IL-2 system constructs

Abstract

The present invention relates, in part, to chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, that contain interleukin-2 or variants thereof, and their use as therapeutic agents.

Description

[Technical Field]

[0001] The present invention relates in part to chimeric proteins or chimeric protein complexes (including Fc-based chimeric protein complexes) comprising interleukin-2 (IL-2) or variants thereof, and their use as therapeutic agents.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 63 / 175,827, filed April 16, 2021, and 63 / 297,330, filed January 7, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety. Computer-readable copy of the Sequence Listing (File name: ORN-080PC_ST25, Created: April 14, 2022, File size: 1,151,315 bytes). [Background technology]

[0004] Interleukin 2 (IL-2) belongs to a family of cytokines, each of which contains a four-alpha helix bundle. IL-2 signals through the IL-2 receptor, a complex consisting of two or three polypeptide chains: an alpha and / or beta chain and a gamma chain.

[0005] IL-2 is approved for cancer treatment in high-dose regimens. Exemplary cancers treated with IL-2 include renal cell (kidney) carcinoma and melanoma (a type of skin cancer).

[0006] IL-2 stimulates the proliferation and activation of immune cells through a receptor-signaling complex containing the alpha receptor polypeptide chain (IL-2Rα, CD25) and / or the beta receptor polypeptide chain (IL-2Rβ, CD122) and the common gamma receptor polypeptide chain (γc, CD132), but its effects are pleiotropic and context-dependent. At high doses, IL-2 binds to the heterodimeric IL-2Rβγ receptor, thereby mediating tumor-killing CD8 + IL-2 also binds with greater affinity to its heterotrimeric receptor IL-2Rαβγ, thereby promoting the growth of immunosuppressive CD4 T cells that express high constitutive levels of IL-2Rα. + CD25 + Increases regulatory T cells (Tregs). Increased Tregs may be an undesirable effect of IL-2 on cancer immunotherapy. Furthermore, activation of IL-2Rαβγ on endothelial cells may lead to vascular leak syndrome (VLS).

[0007] Conversely, CD8 + T cell stimulation is achieved by the autoreactive CD8 + This represents an undesirable effect of IL-2 in the treatment of autoimmune diseases where stimulation of Tregs by IL-2 to suppress T cells is desirable. Due to its global, pleiotropic effects on multiple cell types, IL-2 therapy suffers from significant side effects and systemic toxicity, including vascular leak syndrome (VLS), which limits its therapeutic potential.

[0008] Receptor-selective IL-2 constructs have been created (e.g., constructs that lack or attenuate interactions with IL-2Rα and IL-2Rβ). However, these constructs still suffer from side effects and toxicity. While some of these problems can be ameliorated by reducing the dose (e.g., by extending the half-life), dose reduction alone does not solve the cell selectivity problem. Therefore, targeting IL-2Rβγ (e.g., CD8 in the treatment of cancer) is a promising approach. +There remains a need for IL-2-based agents with reduced cellular side effects (non-specific effects) and lower systemic toxicity for use in IL-2Rαβγ targeting (e.g., for T cell activation, activation of helper T cells in the treatment of cancer, and activation of NK cells in the treatment of cancer) and IL-2Rαβγ targeting (e.g., for Treg stimulation in autoimmune diseases). Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there remains a need for safe and effective IL-2-based therapeutics with improved target selectivity, pharmacokinetic and therapeutic properties, and minimal toxicity profiles. [Means for solving the problem]

[0010] Thus, in some aspects, the present invention relates to chimeric protein complexes, including chimeric proteins and Fc-based chimeric protein complexes, comprising modified IL-2 as a signaling factor. In some embodiments, the modified IL-2 is modified wild-type IL-2 having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified IL-2 is modified neoleukin having the amino acid sequence of SEQ ID NO: 2.

[0011] In some embodiments, the modified IL-2 signaling factor is selected from (i) a mutation selected from D20E, D20F, D20G, D20H, D20I, D20K, D20L, and D20V; or (ii) a mutation selected from N88G, N88E, N88K, N88Q, and N88V; or (iii) a mutation selected from Q126G, Q126A, Q126E, Q126F, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126. or (iv) a mutation selected from (i) and (iii) or a mutation selected from (ii) and (iii); or (v) a mutation selected from R38A / F42K / N88G / C125A, F42K / C125A, D20E / C125A, D20F / C125A, D20G / C125A, D20H / C125A, D20I / C125A, D20K / C125A, D20L / C125A, D20N / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C125A, N88D / C125A, N88E / C125A, N88F / C125A, N88G / C125A, N88H / C125A, N88I / C125A, N88K / C125A, N88H / C125A, N88I / C125A, N88K / C125A, N88L / C125A, N88N / C125A, N88S / C125A, N88T / C125A, N88V / C125A, N88D / C125A, N88E / C125A, N88F / C125A, N88G / C125A, N88H / C125A, N88I ... 25A, N88G / C125A, N88E / C125A, N88H / C125A, N88I / C125A, N88K / C125A, N88Q / C125A, N88R / C125A, N88T / C125A, N88V / C125A, D20E / R38A / F42K, D20E / R38A / F42K / C125A, D20V / R38A / F42K, D20V / R38A / F42K / C125A, R3 8A / F42Y / E62A / C125A, R38A / F42Y / Y45A / E62A, F42Y / Y45A / L72G, R38A / and one or more mutations selected from F42Y / Y45A / E62A / C125A, F42Y / Y45A / L72G / C125A, R38A / F42K / N88G, R38A / F42K / Q126G, R38A / F42K / Q126I, R38A / F42K / Q126Y, R38A / F42K / Q126G / C125A, R38A / F42K / Q126I / C125A, R38A / F42K / Q126Y / C125A, R38A / F42K / E62A / N88G, and R38A / F42K / E62A / N88G / C125A.

[0012] In some embodiments, the modified IL-2 signaling factor comprises a mutation at one or more amino acid residues selected from R38, F42, E62, C125, Y45, L72, D20, N88, and Q126 relative to SEQ ID NO: 1. In such embodiments, the modified IL-2 signaling factor comprises a mutation at one or more amino acid residues selected from R38A, F42K, F42Y, E62A, C125A, Y45A, L72G, D20E, D20F, D20G, D20H, D20I, D20K, D20L, D20N, D20V, N88G, N88E, N88I, N88K, N88Q, N88R, N88V, R38A / F42Y / E62A / C125A, F42Y / Y45A / L72G / C125A, F42K / C 125A, R38A / F42K / C125A, D20E / C125A, D20G / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C125A, N88G / C125A, N88H / C125A, N88Q / C125A, N88T / C 125A, D20E / R38A / F42K / C125A, D20V / R38A / F42K / C125A, R38A / F42K / N88A / C125A, Contains one or more mutations selected from R38A / F42K / N88G / C125A, Q126A, Q126E, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126Y.

[0013] In various embodiments, the modified IL-2 signaling factor is selected from the group consisting of D20V, D20E, N88G, F42K / C125A, D20E / C125A, D20G / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C125A, N88G / C125A, N88H / C125A, N88Q / C125A, N88T / C125A, D20E / R38A / F42K, D20E / R38A / F42K / C125A, D20V / R38A / F42K ... and one or more mutations selected from 2K / C125A, D20V / R38A / F42K / E62A, D20V / R38A / F42K / E62A / C125A, R38A / F42K / N88G, R38A / F42K / N88G / C125A, R38A / F42K / E62A / N88G, R38A / F42K / E62A / N88G / C125A, Q126A, Q126E, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126Y.

[0014] In certain embodiments, the modified IL-2 signaling agent has one or more of the following sequences relative to SEQ ID NO: 1: R38A / F42Y / E62A / C125A, F42Y / Y45A / L72G / C125A, F42K / C125A, D20E / C125A, D20G / C125A, D20S / C125A, D20T / C125A, D20V / C125A, and one or more mutations selected from N88A / C125A, N88D / C125A, N88G / C125A, N88H / C125A, N88Q / C125A, N88T / C125A, D20E / R38A / F42K / C125A, D20V / R38A / F42K / C125A, and R38A / F42K / N88G / C125A.

[0015] In some embodiments, the modified IL-2 signaling factor is a modified neoleukin comprising the amino acid sequence of SEQ ID NO: 2. In such embodiments, the modified IL-2 signaling factor comprising SEQ ID NO: 2 comprises one or more mutations at amino acid residues D15 and / or N40. For example, in some embodiments, the modified IL-2 signaling factor comprising SEQ ID NO: 2 comprises D15T, D15H, N40I, N40G, or N40R.

[0016] In some embodiments, the one or more mutations or modifications improve the safety profile of the modified IL-2, e.g., compared to wild-type IL-2. In some embodiments, the one or more mutations or modifications reduce the biological activity of the modified IL-2, e.g., compared to wild-type IL-2. For example, the one or more mutations or modifications may reduce the affinity of the modified IL-2 for a target receptor. In some embodiments, the target receptor is the heterotrimeric receptor IL-2Rαβγ composed of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits. In some embodiments, the target receptor is the heterodimeric receptor IL-2Rβγ composed of IL-2Rβ and IL-2Rγ subunits. In some embodiments, the modified IL-2 comprises one or more mutations or modifications that reduce its affinity for the IL-2Rα subunit. In another embodiment, the modified IL-2 comprises one or more mutations or modifications that reduce its affinity for IL-2Rβ. In yet another embodiment, the modified IL-2 comprises one or more mutations or modifications that reduce its affinity for IL-2Rγ. In certain embodiments, the modified IL-2 comprises one or more mutations or modifications that reduce its affinity for any combination of IL-2Rα, IL-2Rβ, and IL-2Rγ (e.g., one or more mutations or modifications that reduce the affinity of the modified IL-2 for IL-2Rα, IL-2Rβ, and IL-2Rγ, or for IL-2Rα and IL-2Rβ, or for IL-2Rγ and IL-2Rβ, or for IL-2Rα and IL-2Rγ).

[0017] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rα and IL-2Rβ, and optionally the modified IL-2 is selected from the group consisting of D20E / R38A / F42K, D20E / R38A / F42K / C125A, D20V / R38A / F42K, D20V / R38A / F42K / C125A , D20V / R38A / F42K / E62A, D20V / R38A / F42K / E62A / C125A, R38A / F42K / N88G, R38A / F42K / N88G / C125A, R38A / F42K / E62A / N88G, and R38A / F42K / E62A / N88G / C125A.

[0018] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rα and IL-2Rγ, and optionally, the modified IL-2 comprises one or more mutations selected from R38A / F42K / Q126G, R38A / F42K / Q126I, and R38A / F42K / Q126Y.

[0019] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rβ, and optionally the modified IL-2 is selected from the group consisting of D20E, D20F, D20G, D20H, D20I, D20K, D20L, D20V, N88G, N88E, N88K, N88Q, N88V, D20E / C125A, D20F / C125A, D20G / C125A, D20H / C125A, D20I / C125A, D20L / C125A and N88V / C125A, D20K / C125A, D20N / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C125A, N88G / C125A, N88E / C125A, N88H / C125A, N88I / C125A, N88K / C125A, N88Q / C125A, N88R / C125A, N88T / C125A, and N88V / C125A.

[0020] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rγ, and optionally the modified IL-2 comprises one or more mutations selected from Q126A, Q126E, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126Y.

[0021] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rβγ, and optionally the modified IL-2 is selected from the group consisting of D20E / Q126A, D20E / Q126D, D20E / Q126E, D20E / Q126F, D20E / Q126G, D20E / Q126H, D20E / Q126I, D20E / Q126K, D20E / Q126L, D20E / Q126M, D20E / Q126N, D20E / Q126P, D20E / Q126R, D20E / Q126R 6S, D20E / Q126T, D20E / Q126V, D20E / Q126W, D20E / Q126Y, D20V / Q126A, D20V / Q126D, D20V / Q126E, D20V / Q126F, D20V / Q126G, D20V / Q12 6H, D20V / Q126I, D20V / Q126K, D20V / Q126L, D20V / Q126M, D20V / Q126N, D20V / Q126P, D20V / Q126R, D20V / Q126S, D20V / Q126T, D20V / Q12 6V, D20V / Q126W, D20V / Q126Y, N88A / Q126A, N88A / Q126D, N88A / Q126E, N88A / Q126F, N88A / Q126G, N88A / Q126H, N88A / Q126I, N88A / Q12 6K, N88A / Q126L, N88A / Q126M, N88A / Q126N, N88A / Q126P, N88A / Q126R, N88A / Q126S, N88A / Q126T, N88A / Q126V, N88A / Q126W, N88A / Q12 and N88G / Q126Y, N88G / Q126A, N88G / Q126D, N88G / Q126E, N88G / Q126F, N88G / Q126G, N88G / Q126H, N88G / Q126I, N88G / Q126K, N88G / Q126L, N88G / Q126M, N88G / Q126N, N88G / Q126P, N88G / Q126R, N88G / Q126S, N88G / Q126T, N88G / Q126V, N88G / Q126W, and N88G / Q126Y. In a further embodiment, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rβγ and increased selectivity for activated CD8 cells.

[0022] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rβ and is selected from the group consisting of D20E, D20F, D20G, D20H, D20I, D20K, D20L, D20N, D20V, N88G, N88E, N88I, N88K, N88Q, N88R, N88V, D20E / C125A, D20F / C125A, D20G / C125A, D20H / C125A, D20I / C125A, D20K / C125A, D20L / C125A, D20N / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C125A, N88G / C125A, N88H / C125A, N88Q / C125A, and N88T / C125A, and does not contain mutations that reduce affinity or biological activity for the IL-2R alpha subunit (IL-2Rα). In further embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rβ, and exhibits increased selectivity for activated CD8 cells and decreased selectivity for Tregs.

[0023] In some embodiments, the target receptor is the heterotrimeric receptor IL-2Rαβγ composed of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits. In some embodiments, the target receptor is the heterodimeric receptor IL-2Rβγ composed of IL-2Rβ and IL-2Rγ subunits.

[0024] In some embodiments, the IL-2 has one or more mutations or modifications that reduce or eliminate the activity or affinity of the modified IL-2 for the IL-2R alpha chain; and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R that lacks an alpha chain. In some embodiments, the mutations or modifications that reduce the activity or affinity of the modified IL-2 and / or attenuate the modified IL-2 can be restored at the target cell or site of therapeutic action via a targeting moiety linked to the modified IL-2.

[0025] In some embodiments, the IL-2 has one or more mutations or modifications, i.e., one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R that lacks an alpha chain, and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R that includes an alpha chain, and such mutations or modifications are attenuating mutations whose activity or affinity can be restored by induction of proximity to a target or site of therapeutic action (e.g., a cell or extracellular matrix) via a targeting moiety.

[0026] In various embodiments, the present invention contemplates a chimeric protein, chimeric protein complex, or Fc-based chimeric protein complex comprising a modified IL-2 signaling factor comprising a glycosylation mutation. In some embodiments, the glycosylation mutation reduces or eliminates glycosylation of the modified IL-2 signaling factor. In some embodiments, the glycosylation mutation is a T3 substitution, wherein the mutation is one of T3A, T3F, T3H, T3L, T3V, and T3Y. In some embodiments, the glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2.

[0027] In some embodiments, the chimeric protein includes one or more additional signaling factors, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, which may be modified.

[0028] In some embodiments, the chimeric protein comprises one or more targeting moieties described herein (e.g., on IL-2) that allow for restoration of weakened or attenuated activity. In some embodiments, the chimeric protein comprises 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) present on one or more immune cells, which may include, but are not limited to, T cells, Tregs, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor or tumor-associated macrophages (e.g., M1 or M1 macrophages), B cells, and dendritic cells. In some embodiments, the immune cells are T cells and the targeting moiety targets CD8. In embodiments, the immune cells are Tregs and the targeting moiety targets CTLA4. In embodiments, the immune cell is an NK cell and the targeting moiety is NKp46.

[0029] In some embodiments, the recognition domain specifically binds to a target of interest (e.g., an antigen, receptor) to effectively recruit one or more immune cells. In some embodiments, the target of interest (e.g., an antigen, receptor) may be present on one or more tumor cells. In some embodiments, the chimeric protein may recruit immune cells, e.g., immune cells capable of killing and / or suppressing tumor cells, to a site of action (such as, by way of non-limiting example, the tumor microenvironment). In some embodiments, the recognition domain binds to a target of interest present in a normal tissue of interest. In some embodiments, the chimeric protein may bind to a target cell and direct the chimeric protein to localize to the tissue cells of interest, while also being capable of binding to a second, different cell type (e.g., an immune cell) to confer a tissue-localized biological effect mediated by the modified IL-2. In some embodiments, the recognition domain specifically binds to a target of interest (e.g., an antigen, receptor) that is part of a non-cellular structure.

[0030] In various embodiments, the chimeric proteins of the present technology have improved cell targeting selectivity, safety, and / or therapeutic activity and / or pharmacokinetic profile (e.g., increased serum half-life) compared to non-targeted modified IL-2.

[0031] In some embodiments, the chimeric proteins of the present technology are single-chain polypeptides.

[0032] In some embodiments, the chimeric protein of the present technology is a protein complex comprising two or more polypeptide chains. In some embodiments, the targeting moiety(s) and signal transduction factor(s) (e.g., modified IL-2) are present on the same polypeptide chain in the protein complex. In some embodiments, the targeting moiety(s) and signal transduction factor(s) (e.g., modified IL-2) are present on different polypeptide chains in the protein complex.

[0033] In various embodiments, the chimeric proteins find use in the treatment of a variety of diseases or disorders, such as cancer, infectious diseases, immune disorders, autoimmune diseases, cardiovascular diseases, wounds, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and many other diseases and disorders, and the invention encompasses a variety of methods of treatment.

[0034] In another aspect, the present invention relates to an Fc-based chimeric protein complex comprising modified IL-2 as a signaling factor. In some embodiments, the Fc-based chimeric protein complex of the present invention comprises a polypeptide having an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to any one of SEQ ID NOs: 291-296, 298-335. In specific embodiments, the Fc-based chimeric protein complex of the present invention comprises a polypeptide having an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to any one of SEQ ID NOs: 292, 293, 294, 299, 301, 310, 311, 312, 315, 316, 319, 320, 325, 328, 332, 333, and 335.

[0035] In some embodiments, the present invention relates to a chimeric protein or Fc-based chimeric protein complex comprising a polypeptide having an amino acid sequence that has at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 290-449, 478-495, or 501-531.

[0036] Figures 1A-F, 2A-H, 3A-H, 4A-D, 5A-F, 6A-J, 7A-D, 8A-F, 9A-J, 10A-F, 11A-L, 12A-L, 13A-F, 14A-L, 15A-L, 16A-J, 17A-J, 18A-F, and 19A-F show various non-limiting, exemplary schematic diagrams of Fc-based chimeric protein complexes of the invention. In some embodiments, each schematic diagram is a composition of the invention. Where applicable in the figures, "TM" refers to a "targeting moiety" as described herein, "SA" refers to a "signaling agent" as described herein,

[0037] [ka]

[0038] is an optional "linker" as described herein, the two parallel long rectangles are, for example, human Fc domains derived from IgG1, IgG2, or IgG4 as described herein, and optionally with effector knockout and / or stabilizing mutations, also as described herein, and the two parallel long rectangles, one with a protrusion and the other with a recess, are, for example, human Fc domains derived from IgG1, IgG2, or IgG4 as described herein, with knob-in-hole and / or ion-pair (a / k / a charge pair, ion bond, or charged residue pair) mutations as described herein, and optionally with effector knockout and / or stabilizing mutations, also as described herein. At least one signaling factor is IL-2 or modified IL-2, or neoleukin or modified neoleukin. [Brief explanation of the drawings]

