Fibroblast activation protein binding agents and use thereof

Abstract

To provide a fibroblast activation protein (FAP) binding agent that targets or binds FAP for the treatment of cancer.SOLUTION: Provided is a fibroblast activation protein (FAP) binding agent comprising a targeting moiety, the targeting moiety comprising three complementarity determining regions (CDR1, CDR2, and CDR3), and each CDR comprising a sequence consisting of a specific amino acid sequence or an amino acid sequence having 1 to 5 amino acid substitutions, deletions, or insertions in any one of the amino acid sequences.SELECTED DRAWING: Figure 24E

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 825,575, filed on March 28, 2019. The content of this provisional application is hereby incorporated by reference in its entirety.

[0002] Field of the Invention The present technology relates, in part, to binding substances that bind fibroblast activation protein (FAP), chimeric proteins and Fc - based chimeric protein complexes, and their use as therapeutic and diagnostic agents.

[0003] Description of the Text File Filed Electronically The content of the following text file filed electronically together with this specification is hereby incorporated by reference in its entirety: Computer - readable format copy of the sequence listing (filename: ORN - 059PC_ST25; creation date: March 26, 2020, file size: 770,048 bytes).

Background Art

[0004] Fibroblasts regulate the structure and function of healthy tissues, are transiently involved in tissue repair after acute inflammation, and further have an abnormal promoting role during chronic inflammatory conditions including cancer. Cancer-associated fibroblasts (CAFs) regulate the tumor microenvironment and influence the behavior of newly formed cells to promote or suppress tumors. Understanding the tumor microenvironment is important for cancer treatment. Fibroblasts express a diverse array of immunomodulatory factors such as cytokines, lipid mediators, and growth factors. Furthermore, fibroblasts present a number of surface and intracellular receptors and the molecular machinery required for responses to exogenous signals. Fibroblasts can be considered an extension of the “professional” immune system in view of the fact that fibroblasts can initiate inflammation. Fibroblasts are involved in a number of normal and pathological processes. Examples of diseases known to be associated with abnormal fibroblasts include cancer, cardiovascular disease, and autoimmune diseases. CAFs are a major stromal component and play an important role in the regulation of the tumor microenvironment and in the influence on the behavior of tumor cells, mainly through the release of proteolytic enzymes, growth factors, and cytokines. Investigations have shown that stromal cancer-promoting and treatment-resistant properties can be attributed to the action of fibroblasts.

[0005] Human fibroblast activation protein (FAP; GenBank accession number AAC51668; NCBI reference sequence: NM004460.3), also known as seprase, is a 170 kDa integral membrane serine peptidase (EC 3.4.21.B28). FAP belongs to the dipeptidyl peptidase IV family and is a homodimer containing two N-glycosylated subunits with a large C-terminal extracellular domain where the catalytic domain of the enzyme is located (Scanlan et al., Proc. Natl. Acad. Sci. USA 91 (1994), 5657-5661). FAP, in its glycosylated form, has both post-prolyl dipeptidyl peptidase activity and gelatinase activity (Sun et al., Protein Expr. Purif. 24 (2002), 274-281). Thus, FAP is a serine protease with both dipeptidyl peptidase activity and endopeptidase activity that cleaves gelatin and type I collagen.

[0006] FAP has a distinctive tissue distribution; its expression is highly upregulated in reactive stromal fibroblasts that exceed 90% of primary and metastatic epithelial tumors, including lung cancer, colorectal cancer, bladder cancer, ovarian cancer, and breast cancer, while it has been shown to be generally absent in normal adult tissues (Rettig et al., Proc. Natl. Acad. Sci. USA 85 (1988), 3110-3114; Garin-Chesa et al, Proc. Natl. Acad. Sci. USA 87 (1990), 7235-7239). Subsequent reports have shown that FAP is expressed not only in stromal fibroblasts but also in some types of malignant cells of epithelial origin, and that FAP expression correlates directly with the malignant phenotype (Jin et al., Anticancer Res. 23 (2003), 3195-3198).

[0007] Due to its expression in many common cancers and limited expression in normal tissues, FAP is a promising antigenic target for imaging, diagnosing, and treating various carcinomas. There remains a need for improved therapies for treating diseases associated with abnormal fibroblasts, such as cancer treatment by modulating CAF function or fibrotic diseases. SUMMARY OF THE INVENTION

[0008] In one aspect, the technology relates to a fibroblast activation protein (FAP) binding substance that targets or binds to FAP. In some embodiments, the FAP binding substance comprises an FAP targeting moiety. The FAP binding substance or FAP targeting moiety can be, for example, a full-length antibody, a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein, a darpin, an anticalin, an adnectin, an aptamer, an Fv, a Fab, a Fab’, an F(ab’) 2, can be a peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule. In some embodiments, the FAP targeting moiety is a single domain antibody (VHH). In some embodiments, the FAP binding agent directly or indirectly modifies a disease microenvironment comprising disease-associated F2 fibroblasts (e.g., a tumor microenvironment comprising disease-associated F2 fibroblasts). In some embodiments, the FAP binding agent directly or indirectly polarizes disease-associated F2 fibroblasts. In some embodiments, the FAP binding agent further comprises signaling agents such as, but not limited to, interferon, interleukin, and tumor necrosis factor, which can be modified to attenuate their activity. In some embodiments, the FAP binding agent comprises an additional targeting moiety that binds to another target of interest (e.g., an antigen or a receptor). In another embodiment, the other target of interest (e.g., an antigen or a receptor) is present on fibroblasts. In some embodiments, the other target of interest (e.g., an antigen or a receptor) is present on fibroblasts in the cancer stroma. In some embodiments, the fibroblast binding agent of the present invention can directly or indirectly recruit immune cells (e.g., dendritic cells) to the site of action (e.g., in a non-limiting example, a tumor microenvironment). In some embodiments, the FAP binding agent of the present invention promotes the presentation of an antigen (e.g., an antigen or a receptor) by immune cells (e.g., dendritic cells, macrophages) in the tumor stroma or directly by fibroblasts.

[0009] In some embodiments, these FAP-binding substances bind to FAP but do not functionally modulate FAP (e.g., do not partially or fully neutralize it). Thus, in some embodiments, the FAP-binding substances of the present invention are used, for example, to directly or indirectly mobilize FAP-expressing cells to a site of interest while still allowing FAP-mediated signaling in the FAP-expressing cells (i.e., binding of the FAP-binding substance does not reduce or eliminate FAP signaling at the site of interest). Conversely, in some embodiments, the FAP-binding substances of the present invention are used, for example, to directly or indirectly mobilize FAP-expressing cells to a site of interest without allowing FAP-mediated signaling in the FAP-expressing cells (i.e., binding of the FAP-binding substance reduces or eliminates FAP signaling at the site of interest). In some embodiments, the FAP targeting moiety is a single domain antibody (VHH).

[0010] In another aspect, the technology relates to a chimeric protein or Fc-based chimeric protein complex having at least one targeting moiety that has, targets, or binds to an FAP-binding substance disclosed herein. In another aspect, the FAP-binding substances, chimeric proteins, or Fc-based chimeric protein complexes disclosed herein are useful in methods of treating various diseases or disorders such as cancer, infectious diseases, inflammatory diseases or conditions, immune abnormalities, fibrotic diseases, and other diseases and disorders.

[0011] In another aspect, one or more targeting moieties (i.e., FAP-binding substances) and one or more signaling substances are attached to the Fc domain to form an Fc-based chimeric protein complex. In some embodiments, the one or more targeting moieties (i.e., FAP-binding substances) and the one or more signaling substances are attached to the Fc domain either directly or via a linker. Such Fc-based chimeric protein complexes surprisingly have a dramatically improved in vivo half-life, particularly in the heterodimeric structures described herein, compared to Fc-lacking chimeras, and are particularly amenable to production and purification. Thus, the Fc-based chimeric protein approach of the present invention results in agents that are particularly suitable for use as therapeutics.

[0012] In another aspect, the technology relates to an FAP-binding substance, a chimeric protein, or an Fc-based chimeric protein complex that is cross-reactive against human, mouse, and cynomolgus monkey. BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

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[0014] Figures 5A - F, 6A - H, 7A - H, 8A - D, 9A - F, 10A - J, 11A - D, 12A - F, 13A - J, 14A - F, 15A - L, 16A - L, 17A - F, 18A - L, 19A - L, 20A - J, 21A - J, 22A - F, 23A - F show examples of various non - limiting schematic diagrams of the Fc - based chimeric protein complexes of the present invention. In some embodiments, each schematic diagram is a composition of the present invention. Where applicable in the figures, "TM" means the "targeting moiety" described herein, "SA" means the "signaling substance" described herein,

Chemical formula

Mode for Carrying Out the Invention

[0015] This technology is based in part on the discovery of fibroblast-binding substances or fibroblast activation protein-binding substances (e.g., antibodies such as VHH, which are non-limiting examples) that recognize, target, or bind to fibroblasts or fibroblast activation protein. In some embodiments, the fibroblast-binding substance of the present invention is part of a chimeric or fusion protein or an Fc-based chimeric protein complex with one or more targeting moieties and / or one or more signaling substances.

[0016] In some embodiments, the fibroblast-binding substance or fibroblast activation protein-binding substance targets F2 fibroblasts. In some embodiments, the fibroblast-binding substance or FAP-binding substance directly or indirectly alters a disease microenvironment (e.g., a tumor microenvironment containing F2 fibroblasts) containing disease-related F2 fibroblasts. In some embodiments, the fibroblast-binding substance or FAP-binding substance directly or indirectly polarizes F2 fibroblasts into F1 fibroblasts, and in some cases, the fibroblasts are disease-related.

[0017] F2 fibroblasts refer to tumor-promoting (or tumor-promoting) cancer-associated fibroblasts (CAFs) (also known as type-II CAFs). F1 fibroblasts refer to tumor-suppressive CAFs (also known as type-I CAFs). Polarization refers to changing the phenotype of a cell, e.g., changing tumorigenic F2 fibroblasts into tumor-suppressive F1 fibroblasts.

[0018] In some embodiments, the fibroblast-binding substance or FAP-binding substance targets an F2 marker. In some embodiments, the fibroblast-binding substance or FAP-binding substance contains an FAP targeting moiety. In some embodiments, the FAP targeting moiety of the fibroblast-binding substance or FAP-binding substance is any of the FAP targeting moieties disclosed herein.

[0019] In some embodiments, the fibroblast-binding substance or FAP-binding substance comprises an amino acid sequence having at least 90% sequence similarity with any one of SEQ ID NOs: 2-42 or 46-86.

[0020] In some embodiments, the fibroblast-binding substance or FAP-binding substance further comprises one or more signaling substances. In some embodiments, the signaling substance is selected from one or more of interferon, interleukin, and tumor necrosis factor, and any of these may be optionally modified.

[0021] In some embodiments, the fibroblast-binding substance or FAP-binding substance further comprising one or more signaling substances directly or indirectly alters a disease microenvironment comprising disease-related F2 fibroblasts (e.g., a tumor microenvironment comprising F2 fibroblasts). In some embodiments, the fibroblast-binding substance or FAP-binding substance further comprising one or more signaling substances directly or indirectly polarizes F2 fibroblasts into F1 fibroblasts.

[0022] In some embodiments, the fibroblast-binding substance or FAP-binding substance further comprises one or more additional targeting moieties. In some embodiments, the one or more additional targeting moieties recognize and optionally modulate tumor antigens. In some embodiments, the one or more additional targeting moieties recognize and optionally modulate antigens on immune cells.

[0023] In some embodiments, the immune cells are selected from T cells, B cells, dendritic cells, macrophages, neutrophils, and NK cells.

[0024] In some embodiments, the fibroblast-binding substance or FAP-binding substance recruits cytotoxic T cells to tumor cells or the tumor environment.

[0025] In some embodiments, the fibroblast binding substance or FAP binding substance binds to FAP, recognizing it without substantially and functionally modulating its activity.

[0026] In another aspect, the technology is based in part on the discovery of substances (e.g., antibodies such as VHH, by way of non-limiting example) that recognize and bind to fibroblast activation protein (FAP). In some embodiments, the FAP binding substances of the invention are part of a chimeric or fusion protein or an Fc-based chimeric protein complex with one or more targeting moieties and / or one or more signaling agents. In some embodiments, these FAP binding substances bind to FAP but do not functionally modulate it. In some embodiments, the FAP binding substance binds to immune cells and can directly or indirectly recruit them to a site in need of a therapeutic effect (e.g., a tumor or tumor microenvironment). In some embodiments, the FAP binding substance enhances tumor antigen presentation to induce an effective anti-tumor immune response.

[0027] In some embodiments, the FAP binding substance modulates tumor antigen presentation. In some embodiments, the FAP binding substance modulates the immune response to avoid or reduce autoimmunity. In some embodiments, the FAP binding substance results in immunosuppression. In some embodiments, the FAP binding substance increases the ratio of Tregs to CD8+ T cells and / or CD4+ T cells in a patient. In some embodiments, the methods of the invention relate to the reduction of autoreactive T cells in a patient.

[0028] In some embodiments, the technology provides pharmaceutical compositions comprising FAP binding substances and their use in the treatment of various diseases including fibrotic diseases. In some embodiments, the technology provides pharmaceutical compositions comprising FAP binding substances and their use in the treatment of various diseases including cancer, autoimmune diseases, and / or neurodegenerative diseases.

[0029] In some embodiments, the FAP-binding substance of the present invention is used to target cancer-associated fibroblasts (CAFs). For example, in various embodiments, the FAP-binding substance of the present invention targets fibroblasts in the tumor stroma in the treatment of cancers, such as epithelial-derived cancers, such as carcinomas. CAFs are the nucleus of the dynamic and opposing interactions that occur among non-cancerous cells, including epithelial cells, extracellular matrix (ECM), and a number of endothelial, adipose, inflammatory, and immune cells that are frequently found in the tumor microenvironment. Therefore, the FAP-binding substance of the present invention provides a way to deliver a very important anti-tumor therapy (e.g., modified cytokines and / or additional targeting moieties described elsewhere in this specification) to the site of interest. In various embodiments, the FAP-binding substance of the present invention targets the stromal microenvironment composed of activated fibroblasts, endothelial cells (ECs) involved in angiogenesis, and extracellular matrix (ECM) that is constantly remodeled to adapt to tumor growth. Thus, for example, in the case of a chimeric (or chimeric complex) having a cytokine and optionally an additional targeting moiety, the FAP-binding substance of the present invention can deliver anti-tumor signals to the stromal microenvironment that is important for tumor development. In various embodiments, the FAP-binding substance is used to target the cell membranes of cells that are essential for the formation of the tumor microenvironment in primary tumors or metastases, such as cancer-associated fibroblasts, MSCs, selected cancer cells, and endothelial cells.

[0030] FAP-binding substance Fibroblast activation protein (FAP) is a 170 kDa melanoma membrane-bound gelatinase belonging to the serine protease family. FAP is selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas. FAP is thought to be involved in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis.

[0031] In some embodiments, the FAP-binding substance of the present invention is a protein-based substance that can specifically bind to FAP. In some embodiments, the FAP-binding substance is a protein-based substance that can specifically bind to FAP without functional modulation (e.g., partial or complete neutralization) of FAP.

[0032] In some embodiments, the FAP-binding substance of the present technology comprises a targeting moiety having an antigen recognition domain that recognizes an epitope present on FAP. In some embodiments, the antigen recognition domain recognizes one or more linear epitopes present on FAP. As used herein, a linear epitope refers to any continuous sequence of amino acids present on FAP. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on FAP. As used herein, a conformational epitope refers to a portion (which may be discontinuous) of one or more amino acids that forms a three-dimensional surface with characteristics and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0033] In some embodiments, the FAP-binding substance of the present technology can bind to the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other natural or synthetic analog, variant, or mutant of human FAP. In some embodiments, the FAP-binding substance of the present technology can bind to any form of human FAP, including monomers, dimers, heterodimers, multimers, and associated forms. In one embodiment, the FAP-binding substance binds to monomeric FAP. In another embodiment, the FAP-binding substance binds to dimeric FAP. In a further embodiment, the FAP-binding substance binds to glycosylated FAP, which may be monomeric or dimeric.

[0034] In certain embodiments, the FAP-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on human FAP. In some embodiments, human FAP comprises the following amino acid sequence: MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLPTKYALWWSPNGKFLAYAEFNDKDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSRPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD (SEQ ID NO: 1).

[0035] In some embodiments, the FAP-binding substance of the present invention comprises a targeting moiety capable of specific binding. In some embodiments, the FAP-binding substance comprises a targeting moiety having an antigen recognition domain such as an antibody or a derivative thereof. In certain embodiments, the FAP-binding substance comprises a targeting moiety that is an antibody. In some embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., VH) and at least three constant regions (e.g., CHi, CFE, and CHf), and each light chain comprises one variable region (VL) and one constant region (CL). The variable regions determine the specificity of the antibody. Each variable region comprises three highly variable regions known as complementarity-determining regions (CDRs) flanked by four relatively conserved framework regions (FRs). The three CDRs are referred to as CDR1, CDR2, and CDR3 and contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0036] In some embodiments, the FAP-binding substance comprises a targeting moiety that is a derivative or format of an antibody. In some embodiments, the FAP-binding substance of the present invention comprises the following targeting moieties: single-domain antibody, recombinant antibody consisting of only heavy chains (heavy-chain antibody) (VHH), single-chain antibody (scFv), shark antibody consisting of only heavy chains (VNAR), microprotein (cysteine knot protein, knottin), DARPin; tetranectin; affibody; transbody; anticalin; adnectin; affilin; affimer; microbody; aptamer; alterase; plastic antibody; filomer; stradbody; maxibody; evibody; finomer; armadillo repeat protein; knotted domain, avimer, atrimer, probody, immunobody, triomab, tribody, pepbody, waxibody, unibody; duoibody, Fv, Fab, Fab’, F(ab’) 2, a peptidomimetic molecule, or a synthetic molecule. These are incorporated herein by reference in their entirety: U.S. Patent No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Patent No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Patent No. 7,250,297, U.S. Patent No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Patent No. 7,838,629, U.S. Patent No. 7,186,524, U.S. Patent No. 6,004,746, U.S. Patent No. 5,475,096, U.S. Patent Application Publication No. 2004 / 146938, U.S. Patent Application Publication No. 2004 / 157209, U.S. Patent No. 6,994,982, U.S. Patent No. 6,794,144, U.S. Patent Application Publication No. 2010 / 239633, U.S. Patent No. 7,803,907, U.S. Patent Application Publication No. 2010 / 119446, and / or U.S. Patent No. 7,166,697. See also Storz MAbs. 2011 May-Jun;3(3):310-317.

[0037] In some embodiments, the FAP-binding substance comprises a targeting moiety that is a single-domain antibody, such as a VHH. The VHH can be derived, for example, from organisms that produce VHH antibodies, such as camels, sharks, or the VHH can be a designed VHH. The VHH is a therapeutic protein derived from an antibody that includes the unique structural and functional characteristics of naturally-occurring heavy-chain antibodies. The VHH technology is based on fully functional antibodies from camels that lack a light chain. These heavy-chain antibodies include a single variable domain (VHH) and two constant domains (CH2 and CH3). VHHs are commercially available under the registered trademark NANOBODY or NANOBODIES.

[0038] In one embodiment, the FAP-binding substance comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

[0039] In some embodiments, the VHH comprises a fully human VH domain, such as a Humobody (Crescendo Biologics, Cambridge, UK). In some embodiments, the fully human VH domain, such as a Humobody, is monovalent, divalent, or trivalent. In some embodiments, the fully human VH domain, such as a Humobody, is monospecific or multispecific, such as monospecific, bispecific, or trispecific. Exemplary fully human VH domains, such as Humobodies, are described, for example, in International Publication Nos. 2016 / 113555 and 2016 / 113557. The entire disclosures of these are incorporated herein by reference.

