Interleukin-6 agonist chimeric proteins

Abstract

The present invention relates, in part, to chimeric proteins and Fc-based chimeric protein complexes comprising IL-6 or modified IL-6 and their use as therapeutic agents. The present invention further relates to methods of treatment of various diseases, such as cancer.

Description

[0001] ORN-108PC / 114384-5108

[0002] INTERLEUKIN-6 AGONIST CHIMERIC PROTEINS

[0003] FIELD

[0004] The present invention relates, in part, to chimeric proteins, or chimeric protein complexes (including Fc-based chimeric protein complexes) comprising interleukin-6 (IL-6), or variants thereof, and their use as therapeutic agents.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 703,301, filed October 4, 2024, the entire contents of which are hereby incorporated by reference in their entirety

[0007] SEQUENCE LISTING

[0008] The contents of the computer readable Sequence Listing in XM L format (“XML Document”) submitted electronically herewith are incorporated herein by reference in their entirety. A computer readable format copy of the Sequence Listing (filename: ORN-108PC_114384-5108.xml, date produced: September 21 , 2025; size: 637,718 bytes) is submitted per 37 C.F.R. §§ 1.831-1.835.

[0009] BACKGROUND

[0010] Interleukins are mostly secreted proteins that act as cytokines to mediate inter-cellular communication during immune responses. Interleukin-6 (IL-6) is a 184 amino acid polypeptide belonging to the class of helical cytokines. IL-6, which was originally identified as a B-cell differentiation factor, is now known to be a multifunctional cytokine that regulates the immune response, hematopoiesis, the acute phase response, and inflammation. Furthermore, IL-6 is an important regulator of the acute phase response, T cell function, and terminal B cell differentiation. Deregulation of IL-6 production is implicated in the pathology of several disease processes. For example, excessive or inappropriate production of this cytokine may be involved in a variety of autoimmune and neoplastic disorders.

[0011] Since its discovery more than 40 years ago, the IL-6 pathway has emerged as a pivotal pathway involved in immune regulation in health and dysregulation in many diseases. Targeting the IL-6 pathway may provide various innovative therapeutic approaches for a variety of disorders and diseases.

[0012] Accordingly, there remains a need for safe and effective agonistic IL-6-based therapeutics with improved target selectivity, pharmacokinetic and therapeutic properties and minimal toxicity profiles.

[0013] SUMMARY

[0014] In some aspects, the present invention relates to chimeric proteins and chimeric protein complexes, including Fc- based chimeric protein complexes, comprising an interleukin-6 (IL-6) or a modified IL-6 as a signaling agent. In some embodiments, the modified IL-6 is a modified wild type IL-6, wherein the wild type IL-6 has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified IL-6 has one or more substitution mutations at a position selected from F74, L174, L178, R179, A180, and R182, where the residue numbering is based on SEQ

[0015] 1

[0016] DB1 / 162517863.2 ORN-108PC / 114384-5108

[0017] ID NO: 431 (mature IL-6 after signal peptide cleavage after position 28). In some embodiments, one or more of these mutations produce a modified human IL-6 with reduced binding activity and / or affinity for IL-6 receptor (IL- 6Ra) and reduced biological activity. In some embodiments, the modified IL-6 comprises a truncation of at least one, two, three, four, five, six, seven, eight, nine, or ten C-terminal amino acids, relative to SEQ ID NO: 431.

[0018] In some aspects, the present invention provides a chimeric protein comprising: (a) an IL-6 or a modified IL-6, and (b) one or more targeting moieties, said targeting moieties comprising recognition domains which specifically bind to an antigen or receptor of interest; wherein the IL-6 or modified IL-6 and the one or more targeting moieties are optionally connected with one or more linkers.

[0019] In another aspect, the present invention is related to a Fc-based chimeric protein complex comprising: a) an IL-6 or a modified IL-6, and b) one or more targeting moieties, said targeting moieties comprising recognition domains which specifically bind to an antigen or receptor of interest; and c) a Fc domain, the Fc domain optionally having one or more mutations that reduces or eliminates one or more effector functions of the Fc domain, promotes Fc chain pairing in the Fc domain, and / or stabilizes a hinge region in the Fc domain.

[0020] The present invention concerns a target-specific IL-6 agonist having loss-of-function site I mutations that can be restored upon targeting. In embodiments, the reduced agonistic activity of IL-6 is restored when attached to a targeting moiety, said targeting moiety comprising a recognition domain which specifically binds to an antigen or receptor of interest. In embodiments, the target-specific IL-6 agonist comprises one or more site I mutations that reduce IL-6 / IL-6Ra binding.

[0021] The present disclosure concerns, in part, findings that chimeric proteins or chimeric protein complexes (including Fc-based chimeric protein complexes) comprising a modified or mutant IL-6, exhibit substantially reduced IL-6Ra (IL-6 Receptor) activation signaling activity compared to wild type IL-6. This reduced IL-6Ro-activation signaling activity, however, can be induced and / or restored at a target cell when directed to such a cell through a targeting moiety. Surprisingly, the induced IL-6 activity at a target cell, achieved through targeting of chimeric proteins or chimeric protein complexes comprising IL-6, or variants thereof, may be similar or greater at the target cell than that of wild type IL-6. Importantly, the IL-6 chimeric proteins and chimeric protein complexes comprising IL-6 described herein, exhibit substantial and surprising selectivity for target cells versus non-target cells, and substantially more than, for example, achieved with targeted wild type IL-6 chimeric protein(s). In summary, a unique combination of highly potent and highly cell target-selective IL-6Ra-signaling activation can be achieved with IL-6 compositions, and variants thereof, described herein.

[0022] In some embodiments, the IL-6 is modified, i.e., is a variant and comprises one or more mutations in IL-6. In some embodiments, the one or more mutations reduce the biological activity of the IL-6 (sometimes referred to as "attenuated by mutation”). For example, the one or more mutations may reduce the affinity and / or activity of the IL-6 for a therapeutic receptor. In an embodiment, the modified IL-6 comprises one or more mutations that reduce its affinity and / or activity for IL-6Ra. In some embodiments, the loss in affinity and / or activity of the modified IL-6 (“attenuated by mutation”) for a therapeutic receptor, e.g., IL-6Ra, can be induced and restored upon directing or

[0023] DB1 / 162517863.2 2 ORN-108PC / 114384-5108 targeting of the chimeric protein or chimeric protein complex comprising the modified IL-6 to a target cell through a targeting moiety

[0024] In some embodiments, the chimeric proteins and chimeric protein complexes, including Fc-based chimeric protein complexes, comprises one or more additional signaling agents, e.g., without limitation, an interferon, an interleukin, and a tumor necrosis factor, that may be modified. In various embodiments, the chimeric proteins and chimeric protein complexes, including Fc-based chimeric protein complexes, of the invention provides improved safety and / or therapeutic activity and / or pharmacokinetic profiles (e.g., increased serum half-life) compared to an untargeted and / or unmodified IL-6 or an unmodified, wild type IL-6.

[0025] In various embodiments, the chimeric proteins and chimeric protein complexes, including Fc-based chimeric protein complexes, comprise one or more targeting moieties which have recognition domains (e.g. antigen recognition domains, including without limitation various antibody formats, inclusive of single-domain antibodies) which specifically bind to a target (e.g. antigen, receptor) of interest. In various embodiments, the targeting moieties have recognition domains that specifically bind to a target (e.g. antigen, receptor) of interest, including those found on one or more immune cells, which can include, without limitation, T cells, cytotoxic T lymphocytes, T helper cells, T regulatory cells (Tregs), natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor and tumor macrophages (e.g. M1 and M2 macrophages), B cells, B regulatory (Breg) cells, neutrophils, monocytes, myeloid derived cells, and dendritic cells. In various embodiments, the targeting moieties have recognition domains that specifically bind to a target (e.g. antigen, receptor) of interest, including those found on one or more tumor cells, endothelial cells, epithelial cells, mesenchymal cells, stromal cells or other cell types that are characteristic of and / or unique for specific organs and / or tissues, including those specifically associated with disease. In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) of interest and effectively recruit one or more immune cells. In some embodiments, the targets (e.g. antigens, receptors) of interest can be found on one or more tumor cells. In some embodiments, the present chimeric proteins, chimeric protein complexes, including Fc-based chimeric protein complexes, may recruit an immune cell, e.g., an immune cell that can kill and / or suppress a tumor cell, or modulate other immune cells, to a site of action (such as, by way of non-limiting example, the tumor microenvironment). In some embodiments, the present chimeric proteins, chimeric protein complexes, including Fc-based chimeric protein complexes, may modulate an immune cell at a site of action, or recruit an immune cell to a site of action that is associated with an autoimmune disease, inflammatory disease, infection, metabolic and / or cardiovascular disease (such as, by way of non-limiting example, the disease microenvironment). In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) of interest that is part of a non-cel I ular structure.

[0026] In some embodiments, the chimeric proteins or the chimeric protein complexes described herein comprise IL-6 or IL-6 mutants (which is an example of a cytokine / signaling agent that may also be used in the present invention) with reduced biological activity that is coupled to one or more targeting moieties In some embodiments, the chimeric proteins or chimeric protein complexes of the present invention include AcTakines (Activity-on-Target cytokines) that have one or more mutated cytokines that remain inactive en route through the body and only reveal

[0027] DB1 / 162517863.2 3 ORN-108PC / 114384-5108 their full agonistic activity upon target cell binding. In some embodiments, without limitation, the chimeric proteins or chimeric protein complexes target a mutant IL-6 to hepatocytes, adipose stromal cells, NK cells, dendritic cells, or CD8* T cells. In vivo, such chimeric proteins or chimeric protein complexes can potently promote the response of hepatocytes, adipose stromal cells, NK cells, dendritic cells, orCD8* T cells, with a significantly reduced toxicity profile compared to wild-type (WT) IL-6.

[0028] In some embodiments, the targeting moiety binds to one or more of the follow targets: CD8, CTLA4, CD3, CD4, DNAM-1, Nrp1 (neurophilin), TNFR2, GITR, ICOS, CD20, CD70, Clec9a, NKp46, asialoglycoprotein receptor (ASGPR), adipose stromal cells (ASC), PD-1, PD-L1 , PD-L2, SIRPIa, FAP, XCR1, tenascin, prostate-specific membrane antigen (PSMA), and ECM proteins.

[0029] In various embodiments, the present chimeric proteins and chimeric protein complexes, including Fc-based chimeric protein complexes find use in the treatment of various diseases or disorders such as cancer, viral infections, immune disorders, autoimmune diseases, liver regeneration, obesity, cardiovascular diseases, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases and many other diseases and disorders, and the present invention encompasses various methods of treatment.

[0030] Another aspect of the invention is related to a method for treating a subject afflicted with a cancer, comprising administering a chimeric protein or chimeric protein complex as described herein. In some embodiments, the chimeric protein or chimeric protein complex comprises: (i) an IL-6, or a mutant thereof, (ii) one or more targeting moieties, said targeting moieties comprising recognition domains which specifically bind to an antigen or receptor of interest; and (iii) a connector between (i) and (ii), the connector being: (1 ) an Fc domain, the Fc domain optionally having one or more mutations that reduces or eliminates one or more effector functions of the Fc domain, promotes Fc chain pairing in the Fc domain, and / or stabilizes a hinge region in the Fc domain that connects (i) and (ii) and / or (2) a flexible linker that connects (i) and (ii); wherein the mutant IL-6 is characterized by low affinity or activity at the IL-6 receptor (I L-6Ra).

[0031] The present disclosure also concerns, in part, to the finding that chimeric proteins or chimeric protein complexes comprising an IL-6, or a mutant thereof, exhibit substantially reduced IL-6 activity compared to wild type IL-6. This reduced I L-6-activation signaling activity, however, can be induced and / or restored at a target cell when directed to such a cell through a targeting moiety, which binds to an antigen or receptor of interest. Surprisingly, the induced IL-6 activity at a target cell, achieved through targeting of chimeric proteins or chimeric protein complexes comprising IL-6, or variants thereof, may be similar or greater at the target cell than that of wild type IL-6.

[0032] Importantly, the chimeric proteins and chimeric protein complexes comprising mutant IL-6 described herein, exhibit substantial and surprising selectivity for target cells versus non-target cells, and substantially more than, for example, achieved with targeted wild type IL-6 chimeric protein(s). In summary, a unique combination of highly potent and highly cell target-selective signaling activation can be achieved with the chimeric proteins or chimeric protein complexes described herein.

[0033] DB1 / 162517863.2 4 ORN-108PC / 114384-5108

[0034] In embodiments, the chimeric protein or the Fc-based chimeric protein complex of the present application comprises a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one of SEQ ID NO: 414-430, 444, and 448-476. In embodiments, the chimeric protein or the Fc-based chimeric protein complex of the present application comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 414-430, 444, and 448-476.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIGs. 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 illustrative schematics of the Fc-based chimeric protein complexes of the present invention. In embodiments, each schematic is a composition of the present invention. Where applicable in the figures, ”TM” refers to a “targeting moiety” as described herein, “SA” refers to a “signaling agent” as described herein, “l“l” is an optional “linker'’ as described herein, the two long parallel rectangles are human Fc domains, e g. from lgG1 , from lgG2, or from lgG4, as described herein and optionally with effector knock-out and / or stabilization mutations as also described herein, and the two long parallel rectangles with one having a protrusion and the other having an indentation are human Fc domains, e.g. from I gG 1 , from lgG2, or from lgG4 as described herein, with knob-in-hole and / or ionic pair (a / k / a charged pairs, ionic bond, or charged residue pair) mutations as described herein and optionally with effector knock-out and / or stabilization mutations as also described herein.

[0037] FIGs. 1 A-F show illustrative homodimeric 2-chain complexes. These figures show illustrative configurations for the homodimeric 2-chain complexes.

[0038] FIGs. 2A-H show illustrative homodimeric 2-chain complexes with two targeting moieties (TM) (as described herein, more targeting moieties may be present in some embodiments). In embodiments, the position of TM1 and TM2 are interchangeable. In embodiments, the constructs shown in the box (i.e., Figs. 2G and 2H) have signaling agent (SA) between TM1 and TM2 or between TM1 and Fc.

[0039] FIGs. 3A-H show illustrative homodimeric 2-chain complexes with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In embodiments, the position of SA1 and SA2 are interchangeable. In embodiments, the constructs shown in the box ( / .e., Figs. 3G and 3H) have TM between SA1 and SA2 or TM at N- or C-terminus.

[0040] FIGs. 4A-D show illustrative heterodimeric 2-chain complexes with split TM and SA chains, namely the TM on the knob chain of the Fc and the SA on hole chain of the Fc.

[0041] FIGs. 5A-F show illustrative heterodimeric 2-chain complexes with split TM and SA chains, namely with both TMs on the knob chain of the Fc and with SA on hole chain of the Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments) In embodiments, the position of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical.

[0042] DB1 / 162517863.2 5 ORN-108PC / 114384-5108

[0043] FIGs. 6A-J show illustrative heterodimeric 2-chain complexes with split TM and SA chains, namely with TM on the knob chain of the Fc and with a SA on the hole chain of the Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In these orientations and / or configurations, one SA is on the knob chain and one SA is on the hole chain. In embodiments, the position of SA1 and SA2 are interchangeable.

[0044] FIGs. 7A-D show illustrative heterodimeric 2-chain complexes with split TM and SA chains, namely the SA on the knob chain of the Fc and the TM on hole chain of the Fc.

[0045] FIGs. 8A-F show illustrative heterodimeric 2-chain complexes with split TM and SA chains, namely with SA on the knob chain of the Fc and both TMs on hole chain of the Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In embodiments, the position of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical.

[0046] FIGs. 9A-J show illustrative heterodimeric 2-chain complexes with split TM and SA chains, namely with SA on the knob chain of the Fc and TM on hole chain of the Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In these orientations and / or configurations, one SA is on the knob chain and one SA is on the hole chain. In embodiments, the position of SA1 and SA2 are interchangeable.

[0047] FIGs. 10A-F show illustrative heterodimeric 2-chain complexes with TM and SA on the same chain, namely the SA and TM both on the knob chain of the Fc.

[0048] FIGs. 11A-L show illustrative heterodimeric 2-chain complexes with a TM and a SA on the same chain, namely with SA and with TM both on the knob chain of the Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In embodiments, the position of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 can be identical.

[0049] FIGs. 12A-L show illustrative heterodimeric 2-chain complexes with a TM and a SA on the same chain, namely with SA and with TM both on the knob chain of the Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In embodiments, the position of SA1 and SA2 are interchangeable.

[0050] FIGs. 13A-F show illustrative heterodimeric 2-chain complexes with TM and SA on the same chain, namely the SA and TM both on the hole chain of the Fc

[0051] FIGs. 14A-L show illustrative heterodimeric 2-chain complexes with a TM and a SA on the same chain, namely with SA and with TM both on the hole chain of the Fc, with two targeting moieties (as described herein, more targeting moieties are present in some embodiments). In embodiments, the position of TM1 and TM2 are interchangeable. In embodiments, TM1 and TM2 can be identical.

[0052] FIGs. 15A-L show illustrative heterodimeric 2-chain complexes with a TM and a SA on the same chain, namely with SA and with TM both on the hole chain of the Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In embodiments, the position of SA1 and SA2 are interchangeable.

[0053] DB1 / 162517863.2 6 ORN-108PC / 114384-5108

[0054] FIGs. 16A-J show illustrative heterodimeric 2-chain complexes with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments) and with SA on knob Fc and TM on each chain. In embodiments, TM1 and TM2 can be identical.

[0055] FIGs. 17A-J show illustrative heterodimeric 2-chain complexes with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments) and with SA on hole Fc and TM on each chain. In embodiments, TM1 and TM2 can be identical.

[0056] FIGs. 18A-F show illustrative heterodimeric 2-chain complexes with two signaling agents (as described herein, more signaling agents may be present in some embodiments) and with split SA and TM chains: SA on knob and TM on hole Fc.

[0057] FIGs. 19A-F show illustrative heterodimeric 2-chain complexes with two signaling agents (as described herein, more signaling agents may be present in some embodiments) and with split SA and TM chains: TM on knob and SA on hole Fc.

[0058] FIG. 20 depicts a schematic structure of an illustrative target-specific IL-6 agonist (“AcTaleukin-6” or“ALN6”), here termed CD20-ALN6.

[0059] FIG. 21 shows IL-6, Bell 10-IL-6 and CD20-IL-6 driven STAT3 activation in MCF7(-CD20) cells.

[0060] FIG. 22 depicts STAT3 activation by the different CD20-ALN6s on MCF7(-CD20) cells.

[0061] FIG. 23 shows STAT3 activation by CD20-I L-6_del3 (3 most C-terminal residues of human I L-6 (R182-Q183-M 184) were deleted) on MCF7(-CD20) cells.

[0062] FIG. 24 depicts C-reactive protein (CRP) levels in the blood plasma of mice from the different treatment groups: PBS; 25 g Belli O-IL-6; 50 pg Bcll10-IL-6; 100 pg Bell 10-IL-6; 25 pg Bell 10-IL-6 A180L; 50 pg Bell 10-IL-6 A180L; 100 pg Belli O-IL-6 A180L; and 200 pg Bel 110-1 L-6 A180L.

[0063] FIGs. 25A-E depict pSTATI activation by the different CD8-Fc-ALN6s on CD8- and CD8+ cells (PBMC). The mutations at the end of each header relate to SEQ ID NO: 431 . The mutation “del 3” indicates a truncation of three C-terminal amino acids.

[0064] FIGs. 26A-E depict pSTAT3 activation by the different CD8-Fc-ALN6s on CD8- and CD8+ cells (PBMC). Mutants are labelled as above.

[0065] FIG. 27 ranks the tested CD8-Fc-ALN6s according to the on / off target pSTAT 1 activity ratio at 100 ng / ml.

[0066] FIG. 28 ranks the tested CD8-Fc-ALN6s according to the on / off target pSTAT3 activity ratio at 100 ng / ml.

[0067] FIG. 29 shows pSTATI (dots) and pSTAT3 (squares) dose response curves comparing the ASG PR-targeted (filled) and the untargeted (open) Fc-based ALN6 mutants on HepG2 cells.

[0068] FIGs. 30A-B depict sensorgrams of ALN6 mutants binding to IL-6Ra.

[0069] FIG. 31 shows melting temperature curves of ALN6 mutants.

[0070] DB1 / 162517863.2 7 ORN-108PC / 114384-5108

[0071] DETAILED DESCRIPTION

[0072] In one aspect, the present invention provides a chimeric protein or a chimeric protein complex (including an Fc- based chimeric protein complex) that includes an interleukin-6 (IL-6), or a mutant thereof.

[0073] In embodiments, a chimeric protein or Fc-based chimeric protein of the present invention comprises a targetspecific IL-6 agonist having loss-of-function site I mutations that be restored upon targeting. In embodiments, the reduced agonistic activity of IL-6 is restored when attached to a targeting moiety, said targeting moiety comprising a recognition domain which specifically binds to an antigen or receptor of interest. In embodiments, the targetspecific IL-6 agonist comprises one or more site I mutations that reduce I L-6 / 1 L-6Ra binding.

[0074] In various embodiments, the present invention provides a chimeric protein or chimeric protein complex, such as Fc-based chimeric protein complexes, that includes an IL-6, or a mutant thereof, fused to one or more targeting moieties. In some embodiments, without limitation, the chimeric proteins or chimeric protein complexes target a mutant IL-6 to hepatocytes, adipose stromal cells, NK cells, dendritic cells, or CD8+T cells. In vivo, such chimeric proteins or chimeric protein complexes can potently promote the response of hepatocytes, adipose stromal cells, NK cells, dendritic cells, or CD8+T cells to antigens, with a significantly reduced toxicity profile compared to wildtype (WT) IL-6.

[0075] In various embodiments, the present chimeric proteins and chimeric protein complexes, including Fc-based chimeric protein complexes find use in the treatment of various diseases or disorders such as cancer, viral infections, immune disorders, autoimmune diseases, liver regeneration, obesity, cardiovascular diseases, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases and many other diseases and disorders, and the present invention encompasses various methods of treatment.

[0076] In some embodiments, the mutant IL-6 is human IL-6. In some embodiments, the mutant IL-6 has low affinity and / or activity for IL-6 receptor (IL-6Ro). In some embodiments, the mutant IL-6 has substantially reduced or ablated affinity and / or activity for IL-6 receptor. In some embodiments, the low affinity or activity of the mutant IL-6 at the IL-6 receptor is restorable by attachment to one or more targeting moieties or upon inclusion in the chimeric protein complex.

[0077] In an embodiment, the wild type IL-6 has the amino acid sequence of:

[0078] IL-6 (wild type) (SEQ ID NO: 1)

[0079] MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYIL DGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLE FEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQ NQWLQDMTTHLILRSFKEFLQSSLRALRQM.

[0080] In some embodiments, the mutant human IL-6 has an amino acid sequence of at least 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1.

[0081] In some embodiments, the human IL-6 has the amino acid sequence of wildtype human IL-6.

[0082] DB1 / 162517863.2 8 ORN-108PC / 114384-5108

[0083] Signal peptide cleavage resulting in mature IL-6 has been reported at various positions including cleavage after amino acid 27, 28 or 29. Most commonly accepted in literature is the cleavage after position 28 resulting in the mature IL-6 of SED ID NO: 431 :

[0084] PVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAEN NLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTK VLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRAL ROM.

[0085] In some embodiments, the mutant IL-6 has one or more substitution mutations at a position selected from Y31, G35, L57, F74, W157, L174, L178, R179, A180, and R182, where the residue numbering is based on SEQ ID NO: 431. In some embodiments, one or more of these mutations produce a modified human IL-6 with reduced binding activity and / or affinity for IL-6 receptor (IL-6Ra) and reduced biological activity. In some embodiments, the mutant human IL-6 has an amino acid sequence of at least 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 431.

[0086] In some embodiments, the mutation comprises a hydrophilic or hydrophobic amino acid residue In embodiments, the hydrophilic amino acid residue is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K) or an aromatic, polar and positively charged hydrophilic including histidine (H). In some embodiments, the hydrophilic amino acid residue is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the hydrophilic amino acid residue is a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E). In some embodiments, the hydrophobic amino acid is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0087] In some embodiments, the mutant IL-6 has one or more substitution mutations selected from Y31D, G35F, L57A, F74E, W157A, W157G, L174V, L178D, L178Q, L178R, R179A, R179F, R179G, R179I, R179K, R179V, A180L, R182A, R182E, R182F, R182G, R182I, R182K, R182V, R182W, R179K_R182G, L178D_Y31 D_G35F, L178D L57A, L178D _W157A, L178D _W157G, W157G_L178R, a truncation of three C-terminal amino acids, and R179K_truncation of three C-terminal amino acids, where the residue numbering is based on SEQ ID NO: 431. In embodiments, the one or more mutations of the mutant IL-6 is selected from L178D, L178D_Y31 D_G35F, L178DJ.57A, L178D_W157A, R179A, R179G, a truncation of three C-terminal amino acids, R179F, R182E, A180L, and L178D_W157G. In embodiments, the mutant IL-6 comprises a truncation of at least one, two, three, four, five, six, seven, eight, nine, or ten C-terminal amino acids, relative to SEQ ID NO: 431 .

[0088] In various embodiments, the mutations allow for IL-6 to have one or more of attenuated activity such as one or more of reduced binding affinity, reduced endogenous activity, and reduced specific bioactivity relative to unmodified or unmutated, i.e., the wild type form of IL-6 (e.g. comparing IL-6 in a wild type form versus a modified (e.g. mutant) form). In some embodiments, the mutations that attenuate or reduce binding activity or affinity include

[0089] DB1 / 162517863.2 9 ORN-108PC / 114384-5108 those mutations that substantially reduce or ablate binding or activity In some embodiments, the mutations that attenuate or reduce binding or affinity are different than those mutations which substantially reduce or ablate binding or activity. Consequentially, in various embodiments, the mutations allow for IL-6 to have improved safety, e.g. have reduced systemic toxicity, reduced side effects, and reduced off-target effects relative to unmutated, i.e. wild type, IL-6 (e.g. comparing IL-6 in a wild type form versus a modified (e.g. mutant) form).

[0090] In various embodiments, IL-6 is modified to have one or more mutations that reduce its binding affinity or activity for one or more of its receptors. In some embodiments, IL-6 is modified to have one or more mutations that substantially reduce or ablate binding affinity or activity for the receptors. In some embodiments, the activity provided by the wild type IL-6 is agonism at the receptor (e.g. activation of a cellular effect at a site of therapy). For example, the wild type IL-6 may activate its receptor. In such embodiments, the mutations result in the modified IL-6 to have reduced or ablated activating activity at the receptor. For example, the mutations may result in the modified IL-6 to deliver a reduced activating signal to a target cell or the activating signal could be ablated.

[0091] In some embodiments, the activity provided by the wild type IL-6 is antagonism at the receptor (e.g. blocking or dampening of a cellular effect at a site of therapy). For example, the wild type IL-6 may antagonize or inhibit the receptor. In these embodiments, the mutations result in the modified IL-6 to have a reduced or ablated antagonizing activity at the receptor. For example, the mutations may result in the modified IL-6 to deliver a reduced inhibitory signal to a target cell or the inhibitory signal could be ablated.

[0092] In some embodiments, the reduced affinity or activity of IL-6 at the receptor is restorable by attachment with one or more of the targeting moieties. In other embodiments, the reduced affinity or activity of IL-6 at the receptor is not substantially restorable by the activity of one or more of the targeting moieties.

[0093] In various embodiments, the chimeric proteins or chimeric protein complexes of the present invention reduce off- target effects because the IL-6 has mutations that weaken or ablate binding affinity or activity at a receptor. In various embodiments, this reduction in side effects is observed relative with, for example, the wild type IL-6. In various embodiments, the IL-6 is active on target cells because the targeting moiety(ies) compensates for the missing / insufficient binding (e.g., without limitation and / or avidity) required for substantial activation. In various embodiments, the modified IL-6 is substantially inactive en route to the site of therapeutic activity and has its effect substantially on specifically targeted cell types that greatly reduces undesired side effects.

[0094] In various embodiments, substantially reducing or ablating binding or activity at the receptor causes the therapeutic effect of IL-6 to improve as there is a reduced or eliminated sequestration of the therapeutic chimeric proteins away from the site of therapeutic action. For instance, in some embodiments, this obviates the need of high doses of the present chimeric protein or chimeric protein complex compositions that compensate for loss at the other receptor. Such ability to reduce dose further provides a lower likelihood of side effects

[0095] In various embodiments, the modified IL-6 comprises one or more mutations that cause IL-6 to have reduced, substantially reduced, or ablated affinity, e.g. binding (e.g. KD) and / or activation. In various embodiments, the reduced affinity at I L-6's receptor allows for attenuation of activity (inclusive of agonism or antagonism). In such

[0096] DB1 / 162517863.2 10 ORN-108PC / 114384-5108 embodiments, the modified IL-6 has about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% of the affinity for the receptor relative to the wild type IL-6. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower relative to the wild type IL-6.