[0039] [Figure 1A] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1B] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1C]1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1D] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1E] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1F] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 2A] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2B] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2C] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2D] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2E] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2F]1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2G] Shown is an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the construct shown in the box has a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 2H] Shown is an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the construct shown in the box has a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 3A] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3B] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3C] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3D]An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3E] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3F] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3G] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the construct shown in the box has a TM between the Fc and SA1 and / or SA2, the N-terminus or C-terminus of the Fc chain. [Figure 3H] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the construct shown in the box has a TM between the Fc and SA1 and / or SA2, the N-terminus or C-terminus of the Fc chain. [Figure 4A] An example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 4B] An example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 4C]An example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 4D] An example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 5A] 1 shows an example of a heterodimeric two-chain complex with separate TM and SA chains, i.e., with both TM on the knob chain of the Fc and SA on the hole chain of the Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5B] 1 shows an example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5C] 1 shows an example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5D]1 shows an example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5E] 1 shows an example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5F] 1 shows an example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 6A] Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6B]Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6C] Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6D] Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6E] Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6F]Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6G] Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6H] Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6I] Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6J]Examples of heterodimeric two-chain complexes are shown, each with a TM on the knob chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 7A] An example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, is shown. [Figure 7B] An example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, is shown. [Figure 7C] An example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, is shown. [Figure 7D] An example of a heterodimeric two-chain complex with separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, is shown. [Figure 8A] 1 shows an example of a heterodimeric two-chain complex with separate TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be the same. [Figure 8B] 1 shows an example of a heterodimeric two-chain complex with separate TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8C]1 shows an example of a heterodimeric two-chain complex with separate TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8D] 1 shows an example of a heterodimeric two-chain complex with separate TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8E] 1 shows an example of a heterodimeric two-chain complex with separate TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8F] 1 shows an example of a heterodimeric two-chain complex with separate TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 9A]1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9B] 1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9C] 1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9D] 1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9E] 1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9F]1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9G] 1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9H] 1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9I] 1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9J] 1 shows an example of a heterodimeric two-chain complex with a TM on the hole chain of the Fc and two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these configurations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 10A]An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10B] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10C] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10D] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10E] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10F] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 11A] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11B] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11C]1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11D] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11E] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11F] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11G] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11H] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11I] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11J] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11K] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11L] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 12A] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12B] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12C] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12D] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12E] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12F]An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12G] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12H] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12I] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12J] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12K]An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12L] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 13A] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13B] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13C] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13D] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13E] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13F] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 14A]1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14B] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14C] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14D] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14E] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14F] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14G] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14H] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14I] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14J] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14K] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14L] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 15A] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15B] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15C] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15D]An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15E] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15F] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15G] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15H] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15I]An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15J] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15K] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15L] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 16A] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16B]1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16C] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16D] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16E] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16F] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16G] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16H]1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16I] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16J] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17A] An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17B] An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17C] An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17D]An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17E] An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17F] An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17G] An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17H] An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17I] An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17J]An example of a heterodimeric two-chain complex is shown, having two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and having an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 18A] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18B] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18C] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18D] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18E] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18F]An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 19A] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19B] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19C] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19D] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19E] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19F]An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having separate SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 20] Figure 1 shows STAT5 phosphorylation driven by IL-2 and Fc-IL-2 in PBMC lymphocyte populations. [Figure 21A] 1 shows the effect of CD25 knockout mutations and CD8 targeting on STAT5 phosphorylation in lymphocyte populations. [Figure 21B] 1 shows the effect of CD25 knockout mutations and CD8 targeting on STAT5 phosphorylation in lymphocyte populations. [Figure 21C] 1 shows the effect of CD25 knockout mutations and CD8 targeting on STAT5 phosphorylation in lymphocyte populations. [Figure 21D] 1 shows the effect of CD25 knockout mutations and CD8 targeting on STAT5 phosphorylation in lymphocyte populations. [Figure 21E] 1 shows the effect of CD25 knockout mutations and CD8 targeting on STAT5 phosphorylation in lymphocyte populations. [Figure 21F] 1 shows the effect of CD25 knockout mutations and CD8 targeting on STAT5 phosphorylation in lymphocyte populations. [Figure 21G] 1 shows the effect of CD25 knockout mutations and CD8 targeting on STAT5 phosphorylation in lymphocyte populations. [Figure 22A] 1 shows sensorgrams of the binding of IL-2 and Fc-IL-2 fusion protein to biotinylated CD25 in biolayer interferometry. [Figure 22B] 1 shows sensorgrams of the binding of IL-2 and Fc-IL-2 fusion protein to biotinylated CD25 in biolayer interferometry. [Figure 22C] 1 shows sensorgrams of the binding of IL-2 and Fc-IL-2 fusion protein to biotinylated CD25 in biolayer interferometry. [Figure 22D] 1 shows sensorgrams of the binding of IL-2 and Fc-IL-2 fusion protein to biotinylated CD25 in biolayer interferometry. [Figure 22E] 1 shows sensorgrams of the binding of IL-2 and Fc-IL-2 fusion protein to biotinylated CD25 in biolayer interferometry. [Figure 22F] 1 shows sensorgrams of the binding of IL-2 and Fc-IL-2 fusion protein to biotinylated CD25 in biolayer interferometry. [Figure 22G] 1 shows sensorgrams of the binding of IL-2 and Fc-IL-2 fusion protein to biotinylated CD25 in biolayer interferometry. [Figure 23A] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23B] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23C] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23D] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23E] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23F] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23G] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23H]1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23I] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23J] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23K] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23L] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23M] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23N] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23O] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23P] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23Q] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 23R] 1 shows the screening of D20 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24A]1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24B] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24C] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24D] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24E] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24F] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24G] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24H] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24I] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24J] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24K] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24L]1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24M] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24N] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24O] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24P] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24Q] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 24R] 1 shows the screening of N88 mutants against pSTAT4 in CD8+, CD4+CD25-, and CD4+CD25+ PMBCs. [Figure 25A] The effect of beta mutation (here N88G) and CD8 targeting on STAT5 phosphorylation in PBMC subpopulations is shown. [Figure 25B] The effect of beta mutation (here N88G) and CD8 targeting on STAT5 phosphorylation in PBMC subpopulations is shown. [Figure 25C] The effect of beta mutation (here N88G) and CD8 targeting on STAT5 phosphorylation in PBMC subpopulations is shown. [Figure 25D] The effect of beta mutation (here N88G) and CD8 targeting on STAT5 phosphorylation in PBMC subpopulations is shown. [Figure 26A] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 26B] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 26C] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 26D] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 26E] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 26F] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 26G] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 26H] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 26I] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 26J] 1 shows various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in a PMBC population. [Figure 27A] Figure 1 shows binding of CD25 scFv Fc fusion to HEK-Blue IL-2 cells in FACS. [Figure 27B] Figure 1 shows binding of CD25 scFv Fc fusion to HEK-Blue IL-1 cells in FACS. [Figure 28] 1 shows the effect of CD25 scFv Fc fusions on wild-type IL-2 signaling in HEK-Blue IL-2 cells. [Figure 29A]1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29B] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29C] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29D] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29E] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29F] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29G] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29H] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29I] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29J] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29K]1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29L] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 29M] 1 shows the effect of CD25 targeting of ALN2 mutants carrying CD25 mutations or beta mutations on STAT5 phosphorylation in specific PBMC subpopulations. [Figure 30A] STAT5 phosphorylation in PBMC subpopulations by IL-2 and Fc-IL-2 mimetic / neoleukin variants. [Figure 30B] STAT5 phosphorylation in PBMC subpopulations by IL-2 and Fc-IL-2 mimetic / neoleukin variants. [Figure 30C] STAT5 phosphorylation in PBMC subpopulations by IL-2 and Fc-IL-2 mimetic / neoleukin variants. [Figure 30D] STAT5 phosphorylation in PBMC subpopulations by IL-2 and Fc-IL-2 mimetic / neoleukin variants. [Figure 30E] STAT5 phosphorylation in PBMC subpopulations by IL-2 and Fc-IL-2 mimetic / neoleukin variants. [Figure 30F] STAT5 phosphorylation in PBMC subpopulations by IL-2 and Fc-IL-2 mimetic / neoleukin variants. [Figure 30G] STAT5 phosphorylation in PBMC subpopulations by IL-2 and Fc-IL-2 mimetic / neoleukin variants. [Figure 30H] STAT5 phosphorylation in PBMC subpopulations by IL-2 and Fc-IL-2 mimetic / neoleukin variants. [Figure 31] Various bivalent (where VHH1 and VHH2 are identical) or bispecific (where VHH1 and VHH2 represent different targeting domains) knob-in-hole ALN2 configurations are shown. [Figure 32] Various ALN2 constructs are shown. [Figure 33A] Figure 1 shows a comparison of STAT5 phosphorylation by bivalent and bispecific CD8 VHH targeting ALN2 compared to monovalent controls. [Figure 33B] Figure 1 shows a comparison of STAT5 phosphorylation by bivalent and bispecific CD8 VHH targeting ALN2 compared to monovalent controls. [Figure 33C] Figure 1 shows a comparison of STAT5 phosphorylation by bivalent and bispecific CD8 VHH targeting ALN2 compared to monovalent controls. [Figure 33D] Figure 1 shows a comparison of STAT5 phosphorylation by bivalent and bispecific CD8 VHH targeting ALN2 compared to monovalent controls. [Figure 33E] Figure 1 shows a comparison of STAT5 phosphorylation by bivalent and bispecific CD8 VHH targeting ALN2 compared to monovalent controls. [Figure 33F] Figure 1 shows a comparison of STAT5 phosphorylation by bivalent and bispecific CD8 VHH targeting ALN2 compared to monovalent controls. [Figure 33G] 1 shows a comparison of STAT5 phosphorylation by bivalent and bispecific CD8 VHH targeting LN2 compared to monovalent controls. [Figure 34A] STAT5 phosphorylation by different constituents of ALN2 in CD8+ and Treg PBMC subpopulations is shown. [Figure 34B] STAT5 phosphorylation by different constituents of ALN2 in CD8+ and Treg PBMC subpopulations is shown. [Figure 34C] STAT5 phosphorylation by different constituents of ALN2 in CD8+ and Treg PBMC subpopulations is shown. [Figure 34D] STAT5 phosphorylation by different constituents of ALN2 in CD8+ and Treg PBMC subpopulations is shown. [Figure 34E]STAT5 phosphorylation by different constituents of ALN2 in CD8+ and Treg PBMC subpopulations is shown. [Figure 35A] Figure 1 shows that the single peptide ALN2 induced pSTAT5 in CD8+ compared to Treg PBMC subpopulations. [Figure 35B] Figure 1 shows that the single peptide ALN2 induced pSTAT5 in CD8+ compared to Treg PBMC subpopulations. [Figure 35C] Figure 1 shows that the single peptide ALN2 induced pSTAT5 in CD8+ compared to Treg PBMC subpopulations. [Figure 35D] Figure 1 shows that the single peptide ALN2 induced pSTAT5 in CD8+ compared to Treg PBMC subpopulations. [Figure 36A] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36B] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36C] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36D] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36E] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36F] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36G]Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36H] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36I] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36J] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36K] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36L] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 36M] Figure 1 shows STAT5 phosphorylation in CD8+ and CD8- PBMC lymphocyte populations by CD VHH-Fc-IL-2 and its T3 O-glycosylation variant. [Figure 37A] STAT5 phosphorylation by murine CD8-targeted alpha:beta ALN2 mutants in CD8+ and Treg primary mouse splenocytes. [Figure 37B] STAT5 phosphorylation by murine CD8-targeted alpha:beta ALN2 mutants in CD8+ and Treg primary mouse splenocytes. [Figure 37C] STAT5 phosphorylation by murine CD8-targeted alpha:beta ALN2 mutants in CD8+ and Treg primary mouse splenocytes. [Figure 37D]STAT5 phosphorylation by murine CD8-targeted alpha:beta ALN2 mutants in CD8+ and Treg primary mouse splenocytes. [Figure 38] Tumor growth (in mm3) in animals treated with buffer, Fc-ALN2, or CD8-Fc-ALN2 in syngeneic mouse models of colon cancer MC38 and CT26 is shown. [Figure 39] 1 shows the effect of co-treatment with CD8-Fc-ALN2 and anti-PD-1 in the MC38 and CT26 mouse models. [Figure 40A] STAT5 phosphorylation by CD8-targeted Fc gamma consensus Q126 mutant in CD8-positive PBMCs. [Figure 40B] STAT5 phosphorylation by CD8-targeted Fc gamma consensus Q126 mutant in CD8-positive PBMCs. [Figure 40C] STAT5 phosphorylation by CD8-targeted Fc gamma consensus Q126 mutant in CD8-positive PBMCs. [Figure 40D] STAT5 phosphorylation by CD8-targeted Fc gamma consensus Q126 mutant in CD8-positive PBMCs. [Figure 41A] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41B] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41C] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41D] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41E] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41F] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41G] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41H] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41I] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41J] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41K] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41L] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41M] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41N] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41O] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41P] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41Q] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41R] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 41S] 1 shows sensorgrams of the binding of the fusion protein, CD8-Fc-IL-2 and its Q126 mutant, to biotinylated CD25 during BLI. [Figure 42A] pSTAT5 phosphorylation by alpha:gamma variants in CD8+ and Treg PBMC subpopulations from three different donors is shown. [Figure 42B] pSTAT5 phosphorylation by alpha:gamma variants in CD8+ and Treg PBMC subpopulations from three different donors is shown. [Figure 42C] pSTAT5 phosphorylation by alpha:gamma variants in CD8+ and Treg PBMC subpopulations from three different donors is shown. [Figure 42D] pSTAT5 phosphorylation by alpha:gamma variants in CD8+ and Treg PBMC subpopulations from three different donors is shown. [Figure 43A] STAT5 phosphorylation by beta:gamma ALN2 in CD8+ or Treg PBMC subpopulations in the "resting" state or activated with TransAct is shown. [Figure 43B] STAT5 phosphorylation by beta:gamma ALN2 in CD8+ or Treg PBMC subpopulations in the "resting" state or activated with TransAct is shown. [Figure 43C] STAT5 phosphorylation by beta:gamma ALN2 in CD8+ or Treg PBMC subpopulations in the "resting" state or activated with TransAct is shown. [Figure 43D]STAT5 phosphorylation by beta:gamma ALN2 in CD8+ or Treg PBMC subpopulations in the "resting" state or activated with TransAct is shown. [Figure 44] The effect of CD8-Fc-ALN2 (12.5 μg), Fc-ALN2 (10.7 μg), or Fc-IL-2 (10.7 μg) on ​​T cell activation 1 day after a single dose is shown. [Figure 45] Shown is the effect of CD8-Fc-ALN2 (12.5 μg), Fc-ALN2 (10.7 μg), or Fc-IL-2 (10.7 μg), with or without co-treatment with an anti-PD-1 antibody, on T cell activation 7 days after two doses. [Figure 46] The effect of CD8-Fc-ALN2 (25 μg), Fc-ALN2 (21.4 μg), or Fc-IL-2 (21.4 μg) on ​​T cell activation 7 days after a single dose is shown. [Figure 47] 1 shows the effect of three injections of 12.5 μg of CD8-Fc-ALN2 and anti-PD-1 antibody compared to wild-type Fc-IL-2 in the A20 mouse model. [Figure 48] 1 shows the effect of three doses of 12.5 μg CD8-Fc-ALN2 compared to equimolar Fc-ALN2 treatment in a B16F10 mouse model. [Figure 49] 1 shows the effect of two injections of 12.5 μg CD8-Fc-ALN2 compared to equimolar Fc-ALN2 treatment in the Panc02 mouse model. [Figure 50] Figure 1 shows STAT5 phosphorylation in mouse splenocytes (singlet population) comparing wild-type IL-2, PD-L1-targeted ALN2, and non-targeted ALN2. [Figure 51] 1 shows the effect of three doses of 12.5 μg CD8-Fc-ALN2 and anti-PD-1 antibody compared to equimolar Fc-ALN2 treatment in the MC38 mouse model. [Figure 52]1 shows the effect of three doses of 12.5 μg TNCA1-Fc-ALN2 or an equivalent dose of untargeted ALN2, without or with co-treatment with an anti-PD-1 antibody, in the MC38 mouse model. [Figure 53] STAT5 phosphorylation by mouse CD8, or NKp46, or bispecific targeted alpha:beta ALN2 mutants in CD8 and NK primary mouse splenocytes compared to non-targeted IL-2 or wild-type IL-2. [Figure 54] Figure 1 shows upregulation of CD25 (left) and PD-1 (right) by ALN2 mutants R38A_F42K_D20E (top) and R38A_F42K_N88G (bottom) in three PBMC donors. Data are plotted as mean ± standard error of the mean (SEM). [Figure 55] 1 shows the effect of hCD8-Fc-ALN2 on human T cell activation in human immune system (HIS) mice 45 minutes after a single treatment (5 μg). [Figure 56] 1 shows CD25 upregulation in primary mouse splenocytes by murine CD8-targeted or non-targeted ALN2 mutants with various loss-of-function beta mutations. [Figure 57] 1 shows the effects of treatment with 12.5 μg of N88G-based or D20E-based CD8-Fc-ALN2 or equimolar untargeted ALN2 on days 6 and 9 in the MC38 mouse model. [Figure 58] STAT5 phosphorylation in primary mouse splenocytes by ALN2 mutants with various loss-of-function gamma mutations. [Figure 59] 1 shows the effect of three doses of 12.5 or 4.17 μg of the CD8-Fc-ALN2α:gamma mutant combination compared to an equimolar dose of non-targeted Fc-ALN in the MC38 mouse model. [Figure 60] 1 shows the effect of three doses of 4.17 or 1.4 μg of the CD8-Fc-ALN2 beta:gamma mutant combination compared to an equimolar dose of non-targeted Fc-ALN in the MC38 mouse model. [Figure 61]Figure 1 shows signaling of the D20 ALN2 mutant in HEK-Blue CD8 cells versus HEK-Blue NKp46 cells. [Figure 62] Figure 1 shows signaling of the N88 ALN2 mutant in HEK-Blue CD8 cells versus HEK-Blue NKp46 cells. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present technology is based, in part, on the discovery that protein complexes, including targeted chimeric proteins or Fc-based chimeric protein complexes, containing modified IL-2, exhibit beneficial therapeutic properties and reduced side effects. For example, the chimeric proteins of the present technology are highly active and / or have long-lasting effects while eliciting minimal side effects. The present technology provides pharmaceutical compositions containing the chimeric proteins and their use in the treatment of various diseases.

[0041] In some embodiments, the chimeric protein or protein complex having the modified IL-2 is CD8 + It may be advantageous to attenuate the activation of T cells, Thelp cells, or NK cells, which may provide an anti-tumor effect, and these cells primarily express IL-2 receptors β and γ, and the chimeric protein or protein complex inhibits T regs This may be detrimental to regs can provide tumor-promoting immunosuppressive effects), T regshas IL-2 receptors α, β, and γ. Furthermore, in some embodiments, preference for IL-2Rβ and / or IL-2Rγ over IL-2Rα avoids IL-2 side effects such as pulmonary edema and / or vascular leak syndrome (VLS). IL-2-based chimeras are also useful for treating diseases (e.g., autoimmune diseases), for example, when the modified IL-2 is antagonistic at IL-2Rβ and / or IL-2Rγ (e.g., naturally occurring antagonistic activity or antagonistic activity that is the result of one or more mutations, see, e.g., WO 2015 / 007520, the contents of which are incorporated herein by reference in their entirety). For example, the constructs can be used to treat CD8 + may favor attenuating T cell suppression (and thus potentially weakening the immune response), and CD8 + T cells have IL-2 receptors β and γ, and the present constructs are directed against T cells that have IL-2 receptors α, β, and γ. reg Alternatively, in some embodiments, the chimera having the modified IL-2 may be reg agonist activation of CD8 + T cell activation is disfavored. For example, these constructs have use in the treatment of diseases or conditions where immunosuppression would be beneficial, such as autoimmune diseases.

[0042] In some embodiments, the chimeric protein is CD8 + In some embodiments, these constructs comprise a targeting moiety described herein that is directed against T cells, and a modified IL-2 that has reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ, and / or has substantially reduced or eliminated affinity and / or activity for IL-2Rα. + It has activity against T cells, regIn some embodiments, such constructs have increased immunostimulatory effects compared to wild-type IL-2 (e.g., by not stimulating Tregs, without wishing to be bound by theory), while eliminating or reducing systemic side effects and / or toxicity associated with wild-type IL-2 and modified IL-2 protein constructs that preferentially target IL-2Rβγ but do not do so in a cell-selective, targeted, and conditional manner as described herein.

[0043] In some embodiments, the invention relates to a chimeric protein or Fc-based chimeric protein complex comprising a polypeptide having an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 290-449, 478-495, or 501-531. In further embodiments, the chimeric protein or Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence constructed in accordance with one or more of Examples 1-25.

[0044] Modified IL-2 In one aspect, the present technology provides chimeric proteins comprising modified IL-2. In various embodiments, the chimeric proteins of the present technology comprise modified IL-2 as a signaling factor. In various embodiments, the modified IL-2 signaling factor includes a functional derivative, analog, precursor, isoform, splicing variant, or fragment of the modified IL-2. In some embodiments, the modified IL-2 comprises mutations and / or modifications relative to SEQ ID NO: 1, the amino acid sequence of wild-type IL-2. In some embodiments, the modified IL-2 has reduced binding affinity to its target receptor.

[0045] In some embodiments, the modified IL-2 signaling factor has reduced affinity and / or activity for IL-2Rα and / or IL-2Rβ and / or IL-2Rγ. In some embodiments, the modified IL-2 has reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ. In some embodiments, the modified IL-2 has substantially reduced or eliminated affinity and / or activity for IL-2Rα. Such embodiments may be relevant to the treatment of cancer, for example, if the modified IL-2 is antagonistic at IL-2Rβγ.

[0046] In embodiments, the IL-2 has one or more mutations or modifications that bias the IL-2 away from IL-2Rs with an α chain and one or more attenuating mutations that reduce the activity or affinity of the IL-2 in a reversible form at IL-2Rs with only a βγ chain.

[0047] In embodiments, the IL-2 has one or more mutations or modifications that bias the IL-2 away from IL-2Rs that have only a βγ chain, and an attenuating mutation that reduces the activity or affinity of the IL-2 in a form that is reversible at IL-2Rs that have an α chain.

[0048] In embodiments, the IL-2 has one or more mutations or modifications that reduce or eliminate the activity or affinity of the IL-2 for the IL-2Rα chain and / or reduce or eliminate the activity or affinity of the IL-2 for the IL-2Rβγ complex. In some embodiments, the reduced activity or affinity of the IL-2 for the IL-2Rα chain is reversible by inducing its proximity at / to a target (e.g., a cell) via a targeting moiety disclosed herein. In some embodiments, the mutations or modifications that eliminate the activity or affinity of the modified IL-2 for the IL-2Rα chain are not reversible by inducing its proximity at / to a target (e.g., a cell) via a targeting moiety. In some embodiments, the reduced activity or affinity of the modified IL-2 for the IL-2Rβγ complex is reversible by inducing its proximity at / to a target (e.g., a cell) via a targeting moiety disclosed herein. In some embodiments, one or more mutations that reduce the activity or affinity of the modified IL-2 for the IL-2Rα chain and IL-2Rβγ complex, respectively, can both be restored by induction at / proximity to a target (e.g., a cell) via a targeting moiety disclosed herein.

[0049] In embodiments, the IL-2 has one or more mutations or modifications that substantially eliminate the activity or affinity of the IL-2 for the IL-2R alpha chain and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an alpha chainless IL-2R, wherein the reduced activity or affinity is reversible by induction at / proximity to a target (e.g., a cell) via a targeting moiety, and wherein the IL-2 is substantially inactive at an IL-2R on a cell that does not express the target for the targeting moiety, or when contacted with a cell that expresses the target for the targeting moiety, is substantially inactive at an alpha chain-containing IL-2R compared to wild-type IL-2, but is active at an alpha chainless IL-2R and / or a cell that expresses the target for the targeting moiety. In embodiments, the activity or affinity based on the mutation or modification is reduced by about 3-fold, or about 5-fold, or 10-fold, or 30-fold, or 100-fold, or 300-fold, or 500-fold, or 1000-fold compared to the activity or affinity based on a different mutation or modification.

[0050] In embodiments, the IL-2 has one or more mutations or modifications, i.e., one or more mutations or modifications that reduce the activity or affinity of the IL-2 for IL-2Rαβγ and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rα, and such reduced activity or affinity can be restored by inducing proximity to / at a target (e.g., a cell) via a targeting moiety.

