of vascular endothelial growth factor (VEGF). Illustrative VEGFs include, but are not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF, and isoforms thereof, including various isoforms of VEGF-A, such as VEGF 121 , VEGF 121 b. VEGF 145 , VEGF 165 , VEGF 165 b. VEGF 189 and VEGF 206 .

[0346] In one embodiment, the growth factor is a modified version of transforming growth factor (TGF).Illustrative TGFs include, but are not limited to, TGF-α and TGF-β and isoforms thereof, including the various isoforms of TGF-β, including TGFβ1, TGFβ2, and TGFβ3.

[0347] In some embodiments, the additional signaling agent is a modified form of a hormone selected from, but 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, glucocorticoids, mineralocorticoids, epinephrine, norepinephrine, progesterone, insulin, glucagon, pullulan, calcitriol, calciferol, atrial natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor (IGF), leptin, thrombopoietin, erythropoietin (EPO), and angiotensinogen.

[0348] In some embodiments, the additional signaling agent is an immunomodulator, such as one or more of an interleukin, an interferon, and a tumor necrosis factor.

[0349] In some embodiments, the additional signaling agent is an interleukin, including, for example, IL-1β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-14; IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33; IL-35; IL-36 or fragments, variants, analogs or family members thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes and macrophages. Known functions include stimulation of proliferation of immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis of neutrophils and T lymphocytes, and / or inhibition of interferon. Interleukin activity can be determined using assays known in the art: Matthews et al., Lymphokines and Interferons: A Practical Approach, Clemens et al., eds., IRL Press, Washington, DC 1987, pp. 221-225; and Orencole and Dinarello (1989) Cytokine 1, 14-20.

[0350] In some embodiments, the signaling agent is a modified version of an interferon such as type I interferon, type II interferon, and type III interferon. Illustrative interferons include, for example, interferon-α-1, interferon-α-2, interferon-α-4, interferon-α-5, interferon-α-6, interferon-α-7, interferon-α-8, interferon-α-10, interferon-α-13, interferon-α-14, interferon-α-16, interferon-α-17, and interferon-α-21, interferon-β, and interferon-γ, interferon-κ, interferon-ε, interferon-τ, and interferon-ω.

[0351] In various embodiments, the additional signaling agent is a type I interferon. In various embodiments, the type I interferon is selected from IFN-α2, IFNα1, IFN-β, IFN-γ, consensus IFN, IFN-ε, IFN-κ, IFN-τ, IFN-δ, and IFN-v.

[0352] In some embodiments, the additional signaling agent is tumor necrosis factor (TNF) or a protein in the TNF family, including but not limited to TNF-α, TNF-β, LT-β, ​​CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and modified forms of TRAIL.

[0353] In various embodiments, the additional signal transducer is a modified (e.g., mutant) form of a signal transducer with one or more mutations. In various embodiments, the mutation allows the modified signal transducer to have one or more weakened activities relative to the unmodified or unmutated, i.e., wild-type form of a signal transducer, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity or more (e.g., comparing the wild-type form of the same signal transducer to the modified (e.g., mutant) form). In various embodiments, the mutation allows the modified signal transducer to have one or more weakened activities relative to unmodified or unmutated, i.e., unmutated IFNα1, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity or more. In some embodiments, the mutation that weakens or reduces binding or affinity includes those mutations that substantially reduce or eliminate binding or activity. In some embodiments, the mutation that weakens or reduces binding or affinity is different from those mutations that substantially reduce or eliminate binding or activity. As a result, in various embodiments, the mutation allows the signaling agent to be safer, for example, with reduced systemic toxicity, reduced side effects, and reduced off-target effects, relative to an unmutated, i.e., wild-type signaling agent (e.g., comparing a wild-type form versus a modified (e.g., mutant) form of the same signaling agent). In various embodiments, the mutation allows the signaling agent to be safer, for example, with reduced systemic toxicity, reduced side effects, and reduced off-target effects, relative to an unmutated interferon, such as an unmutated sequence of IFNα1.

[0354] In various embodiments, the additional signaling agent is modified to have one or more mutations that reduce its binding affinity or activity to 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 its binding affinity or activity to the receptor. In some embodiments, the activity provided by the wild-type signaling agent is an agonist effect at the receptor (e.g., activating a cellular effect at the treatment site). For example, the wild-type signaling agent can activate its receptor. In such embodiments, the mutation reduces or eliminates the activation activity of the modified signaling agent at the receptor. For example, the mutation can cause the modified signaling agent to deliver a weakened activation signal to the target cell, or can eliminate the activation signal. In some embodiments, the activity provided by the wild-type signaling agent is an antagonist effect at the receptor (e.g., blocking or suppressing a cellular effect at the treatment site). For example, the wild-type signaling agent can antagonize or inhibit the receptor. In these embodiments, the mutation reduces or eliminates the antagonist activity of the modified signaling agent at the receptor. For example, the mutation can cause the modified signal transducer to deliver a weakened inhibitory signal to the target cell, or the inhibitory signal can be eliminated. In various embodiments, the signal transducer has antagonism due to one or more mutations, such as converting an agonist signal transducer into an antagonist signal transducer (e.g., as described in WO 2015 / 007520, the entire contents of which are hereby incorporated by reference), and optionally, such converted signal transducer also carries one or more mutations that reduce its binding affinity or activity to one or more of its receptors, or substantially reduce or eliminate its binding affinity or activity to one or more of its receptors.