[0040] In some embodiments, by way of non-limiting example, the human VHH FAP-binding substance comprises an amino acid sequence selected from the following sequences: 2PE2: QVQLQESGGGSVQVGGSLRLSCADSGSTFTINAMGWYRQAPGKRRDWVAGITSSGVTQYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS (SEQ ID NO: 2) 2PE5: QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTSYPDSVKGRFTISRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 3) 2PE7: QVQLQESGGGLVHAGGSLRLSCADSGSTFSVNAVGWYRQAPGKRRDWVAGITSDGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS (SEQ ID NO: 4) 2PE13: QVQLQESGGGLVQVGGSLRLSCAASGSTFILNAMAWYRQAPGNRRELVAGISSGGDTNYPDSVKGRFTISRDNANNIVYLQMNSLKLEDTAVYYCNLWPPRASPSGRVYWGQGTQVTVSS (SEQ ID NO: 5) 2PE14: QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 6) 2PE17: QVQLQESGGGLVQSGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 7) 2PE19: QVQLQESGGGLVQPGGSLRLSCADSGSTFTINAMAWYRQAPGKRRELVAGISGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS (SEQ ID NO: 8) 2PE20: QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTVSRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 9) 2PE23: QVQLQESGGGLVQPGGSLRLSCADSGSTFSINNAMGWYRQAPGKRRDWVAGITSSGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGTIYWGQGTQVTVSS (SEQ ID NO: 10) 2PE25: QVQLQESGGGLVQVGGSLRLSCAASGSSFIINAMGWYRQAPGKRRELVAGISSDGATHYPDSVKGRFTISRDNAKNIVYLQMNSLKPEDTAVYYCNLWPPRASPSGRVYWGQGTQVTVSS (SEQ ID NO: 11) 2PE27: QVQLQESGGGLVQPGGSLRLSCAASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 12) 2PE28: QVQLQESGGGLVQIGGSLRLSCADSGSTFSINNAMGWYRQAPGKRRDWVAGITSSGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGTIYWGQGTQVTVSS (SEQ ID NO: 13) 2PE29: QVQLQESGGGLVQPGGSLRLSCAASGSTSSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 14) 2PE30: QVQLQESGGGLVQVGGSLRLSCADSGSTFSINNAMGWYRQAPGKRRDWVAGITSSGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS (SEQ ID NO: 15) 2PE32: QVQLQESGGGLVQVGGSLRLSCAASGSTFSINAMGWYRQAPGKRRELVAGISSDDITYYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRGYWGQGTQVTVSS (SEQ ID NO: 16) 2PE33: QVQLQESGGGLVQPGGSLRLSCADSGSTFSINSMGWYRQAPGKRRDWVAGITTDGITKYPDSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRLYWGQGTQVTVSS (SEQ ID NO: 17) 2PE36: QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS (SEQ ID NO: 18) 2PE38: QVQLQESGGGLVQAGESLRLSCAASGSTFTINAMGWYRXAPGKRRDWVAGITSSGVTQYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS (SEQ ID NO: 19) 2PE39: QVQLQESGGGLVQVGGSLRLSCADSGSTFSVNAVGWYRQAPGKRRDWVAGITSDGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS (SEQ ID NO: 20) 2PE40: QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 21) 2PE41: QVQLQESGGGLVQVGGSLRLSCADSGSTFSINSMGWYRQAPGKHRDWVAGITTDGITKYPDSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRLYWGQGTQVTVSS (SEQ ID NO: 22) 2PE42: QVQLQESGGGLVQAGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVRGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS (SEQ ID NO: 23) 2PE43: QVQLQESGGGLVQPGGSLTLACKGSGVELSRSAMAWYQQAPGKRRDWVAGITSSGVTQYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDAAVYYCNLWPPRASPSGRIYWGQGTQVTVSS (SEQ ID NO: 24) 2PE44: QVQLQESGGGLVQPGGSLRLSCAASGSTFSVNAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLIPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 25) 2PE47: QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMDSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS (SEQ ID NO: 26) 2PE49: QVQLQESGGGLVQAGGSLRLSCTASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 27) 2PE55: QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTNHPDSVKGRFTISRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 28) 2PE56: QVQLQESGGGLVQPGGSLRLSCADSGSTFTINAMGWYRQAPGKRRDWVAGITSSGVTQYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS (SEQ ID NO: 29) 2PE58: QVQLQESGGGLVQVGGSLRLSCAASGSTFSINAMGWYRQAPGKRREWVAGISSSGPPHYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPMASPSGAIYWGQGTQVTVSS (SEQ ID NO: 30) 2PE59: QVQLQESGGGLVQPGGSLRLSCAVSGSIFSLNAMAWYRQAPGKRRELVAGISGGSVTNYPDSVKGRFTISRDSTKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 31) 2PE60: QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAMAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 32) 2PE61: QVQLQESGGGLVQPGGSLRLICAASGSTFSGNAMAWYRXAPGKRRELVAGISGGITTYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGLVYWGQGTQVTVSS (SEQ ID NO: 33) 2PE62: QVQLQESGGGLVQAGGSLRLSCADSSGSTFSINAMAWYRQAPGKRRDWVAGITSDSVTKYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIDWGQGTQVTVSS (SEQ ID NO: 34) 2PE63: QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMNSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS (SEQ ID NO: 35) 2PE67: QVQLQESGGGLVQVGGSLRLSCAASGSTFILNAMGWYRQAPGNRRELVAGISSGGDTNYPDSVKGRFTISRDNANNIVYLQMNSLKLEDTAVYYCNLWPPRASPSGRPYWGQGTQVTVSS (SEQ ID NO: 36) 2PE68: QVQLQESGGGLVQPGGSLRLSCAASGSIFSTNAMAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRAPPDGRVYWGQGTQVTVSS (SEQ ID NO: 37) 2PE71: QVQLQESGGGMVQSGRSLRLSCLASVNIVNLNSVGWYRQAPGQQRELVASITSAGSTNYAESVKGRFTISRDNSKNTVYLQMNSLKPSDTAVYYCNLWPPRVSPSGRGYWGQGTQVTVSS (SEQ ID NO: 38) 2PE72: QVQLQESGGGLVQPGGSLRLSCAASGSISSINAMAWYRQAPGRRRELVAGIDGGGVTNYPDSVKGRFTISRDHAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 39) 2PE76: QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFAISRDNAKNIVYLQMDSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS (SEQ ID NO: 40) 2PE83: QVQLQESGGGLVQVGGSLRLSCADSGSTFSINSMGWYRQAPGKRRDWVAGITTDGITKYPDSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRLYWGQGTQVTVSS (SEQ ID NO: 41) 2PE84: QVQLQESGGGLVQAGGSLRLSCAASGSISSLNAMGWYRQAPGKQREWVAGITSGGSTNYADSVKGRFTILRDNAKNTVYLQMSSLKFEDTAVYYCNLWPPRASPSGAVYWGQGTQVTVSS (SEQ ID NO: 42).

[0041] In various exemplary embodiments, the FAP binding substance comprises an amino acid sequence selected from any one of the sequences provided above with or without a terminal histidine tag sequence (i.e., HHHHHH; SEQ ID NO: 43).

[0042] In various exemplary embodiments, the FAP binding substance comprises an amino acid sequence selected from any one of the sequences provided above with or without an HA tag (i.e., YPYDVPDYGS; SEQ ID NO: 44).

[0043] In various exemplary embodiments, the FAP binding substance comprises an amino acid sequence selected from any one of the sequences provided above with or without an AAA linker (i.e., AAA).

[0044] In various exemplary embodiments, the FAP binding substance comprises an amino acid sequence selected from any one of the sequences provided above with or without an AAA linker, an HA tag, and a terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 45).

[0045] In some embodiments, by way of non-limiting example, the human VHH FAP binding substance comprises an amino acid sequence selected from the following sequences: 2PE86: QVQLQESGGGLVQVGGSLRLSCADSGSTFSINAMGWYRQAPGKRRDWVAGITSDGVTKYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRVSPSGRIYWGQGTQVTVSS (SEQ ID NO: 46) 2PE87: QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMDSLKPEDTAAYYCNLWPPRASPSGSIYWGQGTQVTVSS (SEQ ID NO: 47) 2PE88: QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRAPPGGRVYWGQGTQVTVSS (SEQ ID NO: 48) 2PE95: QVQLQESGGGLVQVGGSLRLSCADSGSTFSINAMGWYRQAPGKRRDWVAGITSSGVTKYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS (SEQ ID NO: 49) 3PE1: QVQLQESGGGLVQAGGSLKLSCAGSGSTFSINAMAWYRQAPGERRELVAGISGDNITNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 50) 3PE5: QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAMAWYRQAPGQRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 51) 3PE12: QVQLQESGGGLVQPGGSLRLSCAASGSTFSGNAMAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRVSPGGGVYWGQGTQVTVSS (SEQ ID NO: 52) 3PE21: QVQLQESGGGLVQAGESLRLSCAASGRDFRDNSMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMDSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS (SEQ ID NO: 53) 3PE28: QVQLQESGGGLVQPEGSLRLSCAASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 54) 3PE42: QVQLQESGGGLVQPGESLRLSCAVSGSTSSMNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS (SEQ ID NO: 55) 3PE43: QVQLQESGGGLVQAGGSLRLSCAASGSTFSVNAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS (SEQ ID NO: 56) 3PE44: QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAMAWYRQAPGKRRELVAGISGGDVTHYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 57) 3PE47: QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLIPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 58) 3PE49: QVQLQESGGGLVQPGGSLRLSCAGSGSTFSINAMAWYRQAPGERRELVAGISGDNITNYPNSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 59) 3PE57: QVQLQESGGGLVQPGGSLRLSCAASGSTFSVNAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS (SEQ ID NO: 60) 3PE62: QVQLQESGGGLVQPGGSLRLSCAASGSTFSSNAMAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGGVYWGQGTQVTVSS (SEQ ID NO: 61) 3PE69: QVQLQESGGGLVQPGGSLTLSCTTSEFTLAYFGVGWFRQAPGKRRDWVAGITTDGITKYPDSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRLYWGQGTQVTVSS (SEQ ID NO: 62) 3PE72: QVQLQESGGGLVQAGGSLRLSCAASGSTFSGNAMAWYRRAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRVSPGGRVYWGQGTQVTVSS (SEQ ID NO: 63) 3PE77: QVQLQESGGGLVQPGGSLRLSCADSGSTFTINAMAWYRQAPGKRRELVAGISGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWSQGTQVTVSS (SEQ ID NO: 64) 3PE82: QVQLQESGGGLVQPEGSLRLSCAASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGKGTQVTVSS (SEQ ID NO: 65) 3PE90: QVQLQESGGGLVQVGGSLRLSCAASGSTFSINAMGWYRQAPGKRREWVAGITSGVTHYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS (SEQ ID NO: 66) 3PE92: QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMNSPKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS (SEQ ID NO: 67) 3PE93: QVQLQESGGGLVQPGESLRLSCAVSGSTSSMNAMAWYRQAPGKRRELVAGISGGGATNYPDSMKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS (SEQ ID NO: 68) 3PE94: QVQLQESGGGLVQPEGSLRLSCAASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS (SEQ ID NO: 69) 2PE18: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLSAMGWYRQTPGKQRELVASITSDGRTNYADSVKGRFTISRVNPKRTVYLQMNSLKPDDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 70) 2PE22: QVQLQESGGGLVQPGGSLRLSCADSGSTFGLSAMGWYRQTPGKQRELVASITSDGRTNYADSVKGRFTISRVNPKRTVYLQMNSLKPDDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 71) 2PE26: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVNAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 72) 3PE4: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYSDSVKGRFTISRVNPKRIVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 73) 3PE16: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLSAMGWYRLTPGKQRELVASITSDGRTNYADSVKGRFTISRVNPKRTVYLQMNSLKPDDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 74) 3PE22: QVQLQESGGGLVQPGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVGAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 75) 3PE25: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLTAIGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVNPKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 76) 3PE26: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVGAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 77) 3PE33: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRENPKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 78) 3PE46: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVGAKRTVYLQMNSLRPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 79) 3PE55: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVSAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 80) 3PE61: QVQLQESGGGLVRPGGSLRLSCADSGSTFGLSAMGWYRQSPGKQRELVASIISDGRTNYADSVKGRFTISRVNAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 81) 3PE63: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLSAMGWYRQSPGKQRELVASIISDGRTNYADSVKGRFTISRVNAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 82) 3PE70: QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYSDSVKGRFTISRVTPKRIVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS (SEQ ID NO: 83) 2PE1: QVQLQESGGGLVQAGGSLRLSCAASGSIFGINAVGWYRQAPGKQRELVATFTRGGDINYADSVKGRFTIFRDNAANTVYLQMNSLKAEDTAVYYCNTPPRIGRGYWGQGTQVTVSS (SEQ ID NO: 84) 2PE35: QVQLQESGGGLVQVGGSLRLSCAASGSIFGINAVGWYRQAPGKQRELVATFTRGGDINYADSVKGRFTIFRDNAANTVYLQMNSLKAEDTAVYYCNTPPRIGRGYWGQGTQVTVSS (SEQ ID NO: 85) 3PE11: QVQLQESGGGLVQPGGSLRLSCAASGSIFGINAVGWYRQAPGKQRELVATFTRGGDINYADSVKGRFTIFRDNAANTVYLQMNSLKAEDTAVYYCNTPPRIGRGYWGQGTQVTVSS (SEQ ID NO: 86)

[0046] In some embodiments, by way of non-limiting example, the humanized VHH FAP-binding substance comprises an amino acid sequence selected from the following sequences:

Chemical formula

[0047] In some embodiments, the FAP-binding substance comprises a targeting moiety that 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 the regions in the variable domain that are located between the CDRs. As used herein, "complementary determining region" or "CDR" refers to the variable region in a VHH that comprises an amino acid sequence capable of specifically binding to an antigenic target.

[0048] In some embodiments, the FAP-binding substance comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence. In some embodiments, the FAP-binding substance comprises a VHH having a variable region comprising at least one FR1, FR2, FR3, and / or FR4 sequence.

[0049] In some embodiments, the human FAP-binding substance comprises a CDR1 sequence selected from: ATISSMNSMA (SEQ ID NO: 87); EFTLAYFGVG (SEQ ID NO: 88); ESTFSINAVA (SEQ ID NO: 89); GFIFRSTSMG (SEQ ID NO: 90); GGIFTIGPLG (SEQ ID NO: 91); GRDFRDNSMG (SEQ ID NO: 92); GSIFGINAVG (SEQ ID NO: 93); GSIFSLNAMA (SEQ ID NO: 94); GSIFSMG (SEQ ID NO: 95); GSIFSTNAMA (SEQ ID NO: 96); GSISSINAMA (SEQ ID NO: 97); GSISSLNAMG (SEQ ID NO: 98); GSISSRNAMG (SEQ ID NO: 99); GSSFIINAMG (SEQ ID NO: 100); GSTARLDAMG (SEQ ID NO: 101); GSTFGLGAMG (SEQ ID NO: 102); GSTFGLSAMG (SEQ ID NO: 103); GSTFGLTAIG (SEQ ID NO: 104); GSTFILNAMA (SEQ ID NO: 105); GSTFILNAMG (SEQ ID NO: 106); GSTFSGNAMA (SEQ ID NO: 107); GSTFSINAMA (SEQ ID NO: 108); GSTFSINAMG (SEQ ID NO: 109); GSTFSINAMM (SEQ ID NO: 110); GSTFSINAVA (SEQ ID NO: 111); GSTFSINNAMG (SEQ ID NO: 112); GSTFSINSMG (SEQ ID NO: 113); GSTFSSNAMA (SEQ ID NO: 114); GSTFSVNAVA (SEQ ID NO: 115).

[0050] In some embodiments, the human FAP binding substance comprises a CDR2 sequence selected from the following: AISSGGSTNYAASVKG (SEQ ID NO: 116); AVTSGGVTNYADSVKG (SEQ ID NO: 117); GIATDGRTNYAHSVKG (SEQ ID NO: 118); GIDGGGVTNYPDSVKG (SEQ ID NO: 119); GIDSADITDYARFVKG (SEQ ID NO: 120); GIIGSHSTNYADSVKG (SEQ ID NO: 121); GISGDNITNYPDSVKG (SEQ ID NO: 122); GISGDNITNYPNSVKG (SEQ ID NO: 123); GISGGDVTHYPDSVKG (SEQ ID NO: 124); GISGGGATNYPDSMKG (SEQ ID NO: 125); GISGGGATNYPDSVKG (SEQ ID NO: 126); GISGGGVTNHPDSVKG (SEQ ID NO: 127); GISGGGVTNYPDSVKG (SEQ ID NO: 128); GISGGGVTNYPDSVRG (SEQ ID NO: 129); GISGGGVTSYPDSVKG (SEQ ID NO: 130); GISGGITTYPDSVKG (SEQ ID NO: 131); GISGGSVTNYPDSVKG (SEQ ID NO: 132); GISGGVTNYPDSVKG (SEQ ID NO: 133); GISSDDITYYPDSVKG (SEQ ID NO: 134); GISSDGATHYPDSVKG (SEQ ID NO: 135); GISSGGDTNYPDSVKG (SEQ ID NO: 136); GISSGGVTHYPDSVKG (SEQ ID NO: 137); GISSSGPPHYPDSVKG (SEQ ID NO: 138); GITSDGITNYADSVKG (SEQ ID NO: 139); GITSDGLGNYVDFVKG (SEQ ID NO: 140); GITSDGLGNYVGFAKG (SEQ ID NO: 141); GITSDGVTKYPDSVKG (SEQ ID NO: 142); GITSDGVTNYPDSVKG (SEQ ID NO: 143); GITSDSVTKYPDSVKG (SEQ ID NO: 144).

[0051] In some embodiments, the human FAP binding substance comprises a CDR3 sequence selected from the following: AAVVTAKGMGAIQSRGY (SEQ ID NO: 145); ADSSRGKIYFSNYRSWNY (SEQ ID NO: 146); ARFGIRDF (SEQ ID NO: 147); FWPPLYGRP (SEQ ID NO: 148); FWPPPSDRPI (SEQ ID NO: 149); FWPPPSGRPI (SEQ ID NO: 150); KWPPSVPPN (SEQ ID NO: 151); LWPPMASPSGAIY (SEQ ID NO: 152); LWPPRAPPDGRVY (SEQ ID NO: 153); LWPPRAPPGGRVY (SEQ ID NO: 154); LWPPRASPDGGVY (SEQ ID NO: 155); LWPPRASPDGRVY (SEQ ID NO: 156); LWPPRASPGGGVY (SEQ ID NO: 157); LWPPRASPGGLVY (SEQ ID NO: 158); LWPPRASPGGRVY (SEQ ID NO: 159); LWPPRASPSGAVY (SEQ ID NO: 160); LWPPRASPSGRGY (SEQ ID NO: 161); LWPPRASPSGRID (SEQ ID NO: 162); LWPPRASPSGRIY (SEQ ID NO: 163); LWPPRASPSGRLY (SEQ ID NO: 164); LWPPRASPSGRPY (SEQ ID NO: 165); LWPPRASPSGRVY (SEQ ID NO: 166); LWPPRASPSGSIY (SEQ ID NO: 167); LWPPRASPSGTIY (SEQ ID NO: 168); LWPPRVSPGGGVY (SEQ ID NO: 169); LWPPRVSPGGRVY (SEQ ID NO: 170); LWPPRVSPSGRGY (SEQ ID NO: 171); LWPPRVSPSGRIY (SEQ ID NO: 172); LYPPASSGR (SEQ ID NO: 173); MYRPGTYDY (SEQ ID NO: 174); QWPPRALDA (SEQ ID NO: 175).

[0052] Although non-limiting examples, in some embodiments, the human VHH FAP-binding substance comprises an amino acid sequence selected from the following sequences: 2PE48: QVQLQESGGGLVQPGGSLRLSCAVSGTMLSRNAMGWYRQAPGKPRQWVAGITSDGLGNYVGFAKGRFTISRDNAKNTVYLQMNTLKPDDTAVYHCNFWPPPSGRPIWGQGTQVTVSS (SEQ ID NO: 837) 3PE60: QVQLQESGGGLVQPGGSLRLSCAVSGTMLSRNAMGWYRQAPGKQRQWVAGITSDGLGNYVDFVKGRFTISRDNARNTVYLQMNTLKPDDTAVYYCNFWPPPSDRPIWGQGTQVTVSS (SEQ ID NO: 838) 2PE34: QVQLQESGGGLVQAGGSLRLSCAVSGSTARLDAMGWYRQAPGKQREWVAGIDSADITDYARFVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNKWPPSVPPNWGHGTQVTVSS (SEQ ID NO: 839) 3PE80: QVQLQESGGGLVQAGGSLRLSCAVSGSTARLDAMGWYRQAPGKQREWVAGIDSADITDYARFVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNKWPPSVPPNWGQGTQVTVSS (SEQ ID NO: 840) 2PE54: QVQLQESGGGLVQAGGSLRLSCVHSGGIFTIGPLGWYRQAPGSQRELVATVTNGGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAAVVTAKGMGAIQSRGYWGQGTQVTVSS (SEQ ID NO: 841) 3PE81: QVQLQESGGGLVQAGGSLRLSCAHSGGIFTIGPLGWYRQAPGSQRELVATVTNGGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAAVVTAKGMGAIQSRGYWGQGTQVTVSS (SEQ ID NO: 842) 2PE10: QVQLQESGGGLVQAGGSLRLSCAASGSTFSINAMMWYRQAPGKQRELVASIGSGGNTYYADSVKGRFTISRDNGKSTLYLQMNSLKPEDTAVYYCKMYRPGTYDYWGQGTQVTVSS (SEQ ID NO: 843) 3PE66: QVQLQESGGGWVQPGGSLRLSCAASGSTFSINAMMWYRQAPGKQRELVASIGSGGNTYYADSVKGRFTISRDNGKSTLYLQMNSLKPEDTAVYYCKMYRPGTYDYWGQGTQVTVSS (SEQ ID NO: 844) 2PE31: QVQLQESGGGLVQAGGSLSVSCAASGSIFSMGWFRQAPGKQRELVAAVTSGGVTNYADSVKGRFTISRDNAKNTVYLQMKSLKPEDTAVYYCAADSSRGKIYFSNYRSWNYWGQGTQVTVSS (SEQ ID NO: 845) 2PE79: QVQLQESGGGLVQAGESLRLSCAVSATISSMNSMAWYRQAPGKQREWVAGLETGGRANYVDSVKGRFTISRDNARNTVLLQMNSLKPEDTAVYYCNRWPPLRSSWGQGTQVTVSS (SEQ ID NO: 846) 2PE91: QVQLQESGGGLVQPGESLRLSCAASGSISSRNAMGWYRQAPGKEREWVAGITSDGITNYADSVKGRFTISRDNAKNTVGLQMNSLKPDDTAVYYCNFWPPLYGRPWGQGTQVTVSS (SEQ ID NO: 847) 3PE38: QVQLQESGGGLVQPGGSLRLSCAASGFIFRSTSMGWYRQAPGKQREFVAGIIGSHSTNYADSVKGRFTISRDNAQNAVYLHMNTLKPEDTAVYYCNLYPPASSGRWGKGTQVTVSS (SEQ ID NO: 848) 2PE57: QVQLQESGGGLVQAGGSLRLSCAASLKISSINAMAWYRQAAGKQRELVAGIATDGRTNYAHSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNQWPPRALDAWGQGTQVTVSS (SEQ ID NO: 849) 3PE15: QVQLQESGGGLVQPGGSLRLSCAASGVTFGIGAMGWYRQTPENERELVAAISSGGSTNYAASVKGRFTISRDNAPNTVYLQMNSLKPEDTAIYYCNVRRGLAWYPGWGQGTQVTVSS (SEQ ID NO: 850)

[0053] In some embodiments, the FAP-binding substance comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence. In some embodiments, the FAP-binding substance comprises a VHH having a variable region comprising at least one FR1, FR2, FR3, and / or FR4 sequence.