[0051] In embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the IL-2 for IL-2Rαβγ and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rβγ, wherein the reduced activity or affinity is reversible by induction of proximity at / to a target (e.g., a cell) via a targeting moiety, and wherein the IL-2 is substantially inactive at IL-2R on cells that do not express the target for the targeting moiety, or when contacted with cells that express the target for the targeting moiety, is substantially inactive at IL-2R comprising an alpha chain compared to wild-type IL-2, but is active in cells that express IL-2Rβγ and / or the target for the targeting moiety.

[0052] In embodiments, the IL-2 has a selective (e.g., biased and / or strongly preferential) action at IL-2Rβγ (and not IL-2Rαβγ), but activity at IL-2Rβγ is attenuated by further mutations or modifications to make the IL-2 safe for systemic use (e.g., the IL-2 is primarily active at IL-2Rβγ at the site of therapeutic action, and the IL-2 is directed to the site of therapeutic action via the targeting moiety).

[0053] In embodiments, the IL-2 has a selective (e.g., biased and / or strongly preferential) effect on NK cells and / or CD8+ cells but a substantially non-selective effect on Treg cells, and the selective (e.g., biased and / or strongly preferential) effect on NK cells and / or CD8+ cells is controlled by targeting to reduce or eliminate systemic side effects. For example, in some embodiments, the chimeric protein is not only directed to cells that have an immunostimulatory effect, but also in a controllable manner that focuses the action of IL-2 at the desired therapeutic site. As described herein, such chimeric proteins, in embodiments, have use in the safe stimulation of the immune system for anti-cancer effects.

[0054] In some embodiments, the IL-2 has one or more mutations or modifications, i.e., one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R that does not have an alpha chain, and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R that includes an alpha chain, and such mutations or modifications are attenuating mutations that can restore the activity or affinity based on the attenuating mutation by induction at / proximity to the target (e.g., a cell) via a targeting moiety.

[0055] In embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R lacking an α chain and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R comprising an α chain, wherein such reduced activity or affinity is reversible by induction of proximity at / to a target (e.g., a cell) via a targeting moiety, and wherein the IL-2 is substantially inactive at an IL-2R on a cell that does not express the target for the targeting moiety, or, when contacted with a cell that expresses the target for the targeting moiety, is substantially inactive at an IL-2R lacking an α chain but is substantially inactive at an IL-2R comprising an α chain, compared to wild-type IL-2, but is active in an IL-2R comprising an α chain and / or a cell that expresses the target for the targeting moiety.

[0056] In some embodiments, the IL-2 has one or more mutations or modifications, i.e., one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rβγ and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rα, and such mutations or modifications are attenuating mutations whose activity or affinity can be restored by induction at / proximity to a target (e.g., a cell) via a targeting moiety.

[0057] In some embodiments, the IL-2 has one or more mutations or modifications, i.e., one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rβγ and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rαβγ, and such reduced activity or affinity is reversible by induction at / proximity to the target (e.g., cell) via the targeting moiety.

[0058] In some embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rβγ and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rαβγ, wherein such reduced activity or affinity is reversible by induction of proximity at / to a target (e.g., a cell) via a targeting moiety, and such IL-2 is substantially inactive at IL-2R on cells that do not express the target for the targeting moiety, or, when contacted with cells that express the target for the targeting moiety, is substantially inactive at IL-2Rβγ but substantially inactive at an IL-2R comprising an α chain, compared to wild-type IL-2.

[0059] In embodiments, the modified IL-2 has a selective (e.g., biased and / or strongly preferential) action at IL-2Rαβγ (and not IL-2Rαβγ), but activity at IL-2Rαβγ is attenuated by further mutations or modifications to make the IL-2 safe for systemic use (e.g., the IL-2 is primarily active at IL-2Rαβγ at the site of therapeutic action, and the IL-2 is directed to the site of therapeutic action via the targeting moiety).

[0060] In some embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2Rβγ chain and its normal activity or affinity for the IL-2Rα chain, and the reduced activity or affinity of the modified IL-2 for the IL-2Rβγ chain can be restored by inducing its proximity to / at a target (e.g., a cell) via a targeting moiety.

[0061] In embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rα and its normal activity or affinity for the IL-2Rβγ chain, and the reduced activity or affinity of the modified IL-2 for the IL-2Rα chain is reversible by inducing its proximity to / at a target (e.g., a cell) via a targeting moiety.

[0062] In some embodiments, the IL-2 has one or more mutations or modifications that eliminate the activity or affinity of the modified IL-2 for the IL-2Rα chain and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2Rβγ chain, and the reduced activity or affinity of the modified IL-2 for the IL-2Rβγ chain can be restored by inducing proximity of the modified IL-2 at / to a target (e.g., a cell) via a targeting moiety (e.g., a CD8, CD3, or PD-1 targeting moiety), and is useful for mediating a T cell-mediated immune response (e.g., a CD8 T cell-mediated immune response).

[0063] In some embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2Rα chain and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2Rβγ chain, and the activity of the modified IL-2 for the IL-2Rα and / or IL-2Rβγ chain can be restored by inducing its proximity to or at a target (e.g., a cell) via a targeting moiety (e.g., a CD8, CD3, or PD-1 targeting moiety), and is useful for mediating a T cell-mediated immune response (e.g., a CD8 T cell-mediated immune response).

[0064] In some embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2Rα chain and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2Rβγ chain, and the reduced activity or affinity of the modified IL-2 for the IL-2Rα chain and / or IL-2Rβγ chain can be restored by inducing proximity of the modified IL-2 at / to a target (e.g., a cell) via a targeting moiety, and is useful for mediating a Treg response.

[0065] In some embodiments, the IL-2 has one or more mutations or modifications, i.e., one or more mutations or modifications that eliminate the activity or affinity of the modified IL-2 for the IL-2Rα chain and / or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2Rβγ chain, and the reduced activity or affinity of the modified IL-2 for the IL-2Rβγ chain can be restored by inducing proximity of the modified IL-2 at / to a target (e.g., a cell) via a targeting moiety, and is useful for mediating a Treg response.

[0066] In some embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2Rβγ chain and its normal activity or affinity for the IL-2Rα chain, and the reduced activity or affinity of the modified IL-2 for the IL-2Rβγ chain can be restored by inducing its proximity to / at a target (e.g., a cell) via a targeting moiety, and is useful for mediating a Treg response.

[0067] In embodiments, the IL-2 has a selective (e.g., biased and / or strongly preferential) effect on Treg cells and a substantially non-selective effect on NK cells and / or CD8+ cells, and the selective (e.g., biased and / or strongly preferential) effect on Treg cells is controlled by targeting to reduce or eliminate systemic side effects. For example, the chimeric protein, in some embodiments, is not only directed to cells that have an immunostimulatory effect, but also in a controllable manner that focuses the effect of IL-2 at the desired therapeutic site. As described herein, such chimeric proteins, in embodiments, have use in the safe stimulation of the immune system for anti-autoimmune effects.

[0068] In some embodiments, the modified IL-2 signaling factor is modified wild-type IL-2. Wild-type IL-2 has the following amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 1) It has.

[0069] In some embodiments, the modified IL-2 signaling factor is selected from: (i) a mutation selected from D20E, D20F, D20G, D20H, D20I, D20K, D20L, and D20V; or (ii) a mutation selected from N88G, N88E, N88K, N88Q, and N88V; or (iii) a mutation selected from Q126G, Q126A, Q126E, Q126F, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126Y. or (iv) a mutation selected from (i) and (iii), or a mutation selected from (ii) and (iii); or (v) a mutation selected from R38A / F42K / N88G / C125A, F42K / C125A, D20E / C125A, D20F / C125A, D20G / C125A, D20H / C125A, D20I / C125A, D20K / C125A, D20L / C125A, D20N / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C 125A, N88G / C125A, N88E / C125A, N88H / C125A, N88I / C125A, N88K / C125A , N88Q / C125A, N88R / C125A, N88T / C125A, N88V / C125A, D20E / R38A / F42K , D20E / R38A / F42K / C125A, D20V / R38A / F42K, D20V / R38A / F42K / C125A, R 38A / F42Y / E62A / C125A, R38A / F42Y / Y45A / E62A, F42Y / Y45A / L72G, R38A / F42Y / Y45A / E62A / C125A, F42Y / Y45A / L72G / C125A, R38A / F42K / N88G, R38A / F42K / Q126G, R38A / F42K / Q126I, R38A / F42K / Q126Y, R38A / F42K / Q126G / C125A, R38A / F42K / Q126I / C125A, R38A / F42K / Q126Y / C125A, R38A / F42K / E62A / N88G, and R38A / F42K / E62A / N88G / C125A.

[0070] In some embodiments, the modified IL-2 signaling factor comprises one or more mutations at amino acid residues selected from R38, F42, E62, C125, Y45, L72, D20, N88, and Q126 relative to SEQ ID NO: 1. In such embodiments, the modified IL-2 signaling factor comprises one or more mutations at amino acid residues selected from R38A, F42K, F42Y, E62A, C125A, Y45A, L72G, D20E, D20F, D20G, D20H, D20I, D20K, D20L, D20N, D20V, N88G, N88E, N88I, N88K, N88Q, N88R, N88V, R38A / F42Y / E62A / C125A, F42Y / Y45A / L72G / C125A, F42K / C 125A, R38A / F42K / C125A, D20E / C125A, D20G / C125A, D20S / C125A, D20T / C125A, D20 V / C125A, N88A / C125A, N88D / C125A, N88G / C125A, N88H / C125A, N88Q / C125A, N88T / C125A, D20E / R38A / F42K / C125A, D20V / R38A / F42K / C125A, R38A / F42K / N88A / C125A , R38A / F42K / N88G / C125A, Q126A, Q126E, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126Y.

[0071] In various embodiments, the modified IL-2 signaling factor is selected from the group consisting of D20V, D20E, N88G, F42K / C125A, D20E / C125A, D20G / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C125A, N88G / C125A, N88H / C125A, N88Q / C125A, N88T / C125A, D20E / R38A / F42K, D20E / R38A / F42K / C125A, D20V / R38A / F42K / , D20V / R38A / F42K / , D20V / R38A / F42K / , D20V / R38A / F42K / and one or more mutations selected from 2K / C125A, D20A, R38A / F42K / E62A, D20V / R38A / F42K / E62A / C125A, R38A / F42K / N88G, R38A / F42K / N88G / C125A, R38A / F42K / E62A / N88G, R38A / F42K / E62A / N88G / C125A, Q126A, Q126E, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126Y.

[0072] In certain embodiments, the modified IL-2 signaling agent has one or more of the following sequences relative to SEQ ID NO: 1: R38A / F42Y / E62A / C125A, F42Y / Y45A / L72G / C125A, F42K / C125A, D20E / C125A, D20G / C125A, D20S / C125A, D20T / C125A, D20V / C125A, and one or more mutations selected from N88A / C125A, N88D / C125A, N88G / C125A, N88H / C125A, N88Q / C125A, N88T / C125A, D20E / R38A / F42K / C125A, D20V / R38A / F42K / C125A, and R38A / F42K / N88G / C125A.

[0073] In some embodiments, the modified IL-2 signaling agent comprises a combination of one or more mutations selected from those listed in Table 8.

[0074] Without wishing to be bound by theory, it is believed that the modified IL-2 factors have reduced affinity for the high affinity IL-2 receptor (i.e., IL-2Rαβγ) and maintain affinity for the intermediate affinity IL-2 receptor (i.e., IL-2Rβγ) compared to wild-type IL-2.

[0075] Without wishing to be bound by theory, it is further believed that these modified IL-2 factors have reduced affinity for the IL-2 receptor (e.g., IL-2Rβγ) compared to wild-type IL-2.

[0076] In some embodiments, the modified IL-2 signaling factor having mutations at R38, F42, Y45, and / or E62 is capable of inducing proliferation of effector cells, including CD8+ T cells and NK cells, but not Treg cells and / or endothelial cells. In some embodiments, the modified IL-2 signaling factor having mutations at R38, F42, Y45, and / or E62 is less toxic than wild-type IL-2 factor. Chimeric proteins comprising modified IL-2 factors with substantially reduced affinity and / or activity for IL-2Rα have uses, for example, in oncology.

[0077] In some embodiments, the modified IL-2 factor comprises a mutation at amino acid residue D20. For example, the modified IL-2 factor may comprise D20E, D20G, D20S, D20T, or D20V. In some embodiments, the modified IL-2 factor comprising a mutation at D20 has reduced toxicity compared to wild-type IL-2 factor. In some embodiments, the mutation at D20 reduces toxicity by inhibiting binding to and activation of endothelial cells.

[0078] In some embodiments, the modified IL-2 factor comprises a mutation at amino acid C125. For example, the modified IL-2 factor may comprise the mutation C125A. In some embodiments, a modified IL-2 factor comprising a mutation at C125 is optimal for production of the modified IL-2 factor. In some embodiments, the mutation at C125 promotes stability of the modified IL-2 factor by removing an unpaired cysteine ​​residue. In such embodiments, the modified IL-2 factor may have substantially reduced affinity and / or activity for IL-2Rα, as disclosed, for example, in Carmenate et al. (2013) The Journal of Immunology, 190:6230-6238, the disclosure of which is incorporated herein by reference in its entirety.

[0079] In other embodiments, the modified IL-2 factor may have substantially reduced affinity and / or activity for IL-2Rβ, e.g., as described in WO 2016 / 025385 (the disclosure of which is incorporated herein by reference in its entirety). In such embodiments, the modified IL-2 factor is capable of inducing proliferation of Treg cells but is unable to induce proliferation of effector cells, such as CD8+ T cells and NK cells. Chimeric proteins comprising modified IL-2 factors with substantially reduced affinity and / or activity for IL-2Rβ may find use, for example, in the treatment of autoimmune diseases. In some embodiments, the modified IL-2 factor may comprise a mutation at amino acid residue N88. For example, the modified IL-2 factor may comprise N88A, N88D, N88G, N88H, N88Q, or N88T.

[0080] In some embodiments, the modified IL-2 signaling factor comprises a deletion of Ala at the N-terminus of SEQ ID NO: 1. In some embodiments, the modified IL-2 factor comprises a substitution of cysteine ​​at position 125 of SEQ ID NO: 1 with serine or alanine. In some embodiments, the modified IL-2 factor comprises a deletion of Ala at the N-terminus and a substitution of serine or alanine at position 125 of SEQ ID NO: 1.

[0081] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rα and IL-2Rβ, and optionally the modified IL-2 is selected from the group consisting of D20E / R38A / F42K, D20E / R38A / F42K / C125A, D20V / R38A / F42K, D20V / R38A / F42K / C125A , D20V / R38A / F42K / E62A, D20V / R38A / F42K / E62A / C125A, R38A / F42K / N88G, R38A / F42K / N88G / C125A, R38A / F42K / E62A / N88G, and R38A / F42K / E62A / N88G / C125A.

[0082] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rα and IL-2Rγ, and optionally, the modified IL-2 comprises one or more mutations selected from R38A / F42K / Q126G, R38A / F42K / Q126I, and R38A / F42K / Q126Y.

[0083] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rβ, and optionally the modified IL-2 is selected from the group consisting of D20E, D20F, D20G, D20H, D20I, D20K, D20L, D20V, N88G, N88E, N88K, N88Q, N88V, D20E / C125A, D20F / C125A, D20G / C125A, D20H / C125A, D20I / C125A, D20L / C125A and N88V / C125A, D20K / C125A, D20N / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C125A, N88G / C125A, N88E / C125A, N88H / C125A, N88I / C125A, N88K / C125A, N88Q / C125A, N88R / C125A, N88T / C125A, and N88V / C125A.

[0084] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rβ, and optionally, the modified IL-2 comprises one or more mutations selected from Q126A, Q126E, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126Y.

[0085] In some embodiments, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rβγ, and optionally the modified IL-2 is selected from the group consisting of D20E / Q126A, D20E / Q126D, D20E / Q126E, D20E / Q126F, D20E / Q126G, D20E / Q126H, D20E / Q126I, D20E / Q126K, D20E / Q126L, D20E / Q126M, D20E / Q126N, D20E / Q126P, D20E / Q126R, D20E / Q126R 6S, D20E / Q126T, D20E / Q126V, D20E / Q126W, D20E / Q126Y, D20V / Q126A, D20V / Q126D, D20V / Q126E, D20V / Q126F, D20V / Q126G, D20V / Q12 6H, D20V / Q126I, D20V / Q126K, D20V / Q126L, D20V / Q126M, D20V / Q126N, D20V / Q126P, D20V / Q126R, D20V / Q126S, D20V / Q126T, D20V / Q12 6V, D20V / Q126W, D20V / Q126Y, N88A / Q126A, N88A / Q126D, N88A / Q126E, N88A / Q126F, N88A / Q126G, N88A / Q126H, N88A / Q126I, N88A / Q12 6K, N88A / Q126L, N88A / Q126M, N88A / Q126N, N88A / Q126P, N88A / Q126R, N88A / Q126S, N88A / Q126T, N88A / Q126V, N88A / Q126W, N88A / Q12 and N88G / Q126Y, N88G / Q126A, N88G / Q126D, N88G / Q126E, N88G / Q126F, N88G / Q126G, N88G / Q126H, N88G / Q126I, N88G / Q126K, N88G / Q126L, N88G / Q126M, N88G / Q126N, N88G / Q126P, N88G / Q126R, N88G / Q126S, N88G / Q126T, N88G / Q126V, N88G / Q126W, and N88G / Q126Y. In a further embodiment, the modified IL-2 exhibits reduced affinity and / or activity for IL-2Rβγ and increased selectivity for activated CD8 cells.

[0086] Neoleukin In some embodiments, the modified IL-2 signaling factor is a modified neoleukin, which has the amino acid sequence: PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 2) Includes.

[0087] In such embodiments, the modified IL-2 signaling agent comprising SEQ ID NO:2 has one or more mutations at amino acid residues D15 and / or N40. For example, in some embodiments, the modified IL-2 signaling agent comprising SEQ ID NO:2 comprises D15T, D15H, N40I, N40G, or N40R.

[0088] In some embodiments, the modified IL-2 signaling agent is homologous to SEQ ID NO: 1 or 2 by 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%. 1%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity to the amino acid sequence.

[0089] In various embodiments, the modified IL-2 is modified to have one or more mutations. In some embodiments, the modified IL-2 is modified to have one or more mutations and one or more modifications (e.g., glycosylation). IL-2 contains a threonine O-glycosylation site at position 3 (T3). For example, in some embodiments, the modified IL-2 signaling factor contains a T3 O-glycosylation mutation, where the mutation is one of T3A, T3F, T3H, T3L, T3V, and T3Y. In some embodiments, the modified IL-2 signaling factor contains a T3 O-glycosylation deletion mutation, where the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2 sequence are deleted.

[0090] In some embodiments, the one or more amino acid mutations may be independently selected from a substitution, an insertion, a deletion, and a truncation.

[0091] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0092] "Conservative substitutions" can be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0093] As used herein, a "conservative substitution" is defined as the replacement of an amino acid with another amino acid within the same group of the six standard amino acid groups. For example, replacing Asp with Glu retains a single negative charge in the modified polypeptide. Furthermore, glycine and proline may be substituted for each other based on their ability to disrupt alpha helices.

[0094] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid from a different group of the six standard amino acid groups (1) to (6) above.

[0095] In various embodiments, substitutions also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, C α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general).

[0096] In some embodiments, the mutations enable the modified IL-2 to have one or more attenuated activities, such as one or more of reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to non-mutated IL-2, e.g., wild-type IL-2 (e.g., SEQ ID NO: 1). For example, the one or more attenuated activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to non-mutated, e.g., wild-type IL-2 (e.g., SEQ ID NO: 1), can be for a receptor such as IL-2Rαβγ. As a result, in various embodiments, the mutations enable the modified IL-2 to have reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to non-mutated, e.g., wild-type IL-2.

[0097] In some embodiments, the modified IL-2 is modified to have one or more mutations that reduce its binding affinity or activity for a therapeutic or target receptor, such as IFNAR. In some embodiments, the activity conferred by the modified IL-2 is agonism for a therapeutic or target receptor (e.g., activating a cellular effect at the site of treatment) or agonism for a therapeutic or target receptor (e.g., reducing or eliminating activating a cellular effect at the site of treatment). For example, the modified IL-2 may activate a therapeutic or target receptor. In such embodiments, the mutation results in the modified IL-2 having a reduced activating effect on a therapeutic or target receptor.

[0098] In some embodiments, the reduced affinity or activity for a therapeutic or target receptor is recoverable by binding to a targeting moiety. In other embodiments, the reduced affinity or activity for a therapeutic or target receptor is not substantially recoverable by binding to a targeting moiety. In various embodiments, the chimeric proteins of the present technology reduce off-target effects because the modified IL-2 has a mutation that weakens its binding affinity or activity for a therapeutic or target receptor. In various embodiments, this reduces side effects observed with, for example, wild-type IL-2. In various embodiments, the modified IL-2 is substantially inactive en route to the site of therapeutic action and substantially exerts its effect specifically on the target cell type, which greatly reduces undesirable side effects.

[0099] In various embodiments, the modified IL-2 is a therapeutic agent that has attenuated or reduced affinity, e.g., binding (e.g., K D ) and / or activation (e.g., K A and / or EC 50 In various embodiments, the reduced affinity for the therapeutic receptor allows for attenuated activity and / or signaling from the therapeutic receptor.

[0100] In various embodiments, the modified IL-2 has about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% to 20%, about 20% to 40%, about 50%, about 40% to 60%, about 60% to 80%, or about 80% to 100% affinity for a therapeutic or target receptor (e.g., IL-2Rαβγ or IL-2Rβγ or any one of their subunits IL-2Rα, IL-2Rβ, and / or IL-2Rγ) compared to wild-type IL-2. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower, compared to wild-type IL-2.

[0101] 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 binding data (e.g., Scatchard, The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51: 660-672, 1949) or by reflectance interferometry under flow-through conditions as described by Brecht et al. Biosens Bioelectron 1993;8:387-392, the entire contents of which are incorporated herein by reference. In some embodiments, receptor binding activity is measured by biolayer interferometry (BLI).