[0355] In some embodiments, the reduced affinity or activity at the receptor can be induced or restored by attachment to one or more targeting moieties or upon inclusion in an Fc-based chimeric protein complex disclosed herein. In other embodiments, the reduced affinity or activity at the receptor cannot be substantially induced or restored by the activity of one or more targeting moieties or upon inclusion in an Fc-based chimeric protein complex disclosed herein.

[0356] In various embodiments, the additional signaling agent is active on the target cell because the one or more targeting moieties compensate for the absence / deficiency of binding (e.g., but not limited to, and / or avidity) required for substantial activation. In various embodiments, the modified signaling agent is substantially inactive on the pathway of the therapeutically active site, and its effect is substantially directed to the specifically targeted cell type, thereby greatly reducing undesirable side effects.

[0357] In some embodiments, the additional signaling agent may include one or more mutations that weaken or reduce the binding or affinity to a receptor (i.e., a therapeutic receptor) and one or more mutations that substantially reduce or eliminate the binding or activity at a second receptor. In such embodiments, these mutations may be at the same or different positions (i.e., the same mutation or multiple mutations). In some embodiments, the one or more mutations that reduce the binding and / or activity at one receptor are different from the one or more mutations that substantially reduce or eliminate the binding and / or activity at another receptor. In some embodiments, the one or more mutations that reduce the binding and / or activity at one receptor are the same as the one or more mutations that substantially reduce or eliminate the binding and / or activity at another receptor. In some embodiments, the chimeric protein or chimeric protein complex of the present invention, such as an Fc-based chimeric protein complex, has a modified signaling agent that has both a mutation that weakens the binding and / or activity at the therapeutic receptor and thus allows for a more controlled on-target therapeutic effect (e.g., relative to a wild-type signaling agent) and a mutation that substantially reduces or eliminates the binding and / or activity at another receptor and thus reduces side effects (e.g., relative to a wild-type signaling agent).

[0358] In some embodiments, the substantial reduction or elimination of binding or activity cannot be induced or restored by a targeting moiety or after inclusion in an Fc-based chimeric protein complex disclosed herein. In some embodiments, the substantial reduction or elimination of binding or activity can be induced or restored by a targeting moiety or after inclusion in an Fc-based chimeric protein complex disclosed herein. In various embodiments, substantially reducing or eliminating binding or activity at a second receptor can also prevent adverse effects mediated by another receptor. Alternatively or additionally, substantially reducing or eliminating binding or activity at another receptor improves the therapeutic effect because the reduced or eliminated chelation of the therapeutic chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is away from the site of therapeutic action. For example, in some embodiments, this avoids the need for a high dose of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, that can compensate for the loss at another receptor. This ability to reduce dosage also provides a lower potential for side effects.

[0359] In various embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced, substantially reduced, or eliminated affinity for one or more of its receptors, e.g., binding (e.g., K D ) and / or activation (e.g., when the modified signaling agent is an agonist of its receptor, which can be measured as, for example, K A and / or EC50 ) and / or inhibition (e.g., when the modified signaling agent is an antagonist of its receptor, which can be measured as, for example, K I and / or IC 50 ). In various embodiments, the reduced affinity of the signal transducer receptor allows for attenuation of activity (including agonism or antagonism). In such embodiments, the modified signal transducer has an affinity for the receptor of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% relative to the wild-type signal transducer. 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 relative to the wild-type signaling agent (including but not limited to, relative to unmutated IFNα1).

[0360] In embodiments where the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has a mutation that reduces binding at one receptor and substantially reduces or eliminates binding at a second receptor, the degree of reduction or decrease in the binding affinity of the modified signaling agent for one receptor is less than the substantial reduction or elimination of affinity for the other receptor. In some embodiments, the reduction or decrease in the binding affinity of the modified signaling agent for one receptor is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% lower than the substantial reduction or elimination of affinity for the other receptor. In various embodiments, substantial reduction or elimination refers to a greater reduction in binding affinity and / or activity than reduction or decrease.

[0361] In various embodiments, the additional modified signaling agent comprises one or more mutations that reduce the intrinsic activity of the signaling agent, for example, to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1%, relative to the wild-type signaling agent (including but not limited to, relative to unmutated IFNα1).

[0362] In various embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and / or activity for a receptor for any of the cytokines, growth factors, and hormones described herein.

[0363] In some embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have a reduced affinity for its receptor, lower than the binding affinity of the one or more targeting moieties to its one or more receptors. In some embodiments, this binding affinity difference exists between the signaling agent / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this binding affinity difference allows the signaling agent (e.g., a mutated signaling agent) to have a localized on-target effect and minimizes the off-target effects that form the basis of the side effects observed in the case of wild-type signaling agents. In some embodiments, this binding affinity is at least about 2 times lower, or at least about 5 times, or at least about 10 times, or at least about 15 times, or at least about 25 times lower, or at least about 50 times, or at least about 100 times, or at least about 150 times lower.