[0054] In some embodiments, the human FAP-binding substance comprises a CDR1 sequence selected from the following: GSTFSVNAVG (SEQ ID NO: 851); GSTFTINAMA (SEQ ID NO: 852); GSTFTINAMG (SEQ ID NO: 853); GSTSSINAMA (SEQ ID NO: 854); GSTSSMNAMA (SEQ ID NO: 855); GTMLSRNAMG (SEQ ID NO: 856); GVELSRSAMA (SEQ ID NO: 857); GVTFGIGAMG (SEQ ID NO: 858); LKISSINAMA (SEQ ID NO: 859); SGSTFSINAMA (SEQ ID NO: 860); VNIVNLNSVG (SEQ ID NO: 861).

[0055] In some embodiments, the human FAP-binding substance comprises a CDR2 sequence selected from the following: GITSGGSTNYADSVKG (SEQ ID NO: 862); GITSGVTHYPDSVKG (SEQ ID NO: 863); GITSSGVTKYPDSVKG (SEQ ID NO: 864); GITSSGVTNYPDSVKG (SEQ ID NO: 865); GITSSGVTQYPDSVKG (SEQ ID NO: 866); GITTDGITKYPDSLKG (SEQ ID NO: 867); GLETGGRANYVDSVKG (SEQ ID NO: 868); SIGSGGNTYYADSVKG (SEQ ID NO: 869); SIISDGRTNYADSVKG (SEQ ID NO: 870); SITSAGSTNYAESVKG (SEQ ID NO: 871); SITSDGRTNYADSVKG (SEQ ID NO: 872); SITSGGRTNYADSVKG (SEQ ID NO: 873); SITSGGRTNYSDSVKG (SEQ ID NO: 874); TFTRGGDINYADSVKG (SEQ ID NO: 875); TVTNGGGTYYADSVKG (SEQ ID NO: 876).

[0056] In some embodiments, the human FAP-binding substance comprises a CDR3 sequence selected from the following: RWPPLRSS (SEQ ID NO: 877); TPPRIGRGY (SEQ ID NO: 878); VRRGLAWYPG (SEQ ID NO: 879).

[0057] In some embodiments, the human FAP-binding substance comprises a CDR1 sequence selected from the following: GSTFSIAAVA (SEQ ID NO: 1093); GSTFSIDAVA (SEQ ID NO: 1094); GSTFSIEAVA (SEQ ID NO: 1095); GSTFSIFAVA (SEQ ID NO: 1096); GSTFSIGAVA (SEQ ID NO: 1097); GSTFSIHAVA (SEQ ID NO: 1098); GSTFSIIAVA (SEQ ID NO: 1099); GSTFSIKAVA (SEQ ID NO: 1100); GSTFSILAVA (SEQ ID NO: 1101); GSTFSIPAVA (SEQ ID NO: 1102); GSTFSIQAVA (SEQ ID NO: 1103); GSTFSIRAVA (SEQ ID NO: 1104); GSTFSISAVA (SEQ ID NO: 1105); GSTFSITAVA (SEQ ID NO: 1106); GSTFSIVAVA (SEQ ID NO: 1107); GSTFSIWAVA (SEQ ID NO: 1108); GSTFSIYAVA (SEQ ID NO: 1109); GSTSSANAMA (SEQ ID NO: 1110); GSTSSDNAMA (SEQ ID NO: 1111); GSTSSENAMA (SEQ ID NO: 1112); GSTSSFNAMA (SEQ ID NO: 1113); GSTSSGNAMA (SEQ ID NO: 1114); GSTSSHNAMA (SEQ ID NO: 1115); GSTSSINAMA (SEQ ID NO: 1116); GSTSSKNAMA (SEQ ID NO: 1117); GSTSSLNAMA (SEQ ID NO: 1118); GSTSSNNAMA (SEQ ID NO: 1119); GSTSSPNAMA (SEQ ID NO: 1120); GSTSSQNAMA (SEQ ID NO: 1121); GSTSSRNAMA (SEQ ID NO: 1122); GSTSSSNAMA (SEQ ID NO: 1123); GSTSSTNAMA (SEQ ID NO: 1124); GSTSSVNAMA (SEQ ID NO: 1125); GSTSSWNAMA (SEQ ID NO: 1126); GSTSSYNAMA (SEQ ID NO: 1127); GSTSSAWAMA (SEQ ID NO: 1128); GSTSSDWAMA (SEQ ID NO: 1129).

[0058] In some embodiments, the FAP-binding substance of the present invention is an FAP antibody (e.g., NI-206.82C2, NI-206.59B4, NI-206.22F7, NI-206.27E8, NI-206.12G4) or a portion thereof (VH chain, VL chain, CDR1, CDR2 or CDR3, etc.) as described in International Publication No. WO 2016 / 110598, which is hereby incorporated by reference in its entirety, and the amino acid sequences of the VH chain, VL chain and their CDRs are as follows:

[0059] FAP-binding substance NI-206.82C2 NI-206.82C2 VH: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSVTWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKGRITINPDTSKNQFYLQLKSVTPEDAAVYYCARDSSILYGDYWGQGTLVTVSS (SEQ ID NO: 880) NI-206.82C2 VL: QAVLTQPSSLSASPGASASLTCTLPSGINVGTYRIFWFQQKPGSPPQYLLSYKSDSDNHQGSGVPSRFSGSKDASANAGILLISGLQSEDEADYYCMIWHSSAWVFGGGTKLTVL (SEQ ID NO: 881).

[0060] The FAP-binding substance of the present invention may include CDR1, CDR2, or CDR3 of the VH or VL of NI-206.82C2. The VH chain of NI-206.82C2 includes the following CDRs: CDR1: GDSVSSNSVTWN (SEQ ID NO: 882) CDR2: RTYYRSKWYND (SEQ ID NO: 883) CDR3: DSSILYGDY (SEQ ID NO: 884) The VL chain of NI-206.82C2 includes the following CDRs: CDR1: TLPSGINVGTYRIF (SEQ ID NO: 915) CDR2: KSDSDNH (SEQ ID NO: 916) CDR3: MIWHSSAWV (SEQ ID NO: 917).

[0061] FAP-binding substance NI-206.59B4 NI-206.59B4 VH: QVQLVQSGAEVKKPGASVKVSCKTSGYTFTDYYIHWVRQAPGQGLEWMGWINPNRGGTNYAQKFQGRVTMTRDTSIATAYMELSRLRSDDTAVYYCATASLKIAAVGTFDCWGQGTLVTVSS (SEQ ID NO: 918) NI-206.59B4 VL: SYELTQPPSVSVSPGQTARITCSGDALSKQYAFWFQQKPGQAPILVIYQDTKRPSGIPGRFSGSSSGTTVTLTISGAQADDEADYYCQSADSSGTYVFGTGTKVTVL (SEQ ID NO: 919) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of VH or VL of NI-206.59B4. The VH chain of NI-206.59B4 contains the following CDRs: CDR1: GYTFTDYYIH (SEQ ID NO: 920) CDR2: WINPNRGGTN (SEQ ID NO: 921) CDR3: ASLKIAAVGTFDC (SEQ ID NO: 922) The VL chain of NI-206.82C2 contains the following CDRs: CDR1: SGDALSKQYAF (SEQ ID NO: 923) CDR2: QDTKRPS (SEQ ID NO: 924) CDR3: QSADSSGTYV (SEQ ID NO: 925)

[0062] FAP-binding substance NI-206.22F7 NI-206.22F7 VH: EVQLVETGGGVVQPGRSLRLSCAASGFSFSTHGMYWVRQPPGKGLEWVAVISYDGSDKKYADSVKGRFTISRDNSKNTVYLEMSSVRAEDTALYYCFCRRDAFDLWGQGTMVTVSS (SEQ ID NO: 926) NI-206.22F7 VL: SYVLTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQAPVLVIYEDTKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDSSGNYWVFGGGTEVTVL (SEQ ID NO: 927) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL of NI-206.22F7. The VH chain of NI-206.22F7 contains the following CDRs: CDR1: GFSFSTHGMY (SEQ ID NO: 928) CDR2: VISYDGSDKK (SEQ ID NO: 929) CDR3: RRDAFDL (SEQ ID NO: 930) The VL chain of NI-206.22F7 contains the following CDRs: CDR1: SGDALPKKYAY (SEQ ID NO: 931) CDR2: EDTKRPS (SEQ ID NO: 932) CDR3: YSTDSSGNYWV (SEQ ID NO: 933)

[0063] FAP-binding substance NI-206.27E8 NI-206.27E8 VH: EVQLVESGGGLVEPGGSLRLSCAASGFTFSDAWMNWVRQAPGKGLEWVGRIKTKSDGGTTDYAAPVRGRFSISRDDSKNTLFLEMNSLKTEDTAIYYCFITVIVVSSESPLDHWGQGTLVTVSS (SEQ ID NO: 934) NI-206.27E8 VL: SYELTQPPSVSVSPGQTARITCSGDELPKQYAYWYQQKPGQAPVLVIYKDRQRPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSAYSINTYVIFGGGTKLTVL (SEQ ID NO: 935) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL of NI-206.27E8. The VH chain of NI-206.27E8 contains the following CDRs: CDR1: GFTFSDAWMN (SEQ ID NO: 936) CDR2: RIKTKSDGGTTD (SEQ ID NO: 937) CDR3: TVIVVSSESPLDH (SEQ ID NO: 938) The VL chain of NI-206.27E8 contains the following CDRs: CDR1: SGDELPKQYAY (SEQ ID NO: 939) CDR2: KDRQRPS (SEQ ID NO: 940) CDR3: QSAYSINTYVI (SEQ ID NO: 941)

[0064] FAP-binding substance NI-206.12G4 NI-206.12G4 VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWISYISSGSSYTNYADSVKGRFTISRDNAKKSVYLEVNGLTVEDTAVYYCARVRYGDREMATIGGFDFWGQGTLVTVSS (SEQ ID NO: 942) NI-206.12G4 VL: SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQSPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSGGTSRIFGGGTKLTVL (SEQ ID NO: 943) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL of NI-206.12G4. The VH chain of NI-206.12G4 contains the following CDRs: CDR1: GFTFSDYYMS (SEQ ID NO: 944) CDR2: YISSGSSYTN (SEQ ID NO: 945) CDR3: VRYGDREMATIGGFDF (SEQ ID NO: 946) The VL chain of NI-206.12G4 contains the following CDRs: CDR1: SGDALPKQYAY (SEQ ID NO: 947) CDR2: KDSERPS (SEQ ID NO: 948) CDR3: QSADSGGTSRI (SEQ ID NO: 949)

[0065] FAP-binding substance NI-206.17A6 NI-206.17A6 VH: QVQLQESGPGLVRSTETLSLTCLVSGDSINSHYWSWLRQSPGRGLEWIGYIYYTGPTNYNPSLKSRVSISLGTSKDQFSLKLSSVTAADTARYYCARNKVFWRGSDFYYYMDVWGKGTTVTVSS (SEQ ID NO: 950) NI-206.17A6 VL: EIVLTQSPGTLSLSLGERATLSCRASQSLANNYLAWYQQKPGQAPRLLMYDASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFVTSHHMYIFGQGTKVEIK (SEQ ID NO: 951) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of VH or VL of NI-206.17A6. The VH chain of NI-206.17A6 contains the following CDRs: CDR1: GDSINSHYWS (SEQ ID NO: 952) CDR2: YIYYTGPTN (SEQ ID NO: 953) CDR3: NKVFWRGSDFYYYMDV (SEQ ID NO: 954) The VL chain of NI-206.17A6 contains the following CDRs: CDR1: RASQSLANNYLA (SEQ ID NO: 955) CDR2: DASTRAT (SEQ ID NO: 956) CDR3: QQFVTSHHMYI (SEQ ID NO: 957)

[0066] In some embodiments, the FAP-binding substance of the present invention is an FAP antibody (e.g., humanized F19) or a portion thereof (VH chain, VL chain, CDR1, CDR2, or CDR3, etc.) derived from International Publication No. WO 1999 / 057151 (incorporated herein by reference in its entirety), and the amino acid sequences of the VH chain, VL chain, and their CDRs are as follows: Humanized F19 VH: QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSS (SEQ ID NO: 958) Humanized F19 VL: DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK (SEQ ID NO: 959) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL chain of F19. The VH chain of F19 contains the following CDRs: CDR1: RYTFTEYTIH (SEQ ID NO: 960) CDR2: GINPNNGIPN (SEQ ID NO: 961) CDR3: RRIAYGYDEGHAMDY (SEQ ID NO: 962) The VL chain of F19 contains the following CDRs: CDR1: KSSQSLLYSRNQKNYLA (SEQ ID NO: 963) CDR2: WASTRES (SEQ ID NO: 964) CDR3: QQYFSYPLT (SEQ ID NO: 965)

[0067] In some embodiments, the FAP-binding substance of the present invention is an FAP antibody (e.g., MFP5 mouse, MFP5 humanized variant 1, and MFP5 humanized variant 2) or a portion thereof (VH chain, VL chain, CDR1, CDR2 or CDR3, etc.) from International Publication No. WO2007 / 077173 (incorporated herein by reference in its entirety), and the amino acid sequences of the VH chain, VL chain and their CDRs are as follows:

[0068] FAP-binding substance MP5 (mouse) MFP5 VH (mouse): QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSA (SEQ ID NO: 966) MFP5 VL (mouse): QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKR (SEQ ID NO: 967) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL chain of MFP5 (mouse). The VH chain of MFP5 contains the following CDRs: CDR1: GYTFTNNGIN (SEQ ID NO: 968) CDR2: EIYPRSTNTL (SEQ ID NO: 969) CDR3: TLTAPFAF (SEQ ID NO: 970) The VL chain of MFP5 contains the following CDRs: CDR1: SASSGVNFMH (SEQ ID NO: 971) CDR2: DTSKLAS (SEQ ID NO: 972) CDR3: QQWSFNPPT (SEQ ID NO: 973)

[0069] FAP-binding substance MP5 (humanized variant 1) MFP5 (humanized variant 1) VH: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNNGINWLRQAPGQGLEWMGEIYPRSTNTLYAQKFQGRVTITADRSSNTAYMELSSLRSEDTAVYFCARTLTAPFAFWGQGTLVTVSS (SEQ ID NO: 974) MFP5 (humanized variant 1) VL: QIVLTQSPATLSLSPGERATLSCSASSGVNFMHWYQQKPGQAPRRLIFDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO: 975) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL chain of MFP5 (humanized variant 1). The VH chain of MFP5 (humanized variant 1) contains the following CDRs: CDR1: GYTFTNNGIN (SEQ ID NO: 976) CDR2: EIYPRSTNTL (SEQ ID NO: 977) CDR3: TLTAPFAF (SEQ ID NO: 978) The VL chain of MFP5 (humanized variant 1) contains the following CDRs: CDR1: SASSGVNFMH (SEQ ID NO: 979) CDR2: DTSKLAS (SEQ ID NO: 980) CDR3: QQWSFNPPT (SEQ ID NO: 981)

[0070] FAP-binding substance MP5 (humanized variant 2) MFP5 (humanized variant 2) VH: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNNGINWLRQAPGQGLEWMGEIYPRSTNTLYAQKFQGRVTITADRSSNTAYMELSSLRSEDTAVYFCARTLTAPFAFWGQGTLVTVSS (SEQ ID NO: 982) MFP5 (humanized variant 2) VL: QIVLTQSPATLSLSPGERATLSCSASSGVNFMHWYQQKPGQAPKRLIFDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO: 983) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL chain of MFP5 (humanized variant 2). The VH chain of MFP5 (humanized variant 2) contains the following CDRs: CDR1: GYTFTNNGIN (SEQ ID NO: 984) CDR2: EIYPRSTNTL (SEQ ID NO: 985) CDR3: TLTAPFAF (SEQ ID NO: 986) The VL chain of MFP5 (humanized variant 2) contains the following CDRs: CDR1: SASSGVNFMH (SEQ ID NO: 987) CDR2: DTSKLAS (SEQ ID NO: 988) CDR3: QQWSFNPPT (SEQ ID NO: 989)

[0071] In some embodiments, the FAP-binding substance of the present invention is an FAP antibody (e.g., 4G8, 3F2, 28H1, 29B11, 14B3, and 4B9) or a portion thereof (VH chain, VL chain, CDR1, CDR2, or CDR3, etc.) from International Publication No. WO 2012 / 107417, which is hereby incorporated by reference in its entirety, and the amino acid sequences of the VH chain, VL chain, and their CDRs are as follows:

[0072] FAP-binding substance 4G8 4G8 VH: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS (SEQ ID NO: 990) 4G8 VL: EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKR (SEQ ID NO: 991) The FAP-binding substance of the present invention may comprise CDR1, CDR2, or CDR3 of the VH or VL chain of 4G8. The VH chain of 4G8 contains the following CDRs: CDR1: GFTFSSYAMS (SEQ ID NO: 992) CDR2: AISGSGGSTY (SEQ ID NO: 993) CDR3: GWLGNFDY (SEQ ID NO: 994) The VL chain of 4G8 contains the following CDRs: CDR1: RASQSVSRSYLA (SEQ ID NO: 995) CDR2: GASTRAT (SEQ ID NO: 996) CDR3: QQGQVIPPT (SEQ ID NO: 997)

[0073] FAP-binding substance 3F2 3F2 VH: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS (SEQ ID NO: 998) 3F2 VL: EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKR (SEQ ID NO: 999) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL chain of 3F2. The VH chain of 3F2 contains the following CDRs: CDR1: GFTFSSYAMS (SEQ ID NO: 1000) CDR2: AISGSGGSTY (SEQ ID NO: 1001) CDR3: GWFGGFNY (SEQ ID NO: 1002) The VL chain of 3F2 contains the following CDRs: CDR1: RASQSVTSSYLA (SEQ ID NO: 1003) CDR2: VGSRRAT (SEQ ID NO: 1004) CDR3: QQGIMLPPT (SEQ ID NO: 1005)

[0074] FAP-binding substance 28H1 28H1 VH: QVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS (SEQ ID NO: 1006) 28H1 VL: EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKR (SEQ ID NO: 1007) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL chain of 28H1. The VH chain of 28H1 contains the following CDRs: CDR1: GFTFSSHAMS (SEQ ID NO: 1008) CDR2: AIWASGEQY (SEQ ID NO: 1009) CDR3: GWLGNFDY (SEQ ID NO: 1010) The VL chain of 28H1 contains the following CDRs: CDR1: RASQSVSRSYLA (SEQ ID NO: 1011) CDR2: GASTRAT (SEQ ID NO: 1012) CDR3: QQGQVIPPT (SEQ ID NO: 1013)

[0075] FAP-binding substance 29B11 29B11 VH: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGGITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS (SEQ ID NO: 1014) 29B11 VL: EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKR (SEQ ID NO: 1015) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL chain of 29B11. The VH chain of 29B11 contains the following CDRs: CDR1: GFTFSSYAMS (SEQ ID NO: 1016) CDR2: AIIGSGGITY (SEQ ID NO: 1017) CDR3: GWFGGFNY (SEQ ID NO: 1018) The VL chain of 29B11 contains the following CDRs: CDR1: RASQSVTSSYLA (SEQ ID NO: 1019) CDR2: VGSRRAT (SEQ ID NO: 1020) CDR3: QQGIMLPPT (SEQ ID NO: 1021)

[0076] FAP-binding substance 14B3 14B3 VH: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAILASGAITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS (SEQ ID NO: 1022) 14B3 VL: EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKR (SEQ ID NO: 1023) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL chain of 14B3. The VH chain of 14B3 contains the following CDRs: CDR1: GFTFSSYAMS (SEQ ID NO: 1024) CDR2: AILASGAITY (SEQ ID NO: 1025) CDR3: GWFGGFNY (SEQ ID NO: 1026) The VL chain of 14B3 contains the following CDRs: CDR1: RASQSVTSSYLA (SEQ ID NO: 1027) CDR2: VGSRRAT (SEQ ID NO: 1028) CDR3: QQGIMLPPT (SEQ ID NO: 1029)

[0077] FAP-binding substance 4B9 4B9 VH: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS (SEQ ID NO: 1030) 4B9 VL: EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKR (SEQ ID NO: 1031) The FAP-binding substance of the present invention may contain CDR1, CDR2, or CDR3 of the VH or VL chain of 4B9. The VH chain of 4B9 contains the following CDRs: CDR1: GFTFSSYAMS (SEQ ID NO: 1032) CDR2: AIIGSGASTY (SEQ ID NO: 1033) CDR3: GWFGGFNY (SEQ ID NO: 1034) The VL chain of 4B9 contains the following CDRs: CDR1: RASQSVTSSYLA (SEQ ID NO: 1035) CDR2: VGSRRAT (SEQ ID NO: 1036) CDR3: QQGIMLPPT (SEQ ID NO: 1037).