[0097] In various embodiments, the modified IL-6 comprises one or more mutations that reduce the endogenous activity of IL-6 to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1 %, e.g., relative to the wild type IL-6. In some embodiments, the modified IL-6 comprises one or more mutations that cause IL-6 to have reduced affinity for its receptor that is lower than the binding affinity of the targeting moiety(ies) for its(their) receptor(s). In some embodiments, this binding affinity differential is between I L-6 / receptor and targeting moiety / receptor on the same cell. In some embodiments, this binding affinity differential allows for mutant IL-6 to have localized, on-target effects and to minimize off-target effects that underlie side effects that are observed with wild type IL-6. In some embodiments, this binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold lower, or at least about 25-fold, or at least about 50-fold lower, or at least about 100-fold, or at least about 150-fold.

[0098] Receptor binding activity may be measured using methods known in the art For example, affinity and / or binding activity may be assessed by Scatchard plot analysis and computer-fitting of binding data (e.g. Scatchard, 1949) or by reflectometric interference spectroscopy under flow through conditions, as described by Brecht et al. (1993), the entire contents of all of which are hereby incorporated by reference.

[0099] In various embodiments the modified IL-6 comprises an amino acid sequence that has at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known wild type IL-6 (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%,

[0100] DB1 / 162517863.2 11 ORN-108PC / 114384-5108 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).

[0101] In various embodiments the modified IL-6 comprises an amino acid sequence that has at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with any of amino acid sequences of the IL-6 disclosed herein (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity)

[0102] In various embodiments, the modified IL-6 comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and / or non-conservative substitutions.

[0103] “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0104] As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices.

[0105] As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[0106] DB1 / 162517863.2 12 ORN-108PC / 114384-5108

[0107] In various embodiments, the substitutions may also include non-classical amino acids (e.g. selenocysteine, pyrrolysine, A / -formylmethionine [3-alanine, GABA and 6-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D- isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoro-amino acids, designer amino acids such as p methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general).

[0108] In some embodiments, the reduced affinity or activity of the modified IL-6 'at the therapeutic receptor is inducible or restorable by attachment to a targeting moiety or upon inclusion of a targeting moiety in a chimeric protein or a chimeric protein complex, e.g , a Fc-based chimeric protein complex as disclosed herein. In some embodiments, the activity of IL-6 is reduced or attenuated by virtue of its fusion with another protein, including, in some instances, by fusion with targeting moieties as described herein. In other embodiments, the activity of IL-6 is reduced or attenuated by modifying the IL-6, e g., by introducing mutations as described herein. In some embodiments, attenuation of the activity can be restored by attaching the IL-6 to a targeting moiety or by the action of the attached targeting moiety. In embodiments, the targeting moiety— by virtue of its attachment or by its activity— induces IL- 6’s activity. In some embodiments, the reduced affinity or activity at the receptor is inducible or restorable by attachment with one or more of the targeting moieties as described herein or upon inclusion in the chimeric protein complexes, such as Fc-based chimeric protein complex disclosed herein.

[0109] In embodiments, the chimeric protein or the Fc-based chimeric protein complex of the present application comprises a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one of SEQ ID NO: 414-430, 444, and 448-476. In embodiments, the chimeric protein or the Fc-based chimeric protein complex of the present application comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 414-430, 444, and 448-476. In embodiments, the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex comprises a His tag or do not comprise a His tag In embodiments, the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex comprise a leader sequence or do not comprise a leader sequence.

[0110] Targeting Moiety

[0111] In various embodiments, the chimeric protein or chimeric protein complex of the present invention additionally comprise one or more targeting moieties having recognition domains, which specifically bind to a target (e.g. antigen, receptor) of interest. In some embodiments, the chimeric protein or chimeric protein complex may comprise two, three, four, five, six, seven, eight, nine, ten or more targeting moieties. In illustrative embodiments, the chimeric protein or chimeric protein complex of the invention comprise two or more targeting moieties. In such embodiments, the chimeric protein or chimeric protein complex can target two different cells (e.g. to make a synapse) or the same cell (e.g. to get a more concentrated signaling agent effect).

[0112] DB1 / 162517863.2 13 ORN-108PC / 114384-5108

[0113] In various embodiments, the target (e.g. antigen, receptor) of interest can be found on one or more immune cells, which can include, without limitation, T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor or tumor-associated macrophages (e.g. M1 or M2 macrophages), B cells, Breg cells, dendritic cells, or subsets thereof. In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) of interest and effectively, directly or indirectly, recruit one of more immune cells. In some embodiments, the target (e.g. antigen, receptor) of interest can be found on one or more tumor cells. In some embodiments, the chimeric protein or chimeric protein complex may directly or indirectly recruit an immune cell, e.g., in some embodiments, to a therapeutic site (e.g. a locus with one or more disease cell or cell to be modulated for a therapeutic effect). In some embodiments, the chimeric protein or chimeric protein complex may directly or indirectly recruit an immune cell, e.g. an immune cell that can kill and / or suppress a tumor cell, to a site of action (such as, by way of non-limiting example, the tumor microenvironment).

[0114] In some embodiments, the targeting moiety binds to one or more of the follow targets: CD8, CTLA4, CD3, CD4, DNAM-1 , Nrp1 (neurophilin), TNFR2, GITR, ICOS, CD20, CD70, Clec9a, NKp46, asialoglycoprotein receptor (ASGPR), adipose stromal cells (ASC), PD-1, PD-L1 , PD-L2, SIRPIa, FAP, XCR1, tenascin, prostate-specific membrane antigen (PSMA), and ECM proteins.

[0115] In various embodiments, the chimeric protein or chimeric protein complex have targeting moieties having recognition domains, which specifically bind to a target (e.g. antigen, receptor) which 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, including, without limitation, nucleic acids, inclusive of DNA or RNA, such as, for example, DNA released from necrotic tumor cells or extracellular deposits such as cholesterol.

[0116] In some embodiments, the target (e.g. antigen, receptor) of interest is part of the non-cellular component of the stroma or the extracellular matrix (ECM) or the markers associated therewith As used herein, stroma refers to the connective and supportive framework of a tissue or organ. Stroma may include a compilation of cells such as fibroblasts / myofibroblasts, glial, epithelia, fat, immune, vascular, smooth muscle, and immune cells along with the extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target (e.g. antigen, receptor) of interest is part of the non-cellular component of the stroma such as the extracellular matrix and extracellular molecules. As used herein, the ECM refers to the non-cellular components present within all tissues and organs. The ECM is composed of a large collection of biochemically distinct components including, without limitation, proteins, glycoproteins, proteoglycans, and polysaccharides. These components of the ECM are usually produced by adjacent cells and secreted into the ECM via exocytosis. Once secreted, the ECM components often aggregate to form a complex network of macromolecules. In various embodiments, the chimeric protein or chimeric protein complex of the invention comprises a targeting moiety that recognizes a target (e.g., an antigen or receptor or non- proteinaceous molecule) located on any component of the ECM. Illustrative components of the ECM include, without limitation, the proteoglycans, the non-proteoglycan polysaccharides, fibers, and other ECM proteins or

[0117] DB1 / 162517863.2 14 ORN-108PC / 114384-5108

[0118] 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).

[0119] In some embodiments, the targeting moiety recognizes a target (e.g. antigen, receptor) on ECM proteoglycans. Proteoglycans are glycosylated proteins. The basic proteoglycan unit includes 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 attracts water molecules via osmosis, keeping the ECM and resident cells hydrated. Proteoglycans may also help to trap and store growth factors within the ECM. Illustrative proteoglycans that may be targeted by the chimeric protein or chimeric protein complex of the invention include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In an embodiment, the targeting moiety recognizes a target (e.g. antigen, receptor) on non-proteoglycan polysaccharides such as hyaluronic acid.

[0120] 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 collagens or collagen fibers. Collagens are the most abundant proteins in the ECM. Collagens are present in the ECM as fibrillar proteins and provide structural support to resident cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagens present within the ECM including, without limitation, 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 collagen types VI, VII, or XIII. Elastin fibers provide elasticity to tissues, allowing them to stretch when needed and then return to their original state. In some embodiments, the target moiety recognizes one or more epitopes on elastins or elastin fibers.

[0121] In some embodiments, the targeting moiety recognizes one or more ECM proteins including, but not limited to, a tenascin, a fibronectin, a fibrin, a laminin, or a nidogen / entactin.

[0122] In an embodiment, 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 structures of tenascin proteins include several common motifs ordered in the same consecutive 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 EGF-like and fibronectin type III repeats. Isoform variants also exist particularly with respect to tenascin-C Over 27 splice variants and / or isoforms of tenascin-C are known. In a particular embodiment, the targeting moiety recognizes and binds to tenascin-CA1 . Similarly, tenascin-R also has various splice variants and isoforms Tenascin-R usually exists as dimers or trimers. Tenascin-X is the largest member of the tenascin family and is known to exist as trimers. Tenascin-W exists as trimers. In some embodiments, the targeting moiety recognizes one or more epitopes on a tenascin protein. In some embodiments, the targeting moiety recognizes the monomeric and / or the dimeric and / or the trimeric and / or the hexameric forms of a tenascin protein.

[0123] In an embodiment, the targeting moieties recognize and bind to fibronectin. Fibronectins are glycoproteins that connect cells with collagen fibers in the ECM, allowing cells to move through the ECM. Upon binding to integrins,

[0124] DB1 / 162517863.2 15 ORN-108PC / 114384-5108 fibronectins unfolds to form functional dimers In some embodiments, the targeting moiety recognizes the monomeric and / or the dimeric forms of fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. In illustrative embodiments, the targeting moiety recognizes fibronectin extracellular domain A (EDA) or fibronectin extracellular domain B (EDB). Elevated levels of EDA are associated with various diseases and disorders including psoriasis, rheumatoid arthritis, diabetes, and cancer. In some embodiments, the targeting moiety recognizes fibronectin that contains the EDA isoform and may be utilized to target the chimeric protein or chimeric protein complex to diseased cells including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin that contains the EDB isoform. In various embodiments, such targeting moieties may be utilized to target the chimeric protein or chimeric protein complex to tumor cells including the tumor neovasculature.

[0125] In an embodiment, the targeting moiety recognizes and binds to fibrin. Fibrin is 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 causes the fibrin to polymerize. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes the monomeric as well as the polymerized forms of fibrin.

[0126] In an embodiment, the targeting moiety recognizes and binds to laminin Laminin is a major component of the basal lamina, which is a protein network foundation for cells and organs Laminins are heterotrimeric proteins that contain an a-chain, a [3-chain, and a y-chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes the monomeric, the dimeric as well as the trimeric forms of laminin.

[0127] In an embodiment, the targeting moiety recognizes and binds to a nidogen or entactin. Nidogens / entactins are a family of highly conserved, sulfated glycoproteins. They make up the major structural component of the basement membranes and function to link laminin and collagen IV networks 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.

[0128] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an epitope present on any of the targets described herein In an embodiment, the antigen-recognition domain recognizes one or more linear epitopes present on the protein. As used herein, a linear epitope refers to any continuous sequence of amino acids present on the protein. In another embodiment, the antigen-recognition domain recognizes one or more conformational epitopes present on the protein. As used herein, a conformation epitope refers to one or more sections of amino acids (which may be discontinuous) which form a three-dimensional surface with features and / or shapes and / or tertiary structures capable of being recognized by an antigen recognition domain.

[0129] In various embodiments, the targeting moiety may bind to the full-length and / or mature forms and / or isoforms and / or splice variants and / or fragments and / or any other naturally occurring or synthetic analogs, variants, or mutants of any of the targets described herein. In various embodiments, the targeting moiety may bind to any forms

[0130] DB1 / 162517863.2 16 ORN-108PC / 114384-5108 of the proteins described herein, including monomeric, dimeric, trimeric, tetrameric, heterodimeric, multimeric and associated forms. In various embodiments, the targeting moiety may bind to any post-transl ationally modified forms of the proteins described herein, such as glycosylated and / or phosphorylated forms.

[0131] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes extracellular molecules such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In an embodiment, the DNA is shed into the extracellular space from necrotic or apoptotic tumor cells or other diseased cells.

[0132] 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. The fibro-lipid (fibro-fatty) plaque is characterized by an accumulation of lipid-laden cells underneath the intima of the arteries Beneath the endothelium there is a fibrous cap covering the atheromatous core of the plaque. The core includes lipid-laden cells (macrophages and smooth muscle cells) with elevated tissue cholesterol and cholesterol ester content, fibrin, proteoglycans, collagen, elastin, and cellular debris In advanced plaques, the central core of the plaque usually contains extracellular cholesterol deposits (released from dead cells), which form areas of cholesterol crystals with empty, needle-like clefts At the periphery of the plaque are younger foamy cells and capillaries. A fibrous plaque is also localized under the intima, within the wall of the artery resulting in thickening and expansion of the wall and, sometimes, spotty localized narrowing of the lumen with some atrophy of the muscular layer. The fibrous plaque contains collagen fibers (eosinophilic), precipitates of calcium (hematoxylinophilic) and lipid-laden cells. In some embodiments, the targeting moiety recognizes and binds to one or more of the non-cellular components of these plaques such as the fibrin, proteoglycans, collagen, elastin, cellular debris, and calcium or other mineral deposits or precipitates. In some embodiments, the cellular debris is a nucleic acid, e.g. DNA or RNA, released from dead cells.

[0133] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures found in the brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures located in the amyloid plaques found in the brains of patients with Alzheimer’s disease. For example, the targeting moiety may recognize and bind to the peptide amyloid beta, which is a major component of the amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures located in the brains 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.

[0134] In some embodiments, the chimeric protein or chimeric protein complex of the invention may have two or more targeting moieties that bind to non-cellular structures. In some embodiments, there are two targeting moieties and one targets a cell while the other targets a non-cellular structure In various embodiments, the targeting moieties can directly or indirectly recruit cells, such as disease cells and / or effector cells. In some embodiments, chimeric

[0135] DB1 / 162517863.2 17 ORN-108PC / 114384-5108 protein or chimeric protein complex are capable of, or find use in, methods involving, shifting the balance of immune cells in favor of immune attack of a tumor For instance, chimeric protein or chimeric protein complex can shift the ratio of immune cells at a site of clinical importance in favor of cells that can kill and / or suppress a tumor (e.g. T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g. M1 macrophages), B cells, dendritic cells, or subsets thereof) and in opposition to 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, chimeric protein or chimeric protein complex are capable of increasing a ratio of effector T cells to regulatory T cells.

[0136] For example, in some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with T cells. In some embodiments, the recognition domains directly or indirectly recruit T cells. In an embodiment, the recognition domains specifically bind to effector T cells. In some embodiments, the recognition domain directly or indirectly recruits effector T cells, e.g., in some embodiments, to a therapeutic site (e.g. a locus with one or more disease cell or cell to be modulated for a therapeutic effect). Illustrative effector T cells include cytotoxic T cells (e.g. 0 TCR, CD3", CD8+, CD45RO"); CD4" effector T cells (e.g. a TCR, CD3+, CD4", CCR7+, CD62Lhi, IL 7R / CD127*) CD8" effector T cells (e.g. a|3 TCR, CD3", CD8", CCR7", CD62Lhi, IL7R / CD127"); effector memory T cells (e.g. CD62Llow, CD44", TCR, CD3", IL7R / 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; CD8" effector memory T cells (TEM) including early effector memory T cells (CD27" CD62L-) and late effector memory T cells (CD27- CD62Lj (TemE and TemL, respectively); CD127(")CD25(low / -) effector T cells; CD127( CD25(-) effector T cells; CD8" stem cell memory effector cells (TSCM) (e.g. T cells; CD4"CD45RO"CCR7( ) effector T cells; and effector T cells secreting IL-2, IL-4 and / or IFN-y.

[0137] Illustrative T cell antigens of interest include, for example (and inclusive of the extracellular domains, where applicable): CD8, CD3, SLAMF4, IL-2Ra, 4-1 BB / TNFRSF9, IL-2 R 0, ALCAM, B7-1, IL-4 R, B7-H3, BLAME / SU\MFS, CEACAM1 , IL-6 R, CCR3, IL-7 Ra, CCR4, CXCRI / IL-S RA, CCR5, CCR6, IL-10R a, CCR 7, IL- I 0 R 0, CCRS, IL-12 R 0 1 , CCR9, IL-12 R 0 2, CD2, IL-13 R a 1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrin a 4 / CD49d, CDS, Integrin a E / CD103, CD6, Integrin a M / CD 11 b, CDS, Integrin a X / CD11c, Integrin 0 2 / CDIS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 Ligand / TNFSF5, LAG-3, CD43, LAIR1 , CD45, LAIR2, CDS3, Leukotriene B4-R1 , CDS4 / SLAMF5, NCAM-L1 , CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, Common y Chain / IL-2 R y, Osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1 , L-

[0138] DB1 / 162517863.2 18 ORN-108PC / 114384-5108

[0139] Selectin, CXCR3, SIRP 1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, Thymopoietin, EMMPRIN / CD147, TIM-1 , EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas Ligand / TNFSF6, TIM-4, Fey RIII / CD16, TIM-6, TNFR1 / TNFRSF1A, Granulysin, TNF RIII / TNFRSF1B, TRAIL RI / TNFRSFIOA, ICAM-1 / CD54, TRAIL R2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C,IFN-yR1, TRAILR4 / TNFRSF10D, IFN-y R2, TSLP, IL-1 R1 and TSLP R. In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these illustrative T cell antigens

[0140] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex have a targeting moiety directed against a checkpoint marker expressed on a T cell, e.g. one or more of PD-1 , CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, 0X40, CD27, CD40L, TIM3, and A2aR.

[0141] For example, in some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with B cells In some embodiments, the recognition domains directly or indirectly recruit B cells, e.g., in some embodiments, to a therapeutic site (e g. a locus with one or more disease cell or cell to be modulated for a therapeutic effect). Illustrative 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, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these illustrative B cell antigens

[0142] By way of further example, in some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with Natural Killer cells. In some embodiments, the recognition domains directly or indirectly recruit Natural Killer cells, e.g. , in some embodiments, to a therapeutic site (e.g. a locus with one or more disease cell or cell to be modulated for a therapeutic effect). Illustrative 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-y RI / CD64, MICA, Fc-y RIIB / CD32b, MICB, Fc-y RIIC / CD32c, MULT-1 , Fc-y RIIA / CD32a, Nectin-2 / CD112, Fc-y RIII / CD16, NKG2A, FcRH 1 / 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-1 a, Rae-1 , Rae-1 delta, H60, Rae-1 epsilon, ILT2 / CD85j, Rae-1 y, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1 , ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD 158d and ULBP-3. In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these illustrative NK cell antigens.

[0143] Also, in some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with macrophages / monocytes. In some embodiments, the recognition domains directly or indirectly recruit macrophages / monocytes, e.g., in some embodiments, to a therapeutic site (e.g. a locus with one or more disease cell or cell to be modulated for a therapeutic effect). Illustrative macrophages / monocyte antigens of interest include, for example SIRPI a, B7-1 / CD80, ILT4 / CD85d, B7-H1 , ILT5 / CD85a, Common Chain, Integrin a 4 / CD49d,

[0144] DB1 / 162517863.2 19 ORN-108PC / 114384-5108

[0145] BLAME / SLAMF8, Integrin a X / CDIIc, CCL6 / C10, Integrin 0 2 / CD18, CD155 / PVR, Integrin 0 3 / CD61 , CD31 / PECAM-1, Latexin, CD36 / SR-B3, Leukotriene B4 R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMI R5 / 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-y RI / CD64, Osteopontin, Fc-y RIIB / CD32b, 4 / CD49d, CCR5, Integrin a M / CDII b, CCR8, Integrin a X / CDIIc, CD155 / PVR, Integrin 0 2 / CD18, CD14, Integrin 0 3 / 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 II l / Tissue Factor, LMIR5 / CD300LB, CX3CR1 , CX3CL1 , LMIR6 / CD300LE, CXCR4, LRP-1 , CXCR6, M-CSF R, DEP-1 / CD148, MD-1 , DNAM-1 , MD-2, EMMPRIN / CD147, MMR, Endoglin / CD105, NCAM-L1 , Fc-y RI / CD64, PSGL-1, Fc-y RIIIICD16, RP105, G-CSF R, L-Selectin, GM-CSF R a, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1 , ICAM- 2 / CD102, TREM-I, IL-6 R, TREM-2, CXCRI / IL-8 RA, TREM-3 and TREMLI / TLT-1 . In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these illustrative macrophage / monocyte antigens.

[0146] Also, in some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with dendritic cells. In some embodiments, the recognition domains directly or indirectly recruit dendritic cells, e.g., in some embodiments, to a therapeutic site (e.g. a locus with one or more disease cell or cell to be modulated for a therapeutic effect). Illustrative 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-PI / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1 , TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB Ligand / TN FSF9, IL-12 / IL-23 p40, 4- Amino-1,8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin a 4 / CD49d, Aag, Integrin 0 2 / CD18, AMICA, Langerin, B7-2 / CD86, Leukotriene B4 Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LM I R2 / CD300c, Clq R1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LB CCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1 , CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SU\MF5, MMR, CD97, NCAMLI, CD2F-10 / SLAMF9, Osteoactivin GPNMB, Chern 23, PD- L2, CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC- SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, DEP-1 / CD148, SIGNR4, DLEC / CLEC4C, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-y R1 / CD64, TLR3, Fc-y RIIB / CD32b, TREM-1 , Fc-y RIIC / CD32c, TREM-2, Fc-y RIIA / CD32a, TREM-3, Fc-y RIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102 and Vanilloid R1. In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these illustrative DC antigens.

[0147] DB1 / 162517863.2 20 ORN-108PC / 114384-5108

[0148] In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) on immune cells selected from, but not limited to, megakaryocytes, thrombocytes, erythrocytes, mast cells, basophils, neutrophils, myeloid cells, monocytes, eosinophils, or subsets thereof. In some embodiments, the recognition domains directly or indirectly recruit megakaryocytes, thrombocytes, erythrocytes, mast cells, basophils, neutrophils, myeloid cells, monocytes, eosinophils, or subsets thereof, e.g., in some embodiments, to a therapeutic site (e.g. a locus with one or more disease cell or cell to be modulated for a therapeutic effect) In some embodiments, the immune cell is selected from a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, a mast cell, a monocyte, a red blood cell, myeloid cell, myeloid derived suppressor cell, a NKT cell, and a NK cell, or derivatives thereof.

[0149] In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with megakaryocytes and / or thrombocytes. Illustrative megakaryocyte and / or thrombocyte antigens of interest include, for example, GP llb / llla, GPIb, vWF, PF4, and TSP. In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these illustrative megakaryocyte and / or thrombocyte antigens

[0150] In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with erythrocytes. Illustrative erythrocyte antigens of interest include, for example, CD34, CD36, CD38, CD41 a (platelet glycoprotein llb / llla), CD41 b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA- DR, H2 (MHC-II), and Rhesus antigens. In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these illustrative erythrocyte antigens.

[0151] In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with mast cells. Illustrative mast cells antigens of interest include, for example, SCFR / CD117, FcERI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMAI, FCERIA, FCER2, TPSABI. In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these mast cell antigens.

[0152] In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with basophils. Illustrative basophils antigens of interest include, for example, FceRI, CD203c, CD123, CD13, CD107a, CD107b, and CD164. In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these basophil antigens.

[0153] In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with neutrophils. Illustrative neutrophils 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, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these neutrophil antigens.

[0154] In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with eosinophils. Illustrative eosinophils antigens of interest include, for example, CD35, CD44 and CD69 In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these eosinophil antigens.

[0155] DB1 / 162517863.2 21 ORN-108PC / 114384-5108

[0156] In various embodiments, the recognition domain may bind to any appropriate target, antigen, receptor, or cell surface markers known by the skilled artisan. In some embodiments, the antigen or cell surface marker is a tissuespecific marker. Illustrative tissue-specific markers include, but are not limited to, endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAMI, PROCR, SELE, SELP, TEK, THBD, VCAMI, VWF; smooth muscle cell surface markers such as ACTA2, MYHIO, MYHI 1 , MYH9, MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COLIAI, COL1A2, COL3A1, FAP, PH-4; epithelial cell surface markers such as CDID, K6IRS2, KRTIO, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCI, TACSTDI; neovasculature markers such as CD13, TFNA, Alpha-v beta-3 (avPs), E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN. In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these antigens. In various embodiments, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of cells having these antigens

[0157] In some embodiments, the recognition domains specifically bind to a target (e.g. antigen, receptor) associated with tumor cells. In some embodiments, the recognition domains directly or indirectly recruit tumor cells. For instance, in some embodiments, the direct or indirect recruitment of the tumor cell is to one or more effector cell (e.g. an immune cell as described herein) that can kill and / or suppress the tumor cell

[0158] Tumor cells or cancer cells refer to an uncontrolled growth of cells or tissues and / or an abnormal increase in cell survival and / or inhibition of apoptosis, which interferes with the normal functioning of bodily organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Illustrative tumor cells include, but are not limited to cells of: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal

[0159] DB1 / 162517863.2 22 ORN-108PC / 114384-5108 vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome.

[0160] Tumor cells, or cancer cells also include, but are not limited to, carcinomas, e g. 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 and non-Hodgkin lymphomas, light chain, non-secretory, MGUS, and plasmacytomas), and central nervous system cancers (including, for example, brain (e.g. gliomas (e.g. astrocytoma, oligodendroglioma, and ependymoma), meningioma, pituitary adenoma, and neuromas, and spinal cord tumors (e.g. meningiomas and neurofibroma).

[0161] Illustrative tumor antigens include, but are not limited to, MART-1 / Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal 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, MAGE-family of tumor antigens (e.g., MAGE-A1 , MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE- A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1 , MAGE- 02, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1 , GAGE-2, GAGE-3, GAGE- 4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21 ras, RCAS1, o-fetoprotein, E-cadherin, a-catenin, |3-catenin and y- catenin, p120ctn, gp100 Pmell 17, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, Imp-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-1 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, a targeting moiety of the chimeric protein or chimeric protein complex binds one or more of these tumor antigens. In an embodiment, the chimeric protein or chimeric protein complex binds to HER2. In another embodiment, the chimeric protein or chimeric protein complex binds to PD-L2.

[0162] In some embodiments, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have (i) one or more of the targeting moieties which is directed against an immune cell selected from a T cell, a B cell, a dendritic cell, a macrophage, a NK cell, or subsets thereof and (ii) one or more of the targeting moieties which is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or a variant thereof) described herein. In one embodiment, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have (i) a targeting moiety directed against a T cell (including, without limitation an effector T cell) and (ii) a targeting moiety is directed against a tumor cell, along with any of the

[0163] DB1 / 162517863.2 23 ORN-108PC / 114384-5108 signaling agents described herein. In one embodiment, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have (i) a targeting moiety directed against a B cell and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents described herein. In one embodiment, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have (i) a targeting moiety directed against a dendritic cell and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents described herein. In one embodiment, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have (i) a targeting moiety directed against a macrophage and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents described herein. In one embodiment, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have (i) a targeting moiety directed against a NK cell and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents described herein.

[0164] By way of non-limiting example, in various embodiments, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have (i) a targeting moiety directed against a T cell, for example, mediated by targeting to CD8, SLAMF4, IL-2 R a, 4-1 BB / TNFRSF9, IL-2 R , ALCAM, B7-1, IL-4 R, B7- H3, BLAME / SLAMFS, CEACAM1, IL-6 R, CCR3, IL-7 Ra, CCR4, CXCRI / IL-S RA, CCR5, CCR6, IL-10R a, CCR 7, IL-1 0 R p, CCRS, IL-12 R 1, CCR9, IL-12 R 2, CD2, IL-13 R a 1 , IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrin a 4 / CD49d, CDS, Integrin a E / CD103, CD6, Integrin a M / CD 11 b, CDS, Integrin a X / CD11c, Integrin 0 2 / CDIS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1 , CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 Ligand / TNFSF5, LAG-3, CD43, LAIR1 , CD45, LAIR2, CDS3, Leukotriene B4-R1 , CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, Common y Chain / IL-2 R y, Osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1 , L- Selectin, CXCR3, SIRP 0 1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, Thymopoietin, EMMPRIN / CD147, TIM-1 , EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas Ligand / TNFSF6, TIM-4, Fey RIII / CD16, TIM-6, TNFR1 / TNFRSF1A, Granulysin, TNF RIII / TNFRSF1B, TRAIL RI / TNFRSFIOA, ICAM-1 / CD54, TRAIL R2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF1 OCJFN-yRI , TRAILR4 / TNFRSF10D, IFN-y R2, TSLP, IL-1 R1, or TSLP R; and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or a variant thereof) described herein.