[0364] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be assessed by Scatchard plot analysis and computer fitting of the binding data (e.g., Scatchard, 1949) or by reflectance interferometry spectroscopy under flow-through conditions as described in Brecht et al. (1993), all of which are hereby incorporated by reference in their entirety.

[0365] The amino acid sequences of the wild-type signaling agents described herein are well known in the art. Thus, in various embodiments, the additional modified signaling agents comprise 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%, or at least about 100%, or at least about 101%, or at least about 102%, or at least about 103%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 110%, or at least about 111%, or at least about 112%, or at least about 113%, or at least about 114%, or at least about 115%, or at least about 116%, or at least about 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 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 (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).

[0366] In various embodiments, the additional modified signaling agent comprises a sequence that 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 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%, or at least about 100%, or at least about 101%, or at least about 102%, or at least about 103%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 110%, or at least about 111%, or at least about 112%, or at least about 113%, or at least about 114%, or at least about 115%, or at least about 116%, or at least about 117%, or at least about 118%, or at least about 119%. 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 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 (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 or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0367] In various embodiments, the additional modified signaling agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0368] In some embodiments, the amino acid mutation is an amino acid substitution, and can include conservative and / or non-conservative substitutions as described herein.

[0369] As described herein, the additional modified signaling agents carry mutations that affect affinity and / or activity at one or more receptors. In various embodiments, there is a reduced affinity and / or activity for a therapeutic receptor, such as a receptor through which the desired therapeutic effect (excitation or antagonism) is mediated. In various embodiments, the modified signaling agents carry mutations that substantially reduce or eliminate affinity and / or activity at a receptor, such as a receptor through which the desired therapeutic effect (e.g., due to the heterogeneity of the combination) is not mediated. Receptors of any modified signaling agents as described herein, such as one of cytokines, growth factors, and hormones are known in the art.

[0370] Illustrative mutations that provide reduced affinity and / or activity (e.g., agonism) at the receptor are 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 hereby incorporated by reference. Illustrative mutations that provide reduced affinity and / or activity (e.g., antagonism) at the receptor are found in WO 2015 / 007520, the entire contents of which are hereby incorporated by reference.

[0371] In some embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and / or activity for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a receptor in the tumor necrosis factor receptor (TNFR) superfamily, a TGF-β receptor, a receptor in the immunoglobulin (Ig) superfamily, and / or a receptor in the tyrosine kinase superfamily.

[0372] In various embodiments, the receptor of the additional signaling agent 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 (β subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and receptors for thrombopoietin (TPO), prolactin, and growth hormone. Illustrative 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.

[0373] In various embodiments, the receptor of the other signal transducer is a type II cytokine receptor. Type II cytokine receptor is a multimeric receptor consisting of heterologous subunits, and is a receptor primarily used for interferon. This receptor family includes but is not limited to the receptors of interferon-α, interferon-β and interferon-γ, IL10, IL22 and tissue factor. Illustrative type II cytokine receptors include but are not limited to IFN-α receptors (such as IFNAR1 and IFNAR2), IFN-β receptors, IFN-γ receptors (such as IFNGR1 and IFNGR2) and type II IL receptors.

[0374] In various embodiments, the receptor of the other signal transducer is a G protein coupled receptor. Chemokine receptors are G protein coupled receptors with seven transmembrane structures and coupled to G proteins to carry out signal transduction. Chemokine receptors include but are not limited to CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors and XC chemokine receptors (XCR1). Illustrative 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.

[0375] In various embodiments, the receptor for the additional signaling agent is a TNFR family member.Tumor necrosis factor receptor (TNFR) family members share a cysteine-rich domain (CRD) formed by three disulfide bonds surrounding a CXXCXXC core motif, forming an elongated molecule. Illustrative 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), TNFRSF10D (TNFRSF10D), RANK (TNFRSF11A), osteoprotegerin (TNFRSF11B), and TNFRSF11C (TNFRSF11C). IB), 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).

[0376] In various embodiments, the receptor for the additional signaling agent is a TGF-β receptor. The TGF-β receptor is a one-way serine / threonine kinase receptor. TGF-β receptors include but are not limited to TGFBR1, TGFBR2, and TGFBR3.

[0377] In various embodiments, the receptor for the additional signaling agent is an Ig superfamily receptor. Receptors in the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Receptors in the Ig superfamily include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

[0378] In various embodiments, the receptor of the other signal transducer is a tyrosine kinase superfamily receptor. Receptors in the tyrosine kinase superfamily are well known in the art. There are approximately 58 known receptor tyrosine kinases (RTKs), which are divided into 20 subfamilies. Receptors in the tyrosine kinase superfamily include, but are not limited to, FGF receptors and various isotypes thereof, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0379] In one embodiment, the additional modified signaling 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., 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., IFNAR1 and / or IFNAR2 chains.

[0380] Mutated forms of interferon alpha are known to those skilled in the art. In an illustrative embodiment, the modified signaling agent is an allelic form of IFN-alpha 2a having the amino acid sequence of SEQ ID NO: 233.