[0078] Any one of the FAP-binding substances disclosed herein can be an antibody. The FAP-binding substance that is an antibody can be part of any one of the chimeric proteins or chimeric protein complexes disclosed herein. As used herein, the term "antibody" refers to any immunoglobulin or antibody that specifically binds to an antigen (e.g., human, hamster, cat, mouse, chondrichthyes, or camelid antibody), and any derivative or complex thereof. A wide variety of antibodies are known to those of skill in the art. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, humanized antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies (e.g., single-domain antibodies, camelid antibodies, and chondrichthyes antibodies), chimeric antibodies, cat antibodies, and catized antibodies. A monoclonal antibody is a homogeneous population of antibodies against a specific epitope of an antigen. A polyclonal antibody is a heterogeneous population of antibody molecules contained in the serum of an immunized animal. The term "antibody" also includes antibody derivatives and complexes (e.g., an antibody conjugated to a stabilizing protein, a detectable moiety, or a therapeutic agent).

[0079] Any one of the FAP-binding substances disclosed herein can be an antigen-binding fragment of an antibody. The FAP-binding substance that is an antigen-binding fragment can be part of any one of the chimeric proteins or chimeric protein complexes disclosed herein. An "antigen-binding fragment" is any portion of a full-length antibody that contains at least one variable domain capable of specifically binding to an antigen (the variable domain of a heavy or light chain immunoglobulin of a mammal (e.g., cat, human, hamster, or mouse), a camelid variable antigen-binding domain (VHH) or a chondrichthyes immunoglobulin novel antigen receptor (Ig-NAR) domain). Non-limiting examples of antibody fragments are Fab, Fab’, F(ab’) 2and multispecific antibodies formed from Fv fragments, diabodies, linear antibodies, and antibody fragments. Additional antibody fragments comprising at least one camelid VHH domain or at least one cartilaginous fish Ig-NAR domain include minibodies, microantibodies, subnanobodies, and nanobodies, and any of the other forms of antibodies described, for example, in U.S. Patent Application Publication No. 2010 / 0092470.

[0080] Any one of the FAP-binding substances disclosed herein can be an Fv fragment of an antibody. An FAP-binding substance that is an Fv fragment can be part of any one of the chimeric proteins or chimeric protein complexes disclosed herein. An "Fv fragment" is the minimal antibody fragment that contains the complete target recognition and binding site. This region is composed of a dimer of one heavy chain variable domain and one light chain variable domain held together by a strong non-covalent bond. It is in this arrangement that the three complementarity-determining regions (CDRs) of each variable domain interact to define the target binding site on the surface of the VH-VL dimer.

[0081] Any one of the FAP-binding substances disclosed herein can be a complementarity-determining region (CDR) of an antibody. An FAP-binding substance that is a CDR of an antibody can be part of any one of the chimeric proteins or chimeric protein complexes disclosed herein. The term CDR refers to a region (heavy chain or light chain immunoglobulin) within an immunoglobulin that forms part of the antigen-binding site in an antibody or its antigen-binding fragment. As is known in the prior art, heavy chain and light chain immunoglobulins each contain three CDRs called CDR1, CDR2, and CDR3. In any antibody or antigen-binding fragment, the three CDRs from the heavy chain immunoglobulin and the three CDRs from the light chain immunoglobulin together form the antigen-binding site in the antibody or its antigen-binding fragment. The Kabat database is one system used in the art to number the CDR sequences present in light chain or heavy chain immunoglobulins.

[0082] When combined, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for the antigen), while usually having a lower affinity than the entire binding site, has the ability to recognize and bind the antigen. A "Fab fragment" also includes the constant domain of the light chain and the first constant domain of the heavy chain (CH1). A "Fab' fragment" differs from a "Fab fragment" by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain that contain one or more cysteines from the antibody hinge region. A "F(ab')2 fragment" is originally produced as a pair of "Fab' fragments" that have hinge cysteines between them. Methods for preparing such antibody fragments, such as papain or pepsin digestion, are known to those skilled in the art. For example, an F(ab')2 fragment can be produced by pepsin digestion of an antibody molecule, and Fab can be generated by reducing the disulfide bridges of the F(ab')2 fragment. In some cases, a Fab expression library can be constructed. See, for example, Huse et al., Science, 246:1275, 1989. Once produced, the antibody or its fragment can be tested for recognition of the TNFRSF25 polypeptide using standard immunoassay methods such as ELISA technology, radioimmunoassay, and Western blotting. See Short Protocols in Molecular Biology, Chapter 11, Green Publishing Associates and John Wiley & Sons, Ed. Ausubel et al., 1992.

[0083] The antibody can be of the IgA-, IgD-, IgE-, IgG- or IgM-type, including IgG- or IgM-types such as, but not limited to, IgG1-, IgG2-, IgG3-, IgG4-, IgM1- and IgM2-types. For example, in some cases, the antibody is of the IgG1-, IgG2- or IgG4-type.

[0084] In some embodiments, the antibodies provided herein can be fully human or humanized antibodies. A "human antibody" means an antibody encoded by nucleic acids present in the human genome (e.g., rearranged human immunoglobulin heavy or light chain loci). In some embodiments, human antibodies can be produced in human cell culture (e.g., feline hybridoma cells). In some embodiments, human antibodies can be produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies can be produced in bacterial or yeast cells.

[0085] Human antibodies can avoid certain problems associated with heterologous antibodies, such as antibodies having mouse or rat variable and / or constant regions. For example, because the effector portion is human, it can interact better with other parts of the human immune system and more efficiently destroy target cells, e.g., by complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity. In addition, the human immune system should not recognize this antibody as foreign. Furthermore, the half-life in human circulation is similar to that of naturally occurring human antibodies, allowing for administration at lower doses and less frequent dosing. Methods for preparing humanized antibodies are known in the art.

[0086] As used herein, the term "humanized antibody" refers to a human antibody that contains minimal sequences derived from non-human (e.g., mouse, hamster, rat, rabbit or goat) immunoglobulins. Humanized antibodies are usually human constant regions and / or variable region domains or chimeric or mutant monoclonal antibodies from mouse, rat, hamster, rabbit or other species with specific changes. In non-limiting examples, a humanized antibody is a human antibody (recipient antibody) in which the residues of the hypervariable regions (HVRs) of the recipient antibody are replaced by HVR residues from a non-human species (donor), such as a mouse, rat, rabbit, or goat antibody, that have the desired specificity, affinity, and potency. In some embodiments, the Fv framework residues of the human immunoglobulin can be replaced by the corresponding non-human residues. In some embodiments, a humanized antibody can contain residues not found in either the recipient antibody or the donor antibody. Such modifications can be made, for example, to improve antibody performance.

[0087] In some embodiments, a humanized antibody can contain at least one, usually two, substantially all of the variable domains, with all or substantially all of the hypervariable loops (CDRs) corresponding to those of the non-human immunoglobulin, while at the same time all or substantially all of the framework regions are of human immunoglobulin sequence. A humanized antibody can also contain an immunoglobulin constant (Fc) region, typically at least a portion of the Fc of a human immunoglobulin.

[0088] In some embodiments, the humanized antibodies or antigen-binding fragments provided herein can have reduced or minimal effector function (e.g., compared to the corresponding non-human antibody), such that it does not stimulate effector cell activity to the same extent as the corresponding non-human antibody.

[0089] Techniques for generating humanized antibodies are well known to those skilled in the art. In some embodiments, the controlled rearrangement of antibody domains joined via protein disulfide bonds to form novel artificial protein molecules or "chimeric" antibodies can be utilized (Konieczny et al., Haematologia (Budap.) 14:95, 1981). Using recombinant DNA technology, gene fusions can be constructed between DNA sequences encoding mouse antibody variable light and heavy chain domains and human antibody light and heavy chain constant domains (Morrison et al., Proc Natl Acad Sci USA 81:6851, 1984). For example, DNA sequences encoding the antigen-binding portion or CDRs of a mouse monoclonal antibody can be grafted by molecular means into DNA sequences encoding the frameworks of human heavy and light chains (Jones et al., Nature 321:522, 1986; and Riechmann et al., Nature 332:323, 1988). The recombinant products expressed are called "reshaped" or humanized antibodies and contain the frameworks of human antibody light or heavy chains and the antigen-recognition portions, the CDRs, of mouse monoclonal antibodies.

[0090] Other methods for designing heavy and light chains and for producing humanized antibodies are described, for example, in U.S. Patent Nos. 5,530,101; 5,565,332; 5,585,089; 5,639,641; 5,693,761; 5,693,762; and 5,733,743. Still other methods for humanized antibodies are described, for example, in U.S. Patent Nos. 4,816,567; 4,935,496; 5,502,167; 5,558,864; 5,693,493; 5,698,417; 5,705,154; 5,750,078; and 5,770,403.

[0091] The antibodies disclosed herein can be produced using standard methods. For example, they can be produced recombinantly, purified from biological samples (e.g., heterologous expression systems), or chemically synthesized, and can be used to immunize host animals such as rabbits, chickens, mice, guinea pigs, hamsters, or rats. Various adjuvants that can be used to enhance the immune response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysophosphatidylcholine, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Monoclonal antibodies can be prepared, for example, using FAP and standard hybridoma techniques. In particular, monoclonal antibodies can be obtained by any of the techniques that provide for the production of antibody molecules by continuous cell lines in culture such as those described by Kohler et al. (Nature 256:495, 1975), the human B-cell hybridoma techniques of Kosbor et al. (Immunology Today, 4:72, 1983) or Cote et al. (Proc. Natl. Acad. Sci. USA, 80:2026, 1983), and the EBV hybridoma techniques of Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any of their subclasses. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo.

[0092] In some embodiments, the antibodies provided herein have a heavy chain variable region that includes any one of the VH amino acid sequences disclosed herein. In other embodiments, the antibodies provided herein have a heavy chain variable region that includes any one of the VH amino acid sequences having from 1 to 24 modifications (e.g., substitutions, additions, or deletions) disclosed herein, whereby the amino acid sequence has, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 modifications. In some embodiments, the antibodies provided herein have a light chain variable region that includes any one of the VL amino acid sequences disclosed herein. In other embodiments, the antibodies provided herein have a light chain variable region that includes any one of the VL amino acid sequences having from 1 to 24 modifications (e.g., substitutions, additions, or deletions) disclosed herein, whereby the amino acid sequence has, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 modifications. The present specification also provides antibodies and antigen-binding fragments that include both the heavy chain variable region polypeptide and the light chain variable region polypeptide disclosed herein. The antibodies or antigen-binding fragments disclosed herein may also include, for example, variable region heavy chain framework (FW) sequences juxtaposed between CDRs according to the formula (FW1)-(CDR1)-(FW2)-(CDR2)-(FW3)-(CDR3)-(FW4). In some embodiments, the FW sequences can be human sequences.

[0093] In some embodiments, for example, the FAP-binding substance has up to 5 modifications (substitutions, deletions, or insertions) in any one of the amino acid sequences of the CDRs of the antibodies disclosed herein. For example, the FAP-binding substance includes up to 5 substitutions, deletions, or insertions in any one of the amino acid sequences of CDR1, CDR2, or CDR3 of the antibodies disclosed herein.

[0094] In some embodiments, amino acid substitutions to the antibodies or FAP-binding substances disclosed herein can be made by selecting conservative substitutions that do not vary significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the substitution region, (b) the hydrophobicity or charge of the molecule at the target site, and (c) the bulk of the side chain. For example, natural residues can be classified into several groups based on their side chain properties: (1) hydrophobic amino acids (norleucine, methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine); (5) amino acids that affect chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Non-limiting examples of conservative substitutions include, but are not limited to, substitution of alanine with valine, arginine with lysine, asparagine with glutamine, aspartic acid with glutamic acid, cysteine with serine, glutamine with asparagine, glutamic acid with aspartic acid, glycine with proline, histidine with arginine, isoleucine with leucine, leucine with isoleucine, lysine with arginine, methionine with leucine, phenylalanine with leucine, proline with glycine, serine with threonine, threonine with serine, tryptophan with tyrosine, tyrosine with phenylalanine, and / or valine with leucine. In some embodiments, the amino acid substitutions can be non-conservative, whereby a member of one of the above amino acid classes is exchanged with a member of another class. In some embodiments, the FAP-binding substance has at least 90% identity with any one of the amino acid sequences selected from SEQ ID NOs: 2-42, 46-86, 837-850, 1045-1085, or 1086-1092 or any one of the FAP-binding substances disclosed herein.In some embodiments, the FAP-binding substance has about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with any amino acid selected from SEQ ID NOs: 2-42, 46-86, 837-850, 1045-1085, or 1086-1092 or any one amino acid of the FAP-binding substances disclosed herein.

[0095] In some embodiments, for example, the FAP-binding substance has up to 5 substitutions, deletions, or insertions in any amino acid sequence selected from SEQ ID NOs: 87-175 or 851-879 or in any CDR of the FAP-binding substances disclosed herein. For example, the FAP-binding substance contains up to 5 substitutions, deletions, or insertions (e.g., a total of 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions) in the amino acid sequence of CDR1 selected from, for example, SEQ ID NOs: 87-115, 851-861, 1093-1129, 882, 915, 920, 923, 928, 931, 936, 939, 944, 947, 952, 955, 960, 963, 968, 971, 976, 979, 984, 987, 992, 995, 1000, 1003, 1008, 1011, 1016, 1019, 1024, 1027, 1032, or 1035. Similarly, in another embodiment, the FAP-binding substance contains up to 5 substitutions, deletions, or insertions (e.g., a total of 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions) in the amino acid sequence of CDR2 selected from, for example, SEQ ID NOs: 116-144, 862-876, 883, 916, 921, 924, 929, 932, 937, 940, 945, 948, 953, 956, 961, 964, 969, 972, 977, 980, 985, 988, 993, 996, 1001, 1004, 1009, 1012, 1017, 1020, 1025, 1028, 1033, or 1036. Similarly, in another embodiment, the FAP-binding substance contains up to 5 substitutions, deletions, or insertions (e.g., a total of 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions) in the amino acid sequence of CDR3 selected from, for example, SEQ ID NOs: 145-175, 877-879, 884, 917, 922, 925, 930, 933, 938, 941, 946, 949, 954, 957, 962, 965, 970, 973, 978, 981, 986, 989, 994, 997, 1002, 1005, 1010, 1013, 1018, 1021, 1026, 1029, 1034, 1037. Amino acid substitution refers to the replacement of one or more amino acid residues in a peptide sequence by another residue. Amino acid deletion means the removal of one or more amino acid residues in a peptide sequence.An amino acid insertion refers to the addition of one or more amino acid residues in a peptide sequence.

[0096] In various exemplary embodiments, the mouse FAP binding substance has at least 90% identity with the amino acid sequence of sibrotuzumab.

[0097] In some embodiments, the technology contemplates the use of any natural or synthetic analogs, variants, mutants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the FAP binding substances of the technology described herein. In some embodiments, the amino acid sequence of the FAP binding substance further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0098] In some embodiments, the FAP-binding substance comprises a targeting moiety comprising a sequence that is at least 60% identical to any one of the FAP sequences disclosed herein. For example, the FAP-binding substance may be at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or 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) to any one of the sequences disclosed herein and may comprise a targeting moiety.

[0099] In some embodiments, the FAP-binding substance comprises a targeting moiety comprising an amino acid sequence having one or more amino acid mutations relative to any one of the sequences disclosed herein. In some embodiments, the FAP-binding substance comprises a targeting moiety comprising an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations relative to any one of the sequences disclosed herein. In some embodiments, one or more of the amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0100] In some embodiments, the amino acid mutation is an amino acid substitution and can include conservative and / or non-conservative substitutions.

[0101] "Conservative substitutions" can be made, for example, based on similarities in the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the amino acid residues involved. The 20 natural amino acids can be classified into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0102] As used herein, "conservative substitution" is defined as the replacement of one amino acid by another amino acid described within the same group of the six standard amino acid groups above. For example, the replacement of Asp by Glu retains one negative charge in the polypeptide so modified. Further, glycine and proline can be substituted for each other based on their ability to disrupt α helices.

[0103] As used herein, "non-conservative substitution" is defined as the replacement of one amino acid by another amino acid described within a different group of the six standard amino acid groups (1)-(6) above.

[0104] In some embodiments, the substitution includes non-classical amino acids. Exemplary non-classical amino acids include, but are not limited to, selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, designer amino acids such as fluoroamino acids, β-methylamino acids, C α-methylamino acids, N α-methylamino acids, and amino acid analogs in general.

[0105] In some embodiments, one or more amino acid mutations are present in the CDR of the targeting moiety (e.g., the CDR1, CDR2, or CDR3 region). In another embodiment, one or more amino acid mutations are present in the framework region (FR) of the targeting moiety (e.g., the FR1, FR2, FR3, or FR4 region).

[0106] Modification of the amino acid sequence can be achieved using techniques well known in any such art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.

[0107] In some embodiments, the mutation does not substantially reduce the ability of the FAP-binding substance of the invention to specifically bind to FAP. In some embodiments, the mutation does not substantially reduce the ability of the FAP-binding substance of the invention to specifically bind to FAP and does not functionally regulate (e.g., partially or completely neutralize) FAP.

[0108] In some embodiments, the binding affinity of the FAP-binding substance of the present technology for the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or monomer and / or dimer form and / or any other natural or synthetic analog, variant, or mutant (including monomer and / or dimer form) of human FAP can be characterized by the equilibrium dissociation constant (K D ). In various embodiments, the FAP-binding substance has a K D of about 1 μM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, or about 5 nM, or less than about 1 nM for the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other natural or synthetic analog, variant, or mutant (including monomer and / or dimer form) of human FAP and comprises a targeting moiety that binds at that K

[0109] In some embodiments, the FAP-binding substance comprises a targeting moiety that binds to the antigen of interest, i.e., FAP, but does not functionally modulate (e.g., partially or completely neutralize) it. For example, in some embodiments, the targeting moiety of the FAP-binding substance merely targets the antigen but does not substantially functionally modulate (e.g., partially or completely inhibit, reduce or neutralize) the biological action of the antigen. In some embodiments, the targeting moiety of the FAP-binding substance binds to an epitope that is physically distant from the antigenic site important for its biological activity (e.g., the active site of the antigen).

[0110] Such binding without significant functional modulation is used in some embodiments of the present technology, including methods in which the FAP-binding substance of the present invention is used to directly or indirectly recruit activated immune cells to the required site via an effector antigen. For example, in some embodiments, the FAP-binding substance of the present invention can be used to directly or indirectly recruit dendritic cells to tumor cells via FAP in a method of shrinking or removing a tumor (e.g., the FAP-binding substance can comprise a targeting moiety having an anti-FAP antigen recognition domain and a targeting moiety having a recognition domain for a tumor antigen or receptor (e.g., an antigen recognition domain)). In such embodiments, it is desirable to directly or indirectly recruit dendritic cells without functionally modulating or neutralizing FAP activity. In these embodiments, FAP signaling is an important part of the action of shrinking or removing a tumor.

[0111] In some embodiments, the FAP-binding substance enhances antigen presentation by dendritic cells. For example, in some embodiments, the FAP-binding substance of the present invention can directly or indirectly recruit dendritic cells to tumor cells via FAP, where the tumor antigen is then taken up and presented on the dendritic cells for induction of a strong humoral and cytotoxic T cell response.

[0112] In other embodiments (e.g., in connection with the treatment of cancer, autoimmune or neurodegenerative diseases), the FAP-binding substance comprises a targeting moiety that binds and neutralizes the antigen of interest, i.e., FAP. For example, in some embodiments, the methods of the invention can inhibit or reduce FAP signaling or expression, e.g., to reduce an immune response.

[0113] Chimeras and fusions with signaling substances In some embodiments, the FAP-binding substances of the technology disclosed herein are part of a chimeric or fusion protein or an Fc-based chimeric protein complex with one or more signaling substances. Accordingly, the technology provides, for example, a chimeric or fusion protein or an Fc-based chimeric protein complex comprising a targeting moiety for FAP and one or more signaling substances. In some embodiments, the signaling substance is a wild-type signaling substance or a modified signaling substance.

[0114] In various embodiments, the chimeric or fusion protein or Fc-based chimeric protein complex comprises a wild-type signaling substance with improved target selectivity and safety compared to i) a signaling substance not fused to Fc, or ii) a complex, e.g., but not limited to, a signaling substance that is not a heterodimeric complex. In various embodiments, the chimeric or fusion protein or Fc-based chimeric protein complex comprises a wild-type signaling substance with improved target-selective activity compared to i) a signaling substance not fused to Fc, or ii) a complex, e.g., but not limited to, a signaling substance that is not a heterodimeric complex. In various embodiments, the Fc-based chimeric protein complex enables conditional activity.