[0165] By way of non-limiting example, in various embodiments, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have a targeting moiety directed against (i) a checkpoint marker expressed on a T cell, e.g. one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, 0X40, CD27, CD40L, TIM3, and A2aR and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents described herein.

[0166] In various embodiments, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have one or more targeting moieties directed against PD-1. In some embodiments, the chimeric protein or chimeric protein complex have one or more targeting moieties, which selectively bind a PD-1 polypeptide.

[0167] DB1 / 162517863.2 24 ORN-108PC / 114384-5108

[0168] In some embodiments, the chimeric protein or chimeric protein complex comprise one or more antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind a PD-1 polypeptide.

[0169] In an embodiment, the targeting moiety comprises the anti-PD-1 antibody, nivolumab (aka BMS-936558, MDX- 1106, ONO-4538, OPDIVO), or fragments thereof. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US 8,008,449 and WO 2006 / 121168, the entire disclosures of which are hereby incorporated by reference In illustrative embodiments, nivolumab or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0170] In an embodiment, the targeting moiety comprises the anti-PD-1 antibody pidilizumab (aka CT-011 , hBAT or hBAT- 1), or fragments thereof. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in US 2008 / 0025980 and WO 2009 / 101611 , the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable regions comprising an amino acid sequence selected from SEQ ID NOS: 15-18 of US 2008 / 0025980: SEQ ID No: 15 of US 2008 / 0025980 (SEQ ID NO: 11); SEQ ID No: 16 of US 2008 / 0025980(SEQ ID NO: 12);SEQ ID No: 17 of US 2008 / 0025980 (SEQ ID NO: 13); and SEQ ID No: 18 of US 2008 / 0025980 (SEQ ID NO: 14) ;and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOS: 20-24 of US 2008 / 0025980: SEQ ID No: 20 of US 2008 / 0025980 (SEQ ID NO: 15); SEQ ID No: 21 of US 2008 / 0025980 (SEQ ID NO: 16); SEQ ID No: 22 of US 2008 / 0025980 (SEQ ID NO: 17); SEQ ID No: 23 of US 2008 / 0025980 (SEQ ID NO: 18); and SEQ ID No: 24 of US 2008 / 0025980 (SEQ ID NO: 19).

[0171] In an embodiment, the targeting moiety comprises a light chain comprising SEQ ID NO: 18 of US 2008 / 0025980 (SEQ ID NO: 14) and a heavy chain comprising SEQ ID NO: 22 of US 2008 / 0025980 (SEQ ID NO: 17).

[0172] In an embodiment, the targeting moiety comprises AMP-514 (aka MEDI-0680).

[0173] In an embodiment, the targeting moiety comprises the PD-L2-Fc fusion protein AMP-224, which is disclosed in W02010 / 027827 and WO 2011 / 066342, the entire disclosures of which are hereby incorporated by reference. In such an embodiment, the targeting moiety may include a targeting domain which comprises SEQ ID NO:4 of W02010 / 027827 (SEQ ID NO :20) and / or the B7-DC fusion protein which comprises SEQ ID NO:83 of W02010 / 027827 (SEQ ID NO: 21).

[0174] In an embodiment, the targeting moiety comprises the peptide AUNP 12 or any of the other peptides disclosed in US 2011 / 0318373 or 8,907,053. For example, the targeting moiety may comprise AUNP 12 (i.e., Compound 8 or SEQ ID NO:49 of US 2011 / 0318373) which has the sequence of:

[0175] DB1 / 162517863.2 25 ORN-108PC / 114384-5108

[0176] SEQ ID NO: 22).

[0177] In an embodiment, the targeting moiety comprises the anti-PD-1 antibody 1 E3, or fragments thereof, as disclosed in US 2014 / 0044738, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 1 E3 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

[0178] In an embodiment, the targeting moiety comprises the anti-PD-1 antibody 1 E8, or fragments thereof, as disclosed in US 2014 / 0044738, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 1 E8 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

[0179] In an embodiment, the targeting moiety comprises the anti-PD-1 antibody 1 H3, or fragments thereof, as disclosed in US 2014 / 0044738, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 1 H3 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 light chain variable region comprising the amino acid sequence of SEQ ID NO: 28

[0180] In an embodiment, the targeting moiety comprises a VHH directed against PD-1 as disclosed, for example, in US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the VHHs against PD-1 comprise SEQ ID NOS: 347-351 of US 8,907,065 (SEQ ID No: 347 of US 8,907,065 (SEQ ID NO: 29); SEQ ID No: 348 of US 8,907,065 (SEQ ID NO:30); SEQ ID No: 349 of US 8,907,065 (SEQ ID NO:31); SEQ ID No: 350 of US 8,907,065 (SEQ ID NO:32); and SEQ ID No: 351 of US 8,907,065 (SEQ ID NO:33).

[0181] In an embodiment, the targeting moiety comprises any one of the anti-PD-1 antibodies, or fragments thereof, as disclosed in US2011 / 0271358 and WQ2010 / 036959, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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: 25-29

[0182] DB1 / 162517863.2 26 ORN-108PC / 114384-5108 of US2011 / 0271358 (SEQ ID No: 25 of US2011 / 0271358 (SEQ ID NO:34); SEQ ID No: 26 of US2011 / 0271358 (SEQ ID NO:35); SEQ ID No: 27 of US2011 / 0271358 (SEQ ID NO:36); SEQ ID No: 28 of US2011 / 0271358 (SEQ ID NO:37);and SEQ ID No: 29 of US2011 / 0271358 (SEQ ID NO:38));and / or a light chain comprising an amino acid sequence selected from SEQ ID NOS: 30-33 of US2011 / 0271358 (SEQ ID No: 30 of US2011 / 0271358(SEQ ID NO :39); SEQ ID No: 31 of US2011 / 0271358 (SEQ ID NO :40); SEQ ID No: 32 of US2011 / 0271358 (SEQ ID NO:41); and SEQ ID No: 33 of US2011 / 0271358 (SEQ ID NO:42)).

[0183] In various embodiments, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes comprise one or more antibodies directed against PD-1 , or antibody fragments thereof, selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene Ltd.)

[0184] In various embodiments, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have one or more targeting moieties directed against PD-L1. In some embodiments, the chimeric proteins or chimeric protein complexes have one or more targeting moieties, which selectively bind a PD- L1 polypeptide. In some embodiments, the chimeric proteins or chimeric protein complexes comprise one or more antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind a PD- L1 polypeptide.

[0185] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (aka durvalumab), or fragments 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 comprises 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 WQ / 2016 / 06272, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, 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).

[0186] In illustrative embodiments, the M EDI4736 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 of WQ / 2016 / 06272 (SEQ ID NO:45); and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 of WQ / 2016 / 06272 (SEQ ID NO:46).

[0187] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (aka MPDL3280A, RG7446), or fragments thereof. In illustrative embodiments, atezolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO:47); and / or a light chain comprising the amino acid sequence of (SEQ ID NO:48).

[0188] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody avelumab (aka MSB0010718C), or fragments thereof In illustrative embodiments, avelumab or an antigen-binding fragment thereof for use in the

[0189] DB1 / 162517863.2 27 ORN-108PC / 114384-5108 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).

[0190] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559 (aka 12A4, MDX-1105), or fragments thereof, as disclosed in US 2013 / 0309250 and WQ2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, BMS-936559 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of: (SEQ ID NO:51); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:52).

[0191] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3G10, or fragments thereof, as disclosed in US 2013 / 0309250 and WQ2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 3G10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 53); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 54).

[0192] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10A5, or fragments thereof, as disclosed in US 2013 / 0309250 and W02007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 10A5 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 55); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 56).

[0193] In an embodiment, the targeting moiety comprises the anti-PD-L 1 antibody 5F8, or fragments thereof, as disclosed in US 2013 / 0309250 and W02007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 5F8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 57); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 58).

[0194] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10H10, or fragments thereof, as disclosed in US 2013 / 0309250 and WQ2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 10H10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 59); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 60).

[0195] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1 B12, or fragments thereof, as disclosed in US 2013 / 0309250 and WQ2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 1 B12 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).

[0196] DB1 / 162517863.2 28 ORN-108PC / 114384-5108

[0197] In an embodiment, the targeting moiety comprises the an ti-PD-L 1 antibody 7H 1 , or fragments thereof, as disclosed in US 2013 / 0309250 and W02007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 7H1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 63); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 64).

[0198] In an embodiment, the targeting moiety comprises the anti-PD-L 1 antibody 11 E6, or fragments thereof, as disclosed in US 2013 / 0309250 and W02007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 11 E6 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).

[0199] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 12B7, or fragments thereof, as disclosed in US 2013 / 0309250 and WQ2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 12B7 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 67); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 68).

[0200] In an embodiment, the targeting moiety comprises the anti-PD-L 1 antibody 13G4, or fragments thereof, as disclosed in US 2013 / 0309250 and WQ2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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).

[0201] In an embodiment, the targeting moiety comprises the anti-PD-L 1 antibody 1 E12, or fragments thereof, as disclosed in US 2014 / 0044738, the entire disclosures of which are hereby incorporated by reference In illustrative embodiments, 1 E12 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).

[0202] In an embodiment, the targeting moiety comprises the anti-PD-L 1 antibody 1 F4, or fragments thereof, as disclosed in US 2014 / 0044738, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 1 F4 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).

[0203] In an embodiment, the targeting moiety comprises the anti-PD-L 1 antibody 2G11, or fragments thereof, as disclosed in US 2014 / 0044738, the entire disclosures of which are hereby incorporated by reference In illustrative embodiments, 2G11 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 75); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 76).

[0204] DB1 / 162517863.2 29 ORN-108PC / 114384-5108

[0205] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3B6, or fragments thereof, as disclosed in US 2014 / 0044738, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 3B6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 77); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 78).

[0206] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3D10, or fragments thereof, as disclosed in US 2014 / 0044738 and WO2012 / 145493, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 3D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 79); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 80).

[0207] In an embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in US2011 / 0271358 and WO2010 / 036959, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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: 34-38 of US2011 / 0271358 (SEQ ID No: 34 of US2011 / 0271358 (SEQ ID NO: 81); SEQ ID No: 35 of US2011 / 0271358 (SEQ ID NO: 82); SEQ ID No: 36 of US2011 / 0271358 (SEQ ID NO: 83); SEQ ID No: 37 of US2011 / 0271358 (SEQ ID NO: 84); and SEQ ID No: 38 of US2011 / 0271358 (SEQ ID NO: 85)); and / or a light chain comprising an amino acid sequence selected from SEQ ID Nos: 39-42 of US2011 / 0271358 (SEQ ID No: 39 of US2011 / 0271358 (SEQ ID NO: 86); SEQ ID No: 40 of US2011 / 0271358 (SEQ ID NO: 87); SEQ ID No: 41 of US2011 / 0271358 (SEQ ID NO: 88); and SEQ ID No: 42 of US2011 / 0271358(SEQ ID NO: 89)).

[0208] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4, or fragments thereof, as disclosed in WO 2011 / 066389, US8,779,108, and US2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.7A4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 2 of WO 2011 / 066389 (SEQ ID NO: 90); and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 7 of WO 2011 / 066389 (SEQ ID NO: 91).

[0209] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.9D10, or fragments thereof, as disclosed in WO 2011 / 066389, US8,779,108, and US2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.9D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 12 of WO 2011 / 066389 (SEQ ID NO: 92); and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 17 of WO 2011 / 066389 (SEQ ID NO: 93).

[0210] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9, or fragments thereof, as disclosed in WO 2011 / 066389, US8,779,108, and US2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.14H9 or an antigen-binding fragment thereof for use in

[0211] DB1 / 162517863.2 30 ORN-108PC / 114384-5108 the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 22 of WO 2011 / 066389 (SEQ ID NO: 94); and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 27 of WO 2011 / 066389 (SEQ ID NO: 95).

[0212] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.20A8, or fragments thereof, as disclosed in WO 2011 / 066389, US8,779,108, and US2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.20A8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 32 of WO 2011 / 066389 (SEQ ID NO: 96); and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 37 of WO 2011 / 066389 (SEQ ID NO: 97).

[0213] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.15G8, or fragments thereof, as disclosed in WO 2011 / 066389, US8,779,108, and US2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 3.15G8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 42 of WO 2011 / 066389 (SEQ ID NO: 98); and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 47 of WO 2011 / 066389 (SEQ ID NO: 99).

[0214] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.18G1 , or fragments thereof, as disclosed in WO 2011 / 066389, US8,779,108, and US2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 3.18G1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 52 of WO 2011 / 066389 (SEQ ID NO: 100); and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 57 of WO 2011 / 066389 (SEQ ID NO: 101).

[0215] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4OPT, or fragments thereof, as disclosed in WO 2011 / 066389, US8.779, 108, and US2014 / 0356353, and US2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 of WO 2011 / 066389 (SEQ ID NO:102); and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 67 of WO 2011 / 066389 (SEQ ID NO: 103).

[0216] In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9OPT, or fragments thereof, as disclosed in WO 2011 / 066389, US8,779,108, and US2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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 of WO 2011 / 066389 (SEQ ID NO: 104); and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 77 of WO 2011 / 066389 (SEQ ID NO: 105).

[0217] In an embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WQ2016 / 061142, the entire contents of which are hereby incorporated by reference. In illustrative embodiments,

[0218] DB1 / 162517863.2 31 ORN-108PC / 114384-5108 the antibody or an 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: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of W02016 / 061142 (SEQ ID No: 18 of WO2016 / 061142 (SEQ ID NO:106); SEQ ID No: 30 of WQ2016 / 061142 (SEQ ID NO: 107); SEQ ID No: 38 of WQ2016 / 061142 (SEQ ID NQ:108); SEQ ID No: 46 of WO2016 / 061142 (SEQ ID NQ:109); SEQ ID No: 50 of WQ2016 / 061142 (SEQ ID NO:110); SEQ ID No: 54 of WQ2016 / 061142 (SEQ ID NO:111); SEQ ID No: 62 of WQ2016 / 061142 (SEQ ID NO:112); SEQ ID No: 70 of WQ2016 / 061142 (SEQ ID NO:113); and SEQ ID No: 78 of WQ2016 / 061142 (SEQ ID NO:114)); and / or a light chain comprising an amino acid sequence selected from SEQ ID Nos: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WQ2016 / 061142 (SEQ ID No: 22 of WQ2016 / 061142 (SEQ ID NO:115); SEQ ID No: 26 of WQ2016 / 061142 (SEQ ID NO:116); SEQ ID No: 34 of WQ2016 / 061142 (SEQ ID NO: 117); SEQ ID No: 42 of WQ2016 / 061142 (SEQ ID NO:118);SEQ ID No: 58 of WQ2016 / 061142 (SEQ ID NO:119); SEQ ID No: 66 of WQ2016 / 061142(SEQ ID NQ:120); SEQ ID No: 74 of WQ2016 / 061142 (SEQ ID NO: 121 ); SEQ ID No: 82 of WQ2016 / 061142 (SEQ ID NO: 122); and SEQ ID No: 86 of W02016 / 061142 (SEQ ID NO:123)).

[0219] In an embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WQ2016 / 022630, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WQ2016 / 022630 ( SEQ ID No: 2 of WQ2016 / 022630 (SEQ ID NO: 124); SEQ ID No: 6 of WQ2016 / 022630 (SEQ ID NO: 125); SEQ ID No: 10 of WQ2016 / 022630 (SEQ ID NO: 126); SEQ ID No: 14 of WQ2016 / 022630 (SEQ ID NO:127); SEQ ID No: 18 of WQ2016 / 022630 (SEQ ID NO:128); SEQ ID No: 22 of WQ2016 / 022630 (SEQ ID NO:129); SEQ ID No: 26 of WQ2016 / 022630 (SEQ ID NO: 130); SEQ ID No: 30 of WQ2016 / 022630 (SEQ ID NO:131); SEQ ID No: 34 of W02016 / 022630 (SEQ ID NO: 132); SEQ ID No: 38 of WQ2016 / 022630 (SEQ ID NO:133); SEQ ID No: 42 of WO2016 / 022630 (SEQ ID NO: 134); and SEQ ID No: 46 of WQ2016 / 022630 (SEQ ID NO:135)); and / or a light 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 WQ2016 / 022630 (SEQ ID No: 4 of WO2016 / 022630 (SEQ ID NO: 136); SEQ ID No: 8 of W02016 / 022630 (SEQ ID NO: 137); SEQ ID No: 12 of W02016 / 022630 (SEQ ID NO: 138); SEQ ID No: 16 of W02016 / 022630 (SEQ ID NO: 139); SEQ ID No: 20 of W02016 / 022630 (SEQ ID NO: 140); SEQ ID No: 24 of

[0220] W02016 / 022630 (SEQ ID NO:141); SEQ ID No: 28 of W02016 / 022630 (SEQ ID NO: 142); SEQ ID No: 32 of

[0221] WO2016 / 022630 (SEQ ID NO: 143); SEQ ID No: 36 of W02016 / 022630 (SEQ ID NO: 144); SEQ ID No: 40 of

[0222] W02016 / 022630 (SEQ ID NO: 145); SEQ ID No: 44 of WQ2016 / 022630 (SEQ ID NO: 146); and SEQ ID No: 48 of

[0223] WQ2016 / 022630 (SEQ ID NO: 147)).

[0224] In an embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2015 / 112900, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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 WQ2015 / 112900 (SEQ ID NO:148); SEQ ID No: 50 of WO 2015 / 112900 (SEQ ID NO:149);

[0225] DB1 / 162517863.2 32 ORN-108PC / 114384-5108

[0226] SEQ ID No: 82 of WO 2015 / 112900 (SEQ ID NO:150); and SEQ ID No: 86 of WO 2015 / 112900 (SEO ID NO:151 )); and / or a light 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: 42 of WO2015 / 112900 (SEQ ID NO:152); SEQ ID No: 46 of WO 2015 / 112900: (SEQ ID NO:153); SEQ ID No: 54 of WO 2015 / 112900 (SEQ ID NO:154); SEQ ID No: 58 of WO

[0227] 2015 / 112900 (SEQ ID NO:155); SEQ ID No: 62 of WO 2015 / 112900 (SEQ ID NO:156); SEQ ID No: 66 of WO

[0228] 2015 / 112900 (SEQ ID NO:157); SEQ ID No: 70 of WO 2015 / 112900 (SEQ ID NO:158); SEQ ID No: 74 of WO

[0229] 2015 / 112900 (SEQ ID NO:159); and SEQ ID No: 78 of WO 2015 / 112900 (SEQ ID NO:160)).

[0230] In an embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2010 / 077634 and US 8,217,149, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the anti-PD-L1 antibody or an 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 of WO 2010 / 077634 (SEQ ID NO: 161); and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 21 of WO 2010 / 077634 (SEQ ID NO: 162)

[0231] In an embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies obtainable from the hybridoma accessible under CNCM deposit numbers CNCM 1-4122, CNCM I-4080 and CNCM 1-4081 as disclosed in US 20120039906, the entire disclosures of which are hereby incorporated by reference.

[0232] In an embodiment, the targeting moiety comprises a VHH directed against PD-L1 as disclosed, for example, in US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the VHHs against PD-L1 comprise SEQ ID NOS: 394-399 of US 8,907,065 (SEQ ID No: 394 of US 8,907,065 (SEQ ID NO: 163); SEQ ID No: 395 of US 8,907,065 (SEQ ID NO:164); SEQ ID No: 396 of US 8,907,065 (SEQ ID NO: 165); SEQ ID No: 397 of US 8,907,065 (SEQ ID NO:166); SEQ ID No: 398 of US 8,907,065 (SEQ ID NO:167); and SEQ ID No: 399 of US 8,907,065 (SEQ ID NO: 168)).

[0233] In various embodiments, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have one or more targeting moieties directed against PD-L2. In some embodiments, the chimeric proteins or chimeric protein complexes have one or more targeting moieties which selectively bind a PD- L2 polypeptide. In some embodiments, the chimeric proteins or chimeric protein complexes comprise one or more antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind a PD- L2 polypeptide.

[0234] In an embodiment, the targeting moiety comprises a VHH directed against PD-L2 as disclosed, for example, in US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the VHHs against PD-L2 comprise SEQ ID Nos: 449-455 of US 8,907,065 (SEQ ID No: 449 of US 8,907,065 (SEQ ID NO:169); SEQ ID No: 450 of US 8,907,065 (SEQ ID NQ:170); SEQ ID No: 451 of US 8,907,065 (SEQ ID NO:171); SEQ ID No: 452 of US 8,907,065 (SEQ ID NO:172); SEQ ID No: 453 of US 8,907,065 (SEQ ID NO: 173); SEQ ID No: 454 of US 8,907,065 (SEQ ID NO: 174); and SEQ ID No: 455 of US 8,907,065 (SEQ ID NO:175)).

[0235] DB1 / 162517863.2 33 ORN-108PC / 114384-5108

[0236] In an embodiment, the targeting moiety comprises any one of the anti-PD-L2 antibodies disclosed in US2011 / 0271358 and WO2010 / 036959, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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: 43-47 of US2011 / 0271358 (SEQ ID No: 43 of US2011 / 0271358 (SEQ ID NO:176); SEQ ID No: 44 of US2011 / 0271358 (SEQ ID NO:177); SEQ ID No: 45 of US2011 / 0271358 (SEQ ID NO:178); SEQ ID No: 46 of US2011 / 0271358 (SEQ ID NO: 179); and SEQ ID No: 47 of US2011 / 0271358 (SEQ ID NO: 180)); and / or a light chain comprising an amino acid sequence selected from SEQ ID Nos: 48-51 of US2011 / 0271358 (SEQ ID No: 48 of US2011 / 0271358 (SEQ ID NO:181); SEQ ID No: 49 of US2011 / 0271358 (SEQ ID NO:182); SEQ ID No: 50 of US2011 / 0271358 (SEQ ID NO: 183); and SEQ ID No: 51 of US2011 / 0271358 (SEQ ID NO:184)).

[0237] In various embodiments, the targeting moieties of the invention may comprise a sequence that targets PD-1 , PD- L1 , and / or PD-L2 which is at least about 60%, at least about 61 %, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, 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 to any of the sequences disclosed herein (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99% or about 100% sequence identity with any of the sequences disclosed herein).

[0238] In various embodiments, the targeting moieties of the invention may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity-determining region (CDR), and framework region sequences that target PD-1 , PD-L1, and / or PD-L2 as disclosed herein.

[0239] 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 disclosed in WO 2011 / 066389, US 2008 / 0025980, US 2013 / 0034559, US 8,779,108, US 2014 / 0356353, US 8,609,089, US 2010 / 028330, US 2012 / 0114649, WO 2010 / 027827, WO 2011. / 066342, US 8,907,065, WO 2016 / 062722, WO 2009 / 101611 , WO2010 / 027827, WO 2011 / 066342, WO 2007 / 005874 , WO 2001 / 014556, US2011 / 0271358, WO 2010 / 036959, WO 2010 / 077634, US 8,217,149, US 2012 / 0039906, WO 2012 / 145493, US 2011 / 0318373, U.S. Patent No. 8,779,108, US 20140044738, WO 2009 / 089149, WO 2007 / 00587, WO 2016061142, WO 2016,02263, WO 2010 / 077634, and WO 2015 / 112900, the entire disclosures of which are hereby incorporated by reference.

[0240] DB1 / 162517863.2 34 ORN-108PC / 114384-5108

[0241] In one embodiment, the present chimeric proteins or chimeric protein complexes have (i) a targeting moiety directed against a T cell, for example, mediated by targeting to CD8 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6, or a variant thereof) described herein. In an embodiment, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have a targeting moiety directed against CD8 on T cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0242] In one embodiment, the present chimeric proteins or chimeric protein complexes have (i) a targeting moiety directed against a T cell, for example, mediated by targeting to CD4 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an embodiment, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have a targeting moiety directed against CD4 on T cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0243] In one embodiment, the present chimeric proteins or chimeric protein complexes have (i) a targeting moiety directed against a T cell, for example, mediated by targeting to CD3, CXCR3, CCR4, CCR9, CD70, CD103, or one or more immune checkpoint markers and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or a variant thereof) described herein. In an embodiment, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have a targeting moiety directed against CD3 on T cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0244] In some embodiments, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have one or more targeting moieties directed against CD3 expressed on T cells. In some embodiments, the chimeric protein or chimeric protein complex have one or more targeting moieties, which selectively bind a CD3 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex comprise one or more antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind a CD3 polypeptide.

[0245] In an embodiment, the targeting moiety comprises the anti-CD3 antibody muromonab-CD3 (aka Orthoclone OKT3), or fragments thereof. Muromonab-CD3 is disclosed in U.S. Patent No. 4,361,549 and Wilde et al. (1996) 51 :865-894, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, muromonab-CD3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEO ID NOU 85); and / or a light chain comprising the amino acid sequence of (SEO ID NO: 186).

[0246] In an embodiment, the targeting moiety comprises the anti-CD3 antibody otelixizumab, or fragments thereof. Otelixizumab is disclosed in U.S Patent Publication No. 20160000916 and Chatenoud et al. (2012) 9:372-381, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, otelixizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the

[0247] DB1 / 162517863.2 35 ORN-108PC / 114384-5108 amino acid sequence of: SEQ ID NO: 187; and / or a light chain comprising the amino acid sequence of SEQ ID NO:188.

[0248] In an embodiment, the targeting moiety comprises the anti-CD3 antibody teplizumab (AKA MGA031 and hOKT3y1 (Ala-Ala)), or fragments thereof. Teplizumab is disclosed in Chatenoud et al, (2012) 9:372-381 , the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, teplizumab or an antigenbinding 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 NQ:190.

[0249] In an embodiment, the targeting moiety comprises the anti-CD3 antibody visilizumab (AKA Nuvion®; HuM291), or fragments thereof. Visilizumab is disclosed in U.S. 5,834,597 and WQ2004052397, and Cole eta / ., Transplantation (1999) 68:563-571 , the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 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.

[0250] In an embodiment, the targeting moiety comprises the anti-CD3 antibody foralumab (aka N 1-0401 ), or fragments thereof. In various embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in US20140193399, US 7,728,114, US20100183554, and US 8,551 ,478, the entire disclosures of which are hereby incorporated by reference.

[0251] In illustrative embodiments, the anti-CD3 antibody 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 Nos: 2 and 6 of US 7,728,114 (SEQ ID No: 2 of US 7,728,114 (SEQ ID NO:193) and SEQ ID No: 6 of US 7,728,114 (SEQ ID NO: 194)); and / or a light chain variable region comprising the amino acid sequence of SEQ ID NOs 4 and 8 of US 7,728,114 (SEQ ID No: 4 of US 7,728,114 (SEQ ID NO:195) and SEQ ID No: 8 of US 7,728,114 (SEQ ID NO:196)).

[0252] In an embodiment, the targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 of US 7,728,114 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4 of US 7,728,114. In an embodiment, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in US2016 / 0168247, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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: 6-9 of US2016 / 0168247 (SEQ ID No: 6 of US2016 / 0168247 (SEQ ID NO:197); SEQ ID No: 7 of US2016 / 0168247 (SEQ ID NO:198); SEQ ID No: 8 of US2016 / 0168247 (SEQ ID NO: 199); and SEQ ID No: 9 of US2016 / 0168247 (SEQ ID NQ:200)); and / or a light chain comprising an amino acid sequence selected from SEQ ID Nos: 10-12 of US2016 / 0168247 (SEQ ID No: 10 of US2016 / 0168247 (SEQ ID NQ:201); SEQ ID No: 11 of US2016 / 0168247 (SEQ ID NQ:202); and SEQ ID No: 12 of US2016 / 0168247 (SEQ ID NQ:203)).

[0253] DB1 / 162517863.2 36 ORN-108PC / 114384-5108

[0254] In an embodiment, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in US2015 / 0175699, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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 of US2015 / 0175699 (SEQ ID NO:204); and / or a light chain comprising an amino acid sequence selected from SEQ ID No: 10 of US2015 / 0175699 (SEQ ID NQ:205).