[0381] In illustrative embodiments, the modified signaling agent is an allelic form of IFN-α2b having the amino acid sequence of SEQ ID NO: 234 (which differs from IFN-α2a at amino acid position 23).

[0382] In some embodiments, the IFN-α2 mutant (IFN-α2a or IFN-α2b) is mutated at one or more amino acids at positions 144-154, such as amino acid positions 148, 149, and / or 153. In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from L153A, R149A, and M148A. Such mutants are described, for example, in WO2013 / 107791 and Piehler et al., (2000) J. Biol. Chem, 275: 40425-33, the entire contents of all of which are hereby incorporated by reference.

[0383] In some embodiments, the IFN-α2 mutant has reduced affinity and / or activity for IFNAR1. In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A as described in WO2010 / 030671, the entire contents of which are hereby incorporated by reference.

[0384] In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A as described in WO2008 / 124086, the entire contents of which are hereby incorporated by reference.

[0385] In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from R120E and R120E / K121E as described in WO2015 / 007520 and WO2010 / 030671, the entire contents of which are hereby incorporated by reference. In such embodiments, the IFN-α2 mutant antagonizes wild-type IFN-α2 activity. In such embodiments, the mutant IFN-α2 has reduced affinity and / or activity for IFNAR1, while retaining affinity and / or activity for IFNR2.

[0386] In some embodiments, the human IFN-α2 mutant comprises (1) one or more mutations selected from R120E and R120E / K121E, without wishing to be bound by theory, which mutations can produce an antagonistic effect; and (2) one or more mutations selected from K133A, R144A, R149A and L153A, without wishing to be bound by theory, which mutations allow for attenuation of the effect at, for example, IFNAR2. In one embodiment, the human IFN-α2 mutant comprises R120E and L153A.

[0387] In some embodiments, the human IFN-α2 mutant 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 disclosure of which is hereby incorporated by reference. In some embodiments, the human IFN-α2 mutant comprises the mutations H57Y, E58N, Q61S and / or L30A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations H57Y, E58N, Q61S and / or R33A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations H57Y, E58N, Q61S and / or M148A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations H57Y, E58N, Q61S and / or L153A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations N65A, L80A, Y85A and / or Y89A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations N65A, L80A, Y85A, Y89A and / or D114A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises one or more mutations selected from R144X1, A145X2 and R33A, wherein X1 is selected from A, S, T, Y, L and I, and wherein X2 is selected from G, H, Y, K and D. In some embodiments, relative to the amino acid sequence of SEQ ID NO: 233 or 234, the human IFN-α2 mutant comprises one or more mutations selected from R33A, T106X3, R120E, R144X1 A145X2, M148A, R149A and L153A, wherein X1 is selected from A, S, T, Y, L and I, wherein X2 is selected from G, H, Y, K and D, and wherein X3 is selected from A and E.

[0388] In one embodiment, the additional modified signaling agent is interferon beta. In such embodiments, the modified interferon beta agent also has reduced affinity and / or activity for IFN-α / β receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified interferon beta agent has substantially reduced or eliminated affinity and / or activity for IFN-α / β receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2 chains.

[0389] In illustrative embodiments, the modified additional signaling agent is IFN-β. In various embodiments, the IFN-β encompasses functional derivatives, analogs, precursors, isoforms, splice variants or fragments of IFN-β. In various embodiments, the IFN-β encompasses IFN-β derived from any species. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a modified version of mouse IFN-β. In another embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a modified version of human IFN-β. Human IFN-β is a polypeptide having a molecular weight of approximately 22 kDa comprising 166 amino acid residues. The amino acid sequence of human IFN-β is SEQ ID NO: 277.

[0390] In some embodiments, the human IFN-β is IFN-β-1a, which is a glycosylated form of human IFN-β. In some embodiments, the human IFN-β is IFN-β-1b, which is a non-glycosylated form of human IFN-β, having a Met-1 deletion and a Cys-17 to Ser mutation.

[0391] In various embodiments, the modified IFN-β has one or more mutations, and the one or more mutations reduce the binding or affinity of the modified IFN-β to the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFN-β has reduced affinity and / or activity at IFNAR1. In various embodiments, the modified IFN-β is human IFN-β 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 selected from the substitutions of F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G and R124G. In one embodiment, the modified IFN-β includes F67G mutation. In one embodiment, the modified IFN-β includes K123G mutation. In one embodiment, the modified IFN-β comprises F67G and R71A mutations. In one embodiment, the modified IFN-β comprises L88G and Y92G mutations. In one embodiment, the modified IFN-β comprises Y92G, I95A and N96G mutations. In one embodiment, the modified IFN-β comprises K123G and R124G mutations. In one embodiment, the modified IFN-β comprises F67G, L88G and Y92G mutations. In one embodiment, the modified IFN-β comprises F67S, L88S and Y92S mutations.