[0102] In some embodiments, the diminished activity or affinity for a therapeutic or target receptor can be restored by conjugation with a targeting moiety (e.g., an antibody or antibody format described herein) that has high affinity for the antigen at the site of therapeutic action. Targeting is achieved by conjugating modified IL-2 to the targeting moiety. In one embodiment, the modified IL-2 is conjugated to the targeting moiety via its amino terminus. In another embodiment, the modified IL-2 is conjugated to the targeting moiety via its carboxy terminus. Thus, the chimeric proteins of the present invention, in some embodiments, provide localized, on-target, and controlled therapeutic action at the site of the therapeutic or target receptor.

[0103] In various embodiments, the activity of the modified IL-2 at a therapeutic or target receptor is increased by binding to a targeting moiety, resulting in a high affinity for the antigen at the therapeutically active site (e.g., an antibody or antibody format described herein). The targeting is achieved by linking the modified IL-2 to a targeting moiety. In one embodiment, the modified IL-2 is linked to a targeting moiety via its amino terminus. In another embodiment, the modified IL-2 is linked to a targeting moiety via its carboxy terminus. In this way, the subject chimeric proteins, in some embodiments, provide a localized, targeted, and controlled therapeutic action at a therapeutic or target receptor.

[0104] Therapeutic agents containing interleukins or their variants In embodiments, the present invention provides chimeric proteins or protein complexes comprising one or more targeting moieties that bind to one or more of the following targets: CD8, CTLA4, CD3, CD4, DNAM-1, Nrp1 (neurophilin), TNFR1, TNFR2, GITR, ICOS, CD20, CD70, Clec9a, NKp46, PD-1, PD-L1, PD-L2, SIRP1α, FAP, XCR1, tenascin, and ECM proteins.

[0105] Cell Recruitment of Targeting Moieties In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention further comprises one or more targeting moieties having a recognition domain that specifically binds to a target of interest (e.g., an antigen, a receptor). In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can comprise two, three, four, five, six, seven, eight, nine, ten, or more targeting moieties. In exemplary embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention can comprise two or more targeting moieties. In such embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can target two different cells (e.g., to create a synapse) or the same cell (e.g., to achieve a higher concentration of a signaling factor effect).

[0106] In some embodiments, the target of interest (eg, antigen, receptor) is directed against immune cells and / or organ cells and / or tissue cells.

[0107] In various embodiments, the desired target (e.g., antigen, receptor) can be found on one or more immune cells, which may include, but are not limited to, T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor or tumor-associated macrophages (e.g., M1 or M2 macrophages), B cells, Breg cells, dendritic cells, or a subset thereof. In some embodiments, the recognition domain specifically binds to the desired target (e.g., antigen, receptor) and effectively recruits one or more immune cells directly or indirectly. In some embodiments, the desired target (e.g., antigen, receptor) can be found on one or more tumor cells. In some embodiments, the desired target (e.g., antigen, receptor) can be present in the neovasculature of a tumor. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention can directly or indirectly recruit immune cells to a treatment site (e.g., a site containing one or more diseased cells or cells to be modulated to achieve a therapeutic effect). In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can directly or indirectly recruit immune cells, e.g., immune cells capable of killing and / or suppressing tumor cells, to a site of action (such as, but not limited to, the tumor microenvironment).

[0108] In various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has a targeting moiety with a recognition domain that specifically binds to a target (e.g., an antigen, a receptor) that is part of a non-cellular structure. In some embodiments, the antigen or receptor is not an integral component of an intact cell or cellular structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-proteinaceous non-cellular marker, which includes, but is not limited to, extracellular deposits such as nucleic acids or cholesterol, including DNA or RNA, such as, for example, DNA released from necrotic tumor cells.

[0109] In some embodiments, the target of interest (e.g., antigen, receptor) is part of the noncellular components of the stroma or extracellular matrix (ECM) or a marker associated therewith. As used herein, stroma refers to the connective and supportive framework of a tissue or organ. The stroma may comprise a collection of cells, such as fibroblasts / myofibroblasts / glial cells, epithelial, adipose, immune, vascular, smooth muscle, and immune cells, along with the extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target of interest (e.g., antigen, receptor) is part of the noncellular components of the stroma, such as the extracellular matrix and extracellular molecules. As used herein, ECM refers to the noncellular components present in all tissues and organs. The ECM consists of a large collection of biochemically distinct components, including, but not limited to, proteins, glycoproteins, proteoglycans, and polysaccharides. These ECM components are typically produced by neighboring cells and secreted into the ECM by exocytosis. Once secreted, ECM components often assemble to form a complex network of macromolecules. In various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein of the present invention, comprises a targeting moiety that recognizes a target (e.g., an antigen or receptor or non-protein molecule) located on any component of the ECM, including, but not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers, and other ECM proteins or ECM non-proteins, e.g., polysaccharides and / or lipids, or ECM-associated molecules (e.g., proteins or non-proteins, e.g., polysaccharides, nucleic acids, and / or lipids).

[0110] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on an ECM proteoglycan. Proteoglycans are glycosylated proteins. A basic proteoglycan unit comprises a core protein with one or more covalently attached glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+), which attract water via osmosis and keep the ECM and resident cells hydrated. Proteoglycans can also capture and store growth factors within the ECM. Examples of proteoglycans that can be targeted by the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In certain embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on a non-proteoglycan polysaccharide, such as hyaluronic acid.

[0111] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on ECM fibers. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety recognizes one or more epitopes on collagen or collagen fibers. Collagen is the most abundant protein in the ECM. Collagen exists as a fibrous protein in the ECM and provides structural support to resident cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagen present in the ECM, including, but not limited to, fibrillar collagens (types I, II, III, V, XI), facit collagens (types IX, XII, XIV), short-chain collagens (types VIII, X), basement membrane collagens (type IV), and / or type VI, VII, or XIII collagen. Elastin fibers provide elasticity to tissues, allowing them to stretch as needed and then return to their original state. In some embodiments, the targeting moiety recognizes one or more epitopes on elastin or elastin fibers.

[0112] In some embodiments, the targeting moiety recognizes one or more ECM proteins, including, but not limited to, tenascin, fibronectin, fibrin, laminin, or nidogen / entactin.

[0113] In certain embodiments, the targeting moiety recognizes and binds to tenascin. The tenascin (TN) family of glycoproteins includes at least four members: tenascin-C, tenascin-R, tenascin-X, and tenascin-W. The primary structure of tenascin proteins contains several common motifs ordered in the same contiguous sequence: amino-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain. Each protein member is associated with typical variations in the number and nature of the EGF-like and fibronectin type III repeats. Isoform variants also exist, particularly for tenascin-C. More than 27 splice variants and / or isoforms of tenascin-C are known. In certain embodiments, the targeting moiety recognizes and binds to tenascin-CA1. Similarly, tenascin-R also has various splice variants and isoforms. Tenascin-R typically exists as a dimer or trimer. Tenascin-X is the largest member of the tenascin family and is known to exist as a trimer. Tenascin-W exists as a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on the tenascin protein. In some embodiments, the targeting moiety recognizes monomeric, dimeric, trimeric, and / or hexameric forms of the tenascin protein.

[0114] In some embodiments, the targeting moiety recognizes and binds to fibronectin. Fibronectin is a glycoprotein that connects cells to collagen fibers in the ECM, allowing cells to migrate through the ECM. Upon binding to integrins, fibronectin unfolds to form functional dimers. In some embodiments, the targeting moiety recognizes monomeric and / or dimeric forms of fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. In exemplary embodiments, the targeting moiety recognizes fibronectin extracellular domain A (EDA) or fibronectin extracellular domain B (EDB). Elevated EDA levels are associated with various diseases and disorders, including psoriasis, rheumatoid arthritis, diabetes, and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing the EDA isoform and can be used to target chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing the EDB isoform. In various embodiments, such targeting moieties may be used to direct a chimeric protein or chimeric protein conjugate, such as an Fc-based chimeric protein conjugate, to tumor cells, including tumor neovasculature.

[0115] In certain embodiments, the targeting moiety recognizes and binds to fibrin, another protein substance often found in the matrix network of the ECM. Fibrin is formed by the action of the protease thrombin on fibrinogen, which polymerizes it. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes both monomeric and polymerized forms of fibrin.

[0116] In certain embodiments, the targeting moiety recognizes and binds to laminin. Laminin is a major component of basement membranes and the underlying protein network for cells and organs. Laminin is a heterotrimeric protein comprising an α chain, a β chain, and a γ chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes monomeric, dimeric, and trimeric forms of laminin.

[0117] In certain embodiments, the targeting moiety recognizes and binds to nidogen or entactin. Nidogen / entactin are a family of highly conserved sulfated glycoproteins. They are major structural components of basement membranes and function to connect laminin with the collagen IV network in basement membranes. 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.

[0118] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an epitope present on any of the targets described herein. In certain embodiments, the antigen recognition domain recognizes one or more linear epitopes present on a protein. As used herein, a linear epitope refers to any continuous sequence of amino acids present on a protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on a protein. As used herein, a conformational epitope refers to a portion of one or more amino acids (which may be discontinuous) that forms a three-dimensional surface with characteristics and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0119] In various embodiments, the targeting moiety may bind to the full-length and / or mature and / or isoforms and / or splice variants and / or fragments and / or any other natural or synthetic analogs, variants, or mutants of any of the targets described herein. In various embodiments, the targeting moiety may bind to any form of the protein described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers, and associated forms. In various embodiments, the targeting moiety may bind to any post-translationally modified form of the protein described herein, such as glycosylated and / or phosphorylated forms.

[0120] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an extracellular molecule such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In certain embodiments, DNA is shed into the extracellular space from necrotic or apoptotic tumor cells or other diseased cells.

[0121] 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: Fibro-lipid (fibro-fatty) plaques are characterized by the accumulation of lipid-laden cells beneath the intima of an artery. Beneath the endothelium lies a fibrous cap that covers the atherosclerotic plaque core. The core contains lipid-laden cells (macrophages and smooth muscle cells) with increased tissue cholesterol and cholesterol ester content, fibrin, proteoglycans, collagen, elastin, and necrotic cellular debris. In advanced plaques, the central core of the plaque typically contains extracellular cholesterol deposits (released from dead cells), which form areas of cholesterol crystals with empty needle-like spaces. The periphery of the plaque contains younger foam cells and capillaries. Fibrous plaques are also localized subintima within the arterial wall, resulting in wall thickening and proliferation and, sometimes, scattered, localized narrowing of the lumen, accompanied by slight atrophy of the muscularis. Fibrous plaques contain collagen fibers (eosinophilic), calcium deposits (hematoxylinophilic), and lipid-loaded cells. In some embodiments, the targeting moiety recognizes and binds to one or more noncellular components of these plaques, such as fibrin, proteoglycans, collagen, elastin, necrotic cell debris, and calcium or other mineral deposits or precipitates. In some embodiments, the necrotic cell debris is nucleic acid, e.g., DNA or RNA released from dying cells.

[0122] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures found in brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures localized in amyloid plaques found in the brains of patients with Alzheimer's disease. For example, the targeting moiety recognizes and binds to the peptide amyloid beta. The peptide amyloid beta is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in brain plaques found in patients with Huntington's disease. In various embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in plaques associated with other neurodegenerative or musculoskeletal diseases, such as Lewy body dementia and inclusion body myositis.

[0123] In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention can have two or more targeting moieties that bind to non-cellular structures. In some embodiments, there are two targeting moieties, one that targets cells while the other targets non-cellular structures. In various embodiments, the targeting moieties can directly or indirectly recruit cells, such as disease cells and / or effector cells. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes of the invention, can or find use in methods for shifting the balance of immune cells to engage them in favor of immune attack of tumors. For example, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can shift the ratio of immune cells at clinically important sites in favor of cells capable of killing and / or suppressing tumors (e.g., T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or subsets thereof) and against cells that protect tumors (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs); tumor-associated neutrophils (TANs), M2 macrophages, tumor-associated macrophages (TAMs), or subsets thereof). In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can increase the ratio of effector T cells to regulatory T cells.

[0124] For example, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a T cell. In some embodiments, the recognition domain directly or indirectly recruits a T cell. In certain embodiments, the recognition domain specifically binds to an effector T cell. In some embodiments, the recognition domain directly or indirectly recruits an effector T cell, for example, in some embodiments, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of effector T cells include cytotoxic T cells (e.g., αβ TCR, CD3 + , CD8 + , CD45RO + );CD4 + Effector T cells (e.g., αβTCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL-7R / CD127 + );CD8 + Effector T cells (e.g., αβTCR, CD3 + , CD8 + , CCR7 + , CD62Lhi, IL-7R / CD127 + effector memory T cells (e.g., CD62Llow, CD44 + , TCR, CD3 + , IL-7R / CD127 + , IL-15R + , CCR7low); central memory T cells (e.g., CCR7 + , CD62L + , CD27 + or CCR7hi, CD44 + , CD62Lhi, TCR, CD3 + , IL-7R / CD127 + , IL-15R + );CD62L + Effector T cells; early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L- ) (TemE and TemL, respectively) containing CD8 + Effector memory T cells (TEM); CD127 ( + )CD25(low / -) effector T cells; CD127( - )CD25( - ) Effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca( + )); TH1 effector T cells (e.g., CXCR3 + , CXCR6 + and CCR5 + ; or αβTCR, CD3 + , CD4 + , 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.

[0125] Examples of T cell antigens of interest include, for example, the following (including extracellular domains, if applicable): 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 / ILSRA, CCR5, CCR6, IL-10Rα, CCR7, IL-10Rβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, and lutegrin.α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF 5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common gamma chain / IL-2Rγ, osteopontin, CRACC / SLAMF7, PD-1, C RTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WS X-1, DNAM-1, thymopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγR III / 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, and TSLPR. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary T cell antigens.

[0126] By way of non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety directed against one or more of checkpoint markers expressed on T cells, e.g., PD-1, PD-L1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, Cd27, CD40L, TIM3, and A2aR.

[0127] For example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with a B cell. In some embodiments, the recognition domain directly or indirectly recruits the B cell to, for example, a therapeutic site (e.g., a site containing one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of B cell antigens of interest 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 a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary B cell antigens.

[0128] Further, for example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with natural killer cells. In some embodiments, the recognition domain directly or indirectly recruits natural killer cells, for example, in some embodiments, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of natural killer cell antigens of interest include, for example, TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-epsilon RII, LMIR6 / CD300LE, Fc-γRl / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NK G2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, and ULBP-3. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary NK cell antigens.

[0129] Also, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with macrophages / monocytes. In some embodiments, the recognition domain directly or indirectly recruits macrophages / monocytes, for example, in some embodiments, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of macrophage / monocyte antigens of interest include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin α4 / CD49d, BLAME / SLAMF8, integrin αX / CDllc, CCL6 / C10, integrin β2 / CD18, CD155 / PVR, integrin β3 / CD61, CD31 / PECAM-1, latexin, CD36 / SR-B3, leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, endoglin / CD105, osteoactivin / GPNMB, Fc-γRI / CD64, osteopontin, Fc-γRIIB / CD32b, PD-L2, Fc-γRIIC / CD32c, Siglec-3 / CD33, Fc-γRIIA / CD32a, SIGNR1 / CD209, Fc-γRIII / CD16, SLAM, GM-CSFRα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γRl, TLR4, IFN-γR2, TREM-l, IL-lRII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4, 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-CSFR, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD105, NCAM-L1, Fc-γRI / C These include D64, PSGL-1, Fc-γRIIIICD16, RP105, G-CSFR, L-selectin, GM-CSFRα, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL-6R, TREM-2, CXCR1 / IL-8RA, TREM-3, and TREML1 / TLT-1. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary macrophage / monocyte antigens.

[0130] Also, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with a dendritic cell. In some embodiments, the recognition domain directly or indirectly recruits dendritic cells to, for example, a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of dendritic cell antigens of interest include, for example, CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23p40, 4-Am ino-1, 8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, ClqR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBCCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45 , MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLl, CD2F-10 / SLAMF9, osteoactivin GPNMB, Chern23, PD-L2 , CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209,These include DEP-1 / CD148, SIGNR4, DLEC / CLEC4C, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, and vanilloid R1. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary DC antigens.

[0131] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) on an immune cell selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, myeloid cells, monocytes, eosinophils, or a subset thereof. In some embodiments, the recognition domain localizes the megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, myeloid cells, monocytes, eosinophils, or a subset thereof, for example, in some embodiments, directly or indirectly, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). In some embodiments, the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, mast cells, monocytes, erythrocytes, myeloid cells, myeloid-derived suppressor cells, NKT cells, and NK cells, or derivatives thereof. In some embodiments, the immune cell is a T cell and the targeting moiety targets CD8. In some embodiments, the immune cell is a Treg and the targeting moiety targets CTLA4, hi some embodiments, the immune cell is an NK cell and the targeting moiety is NKp46.

[0132] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with megakaryocytes and / or platelets. Examples of megakaryocyte and / or platelet antigens of interest include, for example, GPIIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary megakaryocyte and / or platelet antigens.

[0133] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with an erythrocyte. Examples of erythrocyte antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb / IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2 (MHC-II), and Rh antigen. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these exemplary erythrocyte antigens.

[0134] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a mast cell. Examples of mast cell antigens of interest include, for example, SCFR / CD117, Fc ε RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMA1, FCERL, FCER2, TPSABl. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these mast cell antigens.

[0135] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a basophil. Examples of basophil antigens of interest include, for example, Fc εRI, CD203c, CD123, CD13, CD107a, CD107b, and CD 164. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these basophil antigens.

[0136] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a neutrophil. Examples of neutrophil antigens of interest include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRIII), CD18 proteins (LFA-1, CR3, and p150, 95), CD45, CD67, and CD177. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these neutrophil antigens.

[0137] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with eosinophils. Examples of eosinophil antigens of interest include, for example, CD35, CD44, and CD69. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these eosinophil antigens.

[0138] In various embodiments, the recognition domain may specifically bind to any suitable target, antigen, receptor, or cell surface marker known to those of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Examples of tissue-specific markers include endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAM1, PROCR, SELE, SELP, TEK, THBD, VCAM1, and VWF; smooth muscle cell surface markers such as ACTA2, MYH1O, MYH11, MYH9, and MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COL1A1, COL1A2, COL3A1, FAP, and PH-4; epithelial cell surface markers such as CD1D, K6IRS2, KRT1O, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCl, and TACSTD1; and CD13, TFNA, and alpha beta 3 (α). V β3), angiogenic markers such as E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these antigens. In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds cells bearing one or more of these antigens.

[0139] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with a tumor cell. In some embodiments, the recognition domain directly or indirectly recruits tumor cells. For example, in some embodiments, the direct or indirect recruitment of tumor cells is to one or more effector cells (e.g., immune cells described herein) that can kill and / or suppress the tumor cells.

[0140] Tumor cells, or cancer cells, refer to uncontrolled proliferation of cells or tissues and / or abnormally increased cell survival and / or abnormally increased inhibition of apoptosis that interferes with the normal functioning of bodily organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. Examples of tumor cells include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney or renal cancer, and tumors of the liver and kidney. cancer), laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lip, tongue, tonsil, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland carcinoma, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulvar cancer, Hodgkin's lymphoma and non-Hodgkin's lymphoma, and lymphomas including B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)), small lymphocytic (SL) NHL, intermediate-grade / follicular These include, but are not limited to, NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (e.g., associated with brain tumors), and cells of Meigs' syndrome.

[0141] Tumor cells or cancer cells also include, but are not limited to, carcinomas, such as various subtypes (including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myeloid, acute lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell), lymphomas and myelomas (including, for example, Hodgkin's and non-Hodgkin's lymphoma, light chain, non-secretory MGUS, and plasmacytoma), and central nervous system cancers (e.g., brain tumors (e.g., gliomas (e.g., astrocytoma, oligodendroglioma, and ependymoma), meningioma, pituitary adenoma, and neuroma), and spinal cord tumors (e.g., meningioma and neurofibroma)).

[0142] Examples of tumor antigens include MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, tumor antigens of the MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, MAGE-A14, MAGE-A15, MAGE-A16, MAGE-A17, MAGE-A18, MAGE-A19, MAGE-A20, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE-A27, MAGE-A28, MAGE-A29, MAGE-A30, MAGE-A31, MAGE-A29, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE-A27, MAGE-A28, MAGE-A29, MAGE-A20, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE- E-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), tumor antigens of the GAGE ​​family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GA GE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, tumor antigens of the Smad family, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1These tumor antigens include, but are not limited to, CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD47, CS1, CD38, ASGPR, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, and BCMA (TNFRSF17). In various embodiments, the targeting moiety of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these tumor antigens. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to HER2. In another embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to PD-L2.

[0143] In some embodiments, the chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention comprise one or more targeting moieties for immune cells selected from T cells, B cells, dendritic cells, neutrophils, myeloid-derived suppressor cells, macrophages, NK cells, or a subset thereof (e.g., modified IL-2), along with one or more targeting moieties for tumor cells. In one embodiment, the chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention comprise a targeting moiety directed against T cells (including but not limited to, effector T cells) along with any of the signaling factors described herein. In one embodiment, the chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, of the present invention comprise a targeting moiety directed against B cells along with any of the signaling factors described herein. In one embodiment, the chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes of the present invention comprise a targeting moiety directed against dendritic cells along with any of the signaling factors described herein. In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety directed to macrophages in combination with any of the signaling factors described herein. In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety directed to NK cells in combination with any of the signaling factors described herein.

[0144] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention may be administered in combination with any of the signaling factors described herein (e.g., modified IL-2), including, for example, 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 / ILSRA, CCR5, CCR6, IL-10Rα, CCR7, IL-10Rβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrin, or other signaling factors.α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, LAG-3, C D43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common gamma chain / 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, EMMPR IN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A , granulysin, TNFRIII / TNFRSF1B, TRAILRl / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, or TSLPR, and have a targeting moiety with directivity towards T cells mediated by targeting.