[0115] In various embodiments, the chimeric or fusion protein or Fc-based chimeric protein complex has improved safety compared to a signaling agent that is not fused to Fc, or a complex, e.g., a signaling agent that is not a heterodimeric complex, such as reduced systemic toxicity, reduced side effects, and reduced off-target effects, including a wild-type signaling agent. In various embodiments, the improved safety means that the chimeric or fusion protein or Fc-based chimeric protein of the invention has lower toxicity (e.g., systemic toxicity and / or tissue / organ-related toxicity) of the wild-type signaling agent compared to a signaling agent that is not fused to Fc, or a complex, e.g., a signaling agent that is not a heterodimeric complex; and / or reduced or substantially eliminated side effects; and / or increased tolerance, reduced or substantially eliminated adverse events; and / or reduced or substantially eliminated; and / or an expanded therapeutic concentration range.

[0116] In some embodiments, the reduced affinity or activity for a receptor can be restored by the binding of one or more targeting moieties described herein or upon inclusion in an Fc-based chimeric protein complex disclosed herein.

[0117] In various embodiments, the chimeric or fusion protein or Fc-based chimeric protein complex has a reduced, substantially reduced or eliminated affinity, e.g., binding (e.g., K D ) and / or activation (e.g., when the modified signaling agent is an agonist of its receptor, e.g., K A and / or EC 50 measurable as) and / or inhibition (e.g., when the modified signaling agent is an antagonist of its receptor, e.g., K I and / or IC 50It includes a wild-type signaling substance that can be measured as such). In various embodiments, the reduced affinity of the signaling substance for the receptor enables attenuation of activity. In such embodiments, the modified signaling substance has an affinity for the receptor of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% - 20%, about 20% - 40%, about 50%, about 40% - 60%, about 60% - 80%, about 80% - 100% compared to a signaling substance that is not fused to Fc, or a complex, such as, but not limited to, a signaling substance that is not a heterodimeric complex. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10 - 50-fold lower, about 50 - 100-fold lower, about 100 - 150-fold lower, about 150 - 200-fold lower, or 200-fold lower compared to a signaling substance that is not fused to Fc, or a complex, such as, but not limited to, a signaling substance that is not a heterodimeric complex.

[0118] In various embodiments, the chimeric or fusion protein or Fc-based chimeric protein complex includes a wild-type signaling substance that has an endogenous activity of the signaling substance reduced by about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1% compared to, for example, a signaling substance that is not fused to Fc, or a complex, such as, but not limited to, a signaling substance that is not a heterodimeric complex.

[0119] In some embodiments, the signaling molecule is modified to have a reduced affinity or activity for one or more of its receptors, which enables attenuation (including agonism or antagonism) of the activity of a chimeric or fusion protein or Fc-based chimeric protein complex, and / or prevents non-specific signaling or unwanted sequestration. In some embodiments, the signaling molecule is an antagonist in its wild-type form and has one or more mutations that weaken its antagonist activity. In some embodiments, the signaling molecule is an antagonist due to one or more mutations, e.g., an agonist signaling molecule is converted to an antagonist signaling molecule, and such a converted signaling molecule may also have one or more mutations that weaken its antagonist activity (e.g., as described in WO 2015 / 007520. The entire content of this patent is incorporated herein by reference).

[0120] Thus, in some embodiments, the signaling molecule is a modified (mutated) form of the signaling molecule having one or more modifications (e.g., mutations). In some embodiments, the mutation enables the modified signaling molecule to have one or more attenuated activities such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity compared to the non-modified, i.e., wild-type form of the signaling molecule (e.g., by comparing the same signaling molecule in the wild-type form and the modified (e.g., mutated) form). In some embodiments, mutations that weaken or reduce binding or affinity include mutations that substantially reduce or eliminate binding or activity. In some embodiments, mutations that weaken or reduce binding or affinity are different from mutations that substantially reduce or eliminate binding or activity. As a result, in some embodiments, the mutation enables the signaling molecule to have improved safety, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the non-modified, i.e., wild-type signaling molecule (e.g., by comparing the same signaling molecule in the wild-type form and the modified (e.g., mutated) form).

[0121] As described herein, a substance can have improved safety by one or more modifications, e.g., mutations. In some embodiments, improved safety means that the chimeric protein or Fc-based chimeric protein complex of the invention has lower toxicity (e.g., systemic toxicity and / or tissue / organ-related toxicity); and / or reduced or substantially eliminated side effects; and / or increased tolerance, reduced or substantially eliminated adverse events; and / or reduced or substantially eliminated; and / or an expanded therapeutic concentration range.

[0122] In some embodiments, the signaling molecule is modified to have one or more mutations that reduce the binding affinity or activity for one or more of its receptors. In some embodiments, the signaling molecule is modified to have one or more mutations that substantially reduce or eliminate the binding affinity or activity for the receptor. In some embodiments, the activity provided by the wild-type signaling molecule is agonism for the receptor (e.g., activation of a cellular effect at the site of treatment). For example, the wild-type signaling molecule can activate its receptor. In such embodiments, the mutation results in a signaling molecule modified to reduce or eliminate the activating effect on the receptor. For example, the mutation can result in a signaling molecule modified to send a reduced activation signal to the target cell, or the activation signal can be eliminated. In some embodiments, the effect provided by the wild-type signaling molecule is antagonism for the receptor (e.g., blocking or suppressing a cellular effect at the site of treatment). For example, the wild-type signaling molecule can antagonize or inhibit the receptor. In these embodiments, the mutation results in a signaling molecule modified to reduce or eliminate the antagonizing activity for the receptor. For example, the mutation can result in a signaling molecule modified to send a reduced inhibitory signal to the target cell, or the inhibitory signal can be eliminated. In some embodiments, the signaling molecule is antagonistic due to one or more mutations, e.g., an agonist signaling molecule is converted to an antagonist signaling molecule (e.g., as described in International Publication No. WO 2015 / 007520, the entire contents of which are incorporated herein by reference), and such a converted signaling molecule optionally also has one or more mutations that reduce its binding affinity or activity for one or more of its receptors, or that substantially reduce or eliminate the binding affinity or activity for one or more of its receptors.

[0123] In some embodiments, the reduced affinity or activity for a receptor can be restored by the binding of one or more targeting moieties described herein (e.g., a targeting moiety for FAP), or upon inclusion into an Fc-based chimeric protein complex disclosed herein. In other embodiments, the reduced affinity or activity for a receptor is not substantially restorable by the action of one or more targeting moieties or upon inclusion into an Fc-based chimeric protein complex disclosed herein.

[0124] In some embodiments, the chimeric proteins or Fc-based chimeric protein complexes of the present technology reduce off-target effects because their signaling moieties have mutations that weaken or eliminate the binding affinity or activity for a receptor. In some embodiments, for example, this reduction is observed in side effects compared to a wild-type signaling moiety. In some embodiments, the signaling moiety is active against a target cell. The reason is that the targeting moiety(ies) compensate for a defective / inadequate binding (e.g., but not limited to, and / or binding strength) required for substantial activation. In some embodiments, the modified signaling moiety is substantially inactive en route to the site of therapeutic action and has its effect substantially against specifically targeted cell types, which greatly reduces unwanted side effects.

[0125] In some embodiments, the signaling molecule can include one or more mutations that weaken or reduce the binding or affinity for one receptor (i.e., the therapeutic receptor) and one or more mutations that substantially reduce or eliminate the binding or activity for a second receptor. In such embodiments, these mutations can be at the same or different positions (i.e., the same mutation or multiple mutations). In some embodiments, the mutation(s) that reduce binding and / or activity for one receptor are different from the mutation(s) that substantially reduce or eliminate binding for another receptor. In some embodiments, the mutation(s) that reduce binding and / or activity for one receptor are the same as the mutation(s) that substantially reduce or eliminate binding for another receptor. In some embodiments, the chimeric protein or Fc-based chimeric protein complex of the invention has a modified signaling molecule having both a mutation that weakens binding and / or activity for the therapeutic receptor and thus enables a more controlled on-target therapeutic effect (e.g., as compared to the wild-type signaling molecule) and a mutation that substantially reduces or eliminates binding and / or activity for another receptor and thus reduces side effects (e.g., as compared to the wild-type signaling molecule).

[0126] In some embodiments, substantial reduction or elimination of binding or activity is not substantially recoverable using a targeting moiety (e.g., a targeting moiety to FAP or any other targeting moiety described herein) or upon inclusion into an Fc-based chimeric protein complex disclosed herein. In some embodiments, substantial reduction or elimination of binding or activity is recoverable by a targeting moiety or upon inclusion into an Fc-based chimeric protein complex disclosed herein. In some embodiments, substantial reduction or elimination of binding or activity to a second receptor may also prevent deleterious effects mediated by other receptors. Alternatively, or in addition, substantial reduction or elimination of binding or activity to other receptors reduces or eliminates sequestration of the therapeutic chimeric protein away from the site of therapeutic action, thereby improving the therapeutic effect. For example, in some embodiments, this obviates the need for high doses of the chimeric protein or Fc-based chimeric protein complex of the invention to compensate for loss at other receptors. The ability to reduce such dosages further reduces the potential for side effects.

[0127] In some embodiments, the modified signaling molecule has a reduced, substantially reduced, or eliminated affinity, e.g., binding (e.g., K D ) and / or activation (e.g., if the modified signaling molecule is an agonist of its receptor, e.g., K A and / or EC 50 measurable as) and / or inhibition (e.g., if the modified signaling molecule is an antagonist of its receptor, e.g., K i and / or IC 50comprising one or more mutations such that it is measurable as). In some embodiments, the reduced affinity for the receptor of the immunomodulatory agent allows for attenuation of activity (including agonism or antagonism). In such embodiments, the modified signaling agent has an affinity for the receptor of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% - 20%, about 20% - 40%, about 50%, about 40% - 60%, about 60% - 80%, about 80% - 100% compared to the wild-type signaling agent. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10 - 50-fold lower, about 50 - 100-fold lower, about 100 - 150-fold lower, about 150 - 200-fold lower, or 200-fold lower compared to the wild-type signaling agent.

[0128] In some embodiments, the modified signaling substance has a mutation that reduces binding to one receptor and substantially reduces or eliminates binding to a second receptor. In some embodiments, the attenuation or reduction of the binding affinity of the modified signaling substance for one receptor is less than the substantial reduction or elimination of the affinity for another receptor. In some embodiments, the attenuation or reduction of the binding affinity of the modified signaling substance for one receptor is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% less than the substantial reduction or elimination of the affinity for another receptor. In some embodiments, substantial reduction or elimination refers to a reduction in binding affinity and / or activity that is greater than the attenuation or reduction.

[0129] In some embodiments, the modified signaling substance comprises one or more mutations that reduce the intrinsic activity of the signaling substance 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% compared to, for example, the wild-type signaling substance.

[0130] In some embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have a reduced affinity for its receptor that is lower than the binding affinity of the targeting moiety for that receptor. In some embodiments, this difference in binding affinity exists between the signaling agent / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity enables the signaling agent, e.g., the mutant signaling agent, to have a localized on-target effect and to minimize off-target effects underlying side effects observed with the 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 lower.

[0131] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be evaluated by reflectometric interference spectroscopy under flow-through conditions, as described by Scatchard plot analysis and computer fitting of binding data (e.g., Scatchard, 1949) or Brecht et al. (1993). The entire contents of these references are incorporated herein by reference.

[0132] In various embodiments, the additional signaling molecule is selected from wild-type or modified forms of cytokines, growth factors, and hormones. Examples of such cytokines, growth factors, and hormones include, but are not limited to, conventional polypeptide hormones such as lymphokines, monokines, human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; protein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor α and tumor necrosis factor β; Müllerian duct inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial cell growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGFα; platelet growth factor; transforming growth factor (TGF) such as TGFα and TGFβ; insulin-like growth factor-I and II; osteogenic factor; interferons such as interferon α, interferon β, and interferon γ (and interferon types I, II, and III); colony stimulating factors (CSF) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL1β, IL1α, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, and IL18; tumor necrosis factors such as TNFα or TNFβ; and other polypeptide factors such as LIF and kit ligand (KL). As used herein, cytokines, growth factors, and hormones include those of natural origin or products from recombinant bacterial, eukaryotic, or mammalian cell culture systems and biologically active equivalents of native sequence cytokines.

[0133] In some embodiments, the additional signaling molecule is a wild-type or modified growth factor selected from, but not limited to, transforming growth factors (TGFs) such as TGF-α and TGFβ, epidermal growth factor (EGF), insulin-like growth factors such as insulin-like growth factor-I and II, fibroblast growth factor (FGF), heregulin, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF).

[0134] In certain embodiments, the growth factor is a wild-type or modified fibroblast growth factor (FGF). Examples of FGF include, but are not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, mouse FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0135] In certain embodiments, the growth factor is a wild-type or modified transforming growth factor (TGF). Examples of TGF include, but are not limited to, TGF-α and TGF-β and their subtypes, including various subtypes of TGF-β including TGFβ1, TGFβ2, and TGFβ3.

[0136] In some embodiments, the additional signaling molecule is, but is not limited to, a wild-type or modified hormone selected from human chorionic gonadotropin, gonadotropin-releasing hormone, androgen, estrogen, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growth hormone-releasing hormone, corticotropin-releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoid, mineralocorticoid, 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.

[0137] In some embodiments, the signaling molecule is one or more immunomodulatory agents, such as interleukin, interferon, and tumor necrosis factor.

[0138] In some embodiments, the signaling molecule is a wild-type interleukin or a modified interleukin, such as, for example, IL1, IL1β, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36 or a fragment, variant, analog, or family member thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulation of the proliferation of immune cells (such as helper T cells, B cells, eosinophils, and lymphocytes), chemotactic effects on neutrophils and T lymphocytes, and / or inhibition of interferons. Interleukin activity can be measured using assays known in the art (Matthews et al., in Lymphokines and Interferons: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20).

[0139] In some embodiments, the signaling molecule is a wild-type interferon or a modified interferon such as interferon types I, II, and III. Examples of interferons include, for example, interferon α-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21, interferon β and interferon γ, interferon κ, interferon ε, interferon τ, interferon δ, IFNν, and interferon ω.

[0140] In some embodiments, the signaling molecule is wild-type tumor necrosis factor (TNF) or a modified form of tumor necrosis factor (TNF) or a protein of the TNF family, including but not limited to TNFα, TNFβ, LTβ, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TRAIL.

[0141] The amino acid sequences of the wild-type signaling substances described in this specification are well-known in the art. Thus, in some embodiments, the modified signaling substance has an amino acid sequence having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or 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) with the known wild-type amino acid sequences of the signaling substances described in this specification.

[0142] In some embodiments, the modified signaling molecule comprises an amino acid sequence having at least about 60%, or at least about 61%, 65%, or at least about 66%, 70%, or at least about 71%, 75%, or at least about 76%, 80%, or at least about 81%, 85%, or at least about 86%, 90%, or at least about 91%, or at least about 62%, or at least about 67%, or at least about 72%, or at least about 77%, or at least about 82%, or at least about 87%, or at least about 92% or at least about 63%, or at least about 68%, or at least about 73%, or at least about 78%, or at least about 83%, or at least about 88%, or at least about 93% or at least about 64%, or at least about 69%, or at least about 74%, or at least about 79%, or at least about 84%, or at least about 89%, or at least about 94% or at least about or at least about or at least about or at least about or at least about or at least about or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity) to any of the amino acid sequences of the signaling molecules described herein.

[0143] In some embodiments, the modified signaling agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutation is an amino acid substitution and can include conservative substitutions and / or non-conservative substitutions, as described elsewhere herein. In some embodiments, the substitution can include non-classical amino acids, as described elsewhere herein.

[0144] As described herein, the modified signaling agent has mutations that affect the affinity and / or activity for one or more receptors. In some embodiments, there is a reduced affinity and / or activity for a therapeutic receptor, e.g., a receptor by which the desired therapeutic effect is mediated (e.g., agonism or antagonism). In some embodiments, the modified signaling agent has mutations that substantially reduce or eliminate the affinity and / or activity for a receptor, e.g., a receptor by which the desired therapeutic effect is not mediated (e.g., as a result of a disrupted binding state). Receptors for modified signaling agents, e.g., one receptor for the cytokines, growth factors, and hormones described herein, are known in the art.

[0145] Examples of mutations that result in reduced affinity and / or activity (e.g., agonist activity) for a receptor are found in International Publication No. WO 2013 / 107791 and International Application No. PCT / EP2017 / 061544 (e.g., with respect to interferon), International Publication No. WO 2015 / 007542 (e.g., with respect to interleukin), and International Publication No. WO 2015 / 007903 (e.g., with respect to TNF), the entire contents of each of which are incorporated herein by reference. Examples of mutations that reduce the affinity and / or activity (e.g., antagonist activity) for a therapeutic receptor are found in International Publication No. WO 2015 / 007520, the entire contents of which are incorporated herein by reference.

[0146] In some embodiments, the modified signaling agent comprises one or more mutations that reduce the affinity and / or activity for type I cytokine receptors, type II cytokine receptors, chemokine receptors, receptors of the tumor necrosis factor receptor (TNFR) superfamily, TGF-beta receptors, receptors of the immunoglobulin (Ig) superfamily, and / or receptors of the tyrosine kinase superfamily of the signaling agent.

[0147] In some embodiments, the receptor for the 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 receptors for thrombopoietin (TPO), prolactin, and growth hormone. Exemplary type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptor.

[0148] In some embodiments, the receptor for the signaling agent is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits and are primarily receptors for interferons. This receptor family includes, but is not limited to, receptors for interferon alpha, interferon 13 and interferon gamma, IL10, IL22, and tissue factor. Examples of type II cytokine receptors include, but are not limited to, IFNα receptor (e.g., IFNAR1 and IFNAR2), IFN3 receptor, IFNγ receptor (e.g., IFNGR1 and IFNGR2), and type II IL receptor.

[0149] In some embodiments, the receptor for the signaling substance is a G protein-coupled receptor. The chemokine receptor is a G protein-coupled receptor having a seven-transmembrane structure and binding to a G protein for signal transduction. Examples of chemokine receptors include, but are not limited to, CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors (XCR1). Examples of chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR7, XCR1, and CX3CR1.

[0150] In some embodiments, the receptor for the signaling substance is a TNFR family member. Tumor necrosis factor receptor (TNFR) family members share a cysteine-rich domain (CRD) formed from three disulfide bonds surrounding a CXXCXXC core motif that creates an elongated molecule. Examples of the tumor necrosis factor receptor family include the following: CD120a (TNFRSF1A), CD120b (TNFRSF1B), lymphotoxin beta receptor (LTBR, TNFRSF3), CD134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSF10A (TNFRSF10A), TNFRSF10B, (TNFRSF10B), TNFRSF10C (TNFRSF10C), TNFRSF10D (TNFRSF10D), RANK (TNFRSF11A), osteoprotegerin (TNFRSF11B), 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). In one embodiment, the TNFR family member is CD120a (TNFRSF1A) or TNF-R1. In another embodiment, the TNFR family member is CD120b (TNFRSF1B) or TNF-R2.

[0151] In some embodiments, the receptor for the signaling substance is a TGF-beta receptor. The TGF-beta receptor is a single-pass transmembrane serine / threonine kinase receptor. TGF-beta receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR3.

[0152] In some embodiments, the receptor for the signaling substance is an Ig superfamily receptor. Receptors of the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Examples of receptors of the Ig superfamily include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

[0153] In some embodiments, the receptor for the signaling substance is a tyrosine kinase superfamily receptor. Receptors of the tyrosine kinase superfamily are well known in the art. There are approximately 58 receptor tyrosine kinases (RTKs) classified into 20 subfamilies. Examples of receptors of the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0154] In some embodiments, the wild-type or modified signaling substance is interferon α. In some embodiments, the modified IFNα substance has reduced affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFNα substance has substantially reduced or eliminated affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFNα substance is a human modified IFNα substance.

[0155] Mutant interferons are known to those skilled in the art. By way of non-limiting example, in some embodiments, the modified signaling substance is allelic IFNα2a having the amino acid sequence of SEQ ID NO: 176.

[0156] Although this is a non-limiting example, in some embodiments, the modified signaling substance is allelic IFNα2b having the amino acid sequence of SEQ ID NO: 177, which differs from IFNα2a at amino acid position 23.

[0157] In some embodiments, the modified IFNα2 substance is a human IFNα2 variant (IFNα2a or IFNα2b). In some embodiments, the human IFNα2 variant (IFNα2a or IFNα2b) is mutated at one or more amino acids at positions 144-154, such as, for example, amino acid positions 148, 149, and / or 153. In some embodiments, the human IFNα2 variant comprises one or more mutations selected from L153A, R149A, and M148A.

[0158] In some embodiments, the IFNα2 variant has a reduced affinity and / or activity for IFNAR1. In some embodiments, the IFNα2 variant is a human IFNα2 variant comprising one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A as described in International Publication No. WO 2010 / 030671. The entire content of this patent is incorporated herein by reference.

[0159] In some embodiments, the IFNα2 variant is a human IFNα2 variant comprising one or more mutations selected from K133A, R144A, R149A, and L153A as described in International Publication No. WO 2008 / 124086. The entire content of this patent is incorporated herein by reference.

[0160] In some embodiments, the IFNα2 variant is a human IFNα2 variant comprising one or more mutations selected from R120E and R120E / K121E as described in International Publication Nos. WO 2015 / 007520 and WO 2010 / 030671. The entire content of these patents is incorporated herein by reference.

[0161] In some embodiments, the IFNα2 variant antagonizes wild-type IFNα2 activity. In some embodiments, the variant IFNα2 has a reduced affinity and / or activity for IFNAR1, but retains activity for IFNAR2.