[0255] In an embodiment, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in US 8,784,821 , the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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: 2, 18, 34, 50, 66, 82, 98 and 114 of US 8,784,821 (SEQ ID No: 2 of US 8,784,821 (SEQ ID NQ:206); SEQ ID No: 18 of US 8,784,821 (SEQ ID NQ:207); SEQ ID No: 34 of US 8,784,821 (SEQ ID NQ:208); SEQ ID No: 50 of US 8,784,821 (SEQ ID NQ:209); SEQ ID No: 66 of US 8,784,821 (SEQ ID NQ:210); SEQ ID No: 82 of US 8,784,821 (SEQ ID NO:211); SEQ ID No: 98 of US 8,784,821 (SEQ ID NO:212); and SEQ ID No: 114 of US 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 US 8,784,821 (SEQ ID No: 10 of US 8,784,821 (SEQ ID NO:214); SEQ ID No: 26 of US 8,784,821 (SEQ ID NO:215); SEQ ID No: 42 of US 8,784,821 (SEQ ID NO:216); SEQ ID No: 58 of US 8,784,821 (SEQ ID NO:217); SEQ ID No: 74 of US 8,784,821 (SEQ ID NO:218); SEQ ID No: 90 of US 8,784,821 (SEQ ID NO:219); SEQ ID No: 106 of US 8,784,821 (SEQ ID NQ:220); and SEQ ID No: 122 of US 8,784,821 (SEQ ID NO:221).

[0256] In an embodiment, the targeting moiety comprises any one of the anti-CD3 binding constructs disclosed in US20150118252, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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 US20150118252 (SEQ ID No: 6 of US20150118252 (SEQ ID NO :222) and SEQ ID No: 86 of US20150118252 (SEQ ID NO:223)) and / or a light chain comprising an amino acid sequence selected from SEQ ID No: 3 of US2015 / 0175699 (SEQ ID No: 3 of US20150118252 (SEQ ID NO:224)).

[0257] In an embodiment, the targeting moiety comprises any one of the anti-CD3 binding proteins disclosed in US2016 / 0039934, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibody or an 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: 6-9 of US2016 / 0039934 (SEQ ID No: 6 of US2016 / 0039934 (SEQ ID NO:225); SEQ ID No: 7 of US2016 / 0039934 (SEQ ID NO:226); SEQ ID No: 8 of US2016 / 0039934 (SEQ ID NO:227); and SEQ ID No: 9 of US2016 / 0039934 (SEQ ID NO:228)); and / or a light chain comprising an amino acid sequence selected from SEQ ID Nos: 1-4 of US2016 / 0039934 (SEQ ID No: 1 of US2016 / 0039934 (SEQ ID NO:229); SEQ ID No: 2 of US2016 / 0039934 (SEQ ID NQ:230); SEQ ID No: 3 of US2016 / 0039934 (SEQ ID NO:231); and SEQ ID No: 4 of US2016 / 0039934 (SEQ ID NO:232)).

[0258] DB1 / 162517863.2 37 ORN-108PC / 114384-5108

[0259] In various embodiments, the targeting moieties of the invention may comprise a sequence that targets CD3 which is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71 %, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, 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 to any of the sequences disclosed herein (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99% or about 100% sequence identity with any of the sequences disclosed herein)

[0260] In various embodiments, the targeting moieties of the invention may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences that target CD3 as disclosed herein. In various embodiments, the targeting moieties of the invention may comprise any heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences of the CD3-specific antibodies including, but not limited to, X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11 D8, 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, inter alia, described in Tunnacliffe (1989), Int. Immunol. 1 , 546-550, the entire disclosures of which are hereby incorporated by reference.

[0261] Additional antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind or target CD3 are disclosed in US Patent Publication No. 2016 / 0000916, US Patent Nos. 4,361,549, 5,834,597, 6,491,916, 6,406,696, 6,143,297, 6,750,325 and International Publication No. WO 2004 / 052397, the entire disclosures of which are hereby incorporated by reference.

[0262] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a T cell, for example, mediated by targeting to PD-1 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein.

[0263] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a B cell, for example, mediated by targeting to CD10, CD19, CD20, CD21 , CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78,

[0264] DB1 / 162517863.2 38 ORN-108PC / 114384-5108

[0265] CD79a / b, CD80, CD81 , CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, or CDw150; and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an embodiment, the chimeric proteins or chimeric protein complexes have a targeting moiety directed against CD20.

[0266] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a B cell, for example, mediated by targeting to CD19, CD20 or CD70 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein.

[0267] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a B cell, for example, mediated by targeting to CD20 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or a variant thereof) described herein. In an embodiment, the chimeric proteins or chimeric protein complexes have a targeting moiety directed against CD20 on B cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells. By way of example, in some embodiments, the CD20 targeting moiety is a recombinant heavy-chain-only antibody (VHH) having the sequence of:

[0268] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS (SEQ ID NO: 288).

[0269] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a NK cell, for example, mediated by targeting to 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-y RI / CD64, MICA, Fc-y Rl I B / CD32b, MICB, Fc-y RIIC / CD32c, MULT-1, Fc-y RIIA / CD32a, Nectin-2 / CD112, Fc-y RIII / 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-1 a, Rae-1 [3, Rae-1 delta, H60, Rae-1 epsilon, ILT2 / CD85), Rae-1 y, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1 , KIR2DL1 , ULBP-1 , KIR2DL3, ULBP-2, KIR2DL4 / CD 158d, or ULBP-3; and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein.

[0270] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a NK cell, for example, mediated by targeting to Kiri alpha, DNAM-1 or CD64 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein.

[0271] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a NK cell, for example, mediated by targeting to KIR1 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an embodiment, the chimeric proteins or chimeric protein complexes have a targeting moiety directed against KIR1 on NK cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0272] DB1 / 162517863.2 39 ORN-108PC / 114384-5108

[0273] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a NK cell, for example, mediated by targeting to TIGIT or KIR1 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an embodiment, the chimeric proteins or chimeric protein complexes have a targeting moiety directed against TIGIT on NK cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0274] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a dendritic cell, for example, mediated by targeting to CLEC-9A, XCR1 , RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-PI / COLEC 12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1 , TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB Ligand / TN FSF9, IL-12 / IL-23 p40, 4- Amino-1,8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin a 4 / CD49d, Aag, Integrin |3 2 / CD18, AMIGA, Langerin, B7-2 / CD86, Leukotriene B4 Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, Clq R1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LB CCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLI, CD2F-10 / SLAMF9, Osteoactivin GPNMB, Chern 23, PD- L2, CLEC-1 , RP105, CLEC-2, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC- SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, DEP-1 / CD148, SIGNR4, DLEC, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-y R1 / CD64, TLR3, Fc-y RIIB / CD32b, TREM-1, Fc-y RIIC / CD32c, TREM-2, Fc-y RIIA / CD32a, TREM-3, Fc-y RIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, or Vanilloid R1 ; and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein.

[0275] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a dendritic cell, for example, mediated by targeting to CLEC-9A, DC-SIGN, CD64, CLEC4A, or DEC205 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an embodiment, the chimeric protein or chimeric protein complex have a targeting moiety directed against CLEC9A on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells

[0276] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a dendritic cell, for example, mediated by targeting to CLEC9A and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an embodiment, the chimeric proteins or chimeric protein complexes have a targeting moiety directed against CLEC9A on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0277] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a dendritic cell, for example, mediated by targeting to XCR1 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an embodiment, the

[0278] DB1 / 162517863.2 40 ORN-108PC / 114384-5108 chimeric proteins or chimeric protein complexes have a targeting moiety directed against XCR1 on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0279] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a dendritic cell, for example, mediated by targeting to RANK and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an embodiment, the chimeric protein or chimeric protein complex have a targeting moiety directed against RANK on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0280] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a monocyte / macrophage, for example, mediated by targeting to SIRP1 a, B7- 1 / CD80, ILT4 / CD85d, B7-H1 , ILT5 / CD85a, Common p Chain, Integrin a 4 / CD49d, BLAME / SLAMF8, Integrin a X / CDIIc, CCL6 / C10, Integrin p 2 / CD18, CD155 / PVR, Integrin p 3 / CD61, CD31 / PECAM-1, Latexin, CD36 / SR-B3, Leukotriene B4 R1 , 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-y RI / CD64, Osteopontin, Fc-y Rl I B / CD32b, PD-L2, Fc-y RIIC / CD32c, Siglec-3 / CD33, Fc- y RIIA / CD32a, SIGNR1 / CD209, Fc-y RIII / CD16, SLAM, GM-CSF R a, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN- y Rl, TLR4, IFN- y R2, TREM-I, IL-I RII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1 , 2B4 / SLAMF 4, IL-10 R a, ALCAM, IL-10 R p, AminopeptidaseN / ANPEP, ILT2 / CD85j, Common p Chain, ILT3 / CD85k, Clq R1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, Integrin a 4 / CD49d, CCR5, Integrin a M / CDII b, CCR8, Integrin a X / CDIIc, CD155 / PVR, Integrin p 2 / CD18, CD14, Integrin p 3 / CD61 , CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, Leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / CD300LF, Coagulation Factor lll / Tissue Factor, LMIR5 / CD300LB, CX3CR1 , CX3CL1 , LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1 / CD148, MD-1 , DNAM-1 , MD-2, EMMPRIN / CD147, MMR, Endoglin / CD105, NCAM-L1, Fc-y RI / CD64, PSGL-1, Fc-y RIIIICD16, RP105, G-CSF R, L-Selectin, GM-CSF R a, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-I, IL-6 R, TREM- 2, CXCRI / I L-8 RA, TREM-3, or TREMLI / TLT-1 ; and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein.

[0281] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a monocyte / macrophage, for example, mediated by targeting to B7-H1, CD31 / PECAM-1, CD163, CCR2, or Macrophage Mannose Receptor CD206 and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein.

[0282] In one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a monocyte / macrophage, for example, mediated by targeting to SIRP1 a and (ii) a targeting moiety is directed against a tumor cell, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an

[0283] DB1 / 162517863.2 41 ORN-108PC / 114384-5108 embodiment, the chimeric protein or chimeric protein complex have a targeting moiety directed against SIRP1 a on macrophage cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0284] In various embodiments, the chimeric protein or chimeric protein complex have one or more targeting moieties directed against a checkpoint marker, 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 one embodiment, the chimeric protein or chimeric protein complex have (i) a targeting moiety directed against a checkpoint marker on a T cell, for example, PD-1 and (ii) a targeting moiety directed against a tumor cell, for example, PD-L1 or PD-L2, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein. In an embodiment, the chimeric protein or chimeric protein complex have a targeting moiety directed against PD-1 on T cells and a second targeting moiety directed against PD-L1 on tumor cells. In another embodiment, the chimeric protein or chimeric protein complex have a targeting moiety directed against PD-1 on T cells and a second targeting moiety directed against PD-L2 on tumor cells.

[0285] In some embodiments, the chimeric protein or chimeric protein complex comprises two or more targeting moieties directed to the same or different immune cells. In some embodiments the chimeric protein or chimeric protein complex have (i) one or more targeting moieties directed against an immune cell selected from a T cell, a B cell, a dendritic cell, a macrophage, a NK cell, or subsets thereof and (ii) one or more targeting moieties directed against either the same or another immune cell selected from a T cell, a B cell, a dendritic cell, a macrophage, a NK cell, or subsets thereof, along with any of the signaling agents (e.g., IL-6 or variant thereof) described herein.

[0286] In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against a T cell and one or more targeting moieties directed against the same or another T cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against a T cell and one or more targeting moieties directed against a B cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against a T cell and one or more targeting moieties directed against a dendritic cell In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties against a T cell and one or more targeting moieties directed against a macrophage. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties against a T cell and one or more targeting moieties directed against a NK cell. For example, in an illustrative embodiment, the chimeric protein or chimeric protein complex may include a targeting moiety against CD8 and a targeting moiety against Clec9A. In another illustrative embodiment, the chimeric protein or chimeric protein complex may include a targeting moiety against CD8 and a targeting moiety against CD3. In another illustrative embodiment, the chimeric protein or chimeric protein complex may include a targeting moiety against CD8 and a targeting moiety against PD-1.

[0287] In various embodiments, the chimeric proteins or the chimeric protein complexes disclosed herein include a CD8 binding agent that is a protein-based agent capable of specific binding to CD8. In various embodiments, the present

[0288] DB1 / 162517863.2 42 ORN-108PC / 114384-5108

[0289] CD8 binding agent is a protein-based agent capable of specific binding to CD8 without functionally modulating (e.g partial or complete neutralization) CD8.

[0290] In some embodiments, the CDS binding agent comprises a targeting moiety, which is an antibody derivative or format. In some embodiments, the present CD8 binding agent comprises a targeting moiety which is a singledomain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy- chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a DuoBody, a Fv, a Fab, a Fab1, a F(ab')2, a peptide mimetic molecule, or a synthetic molecule, as described in US Patent Nos. or Patent Publication Nos. US 7,417, 130, US 2004 / 132094, US 5,831 ,012, US 2004 / 023334, US 7,250,297, US 6,818,418, US 2004 / 209243, US 7,838,629, US 7, 186,524, US 6,004,746, US 5,475,096, US 2004 / 146938, US 2004 / 157209, US 6,994,982, US 6,794, 144, US 2010 / 239633, US 7,803,907, US 2010 / 119446, and / or US 7, 166,697, the contents of which are hereby incorporated by reference in their entireties. See also, Storz MAbs. 2011 May-Jun; 3(3): 310-317.

[0291] In some embodiments, the CDS binding agent comprises a targeting moiety, which is a single-domain antibody, such as a VHH. The VHH may be derived from, for example, an organism that produces VHH antibody such as a camelid, a shark, or the VHH may be a designed VHH VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3).

[0292] In an embodiment, the CD8 binding agent comprises a VHH. In some embodiments, the VHH is a humanized VHH or camelized VHH.

[0293] DB1 / 162517863.2 43 ORN-108PC / 114384-5108

[0294] In some embodiments, the VHH comprises a fully human V domain, e.g. a HUMABODY (Crescendo Biologies, Cambridge, UK). In some embodiments, fully human V domain, e.g. a HUMABODY is monovalent, bivalent, or trivalent. In some embodiments, the fully human VH domain, e.g. a HUMABODY is mono- or multi-specific such as monospecific, bispecific, or trispecific. Illustrative fully human VH domains, e.g. a HUMABODIES are described in, for example, WO2016 / 113555 and WO2016 / 113557, the entire disclosure of which is incorporated by reference.

[0295] In some embodiments, the CD8 binding agent comprises a targeting moiety which is a VHH comprising a single amino acid chain having four “framework regions" or FRs and three “complementary determining regions” or CDRs. As used herein, “framework region” or “FR” refers to a region in the variable domain, which is located between the CDRs. As used herein, “complementary determining region” or “CDR” refers to variable regions in VHHs that contains the amino acid sequences capable of specifically binding to antigenic targets. In various embodiments, the CD8 binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequences.

[0296] In some embodiments, the targeting moiety comprises anti-CD8 antibody as described in WO 2019033043, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDR H 1 : GFNIKDTYIH (SEQ ID NO: 293); CDR H2: RIDPANDNTLYASKFQG (SEQ ID NO: 294); CDR H2: RIDPANDNTLYARKFQG (SEQ ID NO: 295); CDR H3: GRGYGYYVFDH (SEQ ID NO: 296); or CDR H3: TRGYGYYVFDT (SEQ ID NO: 297).

[0297] In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDR L1 : SISQY (SEQ ID NO: 298); CDR L1 : SISKY (SEQ ID NO: 299); CDR L2: SGSTLQ (SEQ ID NO: 300); CDR L3: HNENPL (SEQ ID NO: 301); CDR L3: HNEFPV (SEQ ID NO: 302); CDR L3: HNEFPP (SEQ ID NO: 303); CDR L3: VNEFPP (SEQ ID NO: 304); CDR L3: VNEFPV (SEQ ID NO: 305).

[0298] In some embodiments, the targeting moiety comprises anti-CD8 antibody as described in WO2019023148, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDR H1 : GFIFSNYG (SEQ ID NO: 306); CDR H2: IWYDGSNK (SEQ ID NO: 307); CDR H3: ARSYDMLTGSGDYYGL (SEQ ID NO: 308). In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDR L1 : QDITNY (SEQ ID NO: 309); CDR L2: GAS; CDR L3: QQYNNYPLT (SEQ ID NO: 310).

[0299] In some embodiments, the targeting moiety comprises anti-CD8 antibody as described in WO2015184203, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDR H1 : SGYTGTDYNMH (SEQ ID NO: 311); CDR H2: YIYPYTGGTGYNQKFKN (SEQ ID NO: 312); CDR H1 : DFGMN (SEQ ID NO: 313); CDR H2: LIYYDGSNKFY (SEQ ID NO: 314); CDR H3: PHYDGYYHFFDS (SEQ ID NO: 315). In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the

[0300] DB1 / 162517863.2 44 ORN-108PC / 114384-5108 amino acid sequence of CDR L1 : RASESVDSYDNSLMH (SEQ ID NO: 316); CDR L2: LASNLES (SEQ ID NO: 317); CDR L3: QQNNEDPYT (SEQ ID NO: 318); CDR L1 : KGSQDINNYLA (SEQ ID NO: 319); CDR L2: NTDILHT (SEQ ID NO: 320); CDR L3: YQYNNGYT (SEQ ID NO: 321).

[0301] In some embodiments, the targeting moiety comprises anti-CD8 antibody as described in WO2018170096, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDR H1 : GYTFTSY (SEQ ID NO: 322); CDR H2: DPSDNY (SEQ ID NO: 333); CDR H3: PKSAYAFDVGGYAMDY (SEQ ID NO: 334). In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDR L1: RTSENIDSYLT (SEQ ID NO: 335); CDR L2: AATLLAD (SEQ ID NO: 336); CDR L3: QHYYSTPWT (SEQ ID NO: 337).

[0302] In some embodiments, the targeting moiety comprises anti-CD8 antibody as described in WO2014164553, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDR H1 : GFNIKD (SEQ ID NO: 338); CDR H2: RIDPANDNT (SEQ ID NO: 339); CDR H3: GYGYYVFDH (SEQ ID NO: 340). In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDR L1 : RTSRSISQYLA (SEQ ID NO: 341); CDR L2: SGSTLQS (SEQ ID NO: 342); CDR L3: QQHNENPLT (SEQ ID NO: 343).

[0303] In various embodiments, the chimeric proteins or the chimeric protein complexes disclosed herein include a CD4 binding agent that is a protein-based agent capable of specific binding to CD4. In various embodiments, the present CD4 binding agent is a protein-based agent capable of specific binding to CD4 without functionally modulating (e.g partial or complete neutralization) CD4.

[0304] In various embodiments, the present CD4 binding agent comprises a targeting moiety capable of specific binding. In various embodiments, the CD4 binding agent comprises a targeting moiety having an antigen recognition domain such as an antibody or derivatives thereof. In an embodiment, the CD4 binding agent comprises a targeting moiety, which is an antibody. In various embodiments, the antibody is a full-length multimeric protein that includes two heavy chains and two light chains. Each heavy chain includes one variable region (e.g., VH) and at least three constant regions (e.g., CHi, CH? and CHa), and each light chain includes one variable region (VL) and one constant region (CL) The variable regions determine the specificity of the antibody. Each variable region comprises three hypervariable regions also known as complementarity-determining regions (CDRs) flanked by four relatively conserved framework regions (FRs). The three CDRs, referred to as CDR1 , CDR2, and CDR3, contribute to the antibody binding specificity. In some embodiments, the antibody is a chimeric antibody In some embodiments, the antibody is a humanized antibody.

[0305] In some embodiments, the CD4 binding agent comprises a targeting moiety, which is an antibody derivative or format. In some embodiments, the present CD4 binding agent comprises a targeting moiety which is a singledomain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-

[0306] DB1 / 162517863.2 45 ORN-108PC / 114384-5108 chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a DuoBody, a Fv, a Fab, a Fab', a F(ab')2, a peptide mimetic molecule, or a synthetic molecule, as described in US Patent Nos. or Patent Publication Nos. US 7,417,130, US 2004 / 132094, US 5,831 ,012, US 2004 / 023334, US 7,250,297, US 6,818,418, US 2004 / 209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004 / 146938, US 2004 / 157209, US 6,994,982, US 6,794,144, US 2010 / 239633, US 7,803,907, US 2010 / 119446, and / or US 7,166,697, the contents of which are hereby incorporated by reference in their entireties. See also, Storz MAbs. 2011 May-Jun; 3(3): 310-317.

[0307] In some embodiments, the CD4 binding agent comprises a targeting moiety, which is a single-domain antibody, such as a VHH. The VHH may be derived from, for example, an organism that produces VHH antibody such as a camelid, a shark, or the VHH may be a designed VHH VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3).

[0308] In an embodiment, the CD4 binding agent comprises a VHH. In some embodiments, the VHH is a humanized VHH or camelized VHH. In some embodiments, the VHH comprises a fully human V domain, e.g. a HUMABODY (Crescendo Biologies, Cambridge, UK). In some embodiments, fully human V domain, e.g. a HUMABODY is monovalent, bivalent, or trivalent. In some embodiments, the fully human V domain, e.g. a HUMABODY is mono- or multi-specific such as monospecific, bispecific, or trispecific. Illustrative fully human VHdomains, e.g. a HUMABODIES are described in, for example, WO2016 / 113555 and WO2016 / 113557, the entire disclosure of which is incorporated by reference.

[0309] In some embodiments, the CD4 binding agent comprises a targeting moiety which is a VHH comprising a single amino acid chain having four “framework regions” or FRs and three “complementary determining regions” or CDRs. As used herein, “framework region” or “FR” refers to a region in the variable domain, which is located between the CDRs. As used herein, “complementary determining region” or “CDR” refers to variable regions in VHHs that contains the amino acid sequences capable of specifically binding to antigenic targets. In various embodiments, the CD4 binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequences. In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in W02020082045, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1 : GYTFTAHI (SEQ ID NO: 344); CDRH2: IKPQYGAV (SEQ ID NO: 345); or CDRH3: AR. In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1: QGVGSD (SEQ ID NO: 346); CDRL2: HTS; or CDRL3: QVLQF (SEQ ID NO: 347).

[0310] DB1 / 162517863.2 46 ORN-108PC / 114384-5108

[0311] In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in WO2018170096, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1 : GYTFTSN (SEQ ID NO: 348); CDRH2: YPRSGN (SEQ ID NO: 349); or CDRH3: RVPYFDH (SEQ ID NO: 350). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1 : KASQSVGNNVA (SEQ ID NO: 351); CDRL2: YASNRYT (SEQ ID NO: 352); or CDRL3: QQHYSSPFT (SEQ ID NO: 353).

[0312] In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in WO2016156570, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one CDR1 comprising the amino acid sequence of: GYWMY (SEQ ID NO: 354); CDR1 : SYSMG (SEQ ID NO: 355); CDR1 : FNAMG (SEQ ID NO: 356); or CDR1 : VMG. In some embodiments, the anti-CD4 antibody comprises at least one CDR2 comprising the amino acid sequence of CDR2: AISPGGGSTYYPDSVK (SEQ ID NO: 357); CDR2: AISWSGDETSYADSVK (SEQ ID NO: 358); CDR2: TIARAGATKYADSVKG (SEQ ID NO: 359); or CDR2: AVRWSSTGIYYTQYAD (SEQ ID NO: 360). In some embodiments, the anti-CD4 antibody comprises at least one CDR3 comprising the amino acid sequence of CDR3: SLTATHTYEYDY (SEQ ID NO: 361); CDR3: DRWWRPAGLQWDY (SEQ ID NO: 362); CDR3: RVFDLPNDY (SEQ ID NO: 363); or CDR3: DTYNSNPARWDGYDF (SEQ ID NO: 364).

[0313] In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in WO2012145238, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1 : AYVIS (SEQ ID NO: 365); CDRH2: EIYPGSGSSYYNEKFKG (SEQ ID NO: 366); or CDRH3: SGDGSKFVY (SEQ ID NO: 367). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1 : KASQSVDYCGDSYMN (SEQ ID NO: 368); CDRL2: VASNLES (SEQ ID NO: 369); or CDRL3: QQSLQDPPT (SEQ ID NO: 370).

[0314] In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in W02008134046, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1 : GYTFTSYVIH (SEQ ID NO: 371); CDRH2: YINPYNDGTDYDEKFK (SEQ ID NO: 372); or CDRH3: EKDNYATGAWFAY (SEQ ID NO: 373). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1 : KSSQSLLYSTNQKNY (SEQ ID NO: 374); CDRL2: WASTRES (SEQ ID NO: 375); or CDRL3: QQYYSYRT (SEQ ID NO: 376).

[0315] In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in W02009012944, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1 : SYVIH (SEQ ID NO: 377); CDRH1: GFTFSNYAMS (SEQ ID NO: 378); or CDRH2: AISDHSTNTYYP (SEQ ID NO: 379); CDRH3:

[0316] DB1 / 162517863.2 47 ORN-108PC / 114384-5108

[0317] EKDNYATGAWFAY (SEQ ID NO: 380); or CDRH3: ARKYGGDYDPF (SEQ ID NO: 381). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1 : KSSQSLLYSTNQKNYL (SEQ ID NO: 382); CDRL1 : KSSGSLLYSTNQKNYL (SEQ ID NO: 383); CDRL1 : KASQDINNY (SEQ ID NO: 384); CDRL2: WASTRES (SEQ ID NO: 385); CDRL2: YTSTLQPGVPS (SEQ ID NO: 386); CDRL3: QQYYSYRT (SEQ ID NO: 387); or CDRL3: YDNLLF (SEQ ID NO: 388).

[0318] In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in W02004005350, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1 : TFGVH (SEQ ID NO: 389); CDRH1 : TAGVH (SEQ ID NO: 390); or CDRH1 : TFGVA (SEQ ID NO: 391); CDRH2: VIWRSGITDYNVPFMS (SEQ ID NO: 392); CDRH2: VIARSGITDYNVPFMS (SEQ ID NO: 393); CDRH2: VIWASGITDYNVPFMS (SEQ ID NO: 394); CDRH3: NDPGTGFAY (SEQ ID NO: 395); CDRH3: NDPGTGAAY (SEQ ID NO: 396); or CDRH3: NDPGTGFAA (SEQ ID NO: 397). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1 : RASENIYSYLA (SEQ ID NO: 398); CDRL1 : RASENIYSALA (SEQ ID NO: 399); CDRL2: DAKTLAE (SEQ ID NO: 400); CDRL3: QHHYGNPPT (SEQ ID NO: 401); CDRL3: QHAYGNPPT (SEQ ID NO: 402); or CDRL3: QHHAGNPPT (SEQ ID NO: 403).

[0319] In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in W02004083247, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1 : DYVIN (SEQ ID NO: 404); CDRH2: EIYPGSGSDYYNENLKD (SEQ ID NO: 405); or CDRH3: KGENGNSLAFAY (SEQ ID NO: 406). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1 : QSVDYDGDSYMN (SEQ ID NO: 407); CDRL2: AASNLES (SEQ ID NO: 408); or CDRL3: QQSIQDPCT (SEQ ID NO: 409).

[0320] In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in W02014100139, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain comprising the following amino acid sequence:

[0321] Anti-CD4 antibody MV1 , Heavy Chain

[0322] MEWSGVFMFLLSVTAGVHSQVQLQQSGPEWKPGASVKMSCKASGYTFTSYVIHWVRQKP GQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNY ATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 410)

[0323] DB1 / 162517863.2 48 ORN-108PC / 114384-5108

[0324] In some embodiments, the anti-CD4 antibody comprises at least one light chain comprising the following amino acid sequence:

[0325] Anti-CD4 antibody MV1, Light Chain

[0326] MEWSGVFIFLLSVTAGVHSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQ QKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYYRTFGGG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 411)

[0327] In some embodiments, the targeting moiety comprises anti-CD4 antibody as described in W02004083247, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain comprising the following amino acid sequence:

[0328] EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWLRQAPGKGLEWIGVISVKSENYGAN YAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCSASYYRYDVGAFAYGQGTLVTVSS (SEQ ID NO: 412)

[0329] In some embodiments, the anti-CD4 antibody comprises at least one light chain comprising the following amino acid sequence:

[0330] DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQKPGQPPKLLIYLASILESGVPD

[0331] RFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPTFGQGTKVEIK (SEQ ID NO: 413)

[0332] In one embodiment, the chimeric proteins or chimeric protein complexes comprises one or more targeting moieties directed against a B cell and one or more targeting moieties directed against the same or another B cell. In one embodiment, the chimeric proteins or chimeric protein complexes comprises one or more targeting moieties directed against a B cell and one or more targeting moieties directed against a T cell. In one embodiment, the chimeric proteins or chimeric protein complexes comprises one or more targeting moieties directed against a B cell and one or more targeting moieties directed against a dendritic cell. In one embodiment, the chimeric proteins or chimeric protein complexes comprises one or more targeting moieties against a B cell and one or more targeting moieties directed against a macrophage. In one embodiment, the chimeric proteins or chimeric protein complexes comprises one or more targeting moieties against a B cell and one or more targeting moieties directed against a NK cell.