[0145] By way of non-limiting example, in various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise any of the signal transduction factors described herein along with a targeting moiety directed against one or more of checkpoint markers expressed on T cells, e.g., PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

[0146] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties to PD-1. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties that selectively bind a PD-1 polypeptide. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprise one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a PD-1 polypeptide.

[0147] 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 incorporated herein by reference. In an exemplary embodiment, pembrolizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and / or a light chain comprising the amino acid sequence of SEQ ID NO:8.

[0148] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody, nivolvam (also known as BMS-936558, MDX-1106, ONO-4538, Opdivo), or a fragment thereof. Nivolvam (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449 and WO 2006 / 121168, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, nivolvam or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and / or a light chain comprising the amino acid sequence of SEQ ID NO:10.

[0149] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody pidilizumab (also known as CT-011, hBAT, or hBAT-1), or a fragment thereof. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in U.S. Patent Application Publication No. 2008 / 0025980 and WO 2009 / 101611, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a light chain polypeptide comprising an amino acid sequence selected from SEQ ID NOS:15-18 of U.S. Patent Application Publication No. 2008 / 0025980: SEQ ID NO:15 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:11); SEQ ID NO:16 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:12); SEQ ID NO:17 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:13); SEQ ID NO:18 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:14). variable region; and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 20-24 of U.S. Patent Application Publication No. 2008 / 0025980: SEQ ID NO: 20 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 15); SEQ ID NO: 21 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 16); SEQ ID NO: 22 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 17); SEQ ID NO: 23 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 18) and SEQ ID NO: 24 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 19).

[0150] In one embodiment, the targeting moiety comprises a light chain comprising SEQ ID NO: 18 (SEQ ID NO: 14) of U.S. Patent Application Publication No. 2008 / 0025980 and a heavy chain comprising SEQ ID NO: 22 (SEQ ID NO: 17) of U.S. Patent Application Publication No. 2008 / 0025980.

[0151] In certain embodiments, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0152] In some embodiments, the targeting moiety comprises the pd-l2-Fc fusion protein AMP-224, which is disclosed in WO 2010 / 027827 and WO 2011 / 066342. The entire disclosures of these patents are incorporated herein by reference. In such embodiments, the targeting moiety may comprise a targeting domain comprising SEQ ID NO:4 (SEQ ID NO:20) of WO 2010 / 027827 and / or a B7-DC fusion protein comprising SEQ ID NO:83 (SEQ ID NO:21) of WO 2010 / 027827.

[0153] In one embodiment, the targeting moiety comprises a PD-L1-Fc fusion protein and / or a PD-1-Fc fusion protein, as described in WO2019 / 191519.

[0154] In some embodiments, the targeting moiety comprises the peptide AUNP12 or any other peptide disclosed in U.S. Patent Application Publication No. 2011 / 0318373 or U.S. Patent No. 8,907,053. For example, the targeting moiety can comprise AUNP12 (i.e., Compound 8 or SEQ ID NO: 49 of U.S. Patent Application Publication No. 2011 / 0318373), which has the following sequence:

[0155] [ka]

[0156] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E3 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:24.

[0157] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E8, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:26.

[0158] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1H3 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1H3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:27; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:28.

[0159] In certain embodiments, the targeting moiety comprises a VHH directed against PD-1, e.g., as disclosed in U.S. Patent No. 8,907,065 and WO 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In exemplary embodiments, the VHH directed against PD-1 comprises SEQ ID NOS: 347-351 of U.S. Patent No. 8,907,065 (SEQ ID NO: 347 of U.S. Patent No. 8,907,065 (SEQ ID NO: 29); SEQ ID NO: 348 of U.S. Patent No. 8,907,065 (SEQ ID NO: 30); SEQ ID NO: 349 of U.S. Patent No. 8,907,065 (SEQ ID NO: 31); SEQ ID NO: 350 of U.S. Patent No. 8,907,065 (SEQ ID NO: 32); or SEQ ID NO: 351 of U.S. Patent No. 8,907,065 (SEQ ID NO: 33)).

[0160] In some embodiments, the targeting moiety comprises any one of the anti-PD-1 antibodies or fragments thereof, as disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS:25-29 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:25 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:34); SEQ ID NO:26 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:35); SEQ ID NO:27 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:36); SEQ ID NO:28 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:37); U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:38); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 30 to 33 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 30 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 39); SEQ ID NO: 31 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 40); SEQ ID NO: 32 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 41); SEQ ID NO: 33 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 42)).

[0161] In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises one or more antibodies, or antibody fragments thereof, directed against PD-1 selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene Ltd.).

[0162] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties to PD-L1. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties that selectively bind a PD-L1 polypeptide. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprise one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a PD-L1 polypeptide.

[0163] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab), or a fragment thereof. MEDI4736 is selective for PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80 receptors. MEDI4736 and antigen-binding fragments thereof for use in the methods provided herein comprise a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. The sequence of MEDI4736 is disclosed in WO 2016 / 06272, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43; and / or a light chain comprising the amino acid sequence of SEQ ID NO:44.

[0164] In exemplary embodiments, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 (SEQ ID NO:45) of WO 2016 / 06272; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 (SEQ ID NO:46) of WO 2016 / 06272.

[0165] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (also known as MPDL3280A, RG7446), or a fragment thereof. In an exemplary embodiment, atezolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:47; and / or a light chain comprising the amino acid sequence of SEQ ID NO:48.

[0166] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody avelumab (also known as MSB0010718C), or a fragment thereof. In an exemplary embodiment, atezolizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:49; and / or a light chain comprising the amino acid sequence of SEQ ID NO:50.

[0167] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105), or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, BMS-936559 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:51); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:52).

[0168] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3G10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 3G10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:53); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:54).

[0169] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10A5, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 10A5, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:55); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:56).

[0170] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 5F8, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 5F8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:57); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:58).

[0171] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10H10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 10H10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:59); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:60).

[0172] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1B12, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 1B12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:61); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:62).

[0173] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 7H1, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 7H1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:63); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:64).

[0174] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 11E6, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 11E6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:65); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:66).

[0175] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 12B7, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 12B7 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:67); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:68).

[0176] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 13G4, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 13G4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:69); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:70).

[0177] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1E12, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E12, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:71); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:72).

[0178] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1F4, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. 1F4, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:73); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:74).

[0179] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2G11, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 2G11, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:75); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:76).

[0180] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3B6, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 3B6, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:77); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:78).

[0181] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3D10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738 and WO 2012 / 145493. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3D10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:79); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:80).

[0182] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in US Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS: 34-38 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 34 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 81); SEQ ID NO: 35 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 82); SEQ ID NO: 36 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 83); SEQ ID NO: 37 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 84); 358 (SEQ ID NO: 38 (SEQ ID NO: 85)); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 39 to 42 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 39 (SEQ ID NO: 86) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 40 (SEQ ID NO: 87) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 41 (SEQ ID NO: 88) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 42 (SEQ ID NO: 89) of U.S. Patent Application Publication No. 2011 / 0271358).

[0183] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.7A4, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 (SEQ ID NO:90) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:7 (SEQ ID NO:91) of WO 2011 / 066389.

[0184] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.9D10, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.9D10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (SEQ ID NO: 92) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (SEQ ID NO: 93) of WO 2011 / 066389.

[0185] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.14H9, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:22 (SEQ ID NO:94) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 (SEQ ID NO:95) of WO 2011 / 066389.

[0186] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.20A8, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.20A8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:32 (SEQ ID NO:96) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:37 (SEQ ID NO:97) of WO 2011 / 066389.

[0187] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.15G8, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3.15G8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:42 (SEQ ID NO:98) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:47 (SEQ ID NO:99) of WO 2011 / 066389.

[0188] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.18G1, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3.18G1, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:52 (SEQ ID NO:100) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:57 (SEQ ID NO:101) of WO 2011 / 066389.

[0189] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4OPT, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.7A4OPT, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:62 (SEQ ID NO:102) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:67 (SEQ ID NO:103) of WO 2011 / 066389.

[0190] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9OPT, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.14H9OPT, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:72 (SEQ ID NO:104) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:77 (SEQ ID NO:105) of WO 2011 / 066389.

[0191] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 061142, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein are selected from the group consisting of SEQ ID NOs: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of WO 2016 / 061142 (SEQ ID NO: 18 of WO 2016 / 061142 (SEQ ID NO: 106); SEQ ID NO: 30 of WO 2016 / 061142 (SEQ ID NO: 107); SEQ ID NO: 38 of WO 2016 / 061142 (SEQ ID NO: 108); SEQ ID NO: 46 of WO 2016 / 061142 (SEQ ID NO: 109); SEQ ID NO: 50 of WO 2016 / 061142 (SEQ ID NO: 110); SEQ ID NO: 54 of WO 2016 / 061142 (SEQ ID NO: 111); SEQ ID NO: 62 of WO 2016 / 061142 (SEQ ID NO: 112); SEQ ID NO: 70 of WO 2016 / 061142 (SEQ ID NO: 113) and SEQ ID NO: 78 of WO 2016 / 061142 (SEQ ID NO: 114). and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO 2016 / 061142 (SEQ ID NO: 115); SEQ ID NO: 26 of WO 2016 / 061142 (SEQ ID NO: 116); SEQ ID NO: 34 of WO 2016 / 061142 (SEQ ID NO: 117); SEQ ID NO: 4 of WO 2016 / 061142 (SEQ ID NO: 118); 2 (SEQ ID NO:118); SEQ ID NO:58 of WO 2016 / 061142 (SEQ ID NO:119); SEQ ID NO:66 of WO 2016 / 061142 (SEQ ID NO:120); SEQ ID NO:74 of WO 2016 / 061142 (SEQ ID NO:121); SEQ ID NO:82 of WO 2016 / 061142 (SEQ ID NO:122) and SEQ ID NO:86 of WO 2016 / 061142 (SEQ ID NO:123).

[0192] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 022630, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein are selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WO 2016 / 022630 (SEQ ID NO: 2 of WO 2016 / 022630 (SEQ ID NO: 124); SEQ ID NO: 6 of WO 2016 / 022630 (SEQ ID NO: 125); SEQ ID NO: 10 of WO 2016 / 022630 (SEQ ID NO: 126); SEQ ID NO: 11 of WO 2016 / 022630 (SEQ ID NO: 127); SEQ ID NO: 12 of WO 2016 / 022630 (SEQ ID NO: 128); SEQ ID NO: 13 of WO 2016 / 022630 (SEQ ID NO: 139); SEQ ID NO: 14 of WO 2016 / 022630 (SEQ ID NO: 149); SEQ ID NO: 15 of WO 2016 / 022630 (SEQ ID NO: 159); SEQ ID NO: 16 of WO 2016 / 022630 (SEQ ID NO: 169); SEQ ID NO: 17 of WO 2016 / 022630 (SEQ ID NO: 169)). SEQ ID NO: 14 (SEQ ID NO: 127); SEQ ID NO: 18 of WO 2016 / 022630 (SEQ ID NO: 128); SEQ ID NO: 22 of WO 2016 / 022630 (SEQ ID NO: 129); SEQ ID NO: 26 of WO 2016 / 022630 (SEQ ID NO: 130); SEQ ID NO: 30 of WO 2016 / 022630 (SEQ ID NO: 131); SEQ ID NO: 34 of WO 2016 / 022630 (SEQ ID NO: 132); SEQ ID NO: 38 of WO 2016 / 022630 (SEQ ID NO: 133); and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 42 of WO 2016 / 022630 (SEQ ID NO: 134); and SEQ ID NO: 46 of WO 2016 / 022630 (SEQ ID NO: 135)); and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 of WO 2016 / 022630 (SEQ ID NO: 4 of WO 2016 / 022630 (SEQ ID NO: 136); SEQ ID NO: 8 of WO 2016 / 022630 (SEQ ID NO: 137); SEQ ID NO: 12 from WO 2016 / 022630 (SEQ ID NO: 138); SEQ ID NO: 16 from WO 2016 / 022630 (SEQ ID NO: 139); SEQ ID NO: 20 from WO 2016 / 022630 (SEQ ID NO: 140); SEQ ID NO: 24 from WO 2016 / 022630 (SEQ ID NO: 141); SEQ ID NO: 28 from WO 2016 / 022630 (SEQ ID NO: 142); SEQ ID NO: 32 from WO 2016 / 022630 (SEQ ID NO: 143); SEQ ID NO: 36 from WO 2016 / 022630 (SEQ ID NO: 144);a light chain comprising an amino acid sequence selected from SEQ ID NO: 40 of WO 2016 / 022630 (SEQ ID NO: 145); SEQ ID NO: 44 of WO 2016 / 022630 (SEQ ID NO: 146); and SEQ ID NO: 48 of WO 2016 / 022630 (SEQ ID NO: 147);

[0193] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2015 / 112900, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 38, 50, 82, and 86 of WO 2015 / 112900 (SEQ ID NO: 38 of WO 2015 / 112900 (SEQ ID NO: 148); SEQ ID NO: 50 of WO 2015 / 112900 (SEQ ID NO: 149); SEQ ID NO: 82 of WO 2015 / 112900 (SEQ ID NO: 150); and SEQ ID NO: 86 of WO 2015 / 112900 (SEQ ID NO: 151)); and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of WO 2015 / 112900 (SEQ ID NO: 152). and SEQ ID NO: 78 of WO 2015 / 112900 (SEQ ID NO: 160).

[0194] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2010 / 077634 and U.S. Patent No. 8,217,149. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, an anti-PD-L1 or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:20 (SEQ ID NO:161) of WO 2010 / 077634; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:21 (SEQ ID NO:162) of WO 2010 / 077634.

[0195] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies obtainable from the hybridomas available under CNCM Accession Nos. CNCM I-4122, CNCM I-4080, and CNCM I-4081, as disclosed in U.S. Patent Application Publication No. 20120039906, the entire disclosures of which are incorporated herein by reference.

[0196] In some embodiments, the targeting moiety comprises a VHH directed against a PD-L1 antibody, e.g., as disclosed in U.S. Patent No. 8,907,065 and WO 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In exemplary embodiments, the VHH directed against PD-L1 comprises SEQ ID NOS: 394-399 of U.S. Patent No. 8,907,065 (SEQ ID NO: 394 of U.S. Patent No. 8,907,065 (SEQ ID NO: 163); SEQ ID NO: 395 of U.S. Patent No. 8,907,065 (SEQ ID NO: 164); SEQ ID NO: 396 of U.S. Patent No. 8,907,065 (SEQ ID NO: 165); SEQ ID NO: 397 of U.S. Patent No. 8,907,065 (SEQ ID NO: 166); SEQ ID NO: 398 of U.S. Patent No. 8,907,065 (SEQ ID NO: 167); SEQ ID NO: 399 of U.S. Patent No. 8,907,065 (SEQ ID NO: 168)).

[0197] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties to PD-L2. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties that selectively bind a PD-L2 polypeptide. In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprise one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a PD-L2 polypeptide.

[0198] In one embodiment, the targeting moiety comprises a VHH directed against PD-L2, for example, as disclosed in U.S. Patent No. 8,907,065 and WO 2008 / 071447, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, the VHH against PD-L2 comprises SEQ ID NOs:449-455 of U.S. Patent No. 8,907,065 (SEQ ID NO:449 of U.S. Patent No. 8,907,065 (SEQ ID NO:169); SEQ ID NO:450 of U.S. Patent No. 8,907,065 (SEQ ID NO:170); SEQ ID NO:451 of U.S. Patent No. 8,907,065 (SEQ ID NO:171); SEQ ID NO:452 of U.S. Patent No. 8,907,065 (SEQ ID NO:172); SEQ ID NO:453 of U.S. Patent No. 8,907,065 (SEQ ID NO:173); SEQ ID NO:454 of U.S. Patent No. 8,907,065 (SEQ ID NO:174); and SEQ ID NO:455 of U.S. Patent No. 8,907,065 (SEQ ID NO:175)).

[0199] In some embodiments, the targeting moiety comprises any one of the anti-PD-L2 antibodies disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS: 43-47 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 176); SEQ ID NO: 44 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 177); SEQ ID NO: 45 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 178); SEQ ID NO: 46 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 179); U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 179); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 48 to 51 in U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 48 in U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 181); SEQ ID NO: 49 in U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 182); SEQ ID NO: 50 in U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 183); and SEQ ID NO: 51 in U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 184)).

[0200] In various embodiments, the targeting moieties of the invention align with any of the sequences disclosed herein by at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, At least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or may comprise sequences targeting PD-1, PD-L1, and / or PD-L2 that are about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% sequence identity).

[0201] In various embodiments, targeting moieties of the invention can include any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences that target PD-1, PD-L1, and / or PD-L2 as disclosed herein.

[0202] Additional antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind or target PD-1, PD-L1, and / or PD-L2 are described in WO 2011 / 066389, U.S. Patent Application Publication No. 2008 / 0025980, U.S. Patent Application Publication No. 2013 / 0034559, U.S. Patent No. 8,779,108 ... No. 8,779,10 014 / 0356353, U.S. Patent No. 8,609,089, U.S. Patent Application Publication No. 2010 / 028330, U.S. Patent Application Publication No. 2012 / 0114649, WO 2010 / 027827, WO 2011 / 066342, U.S. Patent No. 8,907,065, WO 2016 / 062722, WO 2009 / 101611, WO 2010 / 0278 27, WO 2011 / 066342, WO 2007 / 005874, WO 2001 / 014556, U.S. Patent Application Publication No. 2011 / 0271358, WO 2010 / 036959, WO 2010 / 077634, U.S. Patent No. 8,217,149, U.S. Patent Application Publication No. 2012 / 0039906, WO 2012 / 145493, U.S. and WO 2015 / 112900, the entire disclosures of which are incorporated herein by reference.

[0203] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against T cells, e.g., mediated by targeting to CD8, in combination with any of the signaling factors described herein (e.g., modified IL-2). In certain embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against CD8 on T cells.

[0204] In one embodiment, the targeting moiety directed against CD8 has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFEDYAIGWFRQAPGKGREGVACIRIFDRHTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTATYYCAAGSFFGCTRPEGDMDYFGQGTLVQVQSA (SEQ ID NO: 454) and VHHs comprising:

[0205] In one embodiment, the targeting moiety directed against CD8 has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYVIGWFRQAPGKGREGVACIRIFDRHTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTATYYCAAGSFFGCTRPEGDMDYFGQGTLVQVQSA (SEQ ID NO: 455) and VHHs comprising:

[0206] In one embodiment, the targeting moiety directed against CD8 has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKGREGVACIRIFDRHTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTATYYCAAGSFWGCTRPEGDMDYFGQGTLVQVQSA (SEQ ID NO: 456). and VHHs comprising:

[0207] In various embodiments, the targeting moieties of the invention have a sequence similar to or similar to any of the sequences disclosed herein, such as at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or 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 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% sequence identity), to a sequence that targets CD8.

[0208] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against T cells, e.g., mediated by targeting to CD4, in combination with any of the signaling factors described herein (e.g., modified IL-2). In certain embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against CD4 on T cells.

[0209] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against T cells, e.g., mediated by targeting to CD3, CXCR3, CCR4, CCR9, CD70, CD103, or one or more immune checkpoint markers, along with any of the signal transduction factors described herein (e.g., modified IL-2). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against CD3 on T cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0210] In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties for CD3 expressed on T cells. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises one or more targeting moieties that selectively bind a CD3 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a CD3 polypeptide.

[0211] 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. Pat. No. 4,361,549 and Wilde et al. (1996) 51:865-894, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, muromonab-CD3, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO: 185); and / or a light chain comprising the amino acid sequence of (SEQ ID NO: 186).

[0212] In some embodiments, the targeting moiety comprises the anti-CD3 antibody otelixizumab, or a fragment thereof. Otelixizumab is disclosed in U.S. Patent Application Publication No. 20160000916 and Chatenoud et al. (2012) 9:372-381, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, otelixizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 187; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 188.

[0213] In one embodiment, the targeting moiety comprises the anti-CD3 antibody teplizumab (also known as MGA031 and hOKT3γ1(Ala-Ala)), or a fragment thereof. Teplizumab is disclosed in Chatenoud et al. (2012) 9:372-381, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, teplizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 189; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 190.

[0214] In one embodiment, the targeting moiety comprises the anti-CD3 antibody visilizumab (also known as Nuvion®; HuM291), or a fragment thereof. Visilizumab is disclosed in U.S. Pat. No. 5,834,597 and WO 2004052397 and Cole et al., Transplantation (1999) 68:563-571, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, visilizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 191; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 192.

[0215] In some embodiments, the targeting moiety comprises the anti-CD3 antibody foralarumab (also known as NI-0401), or a fragment thereof. In various embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent Application Publication No. 2014 / 0193399, U.S. Patent No. 7,728,114, U.S. Patent Application Publication No. 2010 / 0183554, and U.S. Patent No. 8,551,478, the entire disclosures of which are incorporated herein by reference.

[0216] In exemplary embodiments, an anti-CD3 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 2 and 6 of U.S. Patent No. 7,728,114 (SEQ ID NO: 2 of U.S. Patent No. 7,728,114 (SEQ ID NO: 193) and SEQ ID NO: 6 of U.S. Patent No. 7,728,114 (SEQ ID NO: 194)); and / or a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 4 and 8 of U.S. Patent No. 7,728,114 (SEQ ID NO: 4 of U.S. Patent No. 7,728,114 (SEQ ID NO: 195) and SEQ ID NO: 8 of U.S. Patent No. 7,728,114 (SEQ ID NO: 196)).