[0392] In some embodiments, the modified IFN-β has one or more mutations, and the one or more mutations reduce the binding or affinity of the modified IFN-β to the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFN-β has reduced affinity and / or activity at IFNAR2. In various embodiments, the modified IFN-β is human IFN-β 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 selected from the substitutions of W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G and Y155G. In one embodiment, the modified IFN-β includes a W22G mutation. In one embodiment, the modified IFN-β includes an L32A mutation. In one embodiment, the modified IFN-β comprises an L32G mutation. In one embodiment, the modified IFN-β comprises an R35A mutation. In one embodiment, the modified IFN-β comprises an R35G mutation. In one embodiment, the modified IFN-β comprises a V148G mutation. In one embodiment, the modified IFN-β comprises an R152A mutation. In one embodiment, the modified IFN-β comprises an R152G mutation. In one embodiment, the modified IFN-β comprises a Y155G mutation. In one embodiment, the modified IFN-β comprises a W22G and R27G mutation. In one embodiment, the modified IFN-β comprises an L32A and R35A mutation. In one embodiment, the modified IFN-β comprises an L151G and R152A mutation. In one embodiment, the modified IFN-β comprises a V148G and R152A mutation.

[0393] In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H, in combination with C17S or C17A.

[0394] In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H, in combination with any other IFN-β mutations described herein.

[0395] The crystal structure of human IFN-β is known and is described in Karpusas et al., (1998) PNAS, 94 (22): 11813-11818. Specifically, it has been shown that the structure of human IFN-β includes five α spirals (i.e. A, B, C, D and E) and four loops (i.e. AB, BC, CD and DE rings) connecting these spirals. In various embodiments, the modified IFN-β has one or more mutations in A, B, C, D, E spirals and / or AB, BC, CD and DE rings, and the one or more mutations reduce the binding affinity or activity of the modified IFN-β at treatment receptors such as IFNAR. Illustrative mutations are described in WO2000 / 023114 and US20150011732, the full contents of which are hereby incorporated by reference. In illustrative embodiments, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In illustrative embodiments, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 28-30, 32, and 33. In illustrative embodiments, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 36, 37, 39, and 42. In illustrative embodiments, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In illustrative embodiments, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 71-73. In illustrative embodiments, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 92, 96, 99, and 100. In illustrative embodiments, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 128, 130, 131, and 134. In illustrative embodiments, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 149, 153, 156, and 159. In some embodiments, the mutant IFN-β comprises SEQ ID NO: 277 and a mutation at W22 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0396] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at R27 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0397] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at W22, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R27, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0398] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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).

[0399] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at R35 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0400] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at L32, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at R35, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0401] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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).

[0402] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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).

[0403] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at F67, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R71, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0404] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0405] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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).

[0406] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at F67, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at L88, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0407] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and 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).

[0408] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at I95, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V); and a mutation at Y92, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0409] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at N96, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and 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).

[0410] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at Y92, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at I95, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), methionine (M), and valine (V); and a mutation at N96, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0411] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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).

[0412] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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).

[0413] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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 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).

[0414] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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).

[0415] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and 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).

[0416] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at L151, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at R152, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0417] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at V148 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0418] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at V148, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R152, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0419] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 277 and a mutation at Y155 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0420] In some embodiments, the present invention relates to a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprising: (a) a modified IFN-β and a modified IL-2 or IL-2 variant disclosed herein, wherein the modified IFN-β has the amino acid sequence SEQ ID NO: 277 and a mutation at position W22, wherein the mutation is an aliphatic hydrophobic residue; and (b) one or more targeting moieties, wherein the one or more targeting moieties comprise a recognition domain that specifically binds to a target antigen or receptor, the modified IFN-β and the one or more targeting moieties are optionally connected to 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.

[0421] Other illustrative IFNβ mutants are provided in PCT / EP2017 / 061544, the entire disclosure of which is incorporated herein by reference.

[0422] In some embodiments, the modified additional signaling 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., IFNGR1 and 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., IFNGR1 and IFNGR2 chains.

[0423] In some embodiments, the modified additional signal transducer is a consensus interferon. By scanning the sequences of several human non-allelic IFN-α subtypes and assigning the most commonly observed amino acids at each corresponding position, a consensus interferon can be generated. The consensus interferon has 20 amino acids different from IFN-α2b in 166 amino acids (88% homology) and is shown to have identity at more than 30% of amino acid positions compared to IFN-β. In various embodiments, the consensus interferon comprises the following amino acid sequence SEQ ID NO: 278.

[0424] 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, ie, SEQ ID NO:279 lacks the initial methionine residue of SEQ ID NO:278.

[0425] In various embodiments, the consensus interferon includes a modified version of the consensus interferon, i.e., a consensus interferon variant, as a signaling agent. In various embodiments, the consensus interferon encompasses a functional derivative, analog, precursor, isoform, splice variant, or fragment of the consensus interferon.

[0426] In one embodiment, 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, all of which are hereby incorporated by reference in their entirety. For example, the consensus interferon variant may 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 one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON2: SEQ ID NO: 280.

[0427] In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON3: SEQ ID NO: 281.

[0428] In one embodiment, the consensus interferon variant comprises the amino acid sequence of any variant 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.

[0429] In another embodiment, the consensus interferon variant may comprise the amino acid sequence of SEQ ID NO:283.