[0333] In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against a dendritic cell and one or more targeting moieties directed against the same or another dendritic cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against a dendritic cell and one or more targeting moieties directed against a T cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against a dendritic cell and one or more targeting moieties directed against a B cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties against a dendritic cell and one or more

[0334] DB1 / 162517863.2 49 ORN-108PC / 114384-5108 targeting moieties directed against a macrophage In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties against a dendritic cell and one or more targeting moieties directed against a NK cell.

[0335] In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against a macrophage and one or more targeting moieties directed against the same or another macrophage. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against a macrophage and one or more targeting moieties directed against a T cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against a macrophage and one or more targeting moieties directed against a B cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties against a macrophage and one or more targeting moieties directed against a dendritic cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties against a macrophage and one or more targeting moieties directed against a NK cell.

[0336] In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against an NK cell and one or more targeting moieties directed against the same or another NK cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against an NK cell and one or more targeting moieties directed against a T cell. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties directed against an NK cell and one or more targeting moieties directed against a B cell In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties against an NK cell and one or more targeting moieties directed against a macrophage. In one embodiment, the chimeric protein or chimeric protein complex comprises one or more targeting moieties against an NK cell and one or more targeting moieties directed against a dendritic cell.

[0337] In one embodiment, the chimeric protein or chimeric protein complex comprises a targeting moiety directed against a tumor cell and a second targeting moiety directed against the same or a different tumor cell. In such embodiments, the targeting moieties may bind to any of the tumor antigens described herein.

[0338] In some embodiments, the chimeric protein or chimeric protein complex of the invention comprises one or more targeting moieties having recognition domains that bind to a target (e.g. antigen, receptor) of interest including those found on one or more cells selected from adipocytes (e.g., white fat cell, brown fat cell), liver lipocytes, hepatic cells, kidney cells (e.g., kidney parietal cell, kidney salivary gland, mammary gland, etc.), duct cells (of seminal vesicle, prostate gland, etc.), intestinal brush border cells (with microvilli), exocrine gland striated duct cells, gall bladder epithelial cells, ductulus efferens nonciliated cells, epididymal principal cells, epididymal basal cells, endothelial cells, ameloblast epithelial cells (tooth enamel secretion), planum semilunatum epithelial cells of vestibular system of ear (proteoglycan secretion), organ of Corti interdental epithelial cells (secreting tectorial membrane covering hair cells), loose connective tissue fibroblasts, corneal fibroblasts (corneal keratocytes),

[0339] DB1 / 162517863.2 50 ORN-108PC / 114384-5108 tendon fibroblasts, bone marrow reticular tissue fibroblasts, nonepithelial fibroblasts, pericytes, nucleus pulposus cells of intervertebral disc, cementoblasts / cementocytes (tooth root bonelike ewan cell secretion), odontoblasts / odontocytes (tooth dentin secretion), hyaline cartilage chondrocytes, fi brocartilage chondrocytes, elastic cartilage chondrocytes, osteoblasts / osteocytes, osteoprogenitor cells (stem cell of osteoblasts), hyalocytes of vitreous body of eye, stellate cells of perilymphatic space of ear, hepatic stellate cells (Ito cell), pancreatic stelle cells, skeletal muscle cells, satellite cells, heart muscle cells, smooth muscle cells, myoepithelial cells of iris, myoepithelial cells of exocrine glands, exocrine secretory epithelial cells (e.g., salivary gland cells, mammary gland cells, lacrimal gland cells, sweat gland cells, sebaceious gland cells, prostate gland cells, gastric glad cells, pancreatic acinar cells, pneumocytes), a hormone secreting cells (e.g., pituitary cells, neurosecretory cells, gut and respiratory tract cells, thyroid gland cells, parathyroid glad cells, adrenal gland cells, Leydig cells of testes, pancreatic islet cells), keratinizing epithelial cells, wet stratified barrier epithelial cells, neuronal cells (e.g., sensory transducer cells, autonomic neuron cells, sense organ and peripheral neuron supporting cells, and central nervous system neurons and glial cells such as interneurons, principal cells, astrocytes, oligodendrocytes, and ependymal cells).

[0340] Targeting Moiety Formats

[0341] In various embodiments, the targeting moiety of the chimeric protein or chimeric protein complex is a protein-based agent capable of specific binding, such as an antibody or derivatives thereof. In an embodiment, the targeting moiety comprises an antibody In various embodiments, the antibody is a full-length multimeric protein that includes two heavy chains and two light chains. Each heavy chain includes one variable region (e.g., V ) and at least three constant regions (e.g., CH 1 , CH2and CH3), and each light chain includes one variable region (VL) and one constant region (C ) The variable regions determine the specificity of the antibody. Each variable region comprises three hypervariable regions also known as complementarity-determining regions (CDRs) flanked by four relatively conserved framework regions (FRs). The three CDRs, referred to as CDR1, CDR2, and CDR3, contribute to the antibody binding specificity. In some embodiments, the antibody is a chimeric antibody In some embodiments, the antibody is a humanized antibody.

[0342] In some embodiments, the targeting moiety comprises antibody derivatives or formats. In some embodiments, the targeting moiety of the chimeric protein or chimeric protein complex is a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; a Microbody; a peptide aptamer; an alterases; a plastic antibodies; a phylomer; a stradobodies; a maxibodies; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; affimers, a DuoBody, a Fv, a Fab, a Fab', a F(ab')2, a peptide mimetic molecule, or a synthetic molecule, as described in US Patent Nos. or Patent Publication Nos. US 7,417,130, US 2004 / 132094, US 5,831 ,012, US 2004 / 023334, US 7,250,297, US 6,818,418, US 2004 / 209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004 / 146938, US 2004 / 157209, US 6,994,982, US 6,794,144, US 2010 / 239633, US 7,803,907, US 2010 / 119446, and / or US

[0343] DB1 / 162517863.2 51 ORN-108PC / 114384-5108

[0344] 7,166,697, the contents of which are hereby incorporated by reference in their entireties. See also, Storz MAbs. 2011 May-Jun; 3(3): 310-317.

[0345] In one embodiment, the targeting moiety comprises a single-domain antibody, such as VHH from, for example, an organism that produces VHH antibody such as a camelid, a shark, or a designed VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies 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 trademark of NANOBODY or NANOBODIES.

[0346] In an embodiment, the targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized VHH or camelized VHH.

[0347] In some embodiments, the VHH comprises a fully human VH domain, e.g. a HUMABODY (Crescendo Biologies, Cambridge, UK). In some embodiments, 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 mono- or multi-specific such as monospecific, bispecific, or trispecific. Illustrative fully human VH domains, e.g. a HUMABODIES are described in, for example, WO 2016 / 113555 and WO2016 / 113557, the entire disclosure of which is incorporated by reference.

[0348] In various embodiments, the targeting moiety of the chimeric protein or chimeric protein complex is a protein-based agent capable of specific binding to a cell receptor, such as a natural ligand for the cell receptor. In various embodiments, the cell receptor is found on one or more immune cells, which can include, without limitation, T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g. M1 macrophages), B cells, dendritic cells, or subsets thereof. In some embodiments, the cell receptor is found on megakaryocytes, thrombocytes, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or subsets thereof.

[0349] In some embodiments, the targeting moiety is a natural ligand such as a chemokine. Illustrative chemokines that may be included in the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex of the invention include, but are not limited to, CCL1 , CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, 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 illustrative embodiment, the targeting moiety may be XCL1 , which is a chemokine that recognizes and binds to the dendritic cell receptor XCR1. In another illustrative embodiment, the targeting moiety is CCL1 , which is a chemokine that recognizes and binds to CCR8. In another illustrative embodiment, the targeting moiety is CCL2, which is a chemokine that recognizes and binds to CCR2 or CCR9. In another illustrative embodiment, the targeting moiety is CCL3, which is a chemokine that recognizes and binds to CCR1 , CCR5, or CCR9. In another illustrative embodiment, the targeting moiety is CCL4, which is a chemokine that recognizes and binds to CCR1 or

[0350] DB1 / 162517863.2 52 ORN-108PC / 114384-5108

[0351] CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL5, which is a chemokine that recognizes and binds to CCR1 or CCR3 or CCR4 or CCR5. In another illustrative embodiment, the targeting moiety is CCL6, which is a chemokine that recognizes and binds to CCR1. In another illustrative embodiment, the targeting moiety is CCL7, which is a chemokine that recognizes and binds to CCR2 or CCR9. In another illustrative embodiment, the targeting moiety is CCL8, which is a chemokine that recognizes and binds to CCR1 or CCR2 or CCR2B or CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL9, which is a chemokine that recognizes and binds to CCR1 . In another illustrative embodiment, the targeting moiety is CCL10, which is a chemokine that recognizes and binds to CCR1. In another illustrative embodiment, the targeting moiety is CCL11 , which is a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR9 In another illustrative embodiment, the targeting moiety is CCL13, which is a chemokine that recognizes and binds to CCR2 orCCR3 or CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL14, which is a chemokine that recognizes and binds to CCR1 or CCR9. In another illustrative embodiment, the targeting moiety is CCL15, which is a chemokine that recognizes and binds to CCR1 or CCR3. In another illustrative embodiment, the targeting moiety is CCL16, which is a chemokine that recognizes and binds to CCR1, CCR2, CCR5, or CCR8. In another illustrative embodiment, the targeting moiety is CCL17, which is a chemokine that recognizes and binds to CCR4. In another illustrative embodiment, the targeting moiety is CCL19, which is a chemokine that recognizes and binds to CCR7 In another illustrative embodiment, the targeting moiety is CCL20, which is a chemokine that recognizes and binds to CCR6. In another illustrative embodiment, the targeting moiety is CCL21 , which is a chemokine that recognizes and binds to CCR7. In another illustrative embodiment, the targeting moiety is CCL22, which is a chemokine that recognizes and binds to CCR4. In another illustrative embodiment, the targeting moiety is CCL23, which is a chemokine that recognizes and binds to CCR1 . In another illustrative embodiment, the targeting moiety is CCL24, which is a chemokine that recognizes and binds to CCR3. In another illustrative embodiment, the targeting moiety is CCL25, which is a chemokine that recognizes and binds to CCR9. In another illustrative embodiment, the targeting moiety is CCL26, which is a chemokine that recognizes and binds to CCR3. In another illustrative embodiment, the targeting moiety is CCL27, which is a chemokine that recognizes and binds to CCR10. In another illustrative embodiment, the targeting moiety is CCL28, which is a chemokine that recognizes and binds to CCR3 or CCR10. In another illustrative embodiment, the targeting moiety is CXCL1, which is a chemokine that recognizes and binds to CXCR1 or CXCR2. In another illustrative embodiment, the targeting moiety is CXCL2, which is a chemokine that recognizes and binds to CXCR2. In another illustrative embodiment, the targeting moiety is CXCL3, which is a chemokine that recognizes and binds to CXCR2. In another illustrative embodiment, the targeting moiety is CXCL4, which is a chemokine that recognizes and binds to CXCR3B. In another illustrative embodiment, the targeting moiety is CXCL5, which is a chemokine that recognizes and binds to CXCR2 In another illustrative embodiment, the targeting moiety is CXCL6, which is a chemokine that recognizes and binds to CXCR1 orCXCR2. In another illustrative embodiment, the targeting moiety is CXCL8, which is a chemokine that recognizes and binds to CXCR1 or CXCR2. In another illustrative embodiment, the targeting moiety is CXCL9, which is a chemokine that recognizes and binds to CXCR3. In another illustrative embodiment, the targeting moiety is CXCL10, which is a chemokine that recognizes and binds to CXCR3. In another illustrative embodiment, the

[0352] DB1 / 162517863.2 53 ORN-108PC / 114384-5108 targeting moiety is CXCL11 , which is a chemokine that recognizes and binds to CXCR3 or CXCR7. In another illustrative embodiment, the targeting moiety is CXCL12, which is a chemokine that recognizes and binds to CXCR4 or CXCR7 In another illustrative embodiment, the targeting moiety is CXCL13, which is a chemokine that recognizes and binds to CXCR5. In another illustrative embodiment, the targeting moiety is CXCL16, which is a chemokine that recognizes and binds to CXCR6. In another illustrative embodiment, the targeting moiety is LDGF- PBP, which is a chemokine that recognizes and binds to CXCR2. In another illustrative embodiment, the targeting moiety is XCL2, which is a chemokine that recognizes and binds to XCR1 . In another illustrative embodiment, the targeting moiety is CX3CL1, which is a chemokine that recognizes and binds to CX3CR1 .

[0353] 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., which is able to bind Flt3). In some embodiments, the targeting moiety is an extracellular domain of Flt3L. In some embodiments, the targeting moiety comprising a Flt3L domain, wherein the Flt3L domain is a single chain dimer, optionally where one Flt3L domain is connoted to the other Flt3L domain via one or more linkers, wherein the linker is aflexible linker. In some embodiments, the targeting moiety of the present invention comprises Flt3L domain, wherein the Flt3L domain is a single chain dimer and an Fc domain, the Fc domain optionally having one or more mutations that reduces or eliminates one or more effector functions of the Fc domain, promotes Fc chain pairing in the Fc domain, and / or stabilizes a 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.

[0354] In various embodiments, the chimeric protein or chimeric protein complex comprises targeting moieties in various combinations. In an illustrative embodiment, the chimeric protein or chimeric protein complex may comprise two targeting moieties, wherein both targeting moieties are antibodies or derivatives thereof In another illustrative embodiment, the chimeric protein or chimeric protein complex may comprise two targeting moieties, wherein both targeting moieties are natural ligands for cell receptors. In a further illustrative embodiment, the chimeric protein or chimeric protein complex may comprise two targeting moieties, wherein one of the targeting moieties is an antibody or derivative thereof, and the other targeting moiety is a natural ligand for a cell receptor

[0355] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex functionally modulates (by way of non-limitation, partially or completely neutralizes) the target (e.g. antigen, receptor) of interest, e.g. substantially inhibiting, reducing, or neutralizing a biological effect that the antigen has. For example, various recognition domains may be directed against one or more tumor antigens that are actively suppressing, or have the capacity to suppress, the immune system of, for example, a patient bearing a tumor. For example, in some embodiments, the chimeric protein or chimeric protein complex functionally modulates immune inhibitory signals (e.g. checkpoint inhibitors), for example, one or more of TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPa, ICOS, CD172a, and TMIGD2. For example, in some embodiments, the chimeric protein or chimeric protein complex is engineered to disrupt, block, reduce, and / or inhibit the transmission of an immune inhibitory signal, by way of non-limiting example, the binding

[0356] DB1 / 162517863.2 54 ORN-108PC / 114384-5108 of PD-1 with PD-L1 or PD-L2 and / or the binding of CTLA-4 with one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A.

[0357] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex binds but does not functionally modulate the target (e.g. antigen, receptor) of interest, e.g. the recognition domain is, or is akin to, a binding antibody. For instance, in various embodiments, the recognition domain simply targets the antigen or receptor but does not substantially inhibit, reduce or functionally modulate a biological effect that the antigen or receptor has. For example, some of the smaller antibody formats described above (e.g. as compared to, for example, full antibodies) have the ability to target hard to access epitopes and provide a larger spectrum of specific binding locales. In various embodiments, the recognition domain binds an epitope that is physically separate from an antigen or receptor site that is important for its biological activity (e.g. the antigen’s active site).

[0358] Such non-neutralizing binding finds use in various embodiments of the present invention, including methods in which the chimeric protein or chimeric protein complex is used to directly or indirectly recruit active immune cells to a site of need via an effector antigen, such as any of those described herein. For example, in various embodiments, the chimeric protein or chimeric protein complex such as Fc-based chimeric protein complex may be used to directly or indirectly recruit cytotoxic T cells via CD8 to a tumor cell in a method of reducing or eliminating a tumor (e.g. the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex may comprise an anti-CD8 recognition domain and a recognition domain directed against a tumor antigen) In such embodiments, it is desirable to directly or indirectly recruit CD8-expressing cytotoxic T cells but not to functionally modulate the CD8 activity. On the contrary, in these embodiments, CD8 signaling is an important piece of the tumor reducing or eliminating effect. By way of further example, in various methods of reducing or eliminating tumors, the chimeric protein or chimeric protein complex is used to directly or indirectly recruit dendritic cells (DCs) via CLEC9A (e.g. the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex may comprise an anti-CLEC9A recognition domain and a recognition domain directed against a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit CLEC9A-expressing DCs but not to functionally modulate the CLEC9A activity. On the contrary, in these embodiments, CLEC9A signaling is an important piece of the tumor reducing or eliminating effect.

[0359] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex binds to XCR1 e.g. on dendritic cells. For instance, the recognition domain, in some embodiments comprises all or part of XCL1 or a non-neutralizing anti-XCR1 agent.

[0360] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex binds to an immune modulatory antigen (e.g. immune stimulatory or immune inhibitory). In various embodiments, the immune modulatory antigen is one or more of 4-1 BB, 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, such immune stimulatory antigens are expressed on a tumor cell. In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex binds but does

[0361] DB1 / 162517863.2 55 ORN-108PC / 114384-5108 not functionally modulate such immune stimulatory antigens and therefore allows recruitment of cells expressing these antigens without the reduction or loss of their potential tumor reducing or eliminating capacity.

[0362] In various embodiments, the recognition domain of the chimeric protein or chimeric protein complex may be in the context of chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex that comprises two recognition domains that have neutralizing activity, or comprises two recognition domains that have non-neutralizing (e.g. binding) activity, or comprises one recognition domain that has neutralizing activity and one recognition domain that has non-neutralizing (e.g. binding) activity.

[0363] Fc Domains

[0364] 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 that are 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 contains three heavy chain constant domains (CH domains 2—4) in each polypeptide chain.

[0365] In some embodiments, the Fc-based chimeric protein of complex the present technology includes a Fc domain. In some embodiments, the Fc domains are from selected from IgG, IgA, IgD, IgM or IgE. In some embodiments, the Fc domains are from selected from lgG1, lgG2, lgG3, or lgG4.

[0366] In some embodiments, the Fc domains are from selected from human IgG, IgA, IgD, IgM or IgE. In some embodiments, the Fc domains are from selected from human IgG 1 , lgG2, lgG3, or lgG4

[0367] In some embodiments, the Fc domains of the Fc-based chimeric protein complex comprise the CH2 and CH3 regions of IgG. In some embodiments, the IgG is human IgG. In some embodiments, the human IgG is selected from lgG1 , lgG2, lgG3, or lgG4.

[0368] In some embodiments, the Fc domains comprise one or more mutations. In some embodiments, the mutation(s) to the Fc domains reduces or eliminates the effector function the Fc domains. In some embodiments, the mutated Fc domain has reduced affinity or binding to a target receptor. By way of example, in some embodiments, the mutation to the Fc domains reduces or eliminates the binding of the Fc domains to FcyR. In some embodiments, the FcyR is selected from FcyRI; FcyRlla, 131 R / R; FcyRlla, 131 H / H, FcyRllb; and FcyRIII. In some embodiments, the mutation to the Fc domains reduces or eliminated binding to complement proteins, such as, e.g., C1q. In some embodiments, the mutation to the Fc domains reduces or eliminated binding to both FcyR and complement proteins, such as, e.g., C1q.

[0369] In some embodiments, the Fc domains comprise the LALA mutation to reduce or eliminate the effector function of the Fc domains. By way of example, in some embodiments, the LALA mutation comprises L234A and L235A substitutions in human IgG (e.g., lgG1) (wherein the numbering is based on the commonly used numbering of the CH2 residues for human lgG1 according to EU convention (PNAS, Edelman et al., 1969; 63 (1) 78-85)).

[0370] DB1 / 162517863.2 56 ORN-108PC / 114384-5108

[0371] In some embodiments, the Fc domains of human IgG comprise a mutation to reduce or eliminate the effector function of the Fc domains. By way of example, in some embodiments, the mutations are selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G, and P331S. In embodiments, the mutations are L234A, L235A, and P331G.

[0372] In some embodiments, the Fc domains comprise the FALA mutation to reduce or eliminate the effector function of the Fc domains. By way of example, in some embodiments, the FALA mutation comprises F234A and L235A substitutions in human lgG4.

[0373] In some embodiments, the Fc domains of human I gG4 comprise a mutation at one or more of F234, L235, K322, D265, and P329 to reduce or eliminate the effector function of the Fc domains. By way of example, in some embodiments, the mutations are selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.

[0374] In some embodiments, the mutation(s) to the Fc domain stabilize a hinge region in the Fc domain. By way of example, in some embodiments, the Fc domain comprises a mutation at S228 of human IgG to stabilize a hinge region In some embodiments, the mutation is S228P.

[0375] In some embodiments, the mutation(s) to the Fc domain promote chain pairing in the Fc domain. In some embodiments, chain pairing is promoted by ionic pairing (a / k / a charged pairs, ionic bond, or charged residue pair).

[0376] In some embodiments, the Fc domain comprises a mutation at one more of the following amino acid residues of IgG to promote of ionic pairing: D356, E357, L368, K370, K392, D399, and K409.

[0377] By way of example, in some embodiments, the human IgG Fc domain comprise one of the mutation combinations in Table 1 to promote of ionic pairing.

[0378] DBl / 162517863.2 57 ORN-108PC / 114384-5108

[0379] DB1 / 162517863.2 58 ORN-108PC / 114384-5108

[0380] In some embodiments, chain pairing is promoted by a knob-in-hole mutations. In some embodiments, the Fc domain comprises one or more mutations to allow for a knob-in-hole interaction in the Fc domain. In some embodiments, a first Fc chain is engineered to express the “knob” and a second Fc chain is engineered to express the complementary “hole.” By way of example, in some embodiments, human IgG Fc domain comprises the mutations of Table 2 to allow for a knob-in-hole interaction.

[0381] In some embodiments, the Fc domains in the Fc-based chimeric protein complexes of the present technology comprise any combination of the above-disclosed mutations. By way of example, in some embodiments, the Fc domain comprises mutations that promote ionic pairing and / or a knob-in-hole interaction. By way of example, in some embodiments, the Fc domain comprises mutations that have one or more of the following properties: promote ionic pairing, induce a knob-in-hole interaction, reduce or eliminate the effector function of the Fc domain, and cause Fc stabilization (e.g. at hinge).

[0382] By way of example, in some embodiments, a human IgG Fc domains comprise mutations disclosed in Table 3, which promote ionic pairing and / or promote a knob-in-hole interaction in the Fc domain.

[0383] DBl / 162517863.2 59 ORN-108PC / 114384-5108

[0384] DB1 / 162517863.2 60 ORN-108PC / 114384-5108

[0385] DB1 / 162517863.2 61 ORN-108PC / 114384-5108

[0386] By way of example, in some embodiments, a human IgG Fc domains comprise mutations disclosed in Table 4, which promote ionic pairing, promote a knob-in-hole interaction, or a combination thereof in the Fc domain. In embodiments, the “Chain 1” and “Chain 2” of Table 4 can be interchanged (e.g. Chain 1 can have Y407T and Chain 2 can have T366Y).

[0387] DBl / 162517863.2 62 ORN-108PC / 114384-5108

[0388] DB1 / 162517863.2 63 ORN-108PC / 114384-5108

[0389] DB1 / 162517863.2 64 ORN-108PC / 114384-5108

[0390] By way of example, in some embodiments, a human IgG Fc domains comprise mutations disclosed in Table 5, which reduce or eliminate FcyR and / or complement binding in the Fc domain. In embodiments, the Table 5 mutations are in both chains.

[0391] DBl / 162517863.2 65 ORN-108PC / 114384-5108

[0392] DB1 / 162517863.2 66 ORN-108PC / 114384-5108

[0393] In some embodiments, the Fc domains in the Fc-based chimeric protein complexes of the present technology are homodimeric, i.e., the Fc region in the chimeric protein complex comprises two identical protein fragments.

[0394] In some embodiments, the Fc domains in the Fc-based chimeric protein complexes of the present technology are heterodimeric, i.e., the Fc domain comprises two non-identical protein fragments.

[0395] In some embodiments, heterodimeric Fc domains are engineered using ionic pairing and / or knob-in-hole mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complexes have a trans orientation / configuration. In a trans orientation / configuration, the targeting moiety and signaling agent, in embodiments, not found on the same polypeptide chain in the present Fc-based chimeric protein complexes.

[0396] In some embodiments, the Fc domains includes or starts with the core hinge region of wild-type human lgG1 , which contains the sequence Cys-Pro-Pro-Cys. In some embodiments, the Fc domains also include the upper hinge, or parts thereof [e.g., DKTHTCPPC; see WO 2009053368), EPKSCDKTHTCPPC, or EPKSSDKTHTCPPC; see Lo et al., Protein Engineering vol.11 no.6 pp.495-500, 1998)).

[0397] Fc-based Chimeric Protein Complexes

[0398] The Fc-based chimeric protein complexes of the present technology comprise at least one Fc domain disclosed herein, at least one signaling agent, e.g. IL-6, (SA) disclosed herein and at least one targeting moiety (TM) disclosed herein.

[0399] It is understood that, the present Fc-based chimeric protein complexes may encompass a complex of two fusion proteins, each comprising an Fc domain. In some embodiments, the Fc-based chimeric protein complex is heterodimeric. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation / configuration. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a cis orientation / configuration.

[0400] DBl / 162517863.2 67 ORN-108PC / 114384-5108

[0401] In some embodiments, heterodimeric Fc domains are engineered using ionic pairing and / or knob-in-hole mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complexes have a trans orientation. In a trans orientation, the targeting moiety and signaling agent are, in embodiments, not found on the same polypeptide chain in the present Fc-based chimeric protein complexes. In a trans orientation, the targeting moiety and signaling agent are, in embodiments, found on separate polypeptide chains in the Fc-based chimeric protein complexes. In a cis orientation, the targeting moiety and signaling agent are, in embodiments, found on the same polypeptide chain in the Fc-based chimeric protein complexes.

[0402] In some embodiments, where more than one targeting moiety is present in the heterodimeric protein complexes described herein, one targeting moiety may be in trans orientation (relative to the signaling agent), whereas another targeting moiety may be in cis orientation (relative to the signaling agent). In some embodiments, the signaling agent and target moiety are on the same ends / sides (N-terminal or C-terminal ends) of an Fc domain. In some embodiments, the signaling agent and targeting moiety are on different sides / ends of a Fc domain (N-terminal and C-terminal ends).

[0403] In some embodiments, where more than one targeting moiety is present in the heterodimeric protein complexes described herein, the targeting moieties may be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case the targeting moieties would be in trans relative to each other, as they are on different Fc chains). In some embodiments, where more than one targeting moiety is present on the same Fc chain, the targeting moieties may be on the same or different sides / ends of a Fc chain (N-terminal or / and C-terminal ends).

[0404] In some embodiments, where more than one signaling agent is present in the heterodimeric protein complexes described herein, the signaling agents may be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case the signaling agents would be in trans relative to each other, as they are on different Fc chains) In some embodiments, where more than one signaling agent is present on the same Fc chain, the signaling agents may be on the same or different sides / ends of a Fc chain (N-terminal or / and C-terminal ends).

[0405] In some embodiments, where more than one signaling agent is present in the heterodimeric protein complexes described herein, one signaling agent may be in trans orientation (as relates to the targeting moiety), whereas another signaling agent may be in cis orientation (as relates to the targeting moiety).

[0406] In some embodiments, the heterodimeric Fc-based chimeric protein complex does not comprise the signaling agent, e.g. IL-6 and targeting moiety on a single polypeptide.

[0407] In some embodiments, the Fc-based chimeric protein has an improved in vivo half-life relative to a chimeric protein lacking an Fc or a chimeric protein, which is not a heterodimeric complex. In some embodiments, the Fc-based chimeric protein has an improved solubility, stability and other pharmacological properties relative to a chimeric protein lacking an Fc or a chimeric protein, which is not a heterodimeric complex.