[0217] In one embodiment, the targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 of U.S. Patent Application Publication No. 7,728,114; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4 of U.S. Patent Application Publication No. 7,728,114. In one embodiment, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent Application Publication No. 2016 / 0168247, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOs:6-9 of U.S. Patent Application Publication No. 2016 / 0168247 (SEQ ID NO:6 (SEQ ID NO:197) of U.S. Patent Application Publication No. 2016 / 0168247; SEQ ID NO:7 (SEQ ID NO:198) of U.S. Patent Application Publication No. 2016 / 0168247; SEQ ID NO:8 (SEQ ID NO:199) of U.S. Patent Application Publication No. 2016 / 0168247. No. 7 (SEQ ID NO: 9 (SEQ ID NO: 200)); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 10-12 of U.S. Patent Application Publication No. 2016 / 0168247 (SEQ ID NO: 10 (SEQ ID NO: 201) of U.S. Patent Application Publication No. 2016 / 0168247; SEQ ID NO: 11 (SEQ ID NO: 202) of U.S. Patent Application Publication No. 2016 / 0168247; SEQ ID NO: 12 (SEQ ID NO: 203) of U.S. Patent Application Publication No. 2016 / 0168247).

[0218] In some embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent Application Publication No. 2015 / 0175699, the entire contents of which are incorporated herein by reference. In exemplary embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO:9 (SEQ ID NO:204) of U.S. Patent Application Publication No. 2015 / 0175699; and / or a light chain comprising an amino acid sequence selected from SEQ ID NO:10 (SEQ ID NO:205) of U.S. Patent Application Publication No. 2015 / 0175699.

[0219] In one embodiment, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent No. 8,784,821, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, and 114 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 2 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 206); SEQ ID NO: 18 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 207); SEQ ID NO: 34 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 208); SEQ ID NO: 50 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 209); SEQ ID NO: 66 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 210); SEQ ID NO: 82 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 211); SEQ ID NO: 98 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 212); and SEQ ID NO: 114 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 213)). and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106 and 122 of U.S. Patent No. 8,784,821 (SEQ ID NO: 10 of U.S. Patent No. 8,784,821 (SEQ ID NO: 214); SEQ ID NO: 26 of U.S. Patent No. 8,784,821 (SEQ ID NO: 215); SEQ ID NO: 42 of U.S. Patent No. 8,784,821 (SEQ ID NO: 216); SEQ ID NO: 58 of U.S. Patent No. 8,784,821 (SEQ ID NO: 217); SEQ ID NO: 74 of U.S. Patent No. 8,784,821 (SEQ ID NO: 218); SEQ ID NO: 90 of U.S. Patent No. 8,784,821 (SEQ ID NO: 219); SEQ ID NO: 106 of U.S. Patent No. 8,784,821 (SEQ ID NO: 220); and SEQ ID NO: 122 of U.S. Patent No. 8,784,821 (SEQ ID NO: 221)).

[0220] In some embodiments, the targeting moiety comprises any one of the anti-CD3 binding constructs disclosed in U.S. Patent Application Publication No. 2015 / 0118252, the entire contents of which are incorporated herein by reference. In exemplary embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 6 and 86 of U.S. Patent Application Publication No. 2015 / 0118252 (SEQ ID NO: 6 of U.S. Patent Application Publication No. 2015 / 0118252 (SEQ ID NO: 222) and SEQ ID NO: 86 of U.S. Patent Application Publication No. 2015 / 0118252 (SEQ ID NO: 223)); and / or a light chain comprising an amino acid sequence selected from SEQ ID NO: 3 of U.S. Patent Application Publication No. 2015 / 0175699 (SEQ ID NO: 3 of U.S. Patent Application Publication No. 2015 / 0118252 (SEQ ID NO: 224)).

[0221] In certain embodiments, the targeting moiety comprises any one of the anti-CD3 binding proteins disclosed in U.S. Patent Application Publication No. 2016 / 0039934, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from SEQ ID NOS: 6-9 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 6 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 225); SEQ ID NO: 7 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 226); SEQ ID NO: 8 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 227); and SEQ ID NO: 9 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 228)). ); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 1 to 4 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 1 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 229); SEQ ID NO: 2 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 230); SEQ ID NO: 3 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 231); and SEQ ID NO: 4 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 232)).

[0222] In various embodiments, the targeting moieties of the invention align with any of the sequences disclosed herein by at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, 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 or about 98%, or about 99%, or about 100% sequence identity).

[0223] In various embodiments, the targeting moieties of the present invention may comprise any combination of CD3-targeting heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences disclosed herein. In various embodiments, a targeting moiety of the invention may comprise any of the heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences of 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 incorporated herein by reference.

[0224] Additional antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind or target CD3 are disclosed in U.S. Patent Application Publication No. 2016 / 0000916, U.S. Patent Nos. 4,361,549, 5,834,597, 6,491,916, 6,406,696, 6,143,297, 6,750,325, and WO 2004 / 052397, the entire disclosures of which are incorporated herein by reference.

[0225] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises any signal transduction factor described herein (e.g., modified 1L-2) along with a targeting moiety directed against T cells, e.g., mediated by targeting to PD-1.

[0226] By way of non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety with directivity to B cells mediated by targeting to, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, or CDw150, in combination with any of the signaling factors described herein (e.g., modified IL-2). In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety directed against CD20.

[0227] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety directed against B cells, e.g., mediated by targeting to CD19, CD20, or CD70, in combination with any of the signaling factors described herein (e.g., modified IL-2).

[0228] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against B cells, e.g., mediated by targeting to CD20, in combination with any of the signal transduction factors described herein (e.g., modified IL-2). In certain embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against CD20 on B cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells. For example, in some embodiments, the CD20 targeting moiety has the sequence: QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS (SEQ ID NO: 288) It is a recombinant heavy chain-only antibody (VHH) having the following structure:

[0229] By way of non-limiting example, in various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention may be combined with any of the signal transduction factors described herein (e.g., modified IL-2), e.g., 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-epsilon RII, LMIR6 / CD300LE, Fc-γRl / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Nectin-2 / CD112, Fc-γRIII / CD16 , NKG2A, FcRH1 / IRTA5, NKG2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD8 and a targeting moiety with directivity for NK cells mediated by targeting to 5j, 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.

[0230] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety with directivity to NK cells, e.g., mediated by targeting to Kir1α, DNAM-1, or CD64, in combination with any of the signal transduction factors described herein (e.g., modified IL-2).

[0231] In one embodiment, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against NK cells, e.g., mediated by targeting to TIGIT or KIR1, along with any of the signal transduction factors described herein (e.g., modified IL-2). In certain embodiments, a chimeric protein complex, such as a chimeric protein or Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against TIGIT on NK cells and a second targeting moiety against PD-L1 or PD-L2 on tumor cells.

[0232] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention may be used in combination with any of the signaling factors described herein (e.g., modified IL-2), e.g., CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, and / or CLEC9A. , SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23p40, 4-Ami no-1, 8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4 Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, ClqR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBCCR7, LMIR6 / CD300LE, CD40 / TNFRS F5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLl, CD2F-10 / SLAMF9, OsteoactivinGPNMB, Chern23, PD-L2, CLEC-1, RP105, CLEC-2, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, D and a targeting moiety with directivity to dendritic cells mediated by targeting to EP-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 vanilloid R1.

[0233] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against dendritic cells, e.g., mediated by targeting to CLEC-9A, DC-SIGN, CD64, CLEC-4A, or DEC205, along with any of the signal transduction factors described herein (e.g., modified IL-2). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against Clec9A on dendritic cells and a second targeting moiety against PD-L1 or PD-L2 on tumor cells.

[0234] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against dendritic cells, e.g., mediated by targeting to Clec9A, in combination with any of the signal transduction factors described herein (e.g., modified IL-2). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against Clec9A on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0235] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against dendritic cells, e.g., mediated by targeting to XCR1, in combination with any of the signal transduction factors described herein (e.g., modified IL-2). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against XCR1 on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0236] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against dendritic cells, e.g., mediated by targeting to RANK, in combination with any of the signal transduction factors described herein (e.g., modified IL-2). In certain embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against RANK on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0237] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention can be used in combination with any of the signaling factors described herein (e.g., modified IL-2), 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, C D31 / PECAM-1, Latexin, CD36 / SR-B3, Leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL 2, Endoglin / CD105, osteoactivin / GPNMB, Fc-γRI / CD64, osteopontin, Fc-γRIIB / CD32b, PD-L2, Fc-γRIIC / CD32c, Siglec-3 / CD33, Fc-γRIIA / CD32a, SIGNR1 / CD209, Fc-γRIII / CD16, SLAM, GM-CSFRα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γRl, TLR4, IFN-γR2, TREM-l, IL-lRII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4, IL-10Rα, ALCAM, IL-10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, ClqR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / CDllb, CCR8, integrin αX / CDllc, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61,CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, Leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / CD300L F, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSFR, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, M and a targeting moiety with directivity for monocytes / macrophages mediated by targeting to MR, endoglin / CD105, NCAM-L1, Fc-γRI / CD64, PSGL-1, Fc-γRIIIICD16, RP105, G-CSFR, L-selectin, GM-CSFRα, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, -6R, TREM-2, CXCR1 / IL-8RA, TREM-3, or TREML1 / TLT-1.

[0238] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety with directivity to monocytes / macrophages, e.g., mediated by targeting to B7-H1, CD31 / PECAM-1, CD163, CCR2, or the macrophage mannose receptor CD206, in combination with any of the signaling factors described herein (e.g., modified IL-2).

[0239] In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against monocytes / macrophages, e.g., mediated by targeting to SIRP1a, in combination with any of the signal transduction factors described herein (e.g., modified IL-2). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against SIRP1a on macrophages and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0240] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties directed against checkpoint markers, e.g., one or more of 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 addition to any signaling factor described herein (e.g., modified IL-2).

[0241] In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises one or more targeting moieties directed against the same or different immune cells. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises one or more targeting moieties directed against an immune cell selected from T cells, B cells, dendritic cells, macrophages, NK cells, or a subset thereof, along with any signal transduction factor described herein (e.g., modified IL-2).

[0242] In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises a targeting moiety directed against tumor cells, in such an embodiment, the targeting moiety can bind to any of the tumor antigens described herein.

[0243] In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention are expressed in adipocytes (e.g., white adipocytes, brown adipocytes), liver lipid cells, liver cells, kidney cells (e.g., kidney parietal cells, kidney salivary gland, mammary gland, etc.), duct cells (e.g., seminal vesicles, prostate), intestinal brush border cells (with microvilli), exocrine gland striate duct cells, gallbladder epithelial cells, small efferent tract non-ciliated cells, epididymal principal cells, epididymal basal cells, endothelial cells, ameloblast epithelial cells (tooth enamel secretion), Crescentic epithelial cells of the vestibular system of the ear (secreting proteoglycans), interdental epithelial cells of the organ of Corti (secreting the tectorial membrane that covers hair cells), loose connective tissue fibroblasts, corneal fibroblasts (keratocytes), tendon fibroblasts, bone marrow reticular fibroblasts, non-epithelial fibroblasts, pericytes, nucleus pulposus cells of the intervertebral disc, cementoblasts / cementocytes (secreting root bone-like Ivan cells), odontoblasts / odontocytes (secreting tooth dentin), hyaline chondrocytes, fibrochondrocytes , elastic chondrocytes, osteoblasts / osteocytes, bone precursor cells (osteoblast stem cells), vitreous cells of the vitreous body of the eye, stellate cells of the perilymphatic space of the ear, hepatic stellate cells (Ito cells), pancreatic stellate cells, skeletal muscle cells, satellite cells, cardiac muscle cells, smooth muscle cells, myoepithelial cells of the iris, myoepithelial cells of exocrine glands, exocrine secretory epithelial cells (e.g., salivary gland cells, mammary gland cells, lacrimal gland cells, sweat gland cells, sebaceous gland cells, prostate cells, gastric gland cells, pancreatic acinar cells, lung cells), hormone-secreting cells (e.g., pituitary cells, neurosecretory cells, intestinal and airway cells, thyroid gland cells, The targeting moiety comprises one or more targeting moieties having a recognition domain that binds to a target of interest (e.g., antigen, receptor), including those found on one or more cells selected from: epithelial cells, parathyroid cells, adrenal cells, Leydig cells of the testes, pancreatic islet cells, keratinizing epithelial cells, moist stratified barrier epithelial cells, neural cells (e.g., sensory transducer cells, autonomic neuron cells, sensory organ and peripheral neural support cells, and central nervous system neurons, and glial cells such as interneurons, principal cells, astrocytes, oligodendrocytes, and ependymal cells).

[0244] Targeted Moiety Format In some embodiments, the targeting moiety of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention is a protein-based agent capable of specific binding, such as an antibody or a derivative thereof. In certain embodiments, the targeting moiety comprises an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain contains one variable region (V L ) and one constant region (C L ) The variable regions determine the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity-determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs are termed CDR1, CDR2, and CDR3 and contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0245] In some embodiments, the targeting moiety comprises an antibody derivative or format. In some embodiments, the targeting moiety of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention is a single domain antibody, a recombinant heavy chain antibody (VHH), a single chain antibody (scFv), a shark heavy chain antibody (VNAR), a microprotein (cysteine ​​knot protein, knottin), a darpin; a tetranectin; an affibody; a transbody; anticalin; an adnectin; an affilin; a microbody; a peptide aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; a shrimpbody; a phylomer; an armadillo repeat protein; a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troibody, a pepbody, a vaccibody, a unibody; an affimer, a duobody, an Fv, a Fab, a Fab', a F(ab'), a peptidomimetic molecule, or a synthetic molecule. These include U.S. Pat. No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Pat. No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Pat. No. 7,250,297, U.S. Pat. No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Pat. No. 7,838,629, U.S. Pat. No. 7,186,524, U.S. Pat. Nos. 6,004,746, 5,475,096, 2004 / 146938, 2004 / 157209, 6,994,982, 6,794,144, 2010 / 239633, 7,803,907, 2010 / 119446, and / or 7,166,697. See also Storz MAbs. 2011 May-Jun;3(3):310-317.

[0246] In one embodiment, the targeting moiety comprises a single domain antibody, such as a VHH derived from an organism that produces VHH antibodies, e.g., camelids, sharks, or an engineered VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional antibodies derived from camelids that lack light chains. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). VHHs are commercially available under the registered trademarks NANOBODY or NANOBODIES.

[0247] In certain embodiments, the targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized or camelized VHH.

[0248] In some embodiments, the VHH is a fully human VHH. H In some embodiments, the fully human VH domain, e.g., a humabody, is monovalent, bivalent, or trivalent. In some embodiments, the fully human VH domain, e.g., a humabody, is monospecific or multispecific, such as monospecific, bispecific, or trispecific. Exemplary fully human VH domains include: H Domains such as the human body are described, for example, in WO 2016 / 113555 and WO 2016 / 113557, the entire disclosures of which are incorporated herein by reference.

[0249] In various embodiments, the targeting moiety of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention is a protein-based substance capable of specifically binding to a cellular receptor, such as a natural ligand for the cellular receptor. In various embodiments, the cellular receptor can be found on one or more immune cells, including, but not limited to, T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or a subset thereof. In some embodiments, the cellular receptor is found on megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof.

[0250] In some embodiments, the targeting moiety is a natural ligand, such as a chemokine. Examples of chemokines that can be included in the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention include, but are not limited to, CCL1, CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL29, CCL30, CCL31, CCL32, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39, CCL40, CCL41, CCL42, CCL43, CCL44, CCL45, CCL46, CCL47, CCL48, CCL49, CCL50, CCL51, CCL52, CCL53, CCL54, CCL55, CCL56, CCL57, CCL58, CCL59, CCL60, CCL61, CCL62, CCL63, CCL64, CCL65, CCL66, CCL67, CCL68, CCL69, CCL70, CCL71, CCL72, CCL73, CCL74, CCL75, CCL76, CCL77, CCL78, CCL79, CCL80, CCL81, CCL82, CCL83, CCL84, CCL85, CCL86, CCL87, CCL88, CCL89, CCL90, CCL91, Examples of targeting moieties include CCL22, CCL23, CCL24, CLL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, HCC-4, and LDGF-PBP. In an exemplary embodiment, the targeting moiety can be XCL1, a chemokine that recognizes and binds to the dendritic cell receptor XCR1. In another exemplary embodiment, the targeting moiety is CCL1, a chemokine that recognizes and binds to CCR8. In another exemplary embodiment, the targeting moiety is CCL2, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL3, a chemokine that recognizes and binds to CCR1, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL4, a chemokine that recognizes and binds to CCR1 or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL5, a chemokine that recognizes and binds to CCR1 or CCR3 or CCR4 or CCR5. In another exemplary embodiment, the targeting moiety is CCL6, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL7, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL8, a chemokine that recognizes and binds to CCR1 or CCR2 or CCR2B or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL9, a chemokine that recognizes and binds to CCR1.In another exemplary embodiment, the targeting moiety is CCL10, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL11, a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL13, a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL14, a chemokine that recognizes and binds to CCR1 or CCR9. In another exemplary embodiment, the targeting moiety is CCL15, a chemokine that recognizes and binds to CCR1 or CCR3. In another exemplary embodiment, the targeting moiety is CCL16, a chemokine that recognizes and binds to CCR1, CCR2, CCR5, or CCR8. In another exemplary embodiment, the targeting moiety is CCL17, a chemokine that recognizes and binds to CCR4. In another exemplary embodiment, the targeting moiety is CCL19, a chemokine that recognizes and binds to CCR7. In another exemplary embodiment, the targeting moiety is CCL20, a chemokine that recognizes and binds to CCR6. In another exemplary embodiment, the targeting moiety is CCL21, a chemokine that recognizes and binds to CCR7. In another exemplary embodiment, the targeting moiety is CCL22, a chemokine that recognizes and binds to CCR4. In another exemplary embodiment, the targeting moiety is CCL23, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL24, a chemokine that recognizes and binds to CCR3. In another exemplary embodiment, the targeting moiety is CCL25, a chemokine that recognizes and binds to CCR9. In another exemplary embodiment, the targeting moiety is CCL26, a chemokine that recognizes and binds to CCR3. In another exemplary embodiment, the targeting moiety is CCL27, a chemokine that recognizes and binds to CCR10. In another exemplary embodiment, the targeting moiety is CCL28, a chemokine that recognizes and binds to CCR3 or CCR10. In another exemplary embodiment, the targeting moiety is CXCL1, a chemokine that recognizes and binds to CXCR1 or CXCR2.In another exemplary embodiment, the targeting moiety is CXCL2, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL3, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL4, a chemokine that recognizes and binds to CXCR3B. In another exemplary embodiment, the targeting moiety is CXCL5, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL6, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL8, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL9, a chemokine that recognizes and binds to CXCR3. In another exemplary embodiment, the targeting moiety is CXCL10, a chemokine that recognizes and binds to CXCR3. In another exemplary embodiment, the targeting moiety is CXCL11, a chemokine that recognizes and binds to CXCR3 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL12, a chemokine that recognizes and binds to CXCR4 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL13, a chemokine that recognizes and binds to CXCR5. In another exemplary embodiment, the targeting moiety is CXCL16, a chemokine that recognizes and binds to CXCR6. In another exemplary embodiment, the targeting moiety is LDGF-PBP, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is XCL2, a chemokine that recognizes and binds to XCR1. In another exemplary embodiment, the targeting moiety is CX3CL1, a chemokine that recognizes and binds to CX3CR1.

[0251] In some embodiments, the targeting moiety is a natural ligand, such as FMS-like tyrosine kinase 3 ligand (Flt3L) or a truncated region thereof (e.g., a region capable of binding Flt3). In some embodiments, the targeting moiety is the extracellular domain of Flt3L. In some embodiments, the targeting moiety comprises a Flt3L domain, wherein the Flt3L domain is a single-chain dimer, optionally wherein one Flt3L domain is linked to another Flt3L domain via one or more linkers, wherein the linkers are flexible linkers. In some embodiments, the targeting moiety of the present invention comprises a Flt3L domain, wherein the Flt3L domain is a single-chain dimer and an Fc domain, optionally comprising one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain. In some embodiments, the targeting moiety recognizes CD20. In some embodiments, the targeting moiety recognizes PD-L1. In some embodiments, the targeting moiety recognizes Clec9A.

[0252] In various embodiments, the chimeric protein complexes, such as the chimeric protein or Fc-based chimeric protein complexes, of the present invention contain various combinations of targeting moieties. In an exemplary embodiment, the chimeric protein complexes, such as the chimeric protein or Fc-based chimeric protein complexes of the present invention, can contain two targeting moieties, both of which are antibodies or derivatives thereof. In another exemplary embodiment, the chimeric protein complexes, such as the chimeric protein or Fc-based chimeric protein complexes of the present invention, can contain two targeting moieties, both of which are natural ligands for cell receptors. In a further exemplary embodiment, the chimeric protein complexes, such as the chimeric protein or Fc-based chimeric protein complexes of the present invention, can contain two targeting moieties, one of which is an antibody or a derivative thereof, and the other of which is a natural ligand for cell receptors.

[0253] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention functionally modulates (including, but not limited to, partially or completely neutralizes) a target of interest (e.g., an antigen, receptor), e.g., substantially inhibits, reduces, or neutralizes the biological effect of the antigen. For example, various recognition domains may be directed against one or more tumor antigens that actively suppress or have the ability to suppress the immune system of tumor-bearing patients. For example, in some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention functionally modulates one or more immunosuppressive signals (e.g., checkpoint inhibitors), such as TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2. For example, in some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention are genetically modified to disrupt, block, reduce, and / or inhibit the transmission of immunosuppressive signals, including, but not limited to, the binding of PD-1 to PD-L1 or PD-L2, and / or the binding of CTLA-4 to one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A.

[0254] In various embodiments, the recognition domain of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention binds but does not functionally modulate a target of interest (e.g., an antigen, a receptor); e.g., the recognition domain is or resembles a binding antibody. For example, in various embodiments, the recognition domain only targets an antigen or receptor without substantially inhibiting, reducing, or functionally modulating the biological action of the antigen or receptor. For example, some of the above-described antibody formats (e.g., compared to whole antibodies) have the ability to target difficult-to-access epitopes, providing a broader range of specific binding surfaces. In various embodiments, the recognition domain binds to an epitope that is physically distant from the antigen or receptor site (e.g., the active site of the antigen) important for its biological activity.