[0430] In some embodiments, the consensus interferon variant may be PEGylated, ie, comprise a PEG moiety. In one embodiment, the consensus interferon variant may comprise a PEG moiety attached to position S156C of SEQ ID NO: 283.

[0431] In some embodiments, the engineered interferon is a variant of human IFN-α2a, with an insertion of Asp at approximately position 41 of the sequence Glu-Glu-Phe-Gly-Asn-Gln (SEQ ID NO: 284) to produce Glu-Glu-Phe-Asp-Gly-Asn-Gln (SEQ ID NO: 285) (this results in renumbering of the sequence relative to the IFN-α2a sequence) and the following mutations: Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser, Asp86Glu, Ile104Thr, Gly106Glu, Thr110Glu, Lys117Asn, Arg125Lys, and Lys136Thr. All embodiments describing the consensus interferon herein are equally applicable to the engineered interferon.

[0432] In some embodiments, the additional modified signal transducer is vascular endothelial growth factor (VEGF). VEGF is a powerful growth factor that plays a major role in physiological and pathological angiogenesis, regulates vascular permeability, and can serve as a growth factor on cells expressing VEGF receptors. Among other functions, additional functions also include stimulating cell migration in macrophage lineages and endothelial cells. There are several members in the VEGF growth factor family, as well as at least three receptors (VEGFR-1, VEGFR-2, and VEGFR-3). Members of the VEGF family can combine with and activate more than one VEGFR type. For example, VEGF-A combines VEGFR-1 and VEGFR-2, while VEGF-C combines VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 activate and regulate angiogenesis, while VEGFR-3 activation is associated with lymphangiogenesis. The main pro-angiogenic signal is generated by VEGFR-2 activation. It is reported that VEGFR-1 activation may be associated with the negative effects in angiogenesis. It has also been reported that VEGFR-1 signal transduction is important for the in vivo tumor progression of VEGFR-1 positive cells (contributing to forming a metastasis niche in bone) via bone marrow origin. Several therapeutic antibodies based on VEGF-A guidance / neutralization have been developed, and these therapies are mainly used in the treatment of various human tumors that rely on angiogenesis. But these therapies are not without side effects. Considering that these therapies work as general non-cell / tissue-specific VEGF / VEGFR interaction inhibitors, this may not be surprising. Therefore, it is desirable that VEGF (e.g., VEGF-A) / VEGFR-2 inhibition is limited to specific target cells (e.g., tumor vascular endothelial cells).

[0433] In some embodiments, the VEGF is VEGF-A, VEGF-B, VEFG-C, VEGF-D, or VEGF-E, and isoforms thereof, including various isoforms of VEGF-A, such as VEGF 121 , VEGF 121 b. VEGF 145 , VEGF 165 , VEGF 165 b. VEGF 189 and VEGF 206 . In some embodiments, the modified signaling agent has a reduced affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In one embodiment, the modified signaling agent has a reduced affinity and / or activity for VEGFR-2 (KDR / Flk-1) and / or has a substantially reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1). Such embodiments can be used, for example, in wound healing methods or treatments of diseases associated with ischemia (without wishing to be bound by theory, these diseases are mediated by the effects of VEGFR-2 on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1 (Flt-1) associated with cancer and proinflammatory activity is avoided. In various embodiments, VEGFR-1 (Flt-1) acts as a decoy receptor, thereby significantly reducing or eliminating affinity at this receptor, thereby avoiding sequestration of the therapeutic agent. In one embodiment, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1), and / or has a substantially 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 signaling agent has a reduced affinity and / or activity for VEGFR-3. Alternatively, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for VEGFR-3.

[0434] Pro-angiogenic therapy is also very important in various diseases (such as ischemic heart disease, hemorrhage, etc.), and includes VEGF-based therapies. The activation of VEGFR-2 has a pro-angiogenic effect (acting on endothelial cells). The activation of VEFGR-1 can stimulate inflammatory cells (including, for example, macrophages) to migrate and cause the high vascular permeability associated with inflammation. The activation of VEFGR-1 can also promote the formation of tumor niches associated with bone marrow. Therefore, in this case, it will be desirable to select a VEGF-based therapeutic agent for VEGFR-2 activation. In addition, it is desirable that cell-specific targeting, for example, endothelial cells.

[0435] In some embodiments, the additional modified signaling agent has reduced affinity and / or activity (e.g., antagonism) for VEGFR-2, and / or has substantially reduced or eliminated affinity and / or activity for VEGFR-1. For example, when targeting tumor vascular endothelial cells via a targeting moiety bound to a tumor endothelial cell marker (e.g., PSMA, etc.), such a construct will specifically inhibit VEGFR-2 activation on such marker-positive cells without activating VEGFR-1 on the way and on the target cells (if the activity is eliminated), thereby eliminating the induction of an inflammatory response. Compared to VEGF-A neutralization therapy, this will provide a more selective and safer anti-angiogenic therapy for many tumor types.

[0436] In some embodiments, the additional modified signal transducer has a reduced affinity and / or activity (e.g., agonism) to VEGFR-2, and / or has a substantially reduced or eliminated affinity and / or activity to VEGFR-1. In some embodiments, by targeting vascular endothelial cells, such constructs promote angiogenesis without inducing an inflammatory response associated with VEGFR-1. Therefore, such constructs will have a targeted pro-angiogenic effect and substantially reduce the risk of side effects caused by systemic activation of VEGFR-2 and VEGR-1.