[0408] DB1 / 162517863.2 68 ORN-108PC / 114384-5108

[0409] Heterodimeric Fc-based chimeric protein complexes are composed of two different polypeptides. In embodiments described herein, the targeting domain is on a different polypeptide than the signaling agent, e.g. IL-6, and accordingly, proteins that contain only one targeting domain copy, and also only one signaling agent, e.g. IL-6, copy can be made (this provides a configuration in which potential interference with desired properties can be controlled). Further, in embodiments, one targeting domain (e.g. VHH) only can avoid cross-linking of the antigen on the cell surface (which could elicit undesired effects in some cases). Further, in embodiments, one signaling agent, e.g. IL-6 may alleviate molecular “crowding” and potential interference with avidity mediated induction or restoration of effector function in dependence of the targeting domain. Further, in embodiments, heterodimeric Fc- based chimeric protein complexes can have two targeting moieties and these can be placed on the two different polypeptides. For instance, in embodiments, the C-terminus 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). Further, in embodiments, heterodimeric Fc-based chimeric protein complexes, e.g. with the targeting domain on a different polypeptide than the signaling agent, e.g. IL-6 (e.g. wild type signaling agent, e.g. wild type IL-6), may favor “cross-linking” of two cell types (e.g. a tumor cell and an immune cell). Further, in embodiments, heterodimeric Fc-based chimeric protein complexes can have two signaling agent, each on different polypeptides to allow more complex effector responses.

[0410] Further, in embodiments, heterodimeric Fc-based chimeric protein complexes, e.g. with the targeting domain on a different polypeptide than the signaling agent, e.g. IL-6, combinatorial diversity of targeting moiety and signaling agent, e.g. IL-6 is provided in a practical manner For instance, in embodiments, polypeptides with any of the targeting moieties described herein can be combined “off the shelf” with polypeptides with any of the signaling agents described herein to allow rapid generation of various combinations of targeting moieties and signaling agents in single Fc-based chimeric protein complexes.

[0411] In some embodiments, the Fc-based chimeric protein complex comprises one or more linkers. In some embodiments, the Fc-based chimeric protein complex includes a linker that connects the Fc domain, signaling agent, e.g. IL-6(s) and targeting moiety(ies). In some embodiments, the Fc-based chimeric protein complex includes a linker that connects each signaling agent, e.g. IL-6 and targeting moiety (or, if more than one targeting moiety, a signaling agent, e.g. IL-6 to one of the targeting moieties). In some embodiments, the Fc-based chimeric protein complex includes a linker that connects each signaling agent, e.g. IL-6 to the Fc domain. In some embodiments, the Fc-based chimeric protein complex includes a linker that connects each targeting moiety to the Fc domain. In some embodiments, the Fc-based chimeric protein complex includes a linker that connects a targeting moiety to another targeting moiety In some embodiments, the Fc-based chimeric protein complex includes a linker that connects a signaling agent, e.g. IL-6 to another signaling agent.

[0412] In some embodiments, a Fc-based chimeric protein complex comprises two or more targeting moieties. In such embodiments, the targeting moieties can be the same targeting moiety or they can be different targeting moieties.

[0413] DB1 / 162517863.2 69 ORN-108PC / 114384-5108

[0414] In some embodiments, a Fc-based chimeric protein complex comprises two or more signaling agents In such embodiments, the signaling agents can be the same targeting moiety or they can be different targeting moieties.

[0415] By way of example, in some embodiments, the Fc-based chimeric protein complex comprise a Fc domain, at least two signaling agents (SA), and at least two targeting moieties (TM), wherein 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 homodimeric.

[0416] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 1A-F.

[0417] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 2A-H.

[0418] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 3A-H.

[0419] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 4A-D.

[0420] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 5A-F.

[0421] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 6A-J.

[0422] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 7A-D.

[0423] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 8A-F.

[0424] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 9A-J.

[0425] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 10A-F

[0426] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 11A-L.

[0427] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 12A-L.

[0428] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 13A-F

[0429] DB1 / 162517863.2 70 ORN-108PC / 114384-5108

[0430] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 14A-L.

[0431] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 15A-L.

[0432] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 16A-J.

[0433] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 17A-J.

[0434] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 18A-F

[0435] In various embodiments, the Fc-based chimeric protein complex takes the form of any of the schematics of Figs. 19A-F

[0436] In some embodiments, the signaling agents are linked to the targeting moieties and the targeting moieties are linked to the Fc domain on the same terminus (see FIGs. 1A-F). In some embodiments, the Fc domain is homodimeric.

[0437] In some embodiments, the signaling agents and targeting moieties are linked to the Fc domain, wherein the targeting moieties and signaling agents are linked on the same terminus (see FIGs. 1A-F). In some embodiments, the Fc domain is homodimeric.

[0438] In some embodiments, the targeting moieties are linked to signaling agents and the signaling agents are linked to the Fc domain on the same terminus (see FIGs. 1A-F). In some embodiments, the Fc domain is homodimeric.

[0439] In some embodiments, the homodimeric Fc-based chimeric protein complex has two or more targeting moieties. In some embodiments, there are four targeting moieties and two signaling agents, the targeting moieties are linked to the Fc domain and the signaling agents are linked to targeting moieties on the same terminus (see FIGS. 2A- H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four targeting moieties and two signaling agents, two targeting moieties are linked to the Fc domain and two targeting moieties are linked to the signaling agents, which are linked to the Fc domain on the same terminus (see FIGS. 2A-H). In some embodiments, the Fc domain is homodimeric In some embodiments, where there are four targeting moieties and two signaling agents, two targeting moieties are linked to each other and one of the targeting moieties of from each pair is linked to the Fc domain on the same terminus and the signaling agents are linked to the Fc domain on the same terminus (see FIGS. 2A-H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four targeting moieties and two signaling agents, two targeting moieties are linked to each other, wherein one of the targeting moieties of from each pair is linked to a signaling agent, e.g. IL-6 and the other targeting moiety of the pair is linked the Fc domain, wherein the targeting moieties linked to the Fc domain are linked on the same terminus (see FIGS. 2A-H). In some embodiments, the Fc domain is homodimeric.

[0440] DB1 / 162517863.2 71 ORN-108PC / 114384-5108

[0441] In some embodiments, the homodimeric Fc-based chimeric protein complex has two or more signaling agents. In some embodiments, where there are four signaling agents and two targeting moieties, two signaling agents are linked to each other and one of the signaling agents of from pair is linked to the Fc domain on the same terminus and the targeting moieties are linked to the Fc domain on the same terminus (see FIGs. 3A-H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four signaling agents and two targeting moieties, two signaling agents are linked to the Fc domain one the same terminus and two of the signaling agents are each linked to a targeting moiety, wherein the targeting moieties are linked to the Fc domain at the same terminus (see FIGs. 3A-H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four signaling agents and two targeting moieties, two signaling agents are linked to each other and one of the signaling agents of from pair is linked to a targeting moiety and the targeting moieties are linked to the Fc domain on the same terminus (see FIGs. 3A-H). In some embodiments, the Fc domain is homodimeric.

[0442] By way of example, in some embodiments, the Fc-based chimeric protein complex comprise a Fc domain, wherein the Fc domain comprises ionic pairing mutation(s) and / or knob-in-hole mutation(s), at least one signaling agent, e.g. IL-6, and at least one targeting moiety, wherein the ionic pairing motif and / or a knob-in-hole motif, signaling agent, e.g. IL-6, and targeting moiety are selected from any of the ionic pairing motif and / or a knob-in-hole motif, signaling agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0443] In some embodiments, the signaling agent, e.g. IL-6 is linked to the targeting moiety, which is linked to the Fc domain (see FIGs 10A-F and 13A-F). In some embodiments, the targeting moiety is linked to the signaling agent, e.g. IL-6, which is linked to the Fc domain (see FIGs. 10A-F and 13A-F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0444] In some embodiments, the signaling agent, e.g. IL-6 and targeting moiety are linked to the Fc domain (see FIGs. 4A-D, 7A-D, 10A-F, and 13A-F). In some embodiments, the targeting moiety and the signaling agent, e.g. IL-6 are linked to different Fc chains on the same terminus (see FIGs. 4A-D and 7A-D). In some embodiments, the targeting moiety and the signaling agent, e.g. IL-6 are linked to different Fc chains on different termini (see FIGs. 4A-D and 7A-D). In some embodiments, the targeting moiety and the signaling agent, e.g. IL-6 are linked to the same Fc chain (see FIGs. 10A-F and 13A-F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0445] In some embodiments, where there are one signaling agent, e.g. IL-6 and two targeting moieties, the signaling agent, e.g. IL-6 is linked to the Fc domain and two targeting moieties can be: 1) linked to each other with one of the targeting moieties linked to the Fc domain; or 2) each linked to the Fc domain (see FIGs. 5A-F, 8A-F, 11A-L, 14A-L, 16A-J, and 17A-J). In some embodiments, the targeting moieties are linked on one Fc chain and the signaling agent, e.g. IL-6 is on the other Fc chain (see FIGs. 5A-F and 8A-F). In some embodiments, the paired

[0446] DB1 / 162517863.2 72 ORN-108PC / 114384-5108 targeting moieties and the signaling agent, e.g. IL-6 are linked to the same Fc chain (see FIGs. 11A-L and 14A-L). In some embodiments, a targeting moiety is linked to the Fc domain and the other targeting moiety is linked to the signaling agent, e.g. IL-6, and the paired targeting moiety is linked to the Fc domain (see FIGs. 11A-L, 14A-L, 16A- J, and 17A-J). In some embodiments, the unpaired targeting moiety and paired targeting moiety are linked to the same Fc chain (see FIGs. 11A-L and 14A-L) In some embodiments, the unpaired targeting moiety and paired targeting moiety are linked to different Fc chains (see FIGs. 16A-J and 17A-J). In some embodiments, the unpaired targeting moiety and paired targeting moiety are linked on the same terminus (see FIGs. 16A-J and 17A-J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0447] In some embodiments, where there are one signaling agent, e.g. IL-6 and two targeting moieties, a targeting moiety is linked to the signaling agent, e.g. IL-6, which is linked to the Fc domain, and the unpaired targeting moiety is linked the Fc domain (see FIGs. 11A-L, 14A-L, 16A-J, and 17A-J). In some embodiments, the paired signaling agent, e.g., IL-6 and unpaired targeting moiety are linked to the same Fc chain (see FIGs. 11A-L and 14A-L). In some embodiments, the paired signaling agent, e.g. IL-6 and unpaired targeting moiety are linked to different Fc chains (see FIGs. 16A-J and 17A-J). In some embodiments, the paired signaling agent, e.g. IL-6 and unpaired targeting moiety are linked on the same terminus (see FIGs. 16A-J and 17A-J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0448] In some embodiments, where there are one signaling agent, e.g. IL-6 and two targeting moieties, the targeting moieties are linked together and the signaling agent, e.g. IL-6 is linked to one of the paired targeting moieties, wherein the targeting moiety not linked to the signaling agent, e.g. IL-6 is linked to the Fc domain (see FIGs. 11A- L and 14A-L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0449] In some embodiments, where there are one signaling agent, e.g. IL-6 and two targeting moieties, the targeting moieties are linked together and the signaling agent, e.g. IL-6 is linked to one of the paired targeting moieties, wherein the signaling agent, e.g. IL-6 is linked to the Fc domain (see FIGs. 11A-L and 14A-L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0450] In some embodiments, where there are one signaling agent, e.g. IL-6 and two targeting moieties, the targeting moieties are both linked to the signaling agent, e.g. IL-6, wherein one of the targeting moieties is linked to the Fc domain (see FIGs. 11A-L and 14A-L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0451] In some embodiments, where there are one signaling agent, e.g. IL-6 and two targeting moieties, the targeting moieties and the signaling agent, e.g. IL-6 are linked to the Fc domain (see FIGs. 16A-J and 17A-J). In some embodiments, the targeting moieties are linked on the terminus (see FIGs. 16A-J and 17A-J). In some

[0452] DB1 / 162517863.2 73 ORN-108PC / 114384-5108 embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0453] In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are linked to the Fc domain on the same terminus and the targeting moiety is linked to the Fc domain (see FIGs. 6A-J and 9A-J). In some embodiments, the signaling agents are linked to the Fc domain on the same Fc chain and the targeting moiety is linked on the other Fc chain (see FIGs. 18A-F and 19A-F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0454] In some embodiments, where there are two signaling agents and one targeting moiety, a signaling agent, e.g. IL- 6 is linked to the targeting moiety, which is linked to the Fc domain and the other signaling agent, e.g. IL-6 is linked to the Fc domain (see FIGs. 6A-J, 9A-J, 12A-L, and 15A-L). In some embodiments, the targeting moiety and the unpaired signaling agent, e.g. IL-6 are linked to different Fc chains (see FIGs. 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling agent, e.g. IL-6 are linked to different Fc chains on the same terminus (see FIGs. 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling agent, e.g. IL-6 are linked to different Fc chains on different termini (see FIGs. 6A-J and 9A-J). In some embodiments, the targeting moiety and the unpaired signaling agent, e.g. IL-6 are linked to the same Fc chains (see FIGs. 12A-L and 15A-L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0455] In some embodiments, where there are two signaling agents and one targeting moiety, the targeting moiety is linked to a signaling agent, e.g. IL-6, which is linked to the Fc domain and the other signaling agent, e.g. IL-6 is linked to the Fc domain (see FIGs. 6A-J and 9A-J). In some embodiments, the paired signaling agent, e.g. IL-6 and the unpaired signaling agent, e.g. IL-6 are linked to different Fc chains (see FIGs. 6A-J and 9A-J). In some embodiments, the paired signaling agent, e.g. IL-6 and the unpaired signaling agent, e.g. IL-6 are linked to different Fc chains on the same terminus (see FIGs. 6A-J and 9A-J). In some embodiments, the paired signaling agent, e.g. IL-6 and the unpaired signaling agent, e.g. IL-6 are linked to different Fc chains on different termini (see FIGs. 6A- J and 9A-J). In some embodiments, the Fc domain is heterodimeric In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0456] In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are linked together and the targeting moiety is linked to one of the paired signaling agents, wherein the targeting moiety is linked to the Fc domain (see FIGs. 12A-L and 15A-L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0457] In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are linked together and one of the signaling agents is linked to the Fc domain and the targeting moiety is linked to the Fc domain (see FIGs. 12A-L, 15A-L, 18A-F, and 19A-F). In some embodiments, the paired signaling agents and targeting moiety are linked to the same Fc chain (see FIGs. 12A-L and 15A-L). In some embodiments, the paired

[0458] DB1 / 162517863.2 74 ORN-108PC / 114384-5108 signaling agents and targeting moiety are linked to different Fc chains (see FIGs 18A-F and 19A-F). In some embodiments, the paired signaling agents and targeting moiety are linked to different Fc chains on the same terminus (see FIGs. 18A-F and 19A-F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0459] In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are both linked to the targeting moiety, wherein one of the signaling agents is linked to the Fc domain (see FIGs. 12A- L and 15A-L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0460] In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are linked together and one of the signaling agents is linked to the targeting moiety and the other signaling agent, e.g. IL-6 is linked to the Fc domain (see FIGs. 12A-L and 15A-L)

[0461] In some embodiments, where there are two signaling agents and one targeting moiety, each signaling agent, e.g. IL-6 is linked to the Fc domain and the targeting moiety is linked to one of the signaling agents (see FIGs. 12A-L and 15A-L). In some embodiments, the signaling agents are linked to the same Fc chain (see FIGs. 12A-L and 15A-L).

[0462] In some embodiments, a targeting moiety or signaling agent, e.g. IL-6 is linked to the Fc domain, comprising one or both of CH2 and CH3 domains, and optionally a hinge region. For example, vectors encoding the targeting moiety, signaling agent, e.g. IL-6, or combination thereof, linked as a single nucleotide sequence to an Fc domain can be used to prepare such polypeptides.

[0463] Additional Signaling Agents

[0464] In one aspect, the present invention provides a chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex comprising one or more signaling agents (for instance, an immune-modulating agent) in addition to the IL-6 or a variant thereof described herein. In illustrative embodiments, the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex may comprise two, three, four, five, six, seven, eight, nine, ten or more signaling agents in addition to the IL-6 or a variant thereof described herein. In various embodiments, the additional signaling agent is modified to have reduced affinity or activity for one or more of its receptors, which allows for attenuation of activity (inclusive of agonism or antagonism) and / or prevents nonspecific signaling or undesirable sequestration of the chimeric proteins or chimeric protein complexes such as Fc- based chimeric protein complex

[0465] In various embodiments, the additional signaling agent is antagonistic in its wild type form and bears one or more mutations that attenuate its antagonistic activity. In various embodiments, the additional signaling agent is antagonistic due to one or more mutations, e.g. an agonistic signaling agent is converted to an antagonistic signaling agent and, such a converted signaling agent, optionally, also bears one or more mutations that attenuate

[0466] DB1 / 162517863.2 75 ORN-108PC / 114384-5108 its antagonistic activity (e.g. as described in WO 2015 / 007520, the entire contents of which are hereby incorporated by reference).

[0467] In various embodiments, the additional signaling agent is selected from modified versions of cytokines, growth factors, and hormones Illustrative examples of such cytokines, growth factors, and hormones include, but are not limited to, lymphokines, monokines, traditional polypeptide hormones, such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-a and tumor necrosis factor-|3; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-a; platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and TGF-|3; insulin-like growth factor-l and -II ; osteo inductive factors; interferons such as, for example, interferon-a, interferon-|3 and interferon-y (and interferon type I, II, and III), colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as, for example, IL-1 , IL-1o, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, and IL-18; a tumor necrosis factor such as, for example, TNF-a or TNF-[3; and other polypeptide factors including, for example, LIF and kit ligand (KL). As used herein, cytokines, growth factors, and hormones include proteins obtained from natural sources or produced from recombinant bacterial, eukaryotic or mammalian cell culture systems and biologically active equivalents of the native sequence cytokines.

[0468] In some embodiments, the additional signaling agent is a modified version of a growth factor selected from, but not limited to, transforming growth factors (TGFs) such as TGF-o and TGF-(3, epidermal growth factor (EGF), insulin-like growth factor such as insulin-like growth factor-l and -II, fibroblast growth factor (FGF), heregulin, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF)

[0469] In an embodiment, the growth factor is a modified version of a fibroblast growth factor (FGF). Illustrative FGFs include, but are not limited to, FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11 , FGF12, FGF13, FGF14, murine FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0470] In an embodiment, the growth factor is a modified version of a vascular endothelial growth factor (VEGF). Illustrative VEGFs include, but are not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF and isoforms thereof including the various isoforms of VEGF-A such as VEGF121, VEGFi2ib, VEGF145, VEGF165, VEGF sb, VEGFisg, and VEGF206.

[0471] In an embodiment, the growth factor is a modified version of a transforming growth factor (TGF). Illustrative TGFs include, but are not limited to, TGF-o and TGF-(3 and subtypes thereof including the various subtypes of TGF-(3 including TGF 1, TGF|32, and TGF 3.

[0472] In some embodiments, the additional signaling agent is a modified version of a hormone selected from, but not limited to, human chorionic gonadotropin, gonadotropin releasing hormone, an androgen, an estrogen, thyroid-

[0473] DB1 / 162517863.2 76 ORN-108PC / 114384-5108 stimulating hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growth hormone releasing hormone, corticotropin-releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoids, mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin, glucagon, amylin, calcitriol, calciferol, atrial-natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor (IGF), leptin, thrombopoietin, erythropoietin (EPO), and angiotensinogen.

[0474] In some embodiments, the additional signaling agent is an immune-modulating agent, e.g. one or more of an interleukin, interferon, and tumor necrosis factor.

[0475] In some embodiments, the additional signaling agent is an interleukin, including for example IL-1 ; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11 ; IL-12; IL-13; IL-14; IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL- 23; IL-24; IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31 ; IL-32; IL-33; IL-35; IL-36 or a fragment, variant, analogue, or family-member thereof. Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulating proliferation of immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis of neutrophils and T lymphocytes, and / or inhibition of interferons. Interleukin activity can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20.

[0476] In some embodiments, the signaling agent is a modified version of an interferon such as interferon types I, II, and III. Illustrative interferons, including for example, interferon-a-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21 , interferon- P and interferon-y, interferon K, interferon s, interferon T, and interferon G5.

[0477] In embodiments, the additional signaling agent is a type I interferon. In embodiments, the type I interferon is selected from IFN-a2, IFNal , IFN-p, IFN-y, Consensus IFN, IFN-e, IFN-K, IFN-T, IFN-5, and IFN-v.

[0478] In some embodiments, the additional signaling agent is a modified version of a tumor necrosis factor (TNF) or a protein in the TNF family, including but not limited to, TNF-a, TNF-p, LT-p, CD40L, CD27L, CD30L, FASL, 4-1 BBL, OX40L, and TRAIL.

[0479] In various embodiments, the additional signaling agent is a modified (e.g. mutant) form of the signaling agent having one or more mutations. In various embodiments, the mutations allow for the modified signaling agent to have one or more of attenuated activity such as one or more of reduced binding affinity, reduced endogenous activity, and reduced specific bioactivity relative to unmodified or unmutated, i.e. the wild type form of the signaling agent (e.g. comparing the same signaling agent in a wild type form versus a modified (e.g. mutant) form). In various embodiments, the mutations allow for the modified signaling agent to have one or more of attenuated activity such as one or more of reduced binding affinity, reduced endogenous activity, and reduced specific bioactivity relative to unmodified or unmutated, i.e. the unmutated IL-6. In some embodiments, the mutations, which attenuate or reduce binding or affinity, include those mutations that substantially reduce or ablate binding or activity. In some

[0480] DB1 / 162517863.2 77 ORN-108PC / 114384-5108 embodiments, the mutations, which attenuate or reduce binding or affinity, are different than those mutations which substantially reduce or ablate binding or activity. Consequentially, in various embodiments, the mutations allow for the signaling agent to be more safe, e.g. have reduced systemic toxicity, reduced side effects, and reduced off- target effects relative to unmutated, i.e. wild type, signaling agent (e.g. comparing the same signaling agent in a wild type form versus a modified (e.g. mutant) form). In various embodiments, the mutations allow for signaling agent to be safer, e.g. have reduced systemic toxicity, reduced side effects, and reduced off-target effects relative to unmutated interferon, e.g. the unmutated sequence of IL-6.

[0481] In various embodiments, the additional signaling agent is modified to have one or more mutations that reduce its binding affinity or activity for one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or ablate binding affinity or activity for the receptors. In some embodiments, the activity provided by the wild type signaling agent is agonism at the receptor (e.g. activation of a cellular effect at a site of therapy) For example, the wild type signaling agent may activate its receptor. In such embodiments, the mutations result in the modified signaling agent to have reduced or ablated activating activity at the receptor. For example, the mutations may result in the modified signaling agent to deliver a reduced activating signal to a target cell or the activating signal could be ablated. In some embodiments, the activity provided by the wild type signaling agent is antagonism at the receptor (e.g. blocking or dampening of a cellular effect at a site of therapy). For example, the wild type signaling agent may antagonize or inhibit the receptor. In these embodiments, the mutations result in the modified signaling agent to have a reduced or ablated antagonizing activity at the receptor. For example, the mutations may result in the modified signaling agent to deliver a reduced inhibitory signal to a target cell or the inhibitory signal could be ablated. In various embodiments, the signaling agent is antagonistic due to one or more mutations, e.g. an agonistic signaling agent is converted to an antagonistic signaling agent (e.g. as described in WO 2015 / 007520, the entire contents of which are hereby incorporated by reference) and, such a converted signaling agent, optionally, also bears one or more mutations that reduce its binding affinity or activity for one or more of its receptors or that substantially reduce or ablate binding affinity or activity for one or more of its receptors.

[0482] In some embodiments, the reduced affinity or activity at the receptor is inducible or restorable by attachment with one or more of the targeting moieties or upon inclusion in the Fc-based chimeric protein complex disclosed herein. In other embodiments, the reduced affinity or activity at the receptor is not substantially inducible or restorable by the activity of one or more of the targeting moieties or upon inclusion in the Fc-based chimeric protein complex disclosed herein.

[0483] In various embodiments, the additional signaling agent is active on target cells because the targeting moiety(ies) compensates for the missing / insufficient binding (e.g., without limitation and / or avidity) required for substantial activation. In various embodiments, the modified signaling agent is substantially inactive en route to the site of therapeutic activity and has its effect substantially on specifically targeted cell types, which greatly reduces undesired side effects.

[0484] DB1 / 162517863.2 78 ORN-108PC / 114384-5108

[0485] In some embodiments, the additional signaling agent may include one or more mutations that attenuate or reduce binding or affinity for one receptor (i.e., a therapeutic receptor) and one or more mutations that substantially reduce or ablate binding or activity at a second receptor. In such embodiments, these mutations may be at the same or at different positions (i.e., the same mutation or multiple mutations) In some embodiments, the mutation(s) that reduce binding and / or activity at one receptor is different than the mutation(s) that substantially reduce or ablate at another receptor. In some embodiments, the mutation(s) that reduce binding and / or activity at one receptor is the same as the mutation(s) that substantially reduce or ablate at another receptor. In some embodiments, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes have a modified signaling agent that has both mutations that attenuate binding and / or activity at a therapeutic receptor and therefore allow for a more controlled, on-target therapeutic effect (e.g. relative wild type signaling agent) and mutations that substantially reduce or ablate binding and / or activity at another receptor and therefore reduce side effects (e.g. relative to wild type signaling agent).

[0486] In some embodiments, the substantial reduction or ablation of binding or activity is not substantially inducible or restorable with a targeting moiety or upon inclusion in the Fc-based chimeric protein complex disclosed herein. In some embodiments, the substantial reduction or ablation of binding or activity is inducible or restorable with a targeting moiety or upon inclusion in the Fc-based chimeric protein complex disclosed herein. In various embodiments, substantially reducing or ablating binding or activity at a second receptor also may prevent deleterious effects that are mediated by the other receptor. Alternatively, or in addition, substantially reducing or ablating binding or activity at the other receptor causes the therapeutic effect to improve as there is a reduced or eliminated sequestration of the therapeutic chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes away from the site of therapeutic action. For instance, in some embodiments, this obviates the need of high doses of the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes that compensate for loss at the other receptor. Such ability to reduce dose further provides a lower likelihood of side effects.

[0487] In various embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced, substantially reduced, or ablated affinity, e.g. binding (e.g. KD) and / or activation (for instance, when the modified signaling agent is an agonist of its receptor, measurable as, for example, KA and / or EC50) and / or inhibition (for instance, when the modified signaling agent is an antagonist of its receptor, measurable as, for example, Ki and / or IC50), for one or more of its receptors. In various embodiments, the reduced affinity at the signaling agent’s receptor allows for attenuation of activity (inclusive of agonism or antagonism). In such embodiments, the modified signaling agent has about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% of the activity and / or affinity for the receptor relative to the wild type signaling agent In some embodiments, the binding activity and / or 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,

[0488] DB1 / 162517863.2 79 ORN-108PC / 114384-5108 about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower relative to the wild type signaling agent (including, by way of non-lim itation, relative to the unmutated IL-6).

[0489] In embodiments wherein the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex has mutations that reduce binding at one receptor and substantially reduce or ablate binding at a second receptor, the attenuation or reduction in binding affinity of a modified signaling agent for one receptor is less than the substantial reduction or ablation in affinity for the other receptor. In some embodiments, the attenuation or reduction in binding affinity of a modified signaling agent for one receptor is less than the substantial reduction or ablation in affinity for the other receptor by about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In various embodiments, substantial reduction or ablation refers to a greater reduction in binding affinity and / or activity than attenuation or reduction.

[0490] In various embodiments, the additional modified signaling agent comprises one or more mutations that reduce the endogenous activity of the signaling agent to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1 %, e.g., relative to the wild type signaling agent (including, by way of non-limitation, relative to the unmutated IL-6).

[0491] In various embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and / or activity for a receptor of any one of the cytokines, growth factors, and hormones as described herein.

[0492] In some embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity for its receptor that is lower than the binding affinity of the targeting moiety(ies) for its(their) receptor(s). In some embodiments, this binding affinity differential is between signaling agent / receptor and targeting moiety / receptor on the same cell. In some embodiments, this binding affinity differential allows for the signaling agent, e.g. mutated signaling agent, to have localized, on-target effects and to minimize off-target effects that underlie side effects that are observed with wild type signaling agent. In some embodiments, this binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold lower, or at least about 25-fold, or at least about 50-fold lower, or at least about 100-fold, or at least about 150-fold.

[0493] Receptor binding activity may be measured using methods known in the art For example, affinity and / or binding activity may be assessed by Scatchard plot analysis and computer-fitting of binding data (e.g. Scatchard, 1949) or by reflectometric interference spectroscopy under flow through conditions, as described by Brecht et al. (1993), the entire contents of all of which are hereby incorporated by reference.