[0162] In some embodiments, the human IFNα2 variant comprises (1) one or more mutations selected from R120E and R120E / K121E (which, without wishing to be bound by theory, create an antagonist effect), and (2) one or more mutations selected from K133A, R144A, R149A, and L153A (which, without wishing to be bound by theory, enable, for example, a weakening effect on IFNAR2). In some embodiments, the human IFNα2 variant comprises R120E and L153A.

[0163] In some embodiments, the human IFNα2 variant comprises one or more mutations selected from L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, as disclosed in International Publication No. WO 2013 / 059885, the entire content of which is incorporated herein by reference. In some embodiments, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or L30A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or R33A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or M148A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or L153A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFNα2 variant comprises the mutations N65A, L80A, Y85A, and / or Y89A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFNα2 variant comprises the mutations N65A, L80A, Y85A, Y89A and / or D114A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFNα2 variant comprises the mutation R144X 1 , A145X 2 , and R33A, wherein X 1 is selected from A, S, T, Y, L, and I, and X 2 is selected from G, H, Y, K, and D. In some embodiments, the variant human IFNα2 has R33A, T106X 3 , R120E, R144X 1 , A145X with reference to the amino acid sequence of SEQ ID NO: 176 or 1772 comprises one or more mutations selected from M148A, R149A, and L153A, and X 1 is selected from A, S, T, Y, L, and I, and X 2 is selected from G, H, Y, K, and D, and X 3 is selected from A and E.

[0164] In some embodiments, the signaling molecule is wild-type interferon α1 or modified interferon α1. In some embodiments, the present invention provides a chimeric protein or an Fc-based chimeric protein complex comprising wild-type IFNα1. In various embodiments, the wild-type IFNα1 comprises the following amino acid sequence: CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHEL IQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAV KKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO: 1042).

[0165] In various embodiments, the chimeric protein or Fc-based chimeric protein complex of the present invention comprises, as the signaling molecule, a modified form of IFNα1, i.e., an IFNα1 variant comprising an IFNα1 mutant. In various embodiments, the IFNα1 variant includes variants, functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of interferon.

[0166] In some embodiments, the IFNα1 interferon is modified to have one or more amino acid mutations at positions L15, A19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157, based on SEQ ID NO: 1042. The mutations can optionally be hydrophobic mutations and can be selected, for example, from alanine, valine, leucine, and isoleucine. In some embodiments, the IFNα1 interferon is modified to have one or more mutations selected from L15A, A19W, R23A, S25A, L30A, L30V, D32A, R33K, R33A, R33Q, H34A, Q40A, C86S, C86A, D115R, L118A, K121A, K121E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E, R145G, R145H, R145I, R145K, R145L, R145N, R145Q, R145S, R145T, R145V, R145Y, A146D, A146E, A146G, A146H, A146I, A146K, A146L, A146M, A146N, A146Q, A146R, A146S, A146T, A146V, A146Y, M149A, M149V, R150A, S153A, L154A, and N157A, based on SEQ ID NO: 1042. In some embodiments, the IFNα1 variant contains one or more mutations selected from L30A / H58Y / E59N_Q62S, R33A / H58Y / E59N / Q62S, M149A / H58Y / E59N / Q62S, L154A / H58Y / E59N / Q62S, R145A / H58Y / E59N / Q62S, D115A / R121A, L118A / R121A, L118A / R121A / K122A, R121A / K122A, and R121E / K122E, based on SEQ ID NO: 1042.

[0167] In some embodiments, IFNα1 is a variant that contains one or more mutations that reduce undesirable disulfide pair formation, and the one or more mutations are, for example, at amino acid positions C1, C29, C86, C99, or C139, based on SEQ ID NO: 1042. In some embodiments, the mutation at position C86 can be, for example, C86S or C86A or C86Y. These C86 variants of IFNα1 are called aggregation mutants by reducing cysteine. In some embodiments, the IFNα1 variant contains mutations at positions C1, C86, and C99, based on SEQ ID NO: 1042.

[0168] In certain embodiments, the wild-type or modified signaling agent is interferon β. In such embodiments, the modified interferon β substance has a reduced affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFNβ substance has a substantially reduced or eliminated affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0169] In an exemplary embodiment, the modified signaling agent is IFNβ. In some embodiments, IFNβ includes functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of IFNβ. In some embodiments, IFNβ includes IFNβ from any species. In one embodiment, the chimeric protein or Fc-based chimeric protein complex includes a modified mouse IFNβ. In another embodiment, the chimeric protein or Fc-based chimeric protein complex includes a modified human IFNβ. Human IFNβ is a polypeptide having a molecular weight of about 22 kDa and containing 166 amino acid residues. The amino acid sequence of human IFNβ is shown as SEQ ID NO: 178.

[0170] In some embodiments, the human IFNβ is IFNβla, which is a glycosylated form of human IFNβ. In some embodiments, the human IFNβ is IFNβlb, which is a non-glycosylated form of human IFNβ having a Met-1 deletion and a mutation of Cys-17 to Ser.

[0171] In some embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity for the IFNAR1 subunit of IFNAR. In some embodiments, the modified IFNβ has a reduced affinity and / or activity for IFNAR1.

[0172] In some embodiments, the modified IFNβ having a reduced affinity and / or activity for IFNAR1 is human IFNβ and has one or more mutations at positions F67, R71, L88, Y92, I95, N96, K123, and R124. In some embodiments, the one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G. In some embodiments, the modified human IFNβ includes the F67G mutation. In some embodiments, the modified human IFNβ includes the K123G mutation. In some embodiments, the modified human IFNβ includes the F67G and R71A mutations. In some embodiments, the modified human IFNβ includes the L88G and Y92G mutations. In some embodiments, the modified human IFNβ includes the Y92G, I95A, and N96G mutations. In some embodiments, the modified human IFNβ includes the K123G and R124G mutations. In some embodiments, the modified human IFNβ includes the F67G, L88G, and Y92G mutations. In some embodiments, the modified human IFNβ includes the F67S, L88S, and Y92S mutations.

[0173] In some embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity for the IFNAR2 subunit of IFNAR. In some embodiments, the modified IFNβ has a reduced affinity and / or activity for IFNAR2.

[0174] In some embodiments, the modified IFNβ having a reduced affinity and / or activity for IFNAR2 is human IFNβ and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. In some embodiments, the one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In some embodiments, the modified human IFNβ comprises the W22G mutation. In some embodiments, the modified human IFNβ comprises the L32A mutation. In some embodiments, the modified human IFNβ comprises the L32G mutation. In some embodiments, the modified human IFNβ comprises the R35A mutation. In some embodiments, the modified human IFNβ comprises the R35G mutation. In some embodiments, the modified human IFNβ comprises the V148G mutation. In some embodiments, the modified human IFNβ comprises the R152A mutation. In some embodiments, the modified human IFNβ comprises the R152G mutation. In some embodiments, the modified human IFNβ comprises the Y155G mutation. In some embodiments, the modified human IFNβ comprises the W22G and R27G mutations. In some embodiments, the modified human IFNβ comprises the L32A and R35A mutations. In some embodiments, the modified human IFNβ comprises the L151G and R152A mutations. In some embodiments, the modified human IFNβ comprises the V148G and R152A mutations.

[0175] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with C175 or MA.

[0176] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with one or more of the other IFNβ mutations described herein.

[0177] The crystal structure of human IFNβ is known and is described in Karpusas et al., (1998) PNAS, 94(22):11813-11818. In particular, the structure of human IFNβ has been shown to include five α-helices (i.e., A, B, C, D, and E) and four loop regions (i.e., AB, BC, CD, and DE loops) that connect these helices. In some embodiments, the modified IFNβ has one or more mutations in the A, B, C, D, E helices and / or the AB, BC, CD, and DE loops that reduce its binding affinity or activity for a therapeutic receptor such as IFNAR. Examples of mutations are described in International Publication No. WO 2000 / 023114 and U.S. Patent Application Publication No. US 2015 / 0011732. The entire contents of these are incorporated herein by reference.

[0178] In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 28-30, 32, and 33. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 36, 37, 39, and 42. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 71-73. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 92, 96, 99, and 100. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 128, 130, 131, and 134. In an exemplary embodiment, the modified IFNβ is a human IFNβ comprising alanine substitutions at amino acid positions 149, 153, 156, and 159. In some embodiments, the variant IFNβ comprises SEQ ID NO: 178 and comprises 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).

[0179] In some embodiments, the variant IFNβ comprises SEQ ID NO: 178 and comprises a mutation at W27, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0180] In some embodiments, the variant IFNβ comprises SEQ ID NO: 178 and comprises 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 further comprises 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).

[0181] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and comprises a mutation at L32, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0182] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and comprises 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).

[0183] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and comprises 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 further comprises 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).

[0184] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and comprises 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).

[0185] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and comprises 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).

[0186] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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 further comprises 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).

[0187] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises a mutation at L88, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0188] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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).

[0189] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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 comprises 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 comprises 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).

[0190] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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 further comprises 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).

[0191] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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 further comprises 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).

[0192] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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 further comprises 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).

[0193] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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 comprises 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 comprises 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).

[0194] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and contains a mutation at K123, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0195] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and contains a mutation at R124, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0196] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and contains a mutation at K123, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and further contains a mutation at R124, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0197] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and contains a mutation at L151, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0198] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178 and contains a mutation at R152, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0199] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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 further comprises 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).

[0200] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises a mutation at V148, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0201] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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 further comprises 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).

[0202] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 178, comprises 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).

[0203] In some embodiments, the technology relates to a chimeric protein or Fc-based chimeric protein complex comprising: (a) a modified IFNβ having the amino acid sequence of SEQ ID NO: 178 and having a mutation at position W22, the mutation being an aliphatic hydrophobic residue; and (b) one or more targeting moieties, wherein the targeting moiety comprises a recognition domain that specifically binds to a target antigen or receptor (e.g., FAP), and the modified IFNβ and the one or more targeting moieties may be optionally linked by one or more linkers. In some embodiments, the mutation at position W22 is an aliphatic hydrophobic residue selected from G, A, L, I, M, and V. In some embodiments, the mutation at position W22 is G.

[0204] Examples of additional IFNβ variants are provided in International Application No. PCT / EP2017 / 061544. The entire disclosure is incorporated herein by reference.

[0205] In some embodiments, the signaling agent is wild-type or modified interferon gamma. In such embodiments, the modified interferon gamma substance has a reduced affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains. In some embodiments, the modified interferon gamma substance has a substantially reduced or eliminated affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains.

[0206] For example, the mutant IFNγ may include truncations, which are non-limiting examples of mutations. In some embodiments, the mutant IFNγ has, for example, a C-terminal truncation of about 5 to about 20 amino acid residues, or about 16 amino acid residues, or about 15 amino acid residues, or about 14 amino acid residues, or about 7 amino acid residues, or about 5 amino acid residues. In some embodiments, the mutant IFNγ has one or more mutations at positions Q1, V5, E9, K12, H19, S20, V22, A23, D24, N25, G26, T27, L30, K108, H111, E112, I114, Q115, A118, E119, and K125. In some embodiments, the mutant IFNγ has one or more substitutions selected from V5E, S20E, V22A, A23G, A23F, D24G, G26Q, H111A, H111D, I114A, Q115A, and A118G. In some embodiments, the mutant IFNγ includes the V22A mutation. In some embodiments, the mutant IFNγ includes the A23G mutation. In some embodiments, the mutant IFNγ includes the D24G mutation. In some embodiments, the mutant IFNγ includes the H111A or H111D mutation. In some embodiments, the mutant IFNγ includes the I114A mutation. In some embodiments, the mutant IFNγ includes the Q115A mutation. In some embodiments, the mutant IFNγ includes the A118G mutation. In some embodiments, the mutant IFNγ includes the A23G and D24G mutations. In some embodiments, the mutant IFNγ includes the I114A and A118G mutations. IFNγ is represented by SEQ ID NO: 1043 below, and all mutations are based on SEQ ID NO: 1043: MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ (SEQ ID NO: 1043).

[0207] In certain embodiments, the wild-type or modified signaling substance is consensus interferon. In some embodiments, the consensus interferon comprises the amino acids of SEQ ID NO: 179.

[0208] In some embodiments, the consensus interferon is modified to have one or more amino acid mutations at positions 33 and / or 145-155, such as amino acid positions 145, 146, 149, 150, and / or 154, relative to SEQ ID NO: 179. In some embodiments, the consensus interferon is modified to have one or more amino acid mutations at positions 33 and / or 145-155, such as amino acid positions 145, 146, 149, 150, and / or 154, relative to SEQ ID NO: 179, and the substitutions are optionally hydrophobic and are selected from alanine, valine, leucine, and isoleucine. In some embodiments, the consensus interferon variant comprises one or more mutations selected from R33A, R145X 1 , A146X 2 , M149A, R150A, and L154A, wherein X 1 is selected from A, S, T, Y, L, and I, and X 2 is selected from G, H, Y, K, and D.

[0209] In some embodiments, the consensus interferon is modified to have a mutation at amino acid position 121 (i.e., K121) relative to SEQ ID NO: 179. In certain embodiments, the consensus interferon comprises the K121E mutation relative to SEQ ID NO: 179.

[0210] In one embodiment, the modified signaling substance includes any consensus interferon variant disclosed in U.S. Patent Nos. 4,695,623, 4,897,471, 5,541,293, and 8,496,921. The entire contents of these patent documents are incorporated herein by reference. For example, the consensus interferon variant may include 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.

[0211] In some embodiments, the wild-type or modified signaling molecule is vascular endothelial growth factor (VEGF). VEGF plays an important role in both physiological and pathological angiogenesis, is a powerful growth factor that regulates vascular permeability and can act as a growth factor on cells expressing the VEGF receptor, and further functions include, in particular, stimulation of cell migration of macrophage lineages and endothelial cells. In addition to at least three receptors (VEGFR1, VEGFR2, and VEGFR3), there are several members of the VEGF growth factor family. Members of the VEGF family can bind and activate two or more types of VEGFR. For example, VEGF-A can bind VEGFR1 and VEGFR2, while VEGF-C can bind VEGFR2 and VEGFR3. VEGFR1 and VEGFR2 activation regulates angiogenesis, and VEGFR3 activation is involved in lymphangiogenesis. Most pro-angiogenic signals are generated from the activation of VEGFR2. It has been reported that VEGFR1 activation may be associated with a negative role in angiogenesis. It has also been reported that VEGFR1 signaling is important for in vivo progression through bone marrow-derived VEGFR1-positive cells in tumors (contributing to the formation of the pre-metastatic microenvironment in bone). Several therapies targeting / neutralizing VEGF-A-based therapeutic antibodies have been developed mainly for use in the treatment of various human tumors that depend on angiogenesis. However, these do not have no side effects. This is not surprising considering that they act as general non-cell / tissue-specific VEGFNEGFR interaction inhibitors. Therefore, it would be desirable to limit VEGF (e.g., VEGF-A)NEGFR-2 inhibition to specific target cells (e.g., tumor vascular endothelial cells).

[0212] In some embodiments, VEGF is these isoforms including VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E and various VEGF-A isoforms such as VEGF121, VEGF121b, VEGF145, VEGF165, VEGF165b, VEGF189, and VEGF206. In some embodiments, the modified signaling agent has a reduced affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In some embodiments, the modified signaling agent has a reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In one embodiment, the modified signaling agent has a reduced affinity and / or activity for VEGFR-2 (KDR / Flk-1) and / or a reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1). Such embodiments are used, for example, in a method of wound healing or treatment of an ischemia-related disease (not intended to be bound by theory, but mediated by the effect of VEGFR-2 on endothelial cell function and angiogenesis). In some embodiments, binding to VEGFR-1 (Flt-1) associated with cancer and pro-inflammatory activity is avoided. In some embodiments, VEGFR-1 (Flt-1) functions as a decoy receptor, thus substantially reducing or eliminating the affinity for this receptor and avoiding sequestration of the therapeutic agent. In one embodiment, the modified signaling agent has a reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or a reduced or eliminated affinity and / or activity for VEGFR-2 (KDR / Flk-1). In some embodiments, VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for VEGFR3. Alternatively, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for VEGFR3.

[0213] Therapies for promoting angiogenesis are also important in various diseases (e.g., ischemic heart disease, bleeding, etc.) and include VEGF-based therapeutic agents. Activation of VEGFR2 is angiogenic (acting on endothelial cells). VEGFR1 can cause stimulation of the migration of inflammatory cells (including, for example, macrophages) and lead to inflammation associated with vascular hyperpermeability. Activation of VEGFR1 can also activate bone marrow associated with tumor microenvironment formation. Therefore, VEGF-based therapeutic agents that are selective for VEGFR2 activation would be desirable in this case. Further, for example, cells that specifically target endothelial cells would be desirable.

[0214] In some embodiments, the modified signaling agent has a reduced affinity and / or activity (e.g., antagonistic nature) for VEGFR-2 and / or a substantially reduced or eliminated affinity and / or activity for VEGFR-1. When targeting tumor vasculature endothelial cells via a targeting moiety that binds to a tumor endothelial cell marker (e.g., PSMA, etc.), such constructs specifically inhibit VEGFR2 activation on such marker-positive cells, but do not activate VEGFR1 en route to and on the target cells (when the activity is eliminated), thus, for example, eliminating the induction of an inflammatory response. This would provide a more selective and safer anti-angiogenic pharmacotherapy for many tumor types compared to VEGF-A neutralizing therapy.

[0215] In some embodiments, the modified signaling agent has a reduced affinity and / or activity (e.g., agonist activity) for VEGFR-2 and / or a substantially reduced or eliminated affinity and / or activity for VEGFR-1. By targeting vascular endothelial cells, in some embodiments, such constructs promote angiogenesis without causing the induction of an inflammatory response associated with VEGFR1. Therefore, such constructs would have a targeted angiogenesis-promoting effect with a substantially reduced risk of side effects resulting from systemic activation of both VEGFR-2 and VEGFR-1.

[0216] In one exemplary embodiment, the wild-type or modified signaling substance is VEGF165 (wild-type) having the amino acids of SEQ ID NO: 180.

[0217] In another exemplary embodiment, the wild-type or modified signaling substance is VEGF165b (wild-type) having the amino acids of SEQ ID NO: 181.

[0218] In these embodiments, the modified signaling substance has a mutation at amino acid 183 (e.g., a substitution mutation at 183, e.g., 183K, 183R, or 183H). While not intending to be bound by theory, such mutations are thought to result in reduced receptor binding affinity. See, e.g., U.S. Patent No. 9,078,860, the entire contents of which are incorporated herein by reference.

[0219] In one embodiment, the wild-type or modified signaling substance is TNFα. TNF is a pleiotropic cytokine with many diverse functions, including the regulation of cell proliferation, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and trafficking, inflammation, and septic shock. It binds to two separate membrane receptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits a very broad expression pattern, while TNFR2 is selectively expressed on specific populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. In response to receptor activation, entirely separate biological pathways are activated, although there is also some overlap. As a general principle, while not wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), and TNFR2 signaling is associated with the activation of cell survival signals (e.g., activation of the NFκB pathway).

[0220] Administration of TNF is a systemic toxicity, which is mainly due to the involvement of TNFR1. However, it should also be noted that activation of TNFR2 is also associated with diverse effects, similar to TNFR1, and control of TNF targeting and activity is important in the development of TNF-based therapeutic agents.

[0221] In some embodiments, the modified signaling substance has a reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2. TNFR1 is expressed in most tissues and is involved in cell death signaling, whereas, in contrast, TNFR2 is involved in cell survival signaling. Thus, in embodiments related to cancer therapies, the modified signaling substance has a reduced affinity and / or activity for TNFR1 and / or a substantially reduced or eliminated affinity and / or activity for TNFR2. In these embodiments, the chimeric protein or Fc-based chimeric protein complex may target cells in which apoptosis is desirable, such as tumor cells or tumor vascular endothelial cells. For example, in embodiments related to methods of promoting cell survival in neurogenesis for the treatment of neurodegenerative disorders, the modified signaling substance has a reduced affinity and / or activity for TNFR2 and / or a substantially reduced or eliminated affinity and / or activity for TNFR1. In other words, the chimeric protein or Fc-based chimeric protein complex of the present invention, in some embodiments, comprises a modified TNFα substance that prioritizes either the death signal or the survival signal.

[0222] In some embodiments, the chimeric protein or Fc-based chimeric protein complex has a modified TNF with reduced affinity and / or activity for TNFR1 and / or substantially reduced or eliminated affinity and / or activity for TNFR2. Such chimeric or Fc-based chimeric protein complexes are, in some embodiments, more potent inducers of apoptosis compared to chimeric or Fc-based chimeric protein complexes having only mutations that result in reduced affinity and / or activity for wild-type TNF and / or TNFR1. Such chimeric or Fc-based chimeric protein complexes are, in some embodiments, used to induce tumor cell death or tumor vascular endothelial cell death (e.g., in the treatment of cancer). Also, in some embodiments, these chimeric or Fc-based chimeric protein complexes avoid or reduce the activation of Treg cells, for example, via TNFR2, and thus further assist TNFR1-mediated anti-tumor activity in vivo.