[0437] In illustrative embodiments, the modified signaling agent is VEGF 165 (wild type), the VEGF 165 It has the amino acid sequence SEQ ID NO:235.

[0438] In another illustrative embodiment, the additional modified signaling agent is VEGF 165b (wild type), the VEGF 165b It has the amino acid sequence SEQ ID NO:236.

[0439] In these embodiments, the modified signaling agent has a mutation at amino acid I83 (e.g., a substitution mutation at I83, such as I83K, I83R, or I83H). Without wishing to be bound by theory, it is believed that such mutations can result in reduced receptor binding affinity. See, for example, U.S. Patent No. 9,078,860, the entire contents of which are hereby incorporated by reference.

[0440] In one embodiment, the additional modified signaling agent is TNF-α. TNF is a pleiotropic cytokine with many different functions, including regulation of cell growth, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and migration, inflammation, and septic shock. It binds to two unique membrane receptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits a very broad expression pattern, while TNFR2 is preferentially expressed on certain populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. Very distinct biological pathways are activated in response to receptor activation, but there is also some overlap. In general, 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 NFkB pathway). Administration of TNF is systemic toxic, and this is primarily due to TNFR1 binding. However, it should be noted that activation of TNFR2 is also associated with a variety of activities, and like TNFRl, control of TNF targeting and activity is important in the context of development of TNF-based therapeutics.

[0441] In some embodiments, the additional modified signaling agent has reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signaling agent 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, while TNFR2, in contrast, is involved in cell survival signaling. Thus, in embodiments directed to methods of treating cancer, the modified signaling agent has reduced affinity and / or activity for TNFR1 and / or has substantially reduced or eliminated affinity and / or activity for TNFR2. In these embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can be targeted to cells in need of apoptosis, such as tumor cells or tumor vascular endothelial cells. In embodiments directed to methods of promoting cell survival, for example, in neurogenesis for the treatment of neurodegenerative disorders, the modified signaling agent has reduced affinity and / or activity for TNFR2 and / or has substantially reduced or abolished affinity and / or activity for TNFR1. In other words, in some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, comprises a modified TNF-α agent that allows for favorable death or survival signaling.

[0442] In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based ch...

Claims

1. A chimeric protein, comprising: (a) a variant interferon α1 (IFNα1), whose amino acid sequence, based on SEQ ID NO: 1, has only the mutations of C86S; C86Y; C86S and A146G; C86S and M149V; C86Y and A146G; or C86Y and M149V, and (b) one or more targeting moieties comprising a recognition domain that specifically binds to a target antigen or receptor; Wherein said IFNα1 and said one or more targeting moieties are optionally linked to one or more linkers.

2. The chimeric protein of claim 1, wherein the IFNα1 is pegylated, one or more targeting moieties are pegylated, or a linker is pegylated.

3. The chimeric protein of claim 1, wherein the one or more mutations of the variant IFNα1 confer reduced affinity for the interferon-α / β receptor (IFNAR). The chimeric protein of claim 3 , wherein the IFNα1 exhibits reduced affinity for IFNAR1.

5. The chimeric protein of claim 3, wherein the IFNα1 exhibits reduced affinity for IFNAR2.

6. The chimeric protein of claim 3, wherein one or more mutations confer reduced affinity that can be restored by attachment to one or more targeting moieties or upon inclusion in an Fc-based chimeric protein complex.

7. The chimeric protein of claim 1, wherein the targeting moiety is directed against tumor cells.

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

9. The chimeric protein of claim 8, wherein the immune cell is selected from the group consisting of 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.

10. The chimeric protein of claim 1, wherein the targeting moiety comprises a recognition domain, and the recognition domain is a full-length antibody or a fragment thereof, a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a Humabody, a heavy chain-only shark antibody (VNAR), a microprotein, a darpin, anticalin, an adnectin, Fv, Fab, Fab' or F(ab')2.

11. The chimeric protein of claim 1, wherein the recognition domain: i) is a natural ligand, optionally comprising Flt3L or a truncation thereof, optionally is an extracellular domain of Flt3L, optionally is a single-chain Flt3L; ii) recognizes CD20; iii) recognizes PD-1 or PD-L1; or iv) recognizes Clec9A.

12. The chimeric protein of claim 1, wherein the recognition domain is a single domain antibody (V HH ), optionally V HH or humanized V HH .

13. The chimeric protein of claim 1, wherein the recognition domain functionally modulates a target antigen or receptor.

14. The chimeric protein of claim 1, wherein the recognition domain binds to a target antigen or receptor but does not functionally modulate the target antigen or receptor.

15. The chimeric protein of claim 1, comprising two or more targeting moieties.

16. A recombinant nucleic acid composition encoding the chimeric protein of any one of claims 1-15.

17. A host cell comprising the recombinant nucleic acid composition of claim 16.

18. An Fc-based chimeric protein complex, comprising: (a) a variant interferon α1 (IFNα1), whose amino acid sequence, based on SEQ ID NO: 1, has only the mutations of C86S; C86Y; C86S and A146G; C86S and M149V; C86Y and A146G; or C86Y and M149V, and (b) one or more targeting moieties comprising a recognition domain that specifically binds to a target antigen or receptor; and (c) Fc domain.