[0255] Such non-neutralizing binding is used in various embodiments of the present invention, including methods in which chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention are used to directly or indirectly recruit active immune cells to a required site via an effector antigen. For example, in various embodiments, chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention can be used to directly or indirectly recruit cytotoxic T cells to tumor cells via CD8 in methods of reducing or eliminating tumors (e.g., the chimeric protein or chimeric protein complex, such as the Fc-based chimeric protein complex, can include an anti-CD8 recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit CD8-expressing cytotoxic T cells but not functionally modulate CD8 activity. In contrast, in these embodiments, CD8 signaling is an important part of the tumor reduction or elimination process. As a further example, in various embodiments, chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention are used to directly or indirectly recruit dendritic cells (DCs) via Clec9A (e.g., a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can include an anti-Clec9A recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit Clec9A-expressing DCs but not functionally modulate Clec9A activity. In contrast, in these embodiments, Clec9A signaling is an important part of tumor reduction or elimination.

[0256] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention binds to XCR1 on dendritic cells, for example. For example, the recognition domain, in some embodiments, comprises all or a portion of XCL1 or a non-neutralizing anti-XCR1 agent.

[0257] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention binds to an immunomodulatory antigen (e.g., an immunostimulatory antigen or an immunosuppressive antigen). In various embodiments, the immunomodulatory antigen is one or more of 4-1BB, OX-40, HVEM, GITR, CD27, CD28, CD30, CD40, ICOS ligand; OX-40 ligand, LIGHT (CD258), GITR ligand, CD70, B7-1, B7-2, CD30 ligand, CD40 ligand, ICOS, ICOS ligand, CD137 ligand, and TL1A. In various embodiments, the immunostimulatory antigen is expressed on tumor cells. In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention binds but does not functionally modulate such immunostimulatory antigens, thus allowing the recruitment of cells expressing these antigens without reducing or losing potential tumor reduction or elimination capabilities.

[0258] In various embodiments, the recognition domains of the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention may be associated with chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, that include two recognition domains with neutralizing activity, or two recognition domains with non-neutralizing (e.g., binding) activity, or one recognition domain with neutralizing activity and one recognition domain with non-neutralizing (e.g., binding) activity.

[0259] Fc domain The fragment crystallizable domain (Fc domain) is the tail region of an antibody that interacts with Fc receptors located on the cell surface of cells involved in the immune system, e.g., B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. In IgG, IgA, and IgD antibody isotypes, the Fc domain is composed of two identical protein fragments derived from the second and third constant domains of the antibody's two heavy chains. In IgM and IgE antibody isotypes, the Fc domain is composed of the three heavy chain constant domains (C) in each polypeptide chain. H domains 2-4).

[0260] In some embodiments, the Fc-based chimeric protein complex of the present technology comprises an Fc domain. In some embodiments, the Fc domain is selected from IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from IgG1, IgG2, IgG3, or IgG4.

[0261] 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.

[0262] 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.

[0263] In some embodiments, the Fc domain comprises one or more mutations. In some embodiments, the mutations to the Fc domain reduce or eliminate an effector function of the Fc domain. In some embodiments, the mutated Fc domain has reduced affinity or binding to a target receptor. For example, in some embodiments, the mutations to the Fc domain reduce or eliminate binding of the Fc domain to an FcγR. In some embodiments, the FcγR is selected from FcγRI; FcγRIIa, 131R / R; FcγRIIa, 131H / H, FcγRIIb; and FcγRIII. In some embodiments, the mutations to the Fc domain reduce or eliminate binding to a complement protein, such as, for example, C1q. In some embodiments, the mutations to the Fc domain reduce or eliminate binding to both an FcγR and a complement protein, such as, for example, C1q.

[0264] In some embodiments, the Fc domain comprises a LALA mutation to reduce or eliminate effector function of the Fc domain. For example, in some embodiments, the LALA mutation comprises L234A and L235A substitutions in human IgG (e.g., IgG1) (numbering based on the commonly used CH2 residue numbering for human IgG1 according to EU regulations (PNAS, Edelman et al., 1969;63(1)78-85)).

[0265] In some embodiments, the Fc domain of human IgG comprises a mutation at position 46 that reduces or eliminates effector function of the Fc domain. For example, in some embodiments, the mutation is selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G, and P331S.

[0266] In some embodiments, the Fc domain comprises a FALA mutation that reduces or eliminates the effector function of the Fc domain, for example, in some embodiments, the FALA mutation comprises F234A and L235A substitutions in human IgG4.

[0267] In some embodiments, the Fc domain of human IgG4 comprises mutations at one or more of F234, L235, K322, D265, and P329 to reduce or eliminate effector function of the Fc domain, for example, in some embodiments, the mutations are selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.

[0268] In some embodiments, the mutation in the Fc domain stabilizes the hinge region of the Fc domain. For example, in some embodiments, the Fc domain contains a mutation at the S228 position of human IgG to stabilize the hinge region. In some embodiments, the mutation is S228P.

[0269] In some embodiments, mutations to the Fc domain promote chain pairing of the Fc domain, hi some embodiments, chain pairing is promoted by ionic pairing (a / k / a charge pairs, ionic bonds, or charged residue pairs).

[0270] In some embodiments, the Fc domain contains mutations at the following amino acid residue positions of another IgG to promote ion pairing: D356, E357, L368, K370, K392, D399, and K409.

[0271] For example, in some embodiments, a human IgG Fc domain contains one of the combinations of mutations in Table 1 to promote ion pairing.

[0272] [Table 1]

[0273] In some embodiments, chain pairing is facilitated by knobs-in-hole mutations. In some embodiments, the Fc domain contains one or more mutations that enable knobs-in-hole interactions in the Fc domain. In some embodiments, a first Fc chain is engineered to express a "knob" and a second Fc chain is engineered to express a complementary "hole." For example, in some embodiments, a human IgG Fc domain contains the mutations in Table 2 that enable knobs-in-hole interactions.

[0274] [Table 2]

[0275] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of the present technology comprises any combination of the mutations disclosed above. For example, in some embodiments, the Fc domain comprises mutations that promote ion-pairing and / or knobs-in-hole interactions. For example, in some embodiments, the Fc domain comprises mutations that have one or more of the following properties: promote ion-pairing, induce knobs-in-hole interactions, reduce or eliminate effector functions of the Fc domain, and provide Fc stabilization (e.g., hinge).

[0276] For example, in some embodiments, a human IgG Fc domain comprises mutations disclosed in Table 3 that promote ion pairing in the Fc domain and / or promote knob-in-hole interactions.

[0277] [Table 3-1]

[0278] [Table 3-2]

[0279] For example, in some embodiments, a human IgG Fc domain contains mutations disclosed in Table 4 that promote ion pairing of the Fc domain and / or promote knob-in-hole interactions, or a combination thereof. In some embodiments, "Chain 1" and "Chain 2" in Table 4 are interchangeable (e.g., Chain 1 can have Y407T and Chain 2 can have T366Y).

[0280] [Table 4-1]

[0281] [Table 4-2]

[0282] [Table 4-3]

[0283] [Table 4-4]

[0284] [Table 4-5]

[0285] For example, in some embodiments, a human IgG Fc domain comprises mutations disclosed in Table 5 that reduce or eliminate FcγR and / or complement binding in the Fc domain. In some embodiments, the mutations in Table 5 are present in both chains.

[0286] [Table 5-1]

[0287] [Table 5-2]

[0288] [Table 5-3]

[0289] In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology is a homodimer, i.e., the Fc region in the chimeric protein complex comprises two identical protein fragments.

[0290] In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology is a heterodimer, i.e., the Fc domain comprises two non-identical protein fragments.

[0291] In some embodiments, the heterodimeric Fc domain is engineered using ion-pairing and / or knobs-in-holes mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans configuration / structure. In a trans configuration / structure, the targeting moiety and the signaling factor, e.g., IL-2, are not found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention, in some embodiments.

[0292] In some embodiments, the Fc domain includes or begins with the core hinge region of wild-type human IgG1, which region comprises the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 276). In some embodiments, the Fc domain also includes the upper hinge, or a portion thereof (e.g., DKTHTCPPC (SEQ ID NO: 451) (see WO 2009053368), EPKSCDKTHTCPPC (SEQ ID NO: 452), or EPKSSDKTHTCPPC (SEQ ID NO: 453) (see Lo et al., Protein Engineering vol. 11 no. 6 pp. 495-500, 1998)).

[0293] Fc-based chimeric protein complex The Fc-based chimeric protein complexes of the present technology comprise at least one Fc domain disclosed herein, at least one signal transduction agent (SA) disclosed herein, such as IL-2, and at least one targeting moiety (TM) disclosed herein.

[0294] It is understood that an Fc-based chimeric protein complex of the present invention may comprise two fusion proteins, each containing an Fc domain.

[0295] In some embodiments, the Fc-based chimeric protein complex is a heterodimer. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans configuration / structure. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a cis configuration / structure.

[0296] In some embodiments, the heterodimeric Fc domain is engineered using ion-pairing and / or knobs-in-holes mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans configuration.

[0297] In the trans configuration, the targeting moiety and the signaling factor are, in some embodiments, not found on the same polypeptide chain in the Fc-based chimeric protein complexes of the invention. In the trans configuration, the targeting moiety and the signaling factor are, in some embodiments, found on separate polypeptide chains in the Fc-based chimeric protein complexes of the invention. In the cis configuration, the targeting moiety and the signaling factor are, in some embodiments, found on the same polypeptide chain in the Fc-based chimeric protein complexes of the invention.

[0298] In some embodiments where two or more targeting moieties are present in the heterodimeric protein complexes described herein, one targeting moiety may be present in a trans configuration (relative to the signaling factor), while another targeting moiety may be present in a cis configuration (relative to the signaling factor). In some embodiments, the signaling factor and targeting moiety are present on the same end / side (N- or C-terminus) of the Fc domain. In some embodiments, the signaling factor and targeting moiety are present on different ends / sides (N- or C-terminus) of the Fc domain.

[0299] In some embodiments where two or more targeting moieties are present in a heterodimeric protein complex described herein, the targeting moieties can be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties will be in trans with respect to each other since they are on different Fc chains). In some embodiments where two or more targeting moieties are present on the same Fc chain, the targeting moieties can be on the same or different sides / ends of the Fc chain (N- or C-terminus).

[0300] In some embodiments where two or more targeting moieties are present in a heterodimeric protein complex described herein, the targeting moieties are found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties must be in trans relative to each other since they are on different Fc chains). In some embodiments where two or more signaling factors are present on the same Fc chain, the signaling factors can be on the same or different sides / ends of the Fc chain (N- or C-terminus).

[0301] In some embodiments where two or more signaling factors are present in the heterodimeric protein complexes described herein, one signaling factor may be present in a trans configuration (relative to the targeting moiety) while another signaling factor may be present in a cis configuration (relative to the targeting moiety).

[0302] In some embodiments, the heterodimeric Fc-based chimeric protein complex does not comprise a signaling factor, e.g., IL-2, and a targeting moiety on a single polypeptide.

[0303] In some embodiments, the Fc-based chimeric protein has improved in vivo half-life compared to a chimeric protein that lacks Fc or is not a heterodimeric complex, hi some embodiments, the Fc-based chimeric protein has improved solubility, stability, and other pharmacological properties compared to a chimeric protein that lacks Fc or is not a heterodimeric complex.

[0304] Heterodimeric Fc-based chimeric protein complexes are composed of two different polypeptides. In some embodiments described herein, the targeting domain is located on a different polypeptide from the signal transduction factor, e.g., IL-2, thus allowing for the creation of proteins containing only one copy of the targeting domain and, similarly, only one type of signal transduction factor, e.g., IL-2 (which can control for potential interference with desirable properties). Furthermore, in some embodiments, the use of only one targeting domain (e.g., VHH) can avoid cross-linking of antigens on the cell surface, which can induce undesirable effects. Furthermore, in some embodiments, the use of only one signal transduction factor, e.g., IL-2, can mitigate molecular "crowding" and potential interference with avidity-mediated induction or recovery of effector function, depending on the targeting domain. Furthermore, in some embodiments, heterodimeric Fc-based chimeric protein complexes can have two targeting moieties, which can be located on two different polypeptides. For example, in some embodiments, the C-termini of both targeting moieties (e.g., VHHs) can be masked to avoid potential autoantibodies or pre-existing antibodies (e.g., VHH autoantibodies or pre-existing antibodies). Furthermore, in some embodiments, a heterodimeric Fc-based chimeric protein complex having a targeting domain on a polypeptide different from, for example, a signaling factor, e.g., IL-2 (e.g., a wild-type signaling factor, e.g., wild-type IL-2), may preferentially "cross-link" two cell types (e.g., tumor cells and immune cells). Furthermore, in some embodiments, a heterodimeric Fc-based chimeric protein complex has two signaling factors, each on a different polypeptide, allowing for more complex effector responses.

[0305] Furthermore, in some embodiments, heterodimeric Fc-based chimeric protein complexes with a variety of combinations of targeting moieties and signaling factors, e.g., IL-2, are provided in a practical manner, e.g., with a targeting domain on a polypeptide that is different from the signaling factor, e.g., IL-2. For example, in some embodiments, a polypeptide having any of the targeting moieties described herein can be combined "off the shelf" with a polypeptide having any of the signaling factors described herein, allowing for the rapid generation of various combinations of targeting moieties and signaling factors in a single Fc-based chimeric protein complex.

[0306] In some embodiments, an Fc-based chimeric protein complex comprises one or more linkers. In some embodiments, an Fc-based chimeric protein complex comprises a linker linking an Fc domain, a signaling factor, e.g., IL-2, and a targeting moiety. In some embodiments, an Fc-based chimeric protein complex comprises a linker linking each signaling factor, e.g., IL-2, and a targeting moiety (or, in the case of two or more targeting moieties, a linker linking a signaling factor, e.g., IL-2, to one of the targeting moieties). In some embodiments, an Fc-based chimeric protein complex comprises a linker linking each signaling factor, e.g., IL-2, to the Fc domain. In some embodiments, an Fc-based chimeric protein complex comprises a linker linking each targeting moiety to the Fc domain. In some embodiments, an Fc-based chimeric protein complex comprises a linker linking a targeting moiety to another targeting moiety. In some embodiments, an Fc-based chimeric protein complex comprises a linker linking a signaling factor, e.g., IL-2, to another signaling factor.

[0307] In some embodiments, the Fc-based chimeric protein complex comprises two or more targeting moieties, which in such embodiments may be the same targeting moiety or different targeting moieties.

[0308] In some embodiments, an Fc-based chimeric protein complex comprises two or more signaling factors, which in such embodiments may be the same or different targeting moieties.

[0309] For 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), where the Fc domain, signaling agents, and targeting moieties are selected from any of the Fc domains, signaling agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is a homodimer.

[0310] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 1A-F.

[0311] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 2A-H.

[0312] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 3A-H.

[0313] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 4A-D.

[0314] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 5A-F.

[0315] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 6A-J.

[0316] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 7A-D.

[0317] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 8A-F.

[0318] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 9A-J.

[0319] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 10A-F.

[0320] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 11A-L.

[0321] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 12A-L.

[0322] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 13A-F.

[0323] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 14A-L.

[0324] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 15A-L.

[0325] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 16A-J.

[0326] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 17A-J.

[0327] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 18A-F.

[0328] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 19A-F.

[0329] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematic diagrams in Figures 20A-E.

[0330] In various embodiments, the Fc-based chimeric protein complex takes the form of the schematic diagram in FIG.

[0331] In various embodiments, the Fc-based chimeric protein complex takes the form of the schematic diagram in FIG.

[0332] In some embodiments, the present invention contemplates Fc-based chimeric protein complexes comprising one or more of the variants in Table 7 and / or one or more constructs listed in the "ALN2 variants" column in Table 7.

[0333] In some embodiments, the signaling agent is linked to a targeting moiety and the targeting moiety is linked on the same end to an Fc domain (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0334] In some embodiments, the signaling agent and targeting moiety are linked to an Fc domain, and the targeting moiety and signaling agent are linked on the same end (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0335] In some embodiments, the targeting moiety is linked to a signaling factor and the signaling factor is linked on the same end to an Fc domain (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0336] In some embodiments, the homodimeric Fc-based chimeric protein complex comprises two or more targeting moieties. In some embodiments, there are four targeting moieties and two signaling factors, and the targeting moieties are linked to the Fc domain and the signaling factors are linked to the targeting moieties on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments, there are four targeting moieties and two signaling factors, and two targeting moieties are linked to the Fc domain and two targeting moieties are linked to the signaling factors, and they are linked to the Fc domain on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments, there are four targeting moieties and two signaling factors, and two targeting moieties are linked to each other, and one targeting moiety from each pair is linked to the Fc domain on the same end, and the signaling factors are linked to the Fc domain on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four targeting moieties and two signaling factors, two targeting moieties are linked to each other, one targeting moiety from each pair is linked to a signaling factor, e.g., IL-2, and the other targeting moiety of the pair is linked to an Fc domain, with the targeting moieties linked to the Fc domain being linked on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer.

[0337] In some embodiments, the homodimeric Fc-based chimeric protein complex comprises two or more signaling factors. In some embodiments where there are four signaling factors and two signaling factors, the two signaling factors are linked to each other, one signaling factor from the pair is linked to the Fc domain on the same end, and the targeting moiety is linked to the Fc domain on the same end (see Figures 3A-H). In some embodiments, the Fc domain is homodimeric. In some embodiments where there are four signaling factors and two signaling factors, the two signaling factors are linked to the Fc domain on the same end, and each of the two signaling factors is linked to a targeting moiety, and the targeting moiety is linked to the Fc domain on the same end (see Figures 3A-H). In some embodiments, the Fc domain is homodimeric. In some embodiments where there are four signaling factors and two signaling factors, the two signaling factors are linked to each other, one signaling factor from the pair is linked to a targeting moiety, and the targeting moiety is linked to the Fc domain on the same end (see Figures 3A-H). In some embodiments, the Fc domain is homodimeric.

[0338] For example, in some embodiments, an Fc-based chimeric protein complex comprises an Fc domain, wherein the Fc domain comprises an ion-pairing mutation and / or a knobs-in-hole mutation, at least one signal transduction agent, e.g., IL-2, and at least one targeting moiety, wherein the ion-pairing motif and / or knobs-in-hole motif, signal transduction agent, e.g., IL-2, and targeting moiety are selected from any of the ion-pairing motifs and / or knobs-in-hole motifs, signal transduction agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises mutations that reduce or eliminate its effector function.

[0339] In some embodiments, a signaling agent, e.g., IL-2, is linked to a targeting moiety, which is linked to an Fc domain (see Figures 10A-F and 13A-F). In some embodiments, a targeting moiety is linked to a signaling agent, e.g., IL-2, which is linked to an Fc domain (see Figures 10A-F and 13A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0340] In some embodiments, the signaling factor, e.g., IL-2, and the targeting moiety are linked to the Fc domain (see Figures 4A-D, 7A-D, 10A-F, and 13A-F). In some embodiments, the targeting moiety and the signaling factor, e.g., IL-2, are linked to different Fc chains on the same terminus (see Figures 4A-D and 7A-D). In some embodiments, the targeting moiety and the signaling factor, e.g., IL-2, are linked to different Fc chains on different termini (see Figures 4A-D and 7A-D). In some embodiments, the targeting moiety and the signaling factor, e.g., IL-2, are linked to the same Fc chain (see Figures 10A-F and 13A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0341] In some embodiments where there is one signaling factor, e.g., IL-2, and two targeting moieties, the signaling factor, e.g., IL-2, 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-F, 8A-F, 11A-L, 14A-L, 16A-J, and 17A-J). In some embodiments, the targeting moiety is linked to one Fc chain, and the signaling factor, e.g., IL-2, is linked to the other Fc chain (see Figures 5A-F and 8A-F). In some embodiments, the paired targeting moiety and signaling factor, e.g., IL-2, are linked to the same Fc chain (see Figures 11A-L and 14A-L). In some embodiments, a targeting moiety is linked to the Fc domain, another targeting moiety is linked to a signaling factor, e.g., IL-2, and a paired targeting moiety is linked to the Fc domain (see Figures 11A-L and 14A-L, 16A-J, and 17A-J). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked to the same Fc chain (see Figures 11A-L and 14A-L). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked to different Fc chains (see Figures 16A-J and 17A-J). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked on the same terminus (see Figures 16A-J and 17A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0342] In some embodiments where there is one signaling factor, e.g., IL-2, and two targeting moieties, the targeting moiety is linked to the signaling factor, e.g., IL-2, which is Fc domain-linked, and the unpaired targeting moiety is linked to the Fc domain (see Figures 11A-L and 14A-L, 16A-J, and 17A-J). In some embodiments, the paired signaling factor, e.g., IL-2, and the unpaired targeting moiety are linked to the same Fc chain (see Figures 11A-L and 14A-L). In some embodiments, the paired signaling factor, e.g., IL-2, and the paired targeting moiety are linked to different Fc chains (see Figures 16A-J and 17A-J). In some embodiments, the paired signaling factor, e.g., IL-2, and the unpaired targeting moiety are linked on the same terminus (see Figures 16A-J and 17A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0343] In some embodiments where there is one signaling factor, e.g., IL-2, and two targeting moieties, the targeting moieties are linked together, the signaling factor, e.g., IL-2, is linked to one of the paired targeting moieties, and the targeting moiety that is not linked to the signaling factor, e.g., IL-2, is linked to the Fc domain (see Figures 11A-L and 14A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0344] In some embodiments where there is one signaling factor, e.g., IL-2, and two targeting moieties, the targeting moieties are linked together, the signaling factor, e.g., IL-2, is linked to one of the paired targeting moieties, and the signaling factor, e.g., IL-2, is linked to the Fc domain (see Figures 11A-L and 14A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0345] In some embodiments where there is one signaling agent, e.g., IL-2, and two targeting moieties, both targeting moieties are linked to the signaling agent, e.g., IL-2, and one of the targeting moieties is linked to the Fc domain (see Figures 11A-L and 14A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0346] In some embodiments where there is one signaling agent, e.g., IL-2, and two targeting moieties, the targeting moiety and signaling agent, e.g., IL-2, are linked to an Fc domain (see Figures 16A-J and 17A-J). In some embodiments, the targeting moieties are linked terminally (see Figures 16A-J and 17A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0347] In some embodiments where there are two signaling factors and one targeting moiety, the signaling factors are linked to the Fc domain on the same end and the targeting moiety is linked to the Fc domain (see Figures 6A-J and 9A-J). In some embodiments, the signaling factors are linked to the Fc domain on the same Fc chain and the targeting moiety is linked on the other Fc chain (see Figures 18A-F and 19A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0348] In some embodiments where there are two signaling factors and one targeting moiety, the signaling factor, e.g., IL-2, is linked to the targeting moiety, which is linked to the Fc domain, and the other signaling factor, e.g., IL-2, is linked to the Fc domain (see Figures 6A-J and 9A-J, 12A-L, and 15A-L). In some embodiments, the targeting moiety and the unpaired signaling factor, e.g., IL-2, are linked to different Fc chains (see Figures 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling factor, e.g., IL-2, are linked to different Fc chains on the same terminus (see Figures 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling factor, e.g., IL-2, are linked to different Fc chains on different termini (see Figures 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling factor, e.g., IL-2, are linked to the same Fc chain (see Figures 12A-L and 15A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises mutations that reduce or eliminate its effector function.