[0223] In some embodiments, the chimeric protein or Fc-based chimeric protein complex has a modified TNF with reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1. Such chimeric or Fc-based chimeric protein complexes are, in some embodiments, more potent activators of cell survival in some cell types, which can be a specific therapeutic goal in various diseases, including, but not limited to, stimulation of neurogenesis. Further, such TNFR2-selective chimeric or Fc-based chimeric protein complexes are also useful in the treatment of autoimmune diseases (e.g., Crohn's disease, diabetes, MS, colitis, etc., and many other diseases described herein). In some embodiments, the chimeric or Fc-based chimeric protein complex is directed to autoreactive T cells. In some embodiments, the chimeric or Fc-based chimeric protein complex promotes Treg cell activation and indirect suppression of cytotoxic T cells.

[0224] In some embodiments, the chimeric or Fc-based chimeric protein complex results in the death of autoreactive T cells, for example, 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 eliminated affinity and / or activity for TNFR1). Without wishing to be bound by theory, these autoreactive T cells have altered apoptosis / survival signals, for example, due to changes in NFκB pathway activity / signal transduction. In some embodiments, the chimeric or Fc-based chimeric protein complex results in the death of autoreactive T cells having damage or alterations in the NFκB pathway and, optionally, altered sensitivity to certain death-inducing signals (e.g., TNFR2 activation) underlying the imbalance in cell death (apoptosis) / survival signal properties.

[0225] In some embodiments, the TNFR2-based chimeric or Fc-based chimeric protein complex has additional therapeutic uses in various autoimmune diseases, particularly diseases including heart disease, demyelinating and neurodegenerative disorders, and infectious diseases.

[0226] In one embodiment, wild-type TNFα has the amino acid sequence of SEQ ID NO: 182.

[0227] In such embodiments, the modified TNFα substance has mutations at one or more of amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, which results in a modified TNFα having reduced receptor-binding affinity. See, for example, U.S. Patent No. 7,993,636. The entire content of this patent is incorporated herein by reference.

[0228] In some embodiments, the modified human TNFα moiety has a mutation at one or more of amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, A109, P113, Y115, E127, N137, D143, A145, and E146 as described in International Publication No. WO 2015 / 007903, the entire content of which is incorporated herein by reference (numbering according to the human TNF sequence, GenBank accession number BAG70306, version BAG70306.1 GI:197692685). In some embodiments, the modified human TNFα moiety has a substitution mutation selected from L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145R, A145T, E146D, E146K, and S147D. In one embodiment, the human TNFα moiety has a mutation selected from Y87Q, Y87L, Y87A, Y87F, and Y87H. In another embodiment, the human TNFα moiety has a mutation selected from I97A, I97Q, and I97S. In a further embodiment, the human TNFα moiety has a mutation selected from Y115A and Y115G. In one embodiment, the human TNFα moiety has the E146K mutation. In one embodiment, the human TNFα moiety has the Y87H and E146K mutations. In one embodiment, the human TNFα moiety has the Y87H and A145R mutations. In one embodiment, the human TNFα moiety has the R32W and S86T mutations. In one embodiment, the human TNFα moiety has the R32W and E146K mutations. In one embodiment, the human TNFα moiety has the L29S and R32W mutations. In one embodiment, the human TNFα moiety has the D143N and A145R mutations. In one embodiment, the human TNFα moiety has the D143N and A145R mutations. In one embodiment, the human TNFα moiety has the A145T, E146D, and S147D mutations.In certain embodiments, the human TNFα moiety has the A145T and S147D mutations.

[0229] In some embodiments, the modified TNFα substance comprises one or more mutations selected from N39Y, S147Y, and Y87H, as described in International Publication No. WO 2008 / 124086. The entire content of this patent is incorporated herein by reference.

[0230] In some embodiments, the modified human TNFα moiety has mutations that confer receptor selectivity as described in International Application No. PCT / IB2016 / 001668. The entire content of this patent is incorporated herein by reference. In some embodiments, the mutation to TNF is TNFR1-selective. In some embodiments, the mutation to TNF that is TNFR1-selective is to one or more of positions R32, S86, and E146. In some embodiments, the mutation to TNF that is TNFR1-selective is one or more of R32W, S86T, and E146K. In some embodiments, the mutation to TNF that is TNFR1-selective is one or more of R32W, R32W / S86T, R32W / E146K, and E146K. In some embodiments, the mutation to TNF is TNFR2-selective. In some embodiments, the mutation to TNF that is TNFR2-selective is to one or more of positions A145, E146, and S147. In some embodiments, the mutation to TNF that is TNFR2-selective is one or more of A145T, A145R, E146D, and S147D. In some embodiments, the mutation to TNF that is TNFR2-selective is one or more of A145R, A145T / S147D, and A145T / E146D / S147D.

[0231] In some embodiments, the wild-type or modified signaling molecule is TNFβ. TNFβ forms homotrimers or heterotrimers with LTβ (LTα1β2). In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2 and / or herpesvirus entry mediator (HEVM) and / or LTβR.

[0232] In one embodiment, wild-type TNFβ has the amino acid sequence of SEQ ID NO: 183.

[0233] In such embodiments, the modified soluble substance may contain mutations at one or more amino acid positions 106-113, which results in a modified TNFβ with reduced receptor binding affinity for TNFR2. In one embodiment, the modified soluble substance has one or more substitution mutations at amino acid positions 106-113. In some embodiments, the substitution mutations are selected from Q107E, Q107D, S106E, S106D, Q107R, Q107N, Q107E / S106E, Q107E / S106D, Q107D / S106E, and Q107D / S106D. In another embodiment, the modified soluble substance has an insertion of about 1 to about 3 amino acids at positions 106-113.

[0234] In some embodiments, the modified substance is a TNF family member (e.g., TNFα, TNFβ), which can be in the form of a single-chain trimer as described in WO 2015 / 007903. The entire content of this patent is incorporated herein by reference.

[0235] In some embodiments, the modifying substance is a TNF family member (e.g., TNFα, TNFβ), which has a reduced affinity and / or activity for TNFR1, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are incorporated herein by reference). In these embodiments, the modifying substance is a TNF family member (e.g., TNFα, TNFβ), which also optionally has a substantially reduced or eliminated affinity and / or activity for TNFR2. In some embodiments, the modifying substance is a TNF family member (e.g., TNFα, TNFβ), which has a reduced affinity and / or activity for TNFR2, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of this patent are incorporated herein by reference). In these embodiments, the modifying substance is a TNF family member (e.g., TNFα, TNFβ), which similarly optionally has a substantially reduced or eliminated affinity and / or activity for TNFR1. Constructs of such embodiments are used, for example, in methods of suppressing TNF responses in a cell-specific manner. In some embodiments, the antagonist TNF family member (e.g., TNFα, TNFβ) is of the single-chain trimer type as described in WO 2015 / 007903.

[0236] In some embodiments, the wild-type or modified signaling substance is TRAIL. In some embodiments, the modified TRAIL substance has a reduced affinity and / or activity for DR4 (TRAIL-RI) and / or DR5 (TRAIL-RII) and / or DcR1 and / or DcR2. In some embodiments, the modified TRAIL substance has a reduced affinity and / or activity for DR4 (TRAIL-RI) and / or DR5 (TRAIL-RII) and / or DcR1 and / or DcR2.

[0237] In one embodiment, wild-type TRAIL has the amino acid sequence of SEQ ID NO: 184.

[0238] In such embodiments, the modified TRAIL substance may contain mutations at amino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209, T214-1220, K224-A226, W231, E236-L239, E249-K251, T261-H264, and H270-E271 (numbering based on the GenBank accession number NP_003801, version 10NP_003801.1, GI: 4507593, human sequence; see above).

[0239] In some embodiments, the modified TRAIL substance may contain one or more mutations that substantially reduce its affinity and / or activity for TRAIL-R1. In such embodiments, the modified TRAIL substance may specifically bind to TRAIL-R2. Examples of mutations include mutations at one or more of amino acid positions Y189, R191, Q193, H264, 1266, and D267. For example, the mutations can be one or more of Y189Q, R191K, Q193R, H264R, 1266L, and D267Q. In one embodiment, the modified TRAIL substance contains the mutations Y189Q, R191K, Q193R, H264R, 1266L, and D267Q.

[0240] In some embodiments, the modified TRAIL substance may include one or more mutations that substantially reduce its affinity and / or activity for TRAIL-R2. In such embodiments, the modified TRAIL substance may specifically bind to TRAIL-R1. Examples of mutations include mutations at one or more of amino acid positions G131, R149, S159, N199, K201, and S215. For example, the mutations may be one or more of G131R, R149I, S159R, N199R, K201H, and S215D. In certain embodiments, the modified TRAIL substance includes the mutations G131R, R149I, S159R, N199R, K201H, and S215D. Additional TRAIL mutations are described, for example, in Trebing et al., (2014) Cell Death and Disease, 5: e1035. The entire disclosures of these are incorporated herein by reference.

[0241] In some embodiments, the wild-type or modified signaling substance is TGFα. In such embodiments, the modified TGFα substance has a reduced affinity and / or activity for the epidermal growth factor receptor (EGFR). In some embodiments, the modified TGFα substance has a substantially reduced or eliminated affinity and / or activity for the epidermal growth factor receptor (EGFR).

[0242] In some embodiments, the wild-type or modified signaling molecule is TGFβ. In such embodiments, the modified signaling molecule has a reduced affinity for TGFBR1 and / or TGFBR2. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for TGFBR1 and / or TGFBR2. In some embodiments, the modified signaling molecule optionally has a substantially reduced or eliminated affinity and / or activity for TGFBR3, which, without intending to be bound by theory, may act as a reservoir of ligand for the TGF beta receptor. In some embodiments, TGFβ preferentially binds TGFBR1 over TGFBR2 or TGFBR2 over TGFBR1. Similarly, without wishing to be bound by theory, LAP may act as a reservoir of ligand for the TGF beta receptor. In some embodiments, the modified signaling molecule has a reduced affinity and / or activity for TGFBR1 and / or TGFBR2 and a substantially reduced or eliminated affinity and / or activity for latent associated peptide (LAP). In some embodiments, such chimeric or Fc-based chimeric protein complexes are used in camurati-engelmann disease or other diseases associated with inappropriate TGFβ signaling.

[0243] In some embodiments, the modifying substance is a TGF family member (e.g., TGFα, TGFβ), which has a reduced affinity and / or activity for one or more of TGFBR1, TGFBR2, TGFBR3, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520. The entire content of this patent is incorporated herein by reference). In these embodiments, the modifying substance is a TGF family member (e.g., TGFα, TGFβ), which also optionally has a substantially reduced or eliminated affinity and / or activity for one or more of TGFBR1, TGFBR2, TGFBR3.

[0244] In some embodiments, the modifying substance is a TGF family member (e.g., TGFα, TGFβ), which has a reduced affinity and / or activity for TGFBR1 and / or TGFBR2, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520. The entire content of this patent is incorporated herein by reference). In these embodiments, the modifying substance is a TGF family member (e.g., TGFα, TGFβ), which also optionally has a substantially reduced or eliminated affinity and / or activity for TGFBR3.

[0245] In some embodiments, the wild-type or modified signaling molecule is an interleukin. In certain embodiments, the wild-type or modified signaling molecule is IL1. In some embodiments, the wild-type or modified signaling molecule is IL1α or IL1β. In some embodiments, the modified signaling molecule has a reduced affinity and / or activity for IL1R1 and / or IL1RAcP. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for IL1R1 and / or IL1RAcP.

[0246] In some embodiments, the modified signaling molecule has a reduced affinity and / or activity for IL1R2. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for IL1R2. In some embodiments, the modified IL1β substance of the invention avoids interaction with IL1R2 and thus substantially reduces its function as a decoy and / or sink for therapeutic agents.

[0247] In certain embodiments, wild-type IL1β has the amino acid sequence of SEQ ID NO: 185.

[0248] IL1β is a pro-inflammatory cytokine and an important immune system regulator. It is a potent activator of CD4 T cell responses, increases the proportion of Th17 cells, and enhances the proliferation of IFNγ- and IL4-producing cells. IL1β is also a potent regulator of CD8+ T cells, enhancing antigen-specific CD8+ T cell proliferation, differentiation, migration to the periphery, and memory. The IL1β receptor includes IL1R1 and IL1R2. Binding to IL1R1 and signaling through IL1R1 constitute the mechanism by which IL1 mediates many of its biological (and pathological) effects. IL1R2 can function as a decoy receptor, thereby reducing the availability of IL1 for interaction and signaling through IL1R1.

[0249] In some embodiments, the modified IL1β has a reduced affinity and / or activity (e.g., agonist activity) for IL1R1. In some embodiments, the modified IL1 has a substantially reduced or eliminated affinity and / or activity for IL1R2. In such embodiments, there is recoverable IL1β / IL1R1 signaling as well as prevention of loss of the therapeutic chimera for ILR2 and as a result a reduction in the dosage of IL1β required (e.g., as compared to a chimera having only a wild-type or attenuated mutation for ILR1). Such constructs are used, for example, in methods of treating cancer, including, for example, stimulating the immune system to initiate an anti-cancer response.

[0250] In some embodiments, the modified IL1β has a reduced affinity and / or activity (e.g., antagonist activity, e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520. The entire content of this patent is incorporated herein by reference) for IL1R1. In some embodiments, the modified IL1β has a substantially reduced or eliminated affinity and / or activity for IL1R2. In such embodiments, the IL1β / IL1R1 signaling is not recoverable and there is also prevention of loss of the therapeutic chimera for ILR2 and as a result a reduction in the dosage of IL1β required (e.g., as compared to a chimera having only a wild-type or attenuated mutation for ILR1). Such constructs are used, for example, in methods of treating autoimmune diseases, including, for example, suppressing the immune system.

[0251] In such embodiments, the modified signaling substance has a deletion of amino acids 52-54, which produces a modified human IL1β having reduced binding affinity and reduced biological activity for type I IL1R. See, for example, International Publication No. WO 1994 / 000491. The entire content of this patent is incorporated herein by reference. In some embodiments, the modified human IL1β has one or more substitution mutations selected from A117G / P118G, R120X, L122A, T125G / L126G, R127G, Q130X, Q131G, K132A, S137G / Q138Y, L145G, H146X, L145A / L147A, Q148X, Q148G / Q150G, Q150G / D151A, M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209X, K209A / K210A, K219X, E221X, E221S / N224A, N224S / K225S, E244K, N245Q (where X can be any change in an amino acid, e.g., a non-conservative change), which show reduced binding to IL1R as described in, for example, International Publication No. WO 2015 / 007542 and International Publication No. WO 2015 / 007536, the entire content of which is incorporated herein by reference (numbering based on GenBank accession number NP_000567, version NP-000567.1, GI:10835145, human IL1β sequence). In some embodiments, the modified human IL1β can have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S, and E221K. In one embodiment, the modified human IL1β includes the mutations Q131G and Q148G. In one embodiment, the modified human IL1β includes the mutations Q148G and K208E. In one embodiment, the modified human IL1β includes the mutations R120G and Q131G. In one embodiment, the modified human IL1β includes the mutations R120G and H146A. In one embodiment, the modified human IL1β includes the mutations R120G and H146N.In certain embodiments, the modified human IL1β comprises the mutations R120G and H146R. In certain embodiments, the modified human IL1β comprises the mutations R120G and H146E. In certain embodiments, the modified human IL1β comprises the mutations R120G and H146G. In certain embodiments, the modified human IL1β comprises the mutations R120G and K208E. In certain embodiments, the modified human IL1β comprises the mutations R120G, F162A, and Q164E.

[0252] In some embodiments, the wild-type or modified signaling agent is IL2. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for IL2Rα and / or IL2Rβ and / or IL2Rγ. In some embodiments, the modified signaling agent has a reduced affinity and / or activity for IL2Rβ and / or IL2Rγ. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for IL2Rα. Such embodiments may be suitable for the treatment of cancer, for example when the modified IL2 is an agonist for IL2Rβ and / or IL2Rγ. For example, the constructs of the present invention prioritize the attenuated activation of CD8+ T cells (which can confer an anti-tumor effect) having IL2 receptor β and γ, and T cells having IL2 receptors α, β, and γ reg(Those that can impart an immunosuppressive effect and a tumor promoting effect) are not prioritized. Further, in some embodiments, the selectivity for IL2Rβ and / or IL2Rγ over IL2Rα avoids IL2 side effects such as pulmonary edema. Also, an IL2-based chimeric or Fc-based chimeric protein complex is useful for treating autoimmune diseases, for example, when the modified IL2 is an antagonist to IL2Rβ and / or IL2Rγ (for example, antagonist activity as a result of natural antagonist activity or one or more mutations, see, for example, WO 2015 / 007520. The entire content of this patent is incorporated herein by reference). For example, the constructs of the present invention prioritize the attenuation of the suppression of CD8+ T cells having IL2 receptor β and γ (thus suppressing the immune response) and do not prioritize Tregs having IL2 receptors α, β, and γ. Alternatively, in some embodiments, a chimeric or Fc-based chimeric protein complex having IL2 prioritizes the activation of Tregs and thus immunosuppression and does not prioritize the activation of CD8+ T cells. For example, these constructs are used in the treatment of diseases that are thought to benefit from disease or immunosuppression, such as autoimmune disorders.

[0253] In some embodiments, the chimeric protein or Fc-based chimeric protein complex has a targeting moiety described herein directed to FAP+ dendritic cells, and a modified IL2 substance having a reduced affinity and / or activity for IL2Rβ and / or IL2Rγ and / or a substantially reduced or eliminated affinity and / or activity for IL2Rα. In some embodiments, these constructs provide targeted FAP+ dendritic cell activity and are normally inactive (or have substantially reduced activity) against Treg cells. In some embodiments, such constructs have an enhanced immunostimulatory effect compared to wild-type IL2 (without wishing to be bound by theory, by not stimulating Tregs), while removing or reducing the systemic toxicity associated with IL2.

[0254] In some embodiments, wild-type IL2 has the amino acid sequence of SEQ ID NO: 186.

[0255] In such embodiments, the modified IL2 substance has one or more mutations at the position of amino acid L72 (L72G, L72A, L725, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), at the position of F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K) and at the position of Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R or Y45K). Without wishing to be bound by theory, these modified IL2 substances are thought to have a reduced affinity for the high-affinity IL2 receptor and to maintain their affinity for the intermediate-affinity IL2 receptor as compared to wild-type IL2. See, for example, U.S. Patent Application Publication No. 2012 / 0244112. The entire content of this patent is incorporated herein by reference.

[0256] In some embodiments, the modified IL-2 substance has one or more mutations at amino acid positions R38, F42, Y45, and E62. For example, the modified IL-2 substance may comprise one or more of R38A, F42A, Y45A, and E62A. In some embodiments, the modified IL2 substance may comprise a mutation at C125. For example, the mutation may be C125S. In such embodiments, the modified IL2 substance may have substantially reduced affinity and / or activity for IL2Rα, as described, for example, in Carmenate et al. (2013) The Journal of Immunology, 190:6230-6238. The entire disclosure of this document is incorporated herein by reference. In some embodiments, a modified IL2 substance having a mutation at R38, F42, Y45, and / or E62 can induce the proliferation of effector cells, including CD8+ T cells and NK cells, but not Treg cells. In some embodiments, a modified IL2 substance having a mutation at R38, F42, Y45, and / or E62 has less toxicity than the wild-type IL2 substance. Chimeric proteins or Fc-based chimeric protein complexes comprising a modified IL2 substance having substantially reduced affinity and / or activity for IL2Rα may find use, for example, in oncology.

[0257] In other embodiments, the modified IL2 substance may have substantially reduced affinity and / or activity for IL-2Rβ, for example, as described in International Publication No. WO 2016 / 025385. The entire disclosure of this patent is incorporated herein by reference. In such embodiments, the modified IL-2 substance may induce the proliferation of Treg cells, but not the proliferation of effector cells such as CD8+ T cells and NK cells. Chimeric proteins or Fc-based chimeric protein complexes comprising a modified IL2 substance having substantially reduced affinity and / or activity for IL-2Rβ may find use, for example, in the treatment of autoimmune diseases. In some embodiments, the modified IL2 substance may have one or more mutations at amino acid positions N88, D20, and / or A126. For example, the modified IL2 substance may comprise one or more of N88R, N88I, N88G, D20H, Q126L, and Q126F.

[0258] In some embodiments, the modified IL2 substance may contain a mutation at D109 or C125. For example, the mutation may be D109C or C125S. In some embodiments, the modified IL-2 having a mutation at D109 or C125 may be utilized for attachment to a PEG moiety.

[0259] In some embodiments, the wild-type or modified signaling substance is IL3. In some embodiments, the modified signaling substance has reduced affinity and / or activity for the IL3 receptor, which is a heterodimer having a specific alpha chain paired with a common beta (betac or CD131) subunit. In some embodiments, the modified signaling substance has substantially reduced or eliminated affinity and / or activity for the IL3 receptor, which is a heterodimer having a specific alpha chain paired with a common beta (betac or CD131) subunit.

[0260] In some embodiments, the wild-type or modified signaling molecule is IL4. In such embodiments, the modified signaling molecule has a reduced affinity and / or activity for the type 1 and / or type 2 IL4 receptors. In such embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for the type 1 and / or type 2 IL4 receptors. The type 1 IL4 receptor is composed of an IL4Rα subunit having a common γ chain and specifically binds IL4. The type 2 IL4 receptor includes an IL4Rα subunit bound to a different subunit known as IL13Rα1. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for the type 2 IL4 receptor.

[0261] In some embodiments, wild-type IL4 has the amino acid sequence of SEQ ID NO: 187.