[0494] DB1 / 162517863.2 80 ORN-108PC / 114384-5108

[0495] The amino acid sequences of the wild type signaling agents described herein are well known in the art. Accordingly, in various embodiments the additional modified signaling agent comprises an amino acid sequence that has at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known wild type amino acid sequences of the signaling agents described herein (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0496] In various embodiments the additional modified signaling agent comprises an amino acid sequence that has at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with any of the sequences disclosed herein (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0497] In various embodiments, the additional modified signaling agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0498] DB1 / 162517863.2 81 ORN-108PC / 114384-5108

[0499] In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and / or non-conservative substitutions as described herein.

[0500] As described herein, the additional modified signaling agents bear mutations that affect affinity and / or activity at one or more receptors. In various embodiments, there is reduced affinity and / or activity at a therapeutic receptor, e.g. a receptor through which a desired therapeutic effect is mediated (e.g. agonism or antagonism) In various embodiments, the modified signaling agents bear mutations that substantially reduce or ablate affinity and / or activity at a receptor, e.g. a receptor through which a desired therapeutic effect is not mediated (e.g. as the result of promiscuity of binding) The receptors of any modified signaling agents, e.g. one of the cytokines, growth factors, and hormones as described herein, are known in the art.

[0501] Illustrative mutations which provide reduced affinity and / or activity (e.g. agonistic) at a receptor are found in WO 2013 / 107791 (e.g. with regard to interferons), WO 2015 / 007542 (e.g. with regard to interleukins), and WO 2015 / 007903 (e.g. with regard to TNF), the entire contents of each of which are hereby incorporated by reference. Illustrative mutations which provide reduced affinity and / or activity (e.g. antagonistic) at a therapeutic receptor are found in WO 2015 / 007520, the entire contents of which are hereby incorporated by reference.

[0502] In some embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and / or activity for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a receptor in the Tumor Necrosis Factor Receptor (TNFR) superfamily, TGF-beta Receptors, a receptor in the immunoglobulin (Ig) superfamily, and / or a receptor in the tyrosine kinase superfamily.

[0503] In various embodiments, the receptor for the additional signaling agent is a Type I cytokine receptor Type I cytokine receptors are known in the art and include, but are not limited to receptors for IL2 (beta-subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11 , IL12, GM-CSF, G-CSF, LIF, CNTF, and also the receptors for Thrombopoietin (TPO), Prolactin, and Growth hormone. Illustrative type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptors.

[0504] In various embodiments, the receptor for the additional signaling agent is a Type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits, and are receptors mainly for interferons. This family of receptors includes, but is not limited to, receptors for interferon-a, interferon-p and interferon-y, IL10, IL22, and tissue factor. Illustrative type II cytokine receptors include, but are not limited to, IFN- a receptor (e.g. IFNAR1 and IFNAR2), I FN- receptor, IFN-y receptor (e.g. IFNGR1 and IFNGR2), and type II IL receptors.

[0505] In various embodiments, the receptor for the additional signaling agent is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors with seven transmembrane structure and coupled to G-protein for signal transduction. Chemokine receptors include, but are not limited to, CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptor (XCR1). Illustrative chemokine receptors include, but are not limited to, CCR1 , CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1 , and CX3CR1.

[0506] DB1 / 162517863.2 82 ORN-108PC / 114384-5108

[0507] In various embodiments, the receptor for the additional signaling agent is a TNFR family member. Tumor necrosis factor receptor (TNFR) family members share a cysteine-rich domain (CRD) formed of three disulfide bonds surrounding a core motif of CXXCXXC creating an elongated molecule. Illustrative tumor necrosis factor receptor family members include: GDI 20a (TNFRSFIA), CD 120b (TNFRSFIB), Lymphotoxin beta receptor (LTBR, TNFRSF3), CD 134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSFIOA (TNFRSFIOA), TNFRSFIOB, (TNFRSFIOB), TNFRSFIOC (TNFRSFIOC), TNFRSFIOD (TNFRSFIOD), RANK (TNFRSFI IA), Osteoprotegerin (TNFRSFI IB), TNFRSF12A (TNFRSF12A), TNFRSF13B (TNFRSF13B), TNFRSF13C (TNFRSF13C), TNFRSF14 (TNFRSF14), Nerve growth factor receptor (NGFR, TNFRSF16), TNFRSF17 (TNFRSF17), TNFRSF18 (TNFRSF18), TNFRSF19 (TNFRSF19), TNFRSF21 (TNFRSF21), and TNFRSF25 (TNFRSF25).

[0508] In various embodiments, the receptor for the additional signaling agent is a TGF-beta receptor TGF-beta receptors are single pass serine / threonine kinase receptors. TGF-beta receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR3.

[0509] In various embodiments, the receptor for the additional signaling agent is an Ig superfamily receptor. Receptors in the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins Receptors in the Ig superfamily include, but are not limited to, interleukin-1 receptors, CSF-1 R, PDGFR (e.g. PDGFRA and PDGFRB), and SCFR.

[0510] In various embodiments, the receptor for the additional signaling agent is a tyrosine kinase superfamily receptor. Receptors in the tyrosine kinase superfamily are well known in the art. There are about 58 known receptor tyrosine kinases (RTKs), grouped into 20 subfamilies. Receptors in the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0511] In an embodiment, the additional modified signaling agent is interferon a. In such embodiments, the modified IFN- a agent has reduced affinity and / or activity for the IFN-o / p receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFN-a agent has substantially reduced or ablated affinity and / or activity for the IFN-a / (3 receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2 chains.

[0512] Mutant forms of interferon a are known to the person skilled in the art. In an illustrative embodiment, the modified signaling agent is the allelic form IFN-a2a having the amino acid sequence of SEQ ID NO:233.

[0513] In an illustrative embodiment, the modified signaling agent is the allelic form IFN-o2b having the amino acid sequence of SEQ ID NO:234 (which differs from IFN-a2a at amino acid position 23).

[0514] In some embodiments, said I FN-a2 mutant (IFN-a2a or I FN-a2b) is mutated at one or more amino acids at positions 144-154, such as amino acid positions 148, 149 and / or 153. In some embodiments, the IFN-a2 mutant comprises one or more mutations selected from L153A, R149A, and M148A. Such mutants are described, for example, in WO2013 / 107791 and Piehler et al., (2000) J. Biol. Chem, 275:40425-33, the entire contents of all of which are hereby incorporated by reference.

[0515] DB1 / 162517863.2 83 ORN-108PC / 114384-5108

[0516] In some embodiments, the IFN-o2 mutants have reduced affinity and / or activity for i FNAR1. In some embodiments, the I FN-a2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A, as described in WO2010 / 030671 , the entire contents of which is hereby incorporated by reference.

[0517] In some embodiments, the IFN-a2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A as described in W02008 / 124086, the entire contents of which is hereby incorporated by reference.

[0518] In some embodiments, the IFN-a2 mutant comprises one or more mutations selected from R120E and R120E / K121 E, as described in W02015 / 007520 and WO2010 / 030671, the entire contents of which are hereby incorporated by reference. In such embodiments, said I FN-a2 mutant antagonizes wildtype IFN-o2 activity. In such embodiments, said mutant IFN-a2 has reduced affinity and / or activity for IFNAR1 while affinity and / or activity of IFNR2 is retained.

[0519] In some embodiments, the human I FN-a2 mutant comprises (1) one or more mutations selected from R120E and R120E / K121 E, which, without wishing to be bound by theory, create an antagonistic effect and (2) one or more mutations selected from K133A, R144A, R149A, and L153A, which, without wishing to be bound by theory, allow for an attenuated effect at, for example, IFNAR2 In an embodiment, the human I FN-a2 mutant comprises R120E and L153A

[0520] In some embodiments, the human I FN-a2 mutant comprises one or more mutations selected from, L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A as disclosed in WO 2013 / 059885, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the human IFN-a2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L30A as disclosed in WO 2013 / 059885 In some embodiments, the human IFN-o2 mutant comprises the mutations H57Y, E58N, Q61S, and / or R33A as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-a2 mutant comprises the mutations H57Y, E58N, Q61S, and / or M148A as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-a2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L153A as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-a2 mutant comprises the mutations N65A, L80A, Y85A, and / or Y89A as disclosed in WO 2013 / 059885. In some embodiments, the human I FN-o2 mutant comprises the mutations N65A, L80A, Y85A, Y89A, and / or D114A as disclosed in WO 2013 / 059885 In some embodiments, the human IFN-a2 mutant comprises one or more mutations selected from R144Xi, A145X2, and R33A, wherein Xi is selected from A, S, T, Y, L, and I, and wherein X2 is selected from G, H, Y, K, and D. In some embodiments, the human IFN-a2 mutant comprises one or more mutations selected from R33A, TIO6X3, R120E, R144Xi A145X2, M148A, R149A, and L153A with respect to amino acid sequence of SEQ ID NO: 233 or 234, wherein Xi is selected from A, S, T, Y, L, and I, wherein X2is selected from G, H, Y, K, and D, and wherein X3is selected from A and E.

[0521] In an embodiment, the additional modified signaling agent is interferon a1 (IFNal). In such embodiments, the modified IFN-a1 agent has reduced affinity and / or activity for the IFN-a / p receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2 chains In some embodiments, the present invention provides a chimeric protein or chimeric protein

[0522] DB1 / 162517863.2 84 ORN-108PC / 114384-5108 complexes, such as Fc-based chimeric protein complexes that include a wild type IFNol . In various embodiments, the wild-type I FNa 1 comprises the following amino acid sequence:

[0523] CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAA WDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEWRAEIMRS LSLSTNLQERLRRKE (SEQ ID NO: 5).

[0524] In various embodiments, the IFNal variant has one or more mutations that reduce its binding to or its affinity for the IFNAR1 subunit of IFNAR. In one embodiment, the IFNal variant has reduced affinity and / or activity at I FNAR1. In some embodiments, the IFNal variant has one or more mutations that reduce its binding to or its affinity for the IFNAR2 subunit of IFNAR. In some embodiments, the IFNal variant has one or more mutations that reduce its binding to or its affinity for both IFNAR1 and IFNAR2 subunits.

[0525] In some embodiments, the IFNal variant has one or more mutations that reduce its binding to or its affinity for IFNAR1 and one or more mutations that substantially reduce or ablate binding to or its affinity for IFNAR2. In some embodiments, chimeric proteins and chimeric protein complexes, such as or Fc-based chimeric protein complexes with such IFNal variant can provide target-selective IFNAR1 activity (e.g. IFNAR1 activity is inducible or restorable via targeting through the targeting moiety or upon inclusion in the Fc-based chimeric protein complex disclosed herein).

[0526] In some embodiments, the IFNal variant has one or more mutations that reduce its binding to or its affinity for IFNAR2 and one or more mutations that substantially reduce or ablate binding to or its affinity for IFNAR1. In some embodiments, chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes with such IFNal variant can provide target-selective IFNAR2 activity (e.g. IFNAR2 activity is inducible or restorable via targeting through the targeting moiety or upon inclusion in the Fc-based chimeric protein complex disclosed herein).

[0527] In some embodiments, the IFNal variant has one or more mutations that reduce its binding to or its affinity for IFNAR1 and one or more mutations that reduce its binding to or its affinity for IFNAR2. In some embodiments, chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes with such IFNal variant can provide target-selective IFNAR1 and / or IFNAR2 activity (e.g IFNAR1 and / IFNAR2 activity is inducible or restorable via targeting through the targeting moiety or upon inclusion in the Fc-based chimeric protein complex disclosed herein).

[0528] In some embodiments, the IFNal variant comprises one or more mutations which reduce undesired disulphide pairings to improve product homogeneity and pharmaceutical properties of the chimeric protein or chimeric protein complexes, while simultaneously maintaining or avoiding substantial loss of IFNAR-activation of the modified IFNal compared to wild type IFNal in the context of chimeric proteins or chimeric protein complexes, including maintaining or avoiding substantial loss of restoration and induction of IFNAR-activation by the modified IFNal when directed or targeted to a target cell through a targeting moiety. In some embodiments, the IFNal is a variant that comprises one or more mutations which reduce undesired disulphide pairings wherein the one or more

[0529] DB1 / 162517863.2 85 ORN-108PC / 114384-5108 mutations are, e.g., at amino acid positions C1 , C29, C86, C99, or C139 with reference to SEQ ID NO: 5. In some embodiments, the mutation at position C86 can be, e.g., C86S or C86A or C86Y. These C86 mutants of I FNct 1 are called reduced cysteine-based aggregation mutants. In some embodiment, the IFNal variant includes mutations at positions C1, 086 and 099 with reference to SEQ ID NO: 5. In embodiments, any of 01 , 086 and 099 made be deleted or substituted.

[0530] In various embodiments, the chimeric protein or chimeric protein complexes, such as Fc-based chimeric protein complexes of the invention comprises a modified version of IFNal, i.e., a IFNal variant including a IFNal mutant, as a signaling agent. In various embodiments, the IFNal variant encompasses mutants, functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of the interferon.

[0531] In some embodiments, the IFNal variant comprises one or more mutations that cause the IFNal variant to have reduced affinity for a receptor. In some embodiments, the IFNal variant’s binding affinity for a receptor is lower than the binding affinity of the targeting moiety for its receptor In some embodiments, this binding affinity differential is between the IFNal variant / receptor and targeting moiety / receptor on the same cell. In some embodiments, this binding affinity, differential allows for the IFNal variant to have localized, on-target effects and to minimize off- target effects that underlie side effects that are observed with wild type IFNal . In some embodiments, this binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold lower, or at least about 25-fold, or at least about 50-fold lower, or at least about 100-fold, or at least about 150-fold less.

[0532] In an illustrative embodiment, the modified additional signaling agent is I FN-|3. In various embodiments, the IFN-P encompasses functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of IFN-p. In various embodiments, the IFN-P encompasses IFN-P derived from any species. In an embodiment, the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex comprises a modified version of mouse IFN-p. In another embodiment, the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex comprises a modified version of human IFN-p. Human IFN-P is a polypeptide with a molecular weight of about 22 kDa comprising 166 amino acid residues. The amino acid sequence of human IFN- P is SEQ ID NO: 277.

[0533] In some embodiments, the human IFN-P is IFN-P-1a, which is a glycosylated form of human IFN-p. In some embodiments, the human IFN-P is IFN-P- 1 b, which is a non-glycosylated form of human IFN-P that has a Met-1 deletion and a Cys-17 to Ser mutation.

[0534] In various embodiments, the modified IFN-p has one or more mutations that reduce its binding to or its affinity for the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFN-p has reduced affinity and / or activity at IFNAR1 . In various embodiments, the modified IFN-p is human IFN-p and has one or more mutations at positions F67, R71 , L88, Y92, 195, N96, K123, and R124. In some embodiments, the one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G. In an embodiment, the modified IFN-P comprises the F67G mutation. In an embodiment, the modified IFN-P comprises the K123G mutation. In an embodiment, the modified IFN-P comprises the F67G and R71 A mutations. In an embodiment, the

[0535] DB1 / 162517863.2 86 ORN-108PC / 114384-5108 modified IFN-p comprises the L88G and Y92G mutations. In an embodiment, the modified IFN-[3 comprises the Y92G, I95A, and N96G mutations. In an embodiment, the modified IFN-p comprises the K123G and R124G mutations. In an embodiment, the modified IFN-p comprises the F67G, L88G, and Y92G mutations. In an embodiment, the modified IFN-p comprises the F67S, L88S, and Y92S mutations.

[0536] In some embodiments, the modified IFN-P has one or more mutations that reduce its binding to or its affinity for the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFN-P has reduced affinity and / or activity at IFNAR2. In various embodiments, the modified IFN-P is human IFN-P and has one or more mutations at positions W22, R27, L32, R35, V148, L151 , R152, and Y155. In some embodiments, the one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In an embodiment, the modified IFN-p comprises the W22G mutation. In an embodiment, the modified IFN-p comprises the L32A mutation. In an embodiment, the modified IFN-p comprises the L32G mutation. In an embodiment, the modified IFN-p comprises the R35A mutation. In an embodiment, the modified IFN-p comprises the R35G mutation. In an embodiment, the modified IFN-p comprises the V148G mutation. In an embodiment, the modified IFN-p comprises the R152A mutation In an embodiment, the modified IFN-p comprises the R152G mutation. In an embodiment, the modified IFN-p comprises the Y155G mutation. In an embodiment, the modified IFN-p comprises the W22G and R27G mutations. In an embodiment, the modified IFN-p comprises the L32A and R35A mutation In an embodiment, the modified IFN-p comprises the L151G and R152A mutations. In an embodiment, the modified IFN-p comprises the V148G and R152A mutations.

[0537] In some embodiments, the modified IFN-p has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFN-p has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with C17S or C17A

[0538] In some embodiments, the modified IFN-P has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141 Y, A142T, E149K, and R152H In combination with any of the other IFN-P mutations described herein.

[0539] The crystal structure of human IFN-P is known and is described in Karpusas et al., (1998) PNAS, 94(22): 11813— 11818. Specifically, the structure of human IFN-P has been shown to include five o-helices ( / .e., A, B, C, D, and E) and four loop regions that connect these helices ( / .e., AB, BC, CD, and DE loops). In various embodiments, the modified IFN-P has one or more mutations in the A, B, C, D, E helices and / or the AB, BC, CD, and DE loops which reduce its binding affinity or activity at a therapeutic receptor such as IFNAR. Illustrative mutations are described in WG2000 / 023114 and US20150011732, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, the modified IFN-P is human IFN-P comprising alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In an illustrative embodiment, the modified IFN-P is human IFN-P comprising alanine substitutions at amino acid positions 28-30, 32, and 33. In an illustrative embodiment, the modified IFN-P is human IFN-P comprising alanine substitutions at amino acid positions 36, 37, 39, and 42. In an illustrative embodiment, the modified IFN-P is human IFN-P comprising alanine substitutions at amino acid positions 64 and

[0540] DB1 / 162517863.2 87 ORN-108PC / 114384-5108

[0541] 67 and a serine substitution at position 68. In an illustrative embodiment, the modified IFN-|3 is human IFN-p comprising alanine substitutions at amino acid positions 71-73. In an illustrative embodiment, the modified IFN-[3 is human IFN-p comprising alanine substitutions at amino acid positions 92, 96, 99, and 100. In an illustrative embodiment, the modified IFN-p is human I FN-p comprising alanine substitutions at amino acid positions 128, 130, 131, and 134. In an illustrative embodiment, the modified IFN-p is human IFN-p comprising alanine substitutions at amino acid positions 149, 153, 156, and 159 In some embodiments, the mutant IFNp comprises SEQ ID NO:277 and a mutation at W22, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0542] In some embodiments, the mutant IFNp comprises SEQ ID NO:277 and a mutation at R27, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0543] In some embodiments, the mutant IFNp comprises SEQ ID NO:277 and a mutation at W22, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) and a mutation at R27, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0544] In some embodiments, the mutant IFNp comprises SEQ ID NO:277 and a mutation at L32, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0545] In some embodiments, the mutant IFNp comprises SEQ ID NO:277 and a mutation at R35, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0546] In some embodiments, the mutant IFNp comprises SEQ ID NO:277 and a mutation at L32, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V) and a mutation at R35, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0547] In some embodiments, the mutant IFNp comprises SEQ ID NO:277 and a mutation at F67, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0548] In some embodiments, the mutant IFNp comprises SEQ ID NO:277 and a mutation at R71 , the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0549] In some embodiments, the mutant IFNp comprises SEQ ID NO:277 and a mutation at F67, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) and a mutation at R71, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0550] DB1 / 162517863.2 88 ORN-108PC / 114384-5108

[0551] In some embodiments, the mutant I FN[3 comprises SEQ ID NO:277 and a mutation at L88, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0552] In some embodiments, the mutant I FN [3 comprises SEQ ID NO:277 and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0553] In some embodiments, the mutant I FN comprises SEQ ID NO:277 and a mutation at F67, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) and a mutation at L88, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V) and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0554] In some embodiments, the mutant IFNJ3 comprises SEQ ID NO:277 and a mutation at L88, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V) and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0555] In some embodiments, the mutant IFNfl comprises SEQ ID NO:277 and a mutation at 195, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V) and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0556] In some embodiments, the mutant I FN[3 comprises SEQ ID NO:277 and a mutation at N96, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0557] In some embodiments, the mutant IFN|3 comprises SEQ ID NO: 277 and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) and a mutation at 195, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V) and a mutation at N96, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0558] In some embodiments, the mutant IFN|3 comprises SEQ ID NO: 277 and a mutation at K123, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0559] DB1 / 162517863.2 89 ORN-108PC / 114384-5108

[0560] In some embodiments, the mutant IFN[3 comprises SEQ ID NO: 277 and a mutation at R124, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0561] In some embodiments, the mutant I FNf3 comprises SEQ ID NO: 711 and a mutation at K123, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) and a mutation at R124, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0562] In some embodiments, the mutant IFNp comprises SEQ ID NO: 277 and a mutation at L151 , the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0563] In some embodiments, the mutant IFN|3 comprises SEQ ID NO: 277 and a mutation at R152, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0564] In some embodiments, the mutant IFN|3 comprises SEQ ID NO: 277 and a mutation at L151 , the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V) and a mutation at R152, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0565] In some embodiments, the mutant I FN[3 comprises SEQ ID NO: 277 and a mutation at 7148, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0566] In some embodiments, the mutant IFN|3 comprises SEQ ID NO: 277 and a mutation at V148, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) and a mutation at R152, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0567] In some embodiments, the mutant I FN[3 comprises SEQ ID NO: 277 and a mutation at Y155, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0568] In some embodiments, the present invention relates to a chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex comprising: (a) a modified IFN-p, having the amino acid sequence of SEQ ID NO: 277 and a mutation at position W22, wherein the mutation is an aliphatic hydrophobic residue and a modified IL-2 or modified IL-2 variant disclosed here; and (b) one or more targeting moieties, said targeting moieties comprising recognition domains which specifically bind to antigens or receptors of interest, the modified IFN- and the one or more targeting moieties are optionally connected with one or more linkers. In various embodiments the

[0569] DB1 / 162517863.2 90 ORN-108PC / 114384-5108 mutation at position W22 is aliphatic hydrophobic residue is selected from G, A, L, I, M, and V. In various embodiments, the mutation at position W22 is G

[0570] Additional illustrative IFNfJ mutants are provided in PCT / EP2017 / 061544, the entire disclosure of which is incorporated by reference herein.

[0571] In some embodiments, the modified additional signaling agent is interferon y. In such embodiments, the modified interferon y agent has reduced affinity and / or activity for the interferon-gamma receptor (IFNGR), i.e., IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon y agent has substantially reduced or ablated affinity and / or activity for the interferon-gamma receptor (IFNGR), i.e., IFNGR1 and / or IFNGR2 chains.

[0572] In some embodiments, the modified additional signaling agent is a consensus interferon. The consensus interferon is generated by scanning the sequences of several human non-allelic IFN-a subtypes and assigning the most frequently observed amino acid in each corresponding position. The consensus interferon differs from I FN-a2b at 20 out of 166 amino acids (88% homology), and comparison with IFN-P shows identity at over 30% of the amino acid positions. In various embodiments, the consensus interferon comprises the following amino acid sequence of SEQ ID NO: 278.

[0573] In some embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO: 279, which differs from the amino acid sequence of SEQ ID NO: 278 by one amino acid, i.e., SEQ ID NO: 279 lacks the initial methionine residue of SEQ ID NO: 278:

[0574] In various embodiments, the consensus interferon comprises a modified version of the consensus interferon, i.e., a consensus interferon variant, as a signaling agent. In various embodiments, the consensus interferon variant encompasses functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of the consensus interferon.

[0575] In an embodiment, the consensus interferon variants are selected form the consensus interferon variants disclosed in U S Patent Nos. 4,695,623, 4,897,471, 5,541 ,293, and 8,496,921 , the entire contents of all of which are hereby incorporated by reference. For example, the consensus interferon variant may comprise the amino acid sequence of IFN-CON2 or IFN-CON3 as disclosed in U.S. Patent Nos 4,695,623, 4,897,471, and 5,541,293. In an embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON2: SEQ ID NO: 280.

[0576] In an embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON3: SEQ ID NO: 281.

[0577] In an embodiment, the consensus interferon variant comprises the amino acid sequence of any one of the variants disclosed in U.S. Patent No. 8,496,921 For example, the consensus variant may comprise the amino acid sequence of: SEQ ID NO: 282.

[0578] In another embodiment, the consensus interferon variant may comprise the amino acid sequence of: SEQ ID NO: 283.

[0579] DB1 / 162517863.2 91 ORN-108PC / 114384-5108

[0580] In some embodiments, the consensus interferon variant may be PEGylated, i.e., comprises a PEG moiety. In an embodiment, the consensus interferon variant may comprise a PEG moiety attached at the S156C position of SEQ ID NO: 283.

[0581] In some embodiments, the engineered interferon is a variant of human IFN-ct2a, with an insertion of Asp at approximately position 41 in the sequence Glu-Glu-Phe-Gly-Asn-GIn (SEQ ID NO: 284) to yield Glu-Glu-Phe-Asp- Gly-Asn-GIn (SEQ ID NO: 285) (which resulted in a renumbering of the sequence relative to IFN-a2a sequence) and the following mutations of Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser, Asp86Glu, lle104Thr, Gly106Glu, Thr110GIu, Lys117Asn, Arg125Lys, and Lys136Thr. All embodiments herein that describe consensus interferons apply equally to this engineered interferon

[0582] In some embodiments, the additional modified signaling agent is vascular endothelial growth factor (VEGF). VEGF is a potent growth factor that plays major roles in physiological but also pathological angiogenesis, regulates vascular permeability and can act as a growth factor on cells expressing VEGF receptors. Additional functions include, among others, stimulation of cell migration in macrophage lineage and endothelial cells. Several members of the VEGF family of growth factors exist, as well as at least three receptors (VEGFR-1 , VEGFR -2, and VEGFR -3). Members of the VEGF family can bind and activate more than one VEGFR type. For example, VEGF-A binds VEGFR-1 and -2, while VEGF-C can bind VEGFR-2 and -3. VEGFR-1 and VEGFR-2 activation regulate angiogenesis while VEGFR-3 activation is associated with lymphangiogenesis. The major pro-angiogenic signal is generated from activation of VEGFR-2. VEGFR-1 activation has been reported to be possibly associated with negative role in angiogenesis. It has also been reported that VEGFR-1 signaling is important for progression of tumors in vivo via bone marrow-derived VEGFR-1 positive cells (contributing to formation of premetastatic niche in the bone). Several therapies based on VEGF-A directed / neutralizing therapeutic antibodies have been developed, primarily for use in treatment of various human tumors relying on angiogenesis These are not without side effects though. This may not be surprising considering that these operate as general, non-cell / tissue specific VEGFA / EGFR interaction inhibitors. Hence, it would be desirable to restrict VEGF (e.g. VEGF-A)A / EGFR-2 inhibition to specific target cells (e.g. tumor vasculature endothelial cells).

[0583] In some embodiments, the VEGF is VEGF-A, VEGF-B, VEFG-C, VEGF-D, or VEGF-E and isoforms thereof including the various isoforms of VEGF-A such as VEGF121, VEGF121 b, VEGF145, VEGF165, VEGFiesb, VEGFiss, and VEGF206. In some embodiments, the modified signaling agent has reduced affinity and / or activity for VEGFR- 1 (Flt-1 ) and / or VEGFR-2 (KDR / Flk-1 ). In some embodiments, the modified signaling agent has substantially reduced or ablated affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1 ). In an embodiment, the modified signaling agent has reduced affinity and / or activity for VEGFR-2 (KDR / Flk-1 ) and / or substantially reduced or ablated affinity and / or activity for VEGFR-1 (Flt-1). Such an embodiment finds use, for example, in wound healing methods or treatment of ischmia-related diseases (without wishing to be bound by theory, mediated by VEGFR-2’s effects on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR- 1 (Flt-1 ), which is linked to cancers and pro-inflammatory activities, is avoided. In various embodiments, VEGFR- 1 (Flt-1 ) acts a decoy receptor and therefore substantially reduces or ablates affinity at this receptor avoids

[0584] DB1 / 162517863.2 92 ORN-108PC / 114384-5108 sequestration of the therapeutic agent. In an embodiment, the modified signaling agent has substantially reduced or ablated affinity and / or activity for VEGFR-1 (Flt-1) and / or substantially reduced or ablated affinity and / or activity for VEGFR-2 (KDR / Flk-1). In some embodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signaling agent has reduced affinity and / or activity for VEGFR-3. Alternatively, the modified signaling agent has substantially reduced or ablated affinity and / or activity for VEGFR-3.