[0349] In some embodiments where there are two signaling factors and one targeting moiety, the targeting moiety is linked to a signaling factor, e.g., IL-2, which is linked to an Fc domain, and the other signaling factor, e.g., IL-2, is linked to an Fc domain (see Figures 6A-J and 9A-J). In some embodiments, the paired signaling factor, e.g., IL-2, and the unpaired signaling factor, e.g., IL-2, are linked to different Fc chains (see Figures 6A-J and 9A-J). In some embodiments, the paired signaling factor, e.g., IL-2, and the unpaired signaling factor, e.g., IL-2, are linked to different Fc chains on the same end (see Figures 6A-J and 9A-J). In some embodiments, the paired signaling factor, e.g., IL-2, and the unpaired signaling factor, e.g., IL-2, are linked to different Fc chains on different ends (see Figures 6A-J and 9A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0350] In some embodiments where there are two signaling factors and one targeting moiety, the signaling factors are linked together, the targeting moiety is linked to one of the paired signaling factors, and the targeting moiety is linked to the Fc domain (see Figures 12A-L and 15A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0351] In some embodiments where there are two signaling factors and one targeting moiety, the signaling factors are linked together, one of the signaling factors is linked to the Fc domain, and the targeting moiety is linked to the Fc domain (see Figures 12A-L and 15A-L, 18A-F, and 19A-F). In some embodiments, the paired signaling factor and targeting moiety are linked to the same Fc chain (see Figures 12A-L and 15A-L). In some embodiments, th...

Claims

1. (a) a modified IL-2 signaling factor, the modified IL-2 signaling factor having one or more mutations or modifications relative to a wild-type IL-2 signaling factor having SEQ ID NO:1 that improves safety and reduces affinity or biological activity for IL-2Rαβγ and / or IL-2Rβ and / or IL-2Rγ and / or IL-2Rα, compared to the wild-type IL-2 signaling factor having SEQ ID NO:1; (b) one or more targeting moieties that comprise a recognition domain that specifically binds to an antigen or receptor of interest; and (c) one or more linkers and / or Fc domains, with or without one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain; Including, the modified IL-2 signaling factor (i) a mutation selected from D20E, D20V, D20F, D20G, D20H, D20I, D20K, and D20L; or (ii) a mutation selected from N88G, N88E, N88K, N88Q, and N88V; or (iii) a mutation selected from Q126G, Q126A, Q126E, Q126F, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126Y; or (iv) a mutation selected from (i) and (iii) or a mutation selected from (ii) and (iii); or (v) R38A / F42K / N88G / C125A, F42K / C125A, D20E / C125A, D20F / C125A, D20G / C125A, D20H / C125A, D20I / C125A, D20K / C125A, D20L / C125A, D20N / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C125A, N88G / C125A, N88E / C125A, N88H / C125A, N88I / C125A, N88K / C125A, N88Q / C125A, N88R / C125A, N88T / C125A, N88V / C125A, D20E / R38A / F42K, D20E / R38A / F42K / C125A, D20V / R38A / F42K, D20V / R38A / F42K / C125A, R38A / F42Y / E62A / C125A, R38A / F42Y / Y45A / E62A, F42Y / Y45A / L72G, R38A / F42Y / Y45A / E62A / C125A, F42Y / Y45A / L72G / C125A, R38A / F42K / N88G, R38A / F42K / Q126G, R38A / F42K / Q126I, R38A / F42K / Q126Y, R38A / F42K / Q126G / C125A, R38A / F42K / Q126I / C125A, R38A / F42K / Q126Y / C125A, R38A / F42K / E62A / N88G, and R38A / F42K / E62A / N88G / C125A One or more mutations selected from A chimeric protein or protein complex or an Fc-based chimeric protein complex comprising:

2. 2. The chimeric protein or protein complex or Fc-based chimeric protein complex of claim 1, wherein the modified IL-2 signaling agent further comprises a glycosylation mutation that reduces or eliminates glycosylation of the modified IL-2 signaling agent, and / or the glycosylation mutation is a T3 substitution selected from T3A, T3F, T3H, T3L, T3V, and T3Y, or the glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2.

3. (a) a modified IL-2 signaling factor, the modified IL-2 signaling factor having one or more mutations or modifications relative to a wild-type IL-2 signaling factor having SEQ ID NO:1 that improves safety and reduces affinity or biological activity for IL-2Rαβγ and / or IL-2Rβ and / or IL-2Rγ and / or IL-2Rα, compared to the wild-type IL-2 signaling factor having SEQ ID NO:1; (b) one or more targeting moieties that comprise a recognition domain that specifically binds to an antigen or receptor of interest; and (c) one or more linkers and / or Fc domains, with or without one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain; Including, The chimeric protein or protein complex or Fc-based chimeric protein complex, wherein said modified IL-2 signaling agent comprises a glycosylation mutation, said mutation reducing or eliminating glycosylation of said modified IL-2 signaling agent, and / or said glycosylation mutation is a T3 substitution selected from T3A, T3F, T3H, T3L, T3V, and T3Y, or said glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of said modified IL-2.

4. (a) a modified IL-2 signaling factor having one or more mutations or modifications relative to the neoleukin signaling factor having SEQ ID NO:2 that improve the safety and reduce the affinity or biological activity of the modified IL-2 signaling factor for IL-2Rαβγ and / or IL-2Rβ and / or IL-2Rγ and / or IL-2Rα compared to the neoleukin signaling factor having SEQ ID NO:2; (b) one or more targeting moieties that comprise a recognition domain that specifically binds to an antigen or receptor of interest; and (c) one or more linkers and / or Fc domains, with or without one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain; Including, The chimeric protein or protein complex or Fc-based chimeric protein complex, wherein the modified IL-2 signaling agent comprises one or more mutations at positions selected from D15 and N40, wherein the mutations are selected or not selected from D15T, D15H, N40I, N40G, and N40R.

5. the modified IL-2 comprises one or more mutations that reduce affinity for the IL-2 receptor chains, and / or exhibits reduced affinity for IL-2Rαβγ, or exhibits reduced affinity for IL-2Rβ, IL-2Rγ, or a combination thereof, or exhibits reduced affinity for IL-2Rα, IL-2Rβ, IL-2Rγ, or a combination thereof, and / or the modified IL-2 exhibits reduced affinity for IL-2Rα and IL-2Rβ, and the modified IL-2 comprises one or more mutations selected from D20E / R38A / F42K, D20E / R38A / F42K / C125A, D20V / R38A / F42K, D20V / R38A / F42K / C125A, D20V / R38A / F42K / E62A, D20V / R38A / F42K / E62A / C125A, R38A / F42K / N88G, R38A / F42K / N88G / C125A, R38A / F42K / E62A / N88G, and R38A / F42K / E62A / N88G / C125A; or the modified IL-2 exhibits reduced affinity for IL-2Rα and IL-2Rγ, and the modified IL-2 comprises one or more mutations selected from R38A / F42K / Q126G, R38A / F42K / Q126I, and R38A / F42K / Q126Y; or The modified IL-2 exhibits reduced affinity for IL-2Rβ, and the modified IL-2 is selected from the group consisting of D20E, D20V, N88G, D20F, D20G, D20H, D20I, D20K, D20L, N88E, N88K, N88Q, N88V, D20E / C125A, D20F / C125A, D20G / C125A, D20H / C125A, D20I / C125A, D20L / C125A, D20K / C125A, D20 or comprising one or more mutations selected from N / C125A, D20S / C125A, D20T / C125A, D20V / C125A, N88A / C125A, N88D / C125A, N88G / C125A, N88E / C125A, N88H / C125A, N88I / C125A, N88K / C125A, N88Q / C125A, N88R / C125A, N88T / C125A, and N88V / C125A; the modified IL-2 exhibits reduced affinity for IL-2Rγ and comprises one or more mutations selected from Q126A, Q126E, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q126S, Q126T, Q126V, Q126W, and Q126Y, or a combination thereof; or The modified IL-2 exhibits reduced affinity for IL-2Rβγ, and the modified IL-2 is selected from the group consisting of D20E / Q126A, D20E / Q126D, D20E / Q126E, D20E / Q126F, D20E / Q126G, D20E / Q126H, D20E / Q126I, D20E / Q126K, D20E / Q126L, D20E / Q126M, D20E / Q126N, D20E / Q126P, D20E / Q126R, D20E / Q126S, D20E / Q126T ... 26V, D20E / Q126W, D20E / Q126Y, D20V / Q126A, D20V / Q126D, D20V / Q126E, D20V / Q126F, D20V / Q126G, D20V / Q126H, D20V / Q126I, D20 V / Q126K, D20V / Q126L, D20V / Q126M, D20V / Q126N, D20V / Q126P, D20V / Q126R, D20V / Q126S, D20V / Q126T, D20V / Q126V, D20V / Q126W, D20V / Q126Y, N88A / Q126A, N88A / Q126D, N88A / Q126E, N88A / Q126F, N88A / Q126G, N88A / Q126H, N88A / Q126I, N88A / Q126K, N88A / Q1 26L, N88A / Q126M, N88A / Q126N, N88A / Q126P, N88A / Q126R, N88A / Q126S, N88A / Q126T, N88A / Q126V, N88A / Q126W, N88A / Q126Y, N88G N88G / Q126A, N88G / Q126D, N88G / Q126E, N88G / Q126F, N88G / Q126G, N88G / Q126H, N88G / Q126I, N88G / Q126K, N88G / Q126L, N88G / Q126M, N88G / Q126N, N88G / Q126P, N88G / Q126R, N88G / Q126S, N88G / Q126T, N88G / Q126V, N88G / Q126W, and N88G / Q126Y, 2. The chimeric protein or protein complex or Fc-based chimeric protein complex of claim 1.

6. the one or more mutations in the IL-2 signaling factor reduce affinity or biological activity, and the affinity or biological activity can be restored by conjugation to one or more targeting moieties; and / or the one or more targeting moieties are directed against tumor cells, endothelial cells, epithelial cells, mesenchymal cells, tumor stroma or stromal cells, and / or ECM; and / or the one or more targeting moieties have directionality towards immune cells and / or organ cells and / or tissue cells, and / or the immune cells are selected from T cells, Tregs, cytotoxic T lymphocytes, helper T cells, B cells, dendritic cells, anti-tumor or tumor associated macrophages (e.g. M1 or M2 macrophages), neutrophils, myeloid suppressor cells, natural killer (NK) cells, and natural killer T (NKT) cells, and / or the immune cells are T cells and the targeting moiety targets CD8, or the immune cells are Tregs and the targeting moiety targets CTLA4, or the immune cells are NK cells and the targeting moiety is NKp46, and / or said targeting moiety comprises a recognition domain which is a full-length antibody, a single domain antibody, a recombinant heavy chain only antibody (VHH), a single chain antibody (scFv), a shark heavy chain only antibody (VNAR), an Fv, a Fab, a Fab', or an F(ab')2, and / or is a humanized VHH, wherein said recognition domain is a VHH, and / or the recognition domain functionally modulates the antigen or receptor of interest, or the recognition domain binds to the antigen or receptor of interest but does not functionally modulate the antigen or receptor, and / or the chimeric protein or protein complex comprises two or more targeting moieties; and / or The one or more targeting moieties bind to one or more of the following targets: CD8, CTLA4, CD3, CD4, DNAM-1, Nrp1 (neurophilin), TNFR1, TNFR2, GITR, ICOS, CD20, CD70, Clec9a, NKp46, PD-1, PD-L1, PD-L2, SIRP1α, FAP, XCR1, tenascin, or an ECM protein; 2. The chimeric protein or protein complex or Fc-based chimeric protein complex of claim 1.

7. and / or and / or comprising two signaling factors and one targeting moiety, two targeting moieties and one signaling factor, two signaling factors and two targeting moieties, three signaling factors, three targeting moieties, or three signaling factors and three targeting moieties; the additional modified signaling factor comprises one or more mutations that reduce its affinity or activity for the receptor compared to the unmutated signaling factor; and / or The one or more mutations allow for attenuation of activity, 2. The chimeric protein or protein complex or Fc-based chimeric protein complex of claim 1.

8. Use of a chimeric protein or protein complex or an Fc-based chimeric protein complex described in claim 1 in the manufacture of a medicament for the treatment of one or more of cancer, infectious diseases, immune disorders, autoimmune diseases, cardiovascular diseases, wounds, ischemia-related diseases, neurodegenerative diseases, and / or metabolic diseases.

9. A recombinant nucleic acid encoding one or more chimeric proteins or protein complexes or Fc-based chimeric protein complexes according to any one of claims 1 to 7.

10. A host cell comprising the recombinant nucleic acid of claim 9.

11. the Fc domain is from IgG, IgA, IgD, IgM or IgE, or is from human IgG, human IgA, human IgD, human IgM or human IgE, and / or the IgG is selected from IgG1, IgG2, IgG3 or IgG4, or from human IgG1, human IgG2, human IgG3 or human IgG4, and / or the Fc chain pairing is facilitated by ion pairing and / or knob-in-hole pairing; or 2. The Fc-based chimeric protein complex of claim 1, wherein the one or more mutations to the Fc domain result in ionic pairing between Fc chains in the Fc domain, or the one or more mutations to the Fc domain result in knob-in-hole pairing in the Fc domain, or the one or more mutations to the Fc domain reduce or eliminate an effector function of the Fc domain.

12. the Fc-based chimeric protein complex is a heterodimer and has a trans orientation / configuration with respect to any of the targeting moieties and signaling factors relative to each other, or with respect to any of the targeting moieties and signaling factors relative to each other, or with respect to any of the targeting moieties and signaling factors relative to each other, or with respect to any of the targeting moieties and signaling factors relative to each other, and / or the Fc comprises L234A, L235A, and K322Q substitutions (according to EU numbering) in human IgG1; and / or the Fc is human IgG1 and contains or does not contain one or more of the following mutations: L234, L235, K322, D265, P329, and P331 (according to EU numbering); and / or and / or the Fc-based chimeric protein complex has the orientation / configuration of any one of Figures 1A-F, 2A-H, 3A-H, 4A-D, 5A-F, 6A-J, 7A-D, 8A-F, 9A-J, 10A-F, 11A-L, 12A-L, 13A-F, 14A-L, 15A-L, 16A-J, 17A-J, 18A-F, and 19A-F, 31, and 32; and / or the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 292, 293, 294, 299, 301, 310, 311, 312, 315, 316, 319, 320, 325, 328, 332, 333, and 335; and / or the chimeric protein or Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 292, 290-291, 293-449, 478-495, or 501-531; The Fc-based chimeric protein complex of claim 11.

13. (a) a modified IL-2 signaling factor, the modified IL-2 signaling factor having a single mutation relative to a wild-type IL-2 signaling factor having SEQ ID NO:1 that improves safety and reduces affinity or biological activity for IL-2Rβ of the modified IL-2 signaling factor compared to the wild-type IL-2 signaling factor having SEQ ID NO:1; (b) one or more targeting moieties that comprise a recognition domain that specifically binds to an antigen or receptor of interest; and (c) one or more linkers and / or Fc domains, with or without one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain; Including, the single mutation is selected from D20E, D20V, N88G, D20F, D20G, D20H, D20I, D20K, D20L, D20N, N88E, N88I, N88K, N88Q, N88R, and N88V; the modified IL-2 signaling factor does not contain a mutation that reduces affinity or biological activity for IL-2Rα; Chimeric proteins or protein complexes or Fc-based chimeric protein complexes.

14. 14. The chimeric protein or protein complex or Fc-based chimeric protein complex of claim 13, wherein the modified IL-2 signaling agent further comprises a glycosylation mutation that reduces or eliminates glycosylation of the modified IL-2 signaling agent, and / or the glycosylation mutation is a T3 substitution selected from T3A, T3F, T3H, T3L, T3V, and T3Y, or the glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2.

15. the one or more mutations in the IL-2 signaling factor reduce affinity or biological activity, and the affinity or biological activity can be restored by conjugation to one or more targeting moieties; and / or the one or more targeting moieties have directionality for tumor cells, endothelial cells, epithelial cells, mesenchymal cells, tumor stroma or stromal cells, and / or ECM, and / or the one or more targeting moieties have directionality for immune cells and / or organ cells and / or tissue cells, and / or the immune cells are selected from T cells, Tregs, cytotoxic T lymphocytes, helper T cells, B cells, dendritic cells, anti-tumor or tumor associated macrophages (e.g. M1 or M2 macrophages), neutrophils, myeloid suppressor cells, natural killer (NK) cells, and natural killer T (NKT) cells, and / or the immune cells are T cells and the targeting moiety targets CD8, or the immune cells are Tregs and the targeting moiety targets CTLA4, or the immune cells are NK cells and the targeting moiety is NKp46, and / or said targeting moiety comprises a recognition domain which is a full length antibody, a single domain antibody, a recombinant heavy chain only antibody (VHH), a single chain antibody (scFv), a shark heavy chain only antibody (VNAR), an Fv, a Fab, a Fab', or an F(ab')2, and / or said recognition domain is a VHH or a humanized VHH, and / or the recognition domain functionally modulates the antigen or receptor of interest, or the recognition domain binds to the antigen or receptor of interest but does not functionally modulate the antigen or receptor, and / or the chimeric protein or protein complex comprises two or more targeting moieties; and / or The one or more targeting moieties bind to one or more of the following targets: CD8, CTLA4, CD3, CD4, DNAM-1, Nrp1 (neurophilin), TNFR1, TNFR2, GITR, ICOS, CD20, CD70, Clec9a, NKp46, PD-1, PD-L1, PD-L2, SIRP1α, FAP, XCR1, tenascin, or an ECM protein; 14. A chimeric protein or protein complex or an Fc-based chimeric protein complex as described in claim 13.

16. and / or the chimeric protein comprises two signaling factors and one targeting moiety, two targeting moieties and one signaling factor, or two signaling factors and two targeting moieties; and / or the chimeric protein comprises three signaling factors, three targeting moieties, or three signaling factors and three targeting moieties; and / or the additional modified signaling factor comprises one or more mutations that reduce its affinity or activity for the receptor compared to the unmutated signaling factor; and / or The one or more mutations allow for attenuation of activity, 14. A chimeric protein or protein complex or an Fc-based chimeric protein complex as described in claim 13.

17. 17. Use of a chimeric protein or protein complex or an Fc-based chimeric protein complex according to any one of claims 13 to 16 in the manufacture of a medicament for use in the treatment of one or more of cancer, an infectious disease, an immune disorder, an autoimmune disease, a cardiovascular disease, a wound, an ischemia-related disease, a neurodegenerative disease, and / or a metabolic disease.

18. A recombinant nucleic acid encoding one or more of the chimeric proteins or protein complexes or Fc-based chimeric protein complexes according to any one of claims 13 to 16.

19. A host cell comprising the recombinant nucleic acid of claim 18.

20. the Fc domain is from IgG, IgA, IgD, IgM or IgE, or is from human IgG, human IgA, human IgD, human IgM or human IgE, and / or the IgG is selected from IgG1, IgG2, IgG3 or IgG4, or from human IgG1, human IgG2, human IgG3 or human IgG4, and / or the Fc chain pairing is facilitated by ion pairing and / or knob-in-hole pairing; or 14. The Fc-based chimeric protein complex of any one of claims 13, wherein the one or more mutations to the Fc domain result in ionic pairing between Fc chains in the Fc domain, or the one or more mutations to the Fc domain result in knob-in-hole pairing in the Fc domain, or the one or more mutations to the Fc domain reduce or eliminate an effector function of the Fc domain.

21. the Fc-based chimeric protein complex is a heterodimer and has a trans orientation / configuration with respect to any of the targeting moieties and signaling factors relative to each other, or with respect to any of the targeting moieties and with respect to each other, or with respect to any of the signaling factors and with respect to each other, or the Fc-based chimeric protein complex is a heterodimer and has a cis orientation with respect to any of the targeting moieties and signaling factors relative to each other, or with respect to any of the targeting moieties and with respect to each other, or with respect to any of the signaling factors and with respect to each other, and / or the Fc comprises L234A, L235A, and K322Q substitutions (according to EU numbering) in human IgG1; and / or the Fc is human IgG1 and contains or does not contain one or more of the following mutations: L234, L235, K322, D265, P329, and P331 (according to EU numbering); and / or and / or the Fc-based chimeric protein complex has the orientation / configuration of any one of Figures 1A-F, 2A-H, 3A-H, 4A-D, 5A-F, 6A-J, 7A-D, 8A-F, 9A-J, 10A-F, 11A-L, 12A-L, 13A-F, 14A-L, 15A-L, 16A-J, 17A-J, 18A-F, and 19A-F, 31, and 32; and / or the Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 292, 293, 294, 299, 301, 310, 311, 312, 315, 316, 319, 320, 325, 328, 332, 333, and 335; and / or the chimeric protein or Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to any one of SEQ ID NOs: 292, 290-291, 293-449, 478-495, or 501-531; 21. The Fc-based chimeric protein complex of claim 20.

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