19. The Fc-based chimeric protein complex of claim 18, wherein the IFNα1 is pegylated, the one or more targeting moieties are pegylated, and / or the Fc domain is pegylated.

20. The Fc-based chimeric protein complex of claim 18, wherein the one or more mutations of the variant IFNα1 confer reduced affinity for interferon-α / β receptor (IFNAR).

21. The Fc-based chimeric protein complex of claim 20, wherein the IFNα1 exhibits reduced affinity for IFNAR1.

22. The Fc-based chimeric protein complex of claim 20, wherein the IFNα1 exhibits reduced affinity for IFNAR2.

23. The Fc-based chimeric protein complex of claim 18, wherein one or more mutations confer reduced affinity that can be restored by attachment to one or more targeting moieties or upon inclusion in the Fc-based chimeric protein complex.

24. The Fc-based chimeric protein complex of claim 18, wherein the targeting moiety is directed against tumor cells.

25. The Fc-based chimeric protein complex of claim 18, wherein the targeting moiety is directed to an immune cell.

26. The Fc-based chimeric protein complex of claim 25, 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.

27. The Fc-based chimeric protein complex of claim 18, wherein the targeting moiety comprises a recognition domain, and the recognition domain is a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a microprotein, a darpin, an anticalin, an adnectin, Fv, Fab, Fab' or F(ab')2.

28. The Fc-based chimeric protein complex of claim 18, wherein the recognition domain: i) is a natural ligand, optionally comprising Flt3L or a truncation thereof, optionally an extracellular domain of Flt3L; ii) recognizes CD20; iii) recognizes PD-1 or PD-L1; or iv) recognizes Clec9A.

29. The Fc-based chimeric protein complex of claim 18, wherein the recognition domain is a single domain antibody (V HH ), optionally V HH or humanized V HH .

30. The Fc-based chimeric protein complex of claim 18, wherein the recognition domain functionally modulates a target antigen or receptor.

31. The Fc-based chimeric protein complex of claim 18, wherein the recognition domain binds to a target antigen or receptor but does not functionally modulate the target antigen or receptor.

32. The Fc-based chimeric protein complex of claim 18, comprising two or more targeting moieties.

33. A recombinant nucleic acid composition encoding one or more Fc-based chimeric protein complexes or their constituent polypeptides according to any one of claims 18 to 32.

34. A host cell comprising the recombinant nucleic acid composition of claim 33.

35. The Fc-based chimeric protein complex of claim 18, wherein the Fc domain is from IgG, IgA, IgD, IgM or IgE.

36. The Fc-based chimeric protein complex of claim 35, wherein the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

37. The Fc-based chimeric protein complex of claim 18, wherein the Fc domain is from human IgG, IgA, IgD, IgM or IgE.

38. The Fc-based chimeric protein complex of claim 37, wherein the human IgG is selected from human IgG1, IgG2, IgG3 or IgG4.

39. The Fc-based chimeric protein complex of claim 18, wherein the Fc chain pairing is promoted by ion pairing and / or knob-into-hole pairing.

40. The Fc-based chimeric protein complex of claim 18, wherein the Fc-based chimeric protein complex is a heterodimer and has a trans orientation / configuration such as with respect to any targeting moiety and signaling agent relative to each other, or any targeting moieties relative to each other, or any signaling agents relative to each other.

41. The Fc-based chimeric protein complex of claim 18, wherein the Fc-based chimeric protein complex is a heterodimer and has a cis orientation as with respect to any targeting moiety and signaling agent relative to each other, or any targeting moieties relative to each other, or any signaling agents relative to each other.

42. The Fc-based chimeric protein complex of claim 18, wherein the Fc domain has a substitution in human IgG1 selected from the group consisting of L234A, L235A, and K322Q according to EU numbering.

43. The Fc-based chimeric protein complex of claim 18, wherein the Fc domain is human IgG1 and contains one or more mutations selected from the group consisting of L234, L235, K322, D265, P329 and P331 according to EU numbering.

44. The Fc-based chimeric protein complex of claim 18, wherein the Fc-based chimeric protein complex consists of a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 290, 291, 293-303.

45. The Fc-based chimeric protein complex of claim 44, wherein the Fc-based chimeric protein complex consists of a first amino acid sequence of SEQ ID NO: 290 and a second amino acid sequence of SEQ ID NO:

291.

46. ​​The Fc-based chimeric protein complex of claim 44, wherein the Fc-based chimeric protein complex consists of a first amino acid sequence of SEQ ID NO: 293 and a second amino acid sequence of any one of SEQ ID NO: 294, 295, 296, 297, 298 or 299.

47. The Fc-based chimeric protein complex of claim 44, wherein the Fc-based chimeric protein complex consists of a first amino acid sequence of any one of SEQ ID NOs: 300, 301, 302, 303, 303 and a second amino acid sequence of SEQ ID NO: 294.

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