[0585] Proangiogenic therapies are also important in various diseases [e.g. ischemic heart disease, bleeding etc.), and include VEGF-based therapeutics. Activation of VEGFR-2 is proangiogenic (acting on endothelial cells). Activation of VEFGR-1 can cause stimulation of migration of inflammatory cells (including, for example, macrophages) and lead to inflammation associated hypervascular permeability. Activation of VEFGR-1 can also promote bone marrow associated tumor niche formation. Thus, VEGF based therapeutic selective for VEGFR-2 activation would be desirable in this case. In addition, cell specific targeting, e.g. to endothelial cells, would be desirable

[0586] In some embodiments, the additional modified signaling agent has reduced affinity and / or activity (e.g. antagonistic) for VEGFR-2 and / or has substantially reduced or ablated affinity and / or activity for VEGFR-1. When targeted to tumor vasculature endothelial cells via a targeting moiety that binds to a tumor endothelial cell marker (e.g. PSMA and others), such construct inhibits VEGFR-2 activation specifically on such marker-positive cells, while not activating VEGFR-1 en route and on target cells (if activity ablated), thus eliminating induction of inflammatory responses, for example. This would provide a more selective and safe anti-angiogenic therapy for many tumor types as compared to VEGF-A neutralizing therapies.

[0587] In some embodiments, the additional modified signaling agent has reduced affinity and / or activity (e.g. agonistic) for VEGFR-2 and / or has substantially reduced or ablated affinity and / or activity for VEGFR-1 . Through targeting to vascular endothelial cells, such construct, in some embodiments, promotes angiogenesis without causing VEGFR-1 associated induction of inflammatory responses. Hence, such a construct would have targeted proangiogenic effects with substantially reduced risk of side effects caused by systemic activation of VEGFR-2 as well as VEGR-1.

[0588] In an illustrative embodiment, the modified signaling agent is VEGF165, which has the amino acid sequence of SEQ ID NO:235.

[0589] In another illustrative embodiment, the additional modified signaling agent is VEGF sb, which has the amino acid sequence of SEQ ID NO:236.

[0590] In these embodiments, the modified signaling agent has a mutation at amino acid I83 (e.g., a substitution mutation at I83, e.g., I83K, I83R, or I83H). Without wishing to be bound by theory, it is believed that such mutations may result in reduced receptor binding affinity. See, for example, U.S. Patent No. 9,078,860, the entire contents of which are hereby incorporated by reference

[0591] In an embodiment, the additional modified signaling agent is TNF-a. TNF is a pleiotropic cytokine with many diverse functions, including regulation of cell growth, differentiation, apoptosis, tumorigenesis, viral replication,

[0592] DB1 / 162517863.2 93 ORN-108PC / 114384-5108 autoimmunity, immune cell functions and trafficking, inflammation, and septic shock It binds to two distinct membrane receptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits a very broad expression pattern whereas TN FR2 is expressed preferentially on certain populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiac myocytes and mesenchymal stem cells. Very distinct biological pathways are activated in response to receptor activation, although there is also some overlap. As a general rule, without wishing to be bound by theory, TNFR1 signaling is associated with induction of apoptosis (cell death) and TNFR2 signaling is associated with activation of cell survival signals (e.g. activation of NFkB pathway). Administration of TNF is systemically toxic, and this is largely due to TNFR1 engagement. However, it should be noted that activation of TNFR2 is also associated with a broad range of activities and, as with TNFR1, in the context of developing TNF based therapeutics, control over TNF targeting and activity is important.

[0593] In some embodiments, the additional modified signaling agent has reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signaling agent has substantially reduced or ablated affinity and / or activity for TNFR1 and / or TNFR2. TNFR1 is expressed in most tissues, and is involved in cell death signaling while, by contrast, TNFR2 is involved in cell survival signaling Accordingly, in embodiments directed to methods of treating cancer, the modified signaling agent has reduced affinity and / or activity for TNFR1 and / or substantially reduced or ablated affinity and / or activity for TNFR2. In these embodiments, the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex may be targeted to a cell for which apoptosis is desired, e.g. a tumor cell or a tumor vasculature endothelial cell. In embodiments directed to methods of promoting cell survival, for example, in neurogenesis for the treatment of neurodegenerative disorders, the modified signaling agent has reduced affinity and / or activity for TNFR2 and / or substantially reduced or ablated affinity and / or activity for TNFR1. Stated another way, the present chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex, in some embodiments, comprise modified TNF-a agent that allows of favoring either death or survival signals.

[0594] In some embodiments, the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex has a modified TNF having reduced affinity and / or activity for TNFR1 and / or substantially reduced or ablated affinity and / or activity for TNFR2. Such a chimera, in some embodiments, is a more potent inducer of apoptosis as compared to a wild type TNF and / or a chimera bearing only mutation(s) causing reduced affinity and / or activity for TNFR1 Such a chimera, in some embodiments, finds use in inducing tumor cell death or a tumor vasculature endothelial cell death (e.g. in the treatment of cancers). Also, in some embodiments, these chimeras avoid or reduce activation of Tregcells via TNFR2, for example, thus further supporting TNFR1-mediated antitumor activity in vivo.

[0595] In some embodiments, the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complex has a modified TNF having reduced affinity and / or activity for TNFR2 and / or substantially reduced or ablated affinity and / or activity for TNFR1 . Such a chimera, in some embodiments, is a more potent activator of cell survival in some cell types, which may be a specific therapeutic objective in various disease settings, including without limitation, stimulation of neurogenesis. In addition, such a TNFR2-favoring chimeras also are useful in the

[0596] DB1 / 162517863.2 94 ORN-108PC / 114384-5108 treatment of autoimmune diseases (e.g. Crohn’s, diabetes, MS, colitis etc. and many others described herein). In some embodiments, the chimera is targeted to auto-reactive T cells In some embodiments, the chimera promotes Treg cell activation and indirect suppression of cytotoxic T cells.

[0597] In some embodiments, the chimera causes the death of auto-reactive T cells, e.g. by activation of TNFR2 and / or avoidance of TNFR1 (e.g. a modified TNF having reduced affinity and / or activity for TNFR2 and / or substantially reduced or ablated affinity and / or activity for TNFR1). Without wishing to be bound by theory these auto-reactive T cells, have their apoptosis / survival signals altered e.g. by NFkB pathway activity / signaling alterations.

[0598] In some embodiments, a TNFR2 based chimera has additional therapeutic applications in diseases, including various autoimmune diseases, heart disease, de-myelinating and neurodegenerative disorders, and infectious disease, among others.

[0599] In an embodiment, the wild type TNF-a has the amino acid sequence of SEQ ID NO:237.

[0600] In such embodiments, the modified TNF-a agent has mutations at one or more amino acid positions 29, 31 , 32, 84, 85, 86, 87, 88, 89, 145, 146 and 147 which produces a modified TNF-a with reduced receptor binding affinity. See, for example, U.S. Patent No. 7,993,636, the entire contents of which are hereby incorporated by reference.

[0601] In some embodiments, the modified human TNF-a moiety has mutations at one or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91 , I97, T105, P106, A109, P113, Y115, E127, N137, D143, and A145, as described, for example, in WO / 2015 / 007903, the entire contents of which is hereby incorporated by reference (numbering according to the human TNF sequence, Genbank accession number BAG70306, version BAG70306.1 Gl: 197692685). In some embodiments, the modified human TNF-a moiety has substitution mutations selected from R32G, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, Y87Q, Y87L, Y87A, Y87F, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G and A145T. In an embodiment, the human TNF-a moiety has a mutation selected from Y87Q, Y87L, Y87A, and Y87F. In another embodiment, the human TNF-a moiety has a mutation selected from I97A, I97Q, and I97S. In a further embodiment, the human TNF-a moiety has a mutation selected from Y115A and Y115G.

[0602] In some embodiments, the modified TNF-a agent has one or more mutations selected from N39Y, S147Y, and Y87H, as described in W02008 / 124086, the entire contents of which is hereby incorporated by reference.

[0603] In an embodiment, the additional modified signaling agent is TNF-(3. TNF- can form a homotrimer or a heterotrimer with LT- (LT-aip2). In some embodiments, the ...

Claims

ORN-108PC / 114384-5108CLAIMSWhat is claimed is:

1. A chimeric protein or protein complex comprising:(a) a modified interleukin-6 (IL-6) comprising an amino acid sequence having at least about 90% identity with SEQ ID NO: 431, and one or more mutations selected from a substitution mutation of an amino acid position selected from L178, Y31, G35, L57, F74, W157, L174, R179, A180, and R182 and / or a truncation mutation of at least three C-terminal amino acids, and(b) one or more targeting moieties, said targeting moieties comprising recognition domains which specifically bind to an antigen or receptor of interest; wherein the modified IL-6 and the one or more targeting moieties are optionally connected with one or more linkers, wherein the one or more mutations confer reduced activity and / or affinity for IL-6 receptor (I L-6Rct).

2. The chimeric protein or protein complex of claim 1, wherein the modified IL-6 comprises an amino acid sequence having at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 431.

3. The chimeric protein or protein complex of claim 1 or 2, wherein the mutation comprises a hydrophilic or hydrophobic amino acid residue.

4. The chimeric protein or protein complex of any one of the above claims, wherein the hydrophilic amino acid residue is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K) or an aromatic, polar and positively charged hydrophilic including histidine (H).

5. The chimeric protein or protein complex of any one of claims 1-3, wherein the hydrophilic amino acid residue is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

6. The chimeric protein or protein complex of any one of claims 1-3, wherein the hydrophilic amino acid residue is a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E).

7. The chimeric protein or protein complex of any one of claims 1-3, wherein the hydrophobic amino acid is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

8. The chimeric protein or protein complex of any one of the above claims, wherein the one or more mutations is selected from L178D, Y31 D, G35F, L57A, F74E, W157A, W157G, L174V, L178Q, L178R, R179A, R179F, R179G, R179I, R179K, R179V, A180L, R182A, R182E, R182F, R182G, R182I, R182K, R182V, R182W, R179K_R182G, L178D_Y31D_G35F, L178D L57A, L178DJ / V157A, L178D W157G, W157G L178R, a159DBl / 162517863.2ORN-108PC / 114384-5108 truncation of three C-terminal amino acids, and R179K_truncation of three C-terminal amino acids, where the residue numbering is based on SEQ ID NO: 431 , optionally wherein the one or more mutations is selected from L 178D , L178D_Y31 D_G35F, L178D_L57A, L178D_W157A, R179A, R179G, a truncation of three C-terminal amino acids, R179F, R182E, A180L, and L178DJ / V157G, optionally wherein the mutation is L178D and wherein the modified IL-6 comprises one or more further mutations selected from L57A, W157A, and W157G.

9. The chimeric protein or protein complex of any one of claims 1-8, wherein the one or more mutations confer reduced activity and / or affinity that is restorable by attachment to one or more targeting moieties.

10. The chimeric protein or protein complex of any one of claims 1-9, wherein the targeting moiety is directed against a tumor cell.

11. The chimeric protein orprotein complex of any one of claims 1-10, wherein the targeting moiety comprises a recognition domain that recognizes and / or binds an antigen or receptor on a tumor cell, endothelial cell, epithelial cell, mesenchymal cell, tumor stroma or adipose stromal cell, ECM and / or immune cell, organ cells, and / or tissue cells.

12. The chimeric protein or protein complex of claim 11 , wherein the immune cell is selected from a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, a mast cell, a monocyte, a red blood cell, myeloid cell, myeloid derived suppressor cell, a NKT cell, and a NK cell, or derivatives thereof.

13. The chimeric protein or protein complex of any one of claims 1-12, wherein the targeting moiety comprises a recognition domain that is a recombinant heavy-chain-only antibody (VHH), a full-length antibody or a fragment thereof, a single-domain antibody, a single-chain antibody (scFv), a Humabody, a shark heavy-chain-only antibody (VNAR), a microprotein (e.g. cysteine knot protein, knottin), a darpin, an anticalin, an adnectin, an aptamer, a Fv, a Fab, a Fab', a F(ab')2, a peptide mimetic molecule, a natural ligand for a receptor, or a synthetic molecule.

14. The chimeric protein or protein complex of claim 13, wherein the recognition domain is a single-domain antibody or a single-chain antibody (scFv).

15. The chimeric protein or protein complex of claim 14, wherein the single domain antibody is a recombinant heavy-chain-only antibody (VHH) or humanized VHH16. The chimeric protein or protein complex of any one of claims 1-15, wherein the recognition domain functionally modulates the antigen or receptor of interest.

17. The chimeric protein or protein complex of any one of claims 1-15, wherein the recognition domain binds but does not functionally modulate the antigen or receptor of interest.

18. The chimeric protein or protein complex of any one of claims 1-17, comprising two or more targeting moieties.DBl / 162517863.2 160ORN-108PC / 114384-510819. The chimeric protein or protein complex of any one of the above claims, wherein the targeting moiety binds to one or more of the follow targets: CD8, CTLA4, CD3, CD4, DNAM-1 , Nrp1 (neurophilin), TNFR2, GITR, ICOS, CD20, CD70, Clec9a, NKp46, asialoglycoprotein receptor (ASGPR), PD-1 , PD-L1 , PD-L2, SIRPIa, FAP, XCR1 , tenascin, prostate-specific membrane antigen (PSMA), or ECM proteins.

20. The chimeric protein or protein complex of any one of claims 1-19, further comprising one or more additional modified signaling agents.21 . The chimeric protein or protein complex of any one of claims 1-20, wherein the connector between (a) and (b) is a flexible linker.

22. The chimeric protein or protein complex of claim 21 , wherein the flexible linker is substantially comprised of glycine and serine residues, optionally wherein i) the flexible linker comprises (Gly4Ser)n, where n is from about 1 to about 8, or ii) the flexible linker comprises one or more of GGS, SEQ ID NO: 432-SEQ ID NO: 443, SEQ ID NO: 477-SEQ ID NO: 483, and SEQ ID NO: 484-SEQ ID NO: 491.

23. The chimeric protein or protein complex of claim 21 , wherein the flexible linker is or comprises GGSGGSGGGGSGGGGS (SEQ ID NO: 257).

24. The chimeric protein or protein complex of claim 21, wherein the flexible linker is or comprises LE, GGGGS (SEQ ID NO:249), (GGGGS)n (n=1-4) (SEQ ID NO:249 -SEQ ID NO:252), (Gly)8 (SEQ ID NO:258), (Gly)6 (SEQ ID NO:259), (EAAAK)n (n=1 -3) (SEQ ID NQ:260 -SEQ ID NO:262), A(EAAAK)nA (n = 2-5) (SEQ ID NO:263 - SEQ ID NO :266), AEAAAKEAAAKA (SEQ ID NO:263), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO :267), PAPAP (SEQ ID NO:268), KESGSVSSEQLAQFRSLD (SEQ ID NO :269), EGKSSGSGSESKST (SEQ ID NO:270), GSAGSAAGSGEF (SEQ ID NO:271), GGS, (GGS)„ (n=2-8) (SEQ ID NO: 477-SEQ ID NO: 483), (GGS)n(n=9-20) (SEQ ID NO: 432-SEQ ID NO: 443) and (XP)n, with X designating any amino acid, e.g., Ala, Lys, or Glu.

25. The chimeric protein or protein complex of any one of claims 1 -24, wherein the chimeric protein is suitable for use in a patient having one or more of: cancer, viral infections, immune disorders, autoimmune diseases, obesity, liver regeneration, cardiovascular diseases, wound, ischemia-related diseases, neurodegenerative diseases, and / or metabolic diseases26. A recombinant nucleic acid encoding one or more chimeric proteins or protein complexes of any one of claims 1-24.

27. A host cell comprising the nucleic acid of claim 26.

28. A pharmaceutical composition comprising one or more chimeric proteins or protein complexes of any one of claims 1-24 and a pharmaceutical excipient29. A method for treating cancer, comprising administering an effective amount of i) the chimeric protein or protein complex of any one of the claims 1 -24; ii) the recombinant nucleic acid of claim 26; iii) the host cell of claim 27; or (iv) the pharmaceutical composition of claim 28, to a patient in need thereof.DBl / 162517863.2 161ORN-108PC / 114384-510830. The method of claim 29, wherein the cancer is selected form one or more of myeloma, multiple myeloma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cancer of the peritoneum, cervical cancer, choriocarcinoma, colon and rectum cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer, gastrointestinal cancer, glioblastoma, hepatic carcinoma, hepatoma, intra-epithelial neoplasm, kidney or renal cancer, larynx cancer, leukemia, liver cancer, lung cancer, small-cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, melanoma, neuroblastoma, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland carcinoma, sarcoma, Kaposi’s sarcoma, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulval cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's Macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia as well as other carcinomas and sarcomas, post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema, and Meigs' syndrome.31 . The method of claim 30, wherein the cancer is multiple myeloma.

32. The chimeric protein of any one of claims 1-24 or the pharmaceutical composition of claim 28 for use as a medicament.

33. Use of the chimeric protein of any one of claims 1-24 or the pharmaceutical composition of claim 28 in the manufacture of a medicament.

34. The chimeric protein or protein complex of any one of claims 1-24 or the pharmaceutical composition of claim 28 for use in the treatment of cancer, viral infections, immune disorders, autoimmune diseases, obesity, liver regeneration, cardiovascular diseases, wound, ischemia-related diseases, neurodegenerative diseases, and / or metabolic diseases.

35. A Fc-based chimeric protein complex comprising:(a) a modified interleukin-6 (IL-6) comprising an amino acid sequence having at least about 90% identity with SEQ ID NO: 431, and one or more mutations selected from a substitution mutation of an amino acid position selected from L178, Y31, G35, L57, F74, W157, L174, R179, A180, and R182 and / or a truncation mutation of at least three C-terminal amino acids, and(b) one or more targeting moieties, said targeting moieties comprising recognition domains which specifically bind to an antigen or receptor of interest; andDBl / 162517863.2 162ORN-108PC / 114384-5108(c) a Fc domain, the Fc domain optionally having one or more mutations that reduces or eliminates one or more effector functions of the Fc domain, promotes Fc chain pairing in the Fc domain, and / or stabilizes a hinge region in the Fc domain, wherein the one or more mutations confer reduced activity and / or affinity for IL-6 receptor (IL-6Ra).

36. The Fc-based chimeric protein complex of claim 35, wherein the modified IL-6 comprises an amino acid sequence having at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 431.

37. The Fc-based chimeric protein complex of claim 35 or 36, wherein the mutation comprises a hydrophilic or hydrophobic amino acid residue.

38. The Fc-based chimeric protein complex of any one of claims 35-37, wherein the hydrophilic amino acid residue is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K) or an aromatic, polar and positively charged hydrophilic including histidine (H).

39. The Fc-based chimeric protein complex of any one of claims 35-37, wherein the hydrophilic amino acid residue is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

40. The Fc-based chimeric protein complex of any one of claims 35-37, wherein the hydrophilic amino acid residue is a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E).41 . The Fc-based chimeric protein complex of any one of claims 35-37, wherein the hydrophobic amino acid is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

42. The Fc-based chimeric protein complex of any one of claims 35-41, wherein the one or more mutations is selected from L178D, Y31D, G35F, L57A, F74E, W157A, W157G, L174V, L178Q, L178R, R179A, R179F, R179G, R179I, R179K, R179V, A180L, R182A, R182E, R182F, R182G, R182I, R182K, R182V, R182W, R179K_R182G, L178D_Y31D_G35F, L178D_L57A, L178DJ / V157A, L178DJ / V157G, W157GJ.178R, a truncation of three C-terminal amino acids, and R179K_truncation of three C-terminal amino acids, where the residue numbering is based on SEQ ID NO: 431 , optionally wherein the one or more mutations is selected from L178D, L178D_Y31 D_G35F, L178D_L57A, L178D_W157A, R179A, R179G, a truncation of three C-terminal amino acids, R179F, R182E, A180L, and L178D_W157G, optionally wherein the mutation is L178D and wherein the modified IL-6 comprises one or more further mutations selected from L57A, W157A, and W157G.DBl / 162517863.2 163ORN-108PC / 114384-510843. The Fc-based chimeric protein complex of any one of claims 35-42, wherein the one or more mutations confer reduced activity and / or affinity that is restorable by attachment to one or more targeting moieties or upon inclusion in the Fc-based chimeric protein complex.

44. The Fc-based chimeric protein complex of any one of claims 35-43, wherein the targeting moiety is directed against a tumor cell.

45. The Fc-based chimeric protein complex of any one of 35-43, wherein the targeting moiety comprise a recognition domain that recognizes and / or binds an antigen or receptor on a tumor cell, endothelial cell, epithelial cell, mesenchymal cell, tumor stroma or adipose stromal cell, ECM and / or immune cell, organ cells, and / or tissue cells.

46. The Fc-based chimeric protein complex of claim 45, wherein the immune cell is selected from a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, a mast cell, a monocyte, a red blood cell, myeloid cell, myeloid derived suppressor cell, a NKT cell, and a NK cell, or derivatives thereof.

47. The Fc-based chimeric protein complex of any one of claims 35-46, wherein the targeting moiety comprises a recognition domain that is a recombinant heavy-chain-only antibody (VHH), a single-domain antibody, a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (e.g. cysteine knot protein, knottin), a darpin, an anticalin, an adnectin, an aptamer, a Fv, a Fab, a Fab', a F(ab')2, a peptide mimetic molecule, a natural ligand for a receptor, or a synthetic molecule.

48. The Fc-based chimeric protein complex of any one of claims 35-47, wherein the recognition domain is a single-domain antibody (VHH), optionally a VHH or a humanized VHH.

49. The Fc-based chimeric protein complex of any one of claims 35-48, wherein the recognition domain functionally modulates the antigen or receptor of interest.

50. The Fc-based chimeric protein complex of any one of claims 35-48, wherein the recognition domain binds but does not functionally modulate the antigen or receptor of interest.51 . The Fc-based chimeric protein complex of any one of claims 35-50, comprising two or more targeting moieties.

52. The Fc-based chimeric protein of any one of claims 35-51 , wherein the targeting moiety binds to one or more of the follow targets: CD8, CTLA4, CD3, CD4, DNAM-1, Nrp1 (neurophilin), TNFR2, GITR, ICOS, CD20, CD70, Clec9a, N Kp46, asialoglycoprotein receptor (ASGPR), PD-1 , PD-L1 , PD-L2, SI RP1 a, FAP, XCR1 , tenascin, prostate-specific membrane antigen (PSMA), or ECM proteins53. The Fc-based chimeric protein complex of any one of claims 35-52, further comprising one or more additional modified signaling agents.

54. The Fc-based chimeric protein complex of any one of claims 35-53, wherein the Fc-based chimeric protein complex comprises two signaling agents or two targeting moieties or two of both.DBl / 162517863.2 164ORN-108PC / 114384-510855. The Fc-based chimeric protein complex of any one of claims 35-54, wherein the Fc-based chimeric protein complex comprises three signaling agents or three targeting moieties or three of both.

56. The Fc-based chimeric protein complex of any one of the claims 35-55, wherein the Fc-based chimeric protein complex is suitable for use in a patient having one or more of: wherein the chimeric protein is suitable for use in a patient having one or more of: cancer, viral infections, immune disorders, autoimmune diseases, obesity, liver regeneration, cardiovascular diseases, wound, ischemia-related diseases, neurodegenerative diseases, and / or metabolic diseases.

57. A recombinant nucleic acid encoding one or more Fc-based chimeric protein complexes of any one of claims 35-55, or a constituent polypeptide thereof.

58. A host cell comprising the nucleic acid of claim 57.

59. A pharmaceutical composition comprising one or more one or more Fc-based chimeric protein complexes of any one of claims 35-55 and a pharmaceutical excipient.

60. A method for treating cancer, comprising administering an effective amount of i) the Fc-based chimeric protein of any one of the claims 35-55; ii) the recombinant nucleic acid of claim 57; iii) the host cell of claim 58; or (iv) the pharmaceutical composition of claim 59, to a patient in need thereof.61 . The method of claim 60, wherein the cancer is selected form one or more of myeloma, multiple myeloma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cancer of the peritoneum, cervical cancer, choriocarcinoma, colon and rectum cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer, gastrointestinal cancer, glioblastoma, hepatic carcinoma, hepatoma, intra-epithelial neoplasm, kidney or renal cancer, larynx cancer, leukemia, liver cancer, lung cancer, small-cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, melanoma, neuroblastoma, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland carcinoma, sarcoma, Kaposi’s sarcoma, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulval cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-deaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's Macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia as well as other carcinomas and sarcomas, post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema, and Meigs' syndrome.

62. The method of claim 61 , wherein the cancer is multiple myeloma.DBl / 162517863.2 165ORN-108PC / 114384-510863. The Fc-based chimeric protein complex of any one of claims 35-55 or the pharmaceutical composition of claim 59 for use as a medicament.

64. Use of the Fc-based chimeric protein complex of any one of claims 35-55 or the pharmaceutical composition of claim 59 in the manufacture of a medicament.

65. The Fc-based chimeric protein complex of any one of claims 35-55 or the pharmaceutical composition of claim 59 for use in the treatment of cancer, viral infections, immune disorders, autoimmune diseases, obesity, liver regeneration, cardiovascular diseases, wound, ischemia-related diseases, neurodegenerative diseases, and / or metabolic diseases.

66. The Fc-based chimeric protein complex of any one of claims 35-55, wherein the Fc domain is from IgG, IgA, IgD, IgM or IgE.

67. The Fc-based chimeric protein complex of claim 66, wherein the IgG is selected from IgG 1 , lgG2, lgG3, or lgG4.

68. The Fc-based chimeric protein complex of any one of claims 35-55, wherein the Fc domain is from human IgG, IgA, IgD, IgM or IgE69. The Fc-based chimeric protein complex of claim 68, wherein the human IgG is selected from human lgG1 , lgG2, lgG3, or lgG4.

70. The Fc-based chimeric protein complex of any one of claims 35-55 or 66-69, wherein the Fc chain pairing is promoted by ionic pairing and / or a knob-in-hole pairing.

71. The Fc-based chimeric protein complex of any one of claims 35-55 or 66-69, wherein the one or more mutations to the Fc domain results in an ionic pairing between the Fc chains in the Fc domain.

72. The Fc-based chimeric protein complex of any one of claims 35-55 or 66-71 , wherein the one or more mutations to the Fc domain results in a knob-in-hole pairing in the Fc domain.

73. The Fc-based chimeric protein complex of any one of claims 35-55 or 66-72, wherein the one or more mutations to the Fc domain results in the reduction or elimination of an effector function of the Fc domain.

74. The Fc-based chimeric protein complex of any one of claims 35-55 or 66-73, wherein the Fc-based chimeric protein complex is a heterodimer and has a trans orientation / configuration, as relates to any targeting moiety and signaling agent, relative to each other, or any targeting moieties relative to each other, or any signaling agents relative to each other.

75. The Fc-based chimeric protein complex of any one of claims 35-55 or 66-74, wherein the Fc-based chimeric protein complex is a heterodimer and has a cis orientation, as relates to any targeting moiety and signaling agent, relative to each other, or any targeting moieties relative to each other, or any signaling agents relative to each other.DBl / 162517863.2 166ORN-108PC / 114384-510876. The Fc-based chimeric protein complex of any one of claims 35-55 or 66-75, wherein the Fc comprises L234A, L235A, and K322Q substitutions in human IgG 1 (according to EU numbering).

77. The Fc-based chimeric protein complex of any one of claims 35-55 or 66-76, wherein the Fc is human lgG1 , and optionally contains one or more mutations of L234, L235, K322, D265, P329, and P331 (according to EU numbering), optionally containing L234A, L235A, and P331G78. The Fc-based chimeric protein complex of any one of claims 35-55 or 66-77, wherein the Fc-based chimeric protein complex has an orientation and / or configuration of any one of FIGs 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.

79. The chimeric protein or protein complex of any one of claims 1-24 or the Fc-based chimeric protein complex of any one of claims 35-55 or 66-78, wherein the chimeric protein or protein complex or Fc-based chimeric protein complex comprises a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one of SEQ ID NO: 450, 414-430, 444, 448-449, and 451-476.

80. The chimeric protein or protein complex or the Fc-based chimeric protein complex of claim 79, wherein the chimeric protein or protein complex or Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 450, 414-430, 444, 448-449, and 451-476.81 . The chimeric protein or protein complex or the Fc-based chimeric protein complex of claim 80, wherein the chimeric protein or protein complex or Fc-based chimeric protein complex comprises a polypeptide having an amino acid sequence selected from one of SEQ ID NO: 445 and 446 and a polypeptide having an amino acid sequence selected from one of SEQ ID NO: 448-476DBl / 162517863.2 167

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