[0262] In such embodiments, the modified IL4 substance has one or more mutations at amino acids R121 (R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W), E122 (E122F), Y124 (Y124A, Y124Q, Y124R, Y124S, Y124T) and S125 (S125A). Without wishing to be bound by theory, it is believed that these modified IL4 substances maintain activity mediated by the type I receptor but significantly reduce the biological activity mediated by other receptors. See, for example, U.S. Patent No. 6,433,157. The entire content of this patent is incorporated herein by reference.

[0263] In some embodiments, the wild-type or modified signaling molecule is IL6. IL6 signals through a cell surface type I cytokine receptor complex that includes a ligand-binding IL6R chain (CD126) and a signaling component gp130. IL6 can also bind to a soluble form of IL6R (sIL6R), which is the extracellular portion of the IL6R. The sIL6R / IL6 complex is involved in neurite outgrowth and neuron survival and can thus be important for nerve regeneration by remyelination. Thus, in some embodiments, the modified signaling molecule has a reduced affinity and / or activity for IL6R / gp130 and / or sIL6R. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for IL6R / gp130 and / or sIL6R.

[0264] In some embodiments, wild-type IL6 has the amino acid sequence of SEQ ID NO: 188.

[0265] In such embodiments, the modified signaling molecule has one or more mutations at amino acids 58, 160, 163, 171 or 177. Without wishing to be bound by theory, these modified IL6 molecules are thought to exhibit reduced binding affinity and reduced biological activity for IL6Rα. See, for example, WO 97 / 10338. The entire contents of this patent are incorporated herein by reference.

[0266] In some embodiments, the wild-type or modified signaling molecule is IL10. In such embodiments, the modified signaling molecule has a reduced affinity and / or activity for IL10 receptor 1 and IL10 receptor 2. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for IL10 receptor 1 and IL10 receptor 2.

[0267] In some embodiments, the wild-type or modified signaling agent is IL11. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for IL11Rα and / or IL11Rβ and / or gp130. In such embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130.

[0268] In some embodiments, the wild-type or modified signaling agent is IL12. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for IL12Rβ1 and / or IL12Rβ2. In such embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for IL12Rβ1 and / or IL12Rβ2.

[0269] In some embodiments, the wild-type or modified signaling agent is IL13. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for the IL4 receptor (IL4Rα) and IL13Rα1. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for the IL4 receptor (IL4Rα) or IL13Rα1.

[0270] In some embodiments, wild-type IL13 has the amino acid sequence of SEQ ID NO: 189.

[0271] In such embodiments, the modified IL13 substance has one or more mutations at amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113, and 114. Without wishing to be bound by theory, these modified IL13 substances are thought to exhibit reduced biological activity. See, for example, International Publication No. WO 2002 / 018422. The entire contents of this patent are incorporated herein by reference.

[0272] In certain embodiments, the signaling substance is wild-type or modified IL15. In some embodiments, the modified IL15 has a reduced affinity and / or activity for the interleukin-15 receptor.

[0273] In certain embodiments, wild-type IL15 has the amino acid sequence of nwvnvisdlkkiedliqsmhidatlytesdvhpsckvtamkcfllelqvislesgdasihdtvenliilannslssngnvtesgckeceeleeknikeflqsfvhivqmfints (SEQ ID NO: 1044).

[0274] In such embodiments, the modified IL15 substance has one or more mutations at amino acids S7, D8, K10, K11, E46, L47, V49, I50, D61, N65, L66, I67, I68, L69, N72, Q108 relative to SEQ ID NO: 1044.

[0275] In some embodiments, the wild-type or modified signaling molecule is IL18. In some embodiments, the modified signaling molecule has a reduced affinity and / or activity for IL18Rα and / or IL18Rβ. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for IL18Rα and / or IL18Rβ. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for IL18Rα type II, an isoform of IL18Rα that lacks the TIR domain required for signaling.

[0276] In some embodiments, wild-type IL18 has the amino acid sequence of SEQ ID NO: 190.

[0277] In such embodiments, the modified IL18 substance may contain one or more mutations in an amino acid or amino acid region selected from Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N27-K129, Q139-M149, K165-K171, R183, and Q190-N191, as described in International Publication No. WO 2015 / 007542, the entire content of which is incorporated herein by reference (numbering based on the GenBank accession number AAV38697, version AAV38697.1, GI: 54696650, human IL18 sequence).

[0278] In some embodiments, the wild-type or modified signaling molecule is IL33. In such embodiments, the modified signaling molecule has a reduced affinity and / or activity for ST2 receptor 1 and IL1RAcP. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for the ST2 receptor and IL-1RAcP.

[0279] In some embodiments, wild-type IL33 has the amino acid sequence of SEQ ID NO: 191.

[0280] In such embodiments, the modified IL-33 substance may contain one or more mutations in amino acids or amino acid regions selected from 1113 - Y122, 5127 - E139, E144 - D157, Y163 - M183, E200, Q215, L220 - C227, and T260 - E269, as described in International Publication No. WO 2015 / 007542, the entire content of which is incorporated herein by reference (numbering based on the human sequence, GenBank accession number NP_254274, version NP254274.1, GI:15559209).

[0281] In some embodiments, the wild-type or modified signaling substance is epidermal growth factor (EGF). EGF is a family of highly potent growth factors. Members include EGF, HB-EGF, and TGFα, amphiregulin, neuregulin, epiregulin, betacellulin, and others. EGF family receptors include EGFR (ErbB1), ErbB2, ErbB3, and ErbB4. These can function as homodimer and / or heterodimer receptor subtypes. Different EGF family members exhibit different selectivities for different receptor subtypes. For example, EGF binds to ErbB1 / ErbB1, ErbB1 / ErbB2, ErbB4 / ErbB2, and several other heterodimer subtypes. HB-EGF has a similar pattern but binds to ErbB4 / 4. Regulation of EGF (EGF-like) growth factor signaling in the positive or negative direction is of interest from a major therapeutic perspective. For example, inhibition of EGFR signaling is of interest in the treatment of various cancers where EGFR signaling constitutes a major growth-promoting signal. Alternatively, stimulation of EGFR signaling is of interest from a therapeutic perspective, for example, in wound healing (acute and chronic), oral mucositis (a major side effect of various cancer therapies, including but not limited to radiation therapy).

[0282] In some embodiments, the modified signaling molecule has a reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4. Such embodiments are used, for example, in methods of treating wounds. In some embodiments, the modified signaling molecule binds to one or more of ErbB1, ErbB2, ErbB3, and ErbB4 and antagonizes the activity of the receptor. In such embodiments, the modified signaling molecule has a reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4 that allows it to be antagonized in a manner in which the activity of the receptor is attenuated. Such embodiments are used, for example, in the treatment of cancer. In one embodiment, the modified signaling molecule has a reduced affinity and / or activity for ErbB1. ErbB1 is a therapeutic target for kinase inhibitors - but in most cases, they have side effects because they are not very selective (e.g., gefitinib, erlotinib, afatinib, brigatinib, and icotinib). In some embodiments, the attenuated antagonistic ErbB1 signaling is more on-target and has fewer side effects than other substances that target the EGF receptor.

[0283] In some embodiments, the modified signaling agent has a reduced affinity and / or activity for ErbB1 (e.g., antagonist activity, e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, e.g., see International Publication No. WO 2015 / 007520, the entire contents of which are incorporated herein by reference) and / or a substantially reduced or eliminated affinity and / or activity for ErbB4 or other subtypes with which it can interact. Specific targeting via the targeting moiety results in cell-selective inhibition (antagonism, e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, e.g., see International Publication No. WO 2015 / 007520, the entire contents of which are incorporated herein by reference) of ErbB1 / ErbB1 receptor activation, while not involving other receptor subtypes that may be associated with inhibitory-related side effects. Thus, in contrast to EGFR kinase inhibitors that inhibit EGFR activity in all cell types in the body, such constructs will provide a cell-selective (e.g., tumor cells having activated EGFR signaling due to receptor amplification, overexpression, etc.) anti-EGFR (ErbB1) drug action with reduced side effects.

[0284] In some embodiments, the modified signaling agent has a reduced affinity and / or activity (e.g., agonist activity) for ErbB4 and / or other subtypes with which it interacts. Targeting specific target cells via the targeting moiety results in selective activation of ErbB1 signaling (e.g., epithelial cells). Such constructs are used, in some embodiments, for the treatment of wounds with reduced side effects (promotion of wound healing), particularly for the treatment of chronic conditions and applications other than topical administration of therapeutic agents (e.g., systemic wound healing).

[0285] In some embodiments, the wild-type or modified signaling agent is insulin or an insulin analog. In some embodiments, the modified insulin or insulin analog has a reduced affinity and / or activity for the insulin receptor and / or the IGF1 or IGF2 receptor. In some embodiments, the modified insulin or insulin analog has a reduced or eliminated affinity and / or activity for the insulin receptor and / or the IGF1 or IGF2 receptor. The attenuated response to the insulin receptor enables control of diabetes, obesity, metabolic disorders, etc., while avoiding cancer-promoting effects by redirecting away from the IGF1 or IGF2 receptor.

[0286] In some embodiments, the wild-type or modified signaling molecule is insulin-like growth factor-I or insulin-like growth factor-II (IGF1 or IGF2). In some embodiments, the modified signaling molecule is IGF1. In such embodiments, the modified signaling molecule has a reduced affinity and / or activity for the insulin receptor and / or the IGF1 receptor. In some embodiments, the modified signaling molecule binds to the IGF1 receptor and antagonizes the activity of the receptor. In such embodiments, the modified signaling molecule has a reduced affinity and / or activity for the IGF1 receptor, thereby allowing it to be antagonized in a form that weakens the activity of the receptor. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for the IGF1 receptor. In some embodiments, the modified signaling molecule has a reduced affinity and / or activity for the IGF2 receptor, thereby allowing it to be antagonized in a form that weakens the activity of the receptor. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for the insulin receptor and thus does not interfere with insulin signaling. In some embodiments, this is applied to cancer treatment. In some embodiments, the substance can prevent the IR isoform A from developing resistance to cancer treatment.

[0287] In certain embodiments, the wild-type or modified signaling agent is EPO. In various embodiments, the modified EPO substance has a reduced affinity and / or activity for the EPO receptor (EPOR) and / or the ephrin receptor (EphR) compared to wild-type EPO or other EPO-based substances described herein. In some embodiments, the modified EPO substance has a substantially reduced or eliminated affinity and / or activity for the EPO receptor (EPOR) and / or the Eph receptor (EphR). Examples of EPO receptors include, but are not limited to, EPOR homodimers or EPOR / CD131 heterodimers. Also included in the EPO receptor is the beta common receptor (βcR). Examples of Eph receptors include, but are not limited to, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6. In some embodiments, the modified EPO protein comprises one or more mutations that confer a reduced affinity for the EPO protein for one or more different EPO or Eph receptors (e.g., but not limited to, heterodimers, heterotrimers, etc. including EPOR-EPHB4, EPOR-βcR-EPOR). Also provided are receptors of EP Patent Publication No. 2492355, including, but not limited to, NEPOR (the entire contents of this patent are incorporated herein by reference).

[0288] The structure of the human EPO protein is predicted to include a four-helix bundle containing helices A, B, C, and D. In various embodiments, the modified EPO includes one or more mutations in four regions of the EPO protein that are important for biological activity, namely, amino acid residues 10-20, 44-51, 96-108, and 142-156. In some embodiments, the one or more mutations are located at residues 11-15, 44-51, 100-108, and 147-151. These residues are localized in helix A (Val11, Arg14, and Tyr15), helix C (Ser100, Arg103, Ser104, and Leu108), helix D (Asn147, Arg150, Gly151, and Leu155), and the A / B connecting loop (residues 42-51). In some embodiments, the modified EPO protein includes mutations in the residues of amino acids 41-52 as well as the residues of amino acids 147, 150, 151, and 155. Without wishing to be bound by theory, mutations in these residues are thought to have a substantial impact on both receptor binding and in vitro biological activity. In some embodiments, the modified EPO protein includes mutations at residues 11, 14, 15, 100, 103, 104, and 108. Without wishing to be bound by theory, mutations in these residues are thought to have a moderate impact on receptor binding activity and a much greater impact on in vitro biological activity. Examples of substitutions include, but are not limited to, one or more of Val11Ser, Arg14Ala, Arg14Gln, Tyr15Ile, Pro42Asn, Thr44Ile, Lys45Asp, Val46Ala, Tyr51Phe, Ser100Glu, Ser100Thr, Arg103Ala, Ser104Ile, Ser104Ala, Leu108Lys, Asn147Lys, Arg150Ala, Gly151Ala, and Leu155Ala.

[0289] In some embodiments, the modified EPO protein includes mutations that affect biological activity but not binding, such as those described in Eliot, et al. Mapping of the Active Site of Recombinant Human Erythropoietin January 15, 1997; Blood: 89(2). The entire content of this reference is incorporated herein by reference.

[0290] In some embodiments, the modified EPO protein includes one or more mutations that include surface residues of the EPO protein involved in receptor contact. Without wishing to be bound by theory, mutations of these surface residues may have little effect on protein folding and are therefore thought to retain some biological activity. Examples of surface residues into which mutations can be introduced include, but are not limited to, residues 147 and 150. In an exemplary embodiment, the mutation is a substitution that includes one or more of N147A, N147K, R150A, and R150E.

[0291] In some embodiments, the modified EPO protein includes one or more mutations at residues N59, E62, L67, and L70, and one or more mutations that affect disulfide bond formation. Without wishing to be bound by theory, these modifications are predicted to affect folding and / or be in buried positions and are therefore thought to indirectly affect biological activity.

[0292] In one embodiment, the modified EPO protein includes the K20E substitution that greatly reduces receptor binding. See, for example, Elliott, et al., (1997) Blood, 89:493 - 502. The entire content of this reference is incorporated herein by reference.

[0293] Additional EPO mutations that can be incorporated into the chimeric EPO proteins of the present invention are disclosed, for example, in Elliott, et al., (1997) Blood, 89:493-502 and Taylor et al., (2010) PEDS, 23(4):251-260. The entire contents of these references are incorporated herein by reference.

[0294] In one embodiment, the chimeric protein or Fc-based chimeric protein complex of the present invention has (i) a targeting moiety for FAP and (ii) a targeting moiety directed to tumor cells, together with any of the wild-type or modified or mutant signaling agents described herein. In certain embodiments, the chimeric protein or Fc-based chimeric protein complex of the present invention has a targeting moiety for FAP on dendritic cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

[0295] In one embodiment, the chimeric protein or Fc-based chimeric protein complex of the present invention has (i) a targeting moiety for FAP and (ii) a targeting moiety directed to a checkpoint inhibitor marker, together with any of the wild-type or modified or mutant interferons described herein. In certain embodiments, the chimeric protein or Fc-based chimeric protein complex of the present invention has a targeting moiety for FAP on dendritic cells and a second targeting moiety for PD-1.

[0296] In some embodiments, the signaling molecule is a toxin or a toxic enzyme. In some embodiments, the toxin or toxic enzyme is derived from plants and bacteria. Examples of toxins or toxic enzymes include, but are not limited to, diphtheria toxin, Pseudomonas toxin, anthrax toxin, ribosome-inactivating proteins (RIPs) such as ricin and saporin, modeccin, abrin, gelonin, and pokeweed antiviral protein. Additional toxins include those disclosed in Mathew et al., (2009) Cancer Sci 100(8):1359-65. The entire disclosure of this document is incorporated herein by reference. In such embodiments, the chimeric protein or Fc-based chimeric protein complex of the present technology can be utilized to induce cell death in a cell-type specific manner. In such embodiments, the toxin can be modified, for example, by mutagenesis, to reduce the affinity and / or activity of the toxin in order to attenuate the effect, as described herein for other signaling molecules.

[0297] Fc domain The fragment crystallizable domain (Fc domain) is the tail region of an antibody that interacts with Fc receptors located on the cell surface of cells involved in the immune system, such as B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. In IgG, IgA, and IgD antibody isotypes, the Fc domain is composed of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody. In IgM and IgE antibody isotypes, the Fc domain contains three heavy chain constant domains (C H domains 2-4) in each polypeptide chain.

[0298] In some embodiments, the Fc-based chimeric protein complex of the present technology includes an Fc domain. In some embodiments, the Fc domain is selected from IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from IgG1, IgG2, IgG3, or IgG4.

[0299] In some embodiments, the Fc domain is selected from human IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from human IgG1, IgG2, IgG3, or IgG4.

[0300] In some embodiments, the Fc domain of the Fc-based chimeric protein complex comprises the CH2 and CH3 regions of IgG. In some embodiments, the IgG is human IgG. In some embodiments, the human IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0301] In some embodiments, the Fc domain comprises one or more mutations. In some embodiments, the mutations to the Fc domain reduce or eliminate the effector function of the Fc domain. In some embodiments, the mutant Fc domain has a reduced affinity or binding to the target receptor. For example, in some embodiments, the mutations to the Fc domain reduce or eliminate the binding of the Fc domain to FcγR. In some embodiments, the FcγR is selected from FcγRI; FcγRIIa, 131R / R; FcγRIIa, 131H / H, FcγRIIb; and FcγRIII. In some embodiments, the mutations to the Fc domain reduce or eliminate the binding to complement proteins such as, for example, C1q. In some embodiments, the mutations to the Fc domain reduce or eliminate the binding to both FcγR and complement proteins such as, for example, C1q.

[0302] In some embodiments, the Fc domain comprises the LALA mutation, which reduces or eliminates the effector function of the Fc domain. For example, in some embodiments, the LALA mutation comprises the L234A and L235A substitutions in human IgG (e.g., IgG1) (numbering is based on the numbering of the CH2 residues commonly used for human IgG1 according to the EU rules (PNAS, Edelman et al., 1969; 63(1)78-85)).

[0303] In some embodiments, the Fc domain of human IgG contains mutations at one or more positions of L234, L235, K322, D265, P329, and P331, reducing or eliminating the effector function of the Fc domain. For example, in some embodiments, the mutations are selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G, and P331S.

[0304] In some embodiments, the Fc domain contains the FALA mutation, reducing or eliminating the effector function of the Fc domain. For example, in some embodiments, the FALA mutation includes the F234A and L235A substitutions in human IgG4.

[0305] In some embodiments, the Fc domain of human IgG4 contains mutations at one or more positions of F234, L235, K322, D265, and P329, reducing or eliminating the effector function of the Fc domain. For example, in some embodiments, the mutations are selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.

[0306] In some embodiments, the mutation to the Fc domain stabilizes the hinge region of the Fc domain. For example, in some embodiments, the Fc domain contains a mutation at the position of S228 of human IgG, stabilizing the hinge region. In some embodiments, the mutation is S228P.

[0307] In some embodiments, the mutation to the Fc domain promotes the chain pairing of the Fc domain. In some embodiments, the chain pairing is promoted by ion pair formation (also known as charge pair, ionic bond, or charged residue pair).

[0308] In some embodiments, the Fc domain comprises mutations at the positions of the following additional IgG amino acid residues to promote ion pair formation: D356, E357, L368, K370, K392, D399, and K409.

[0309] For example, in some embodiments, the human IgG Fc domain comprises one of the combinations of mutations in Table 1 to promote ion pair formation.

Table 1

[0310] In some embodiments, chain pairing is promoted by knob-in-hole mutations. In some embodiments, the Fc domain comprises one or more mutations that enable knob-in-hole interactions in the Fc domain. In some embodiments, the first Fc chain is modified to express a "knob" and the second Fc chain is modified to express a complementary "hole". For example, in some embodiments, the human IgG Fc domain comprises the mutations in Table 2 to enable knob-in-hole interactions.

Table 2

[0311] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of the present technology comprises any combination of the mutations disclosed above. For example, in some embodiments, the Fc domain comprises mutations that promote ion pair formation and / or knob-in-hole interactions. For example, in some embodiments, the Fc domain comprises mutations having one or more of the following properties: promoting ion pair formation, inducing knob-in-hole interactions, reducing or eliminating effector functions of the Fc domain, and providing Fc stabilization (e.g., hinge).

[0312] For example, in some embodiments, the human IgG Fc domain comprises the mutations disclosed in Table 3, which promote ion pair formation in the Fc domain and / or promote knob-in-hole interactions. [Table 3] TIFF2025084801000010.tif48162

[0313] For example, in some embodiments, the human IgG Fc domain comprises the mutations disclosed in Table 4, which promote ion pair formation of the Fc domain and / or promote knob-in-hole interactions, or combinations thereof. In some embodiments, "Chain 1" and "Chain 2" of Table 4 are interchangeable (e.g., Chain 1 can have Y407T and Chain 2 can have T366Y). [Table 4] TIFF2025084801000012.tif238160TIFF2025084801000013.tif238160TIFF2025084801000014.tif194162

[0314] For example, in some embodiments, the human IgG Fc domain comprises the mutations disclosed in Table 5, which reduce or eliminate FcγR and / or complement binding in the Fc domain. In some embodiments, the mutations in Table 5 are present in both chains. [Table 5] TIFF2025084801000016.tif239161TIFF2025084801000017.tif186162

[0315] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of the present technology is a homodimer, i.e., the Fc domain in the chimeric protein complex comprises two identical protein fragments.

[0316] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of the present technology is a heterodimer, i.e., the Fc domain comprises two non-identical protein fragments.

[0317] In some embodiments, the heterodimeric Fc domain is modified using the ion pair formation and / or knob-in-hole mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation / structure. In the trans orientation / structure, the targeting moiety and the signaling agent are, in some embodiments, not found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention.

[0318] In some embodiments, the heterodimeric Fc domain is modified using the ion pair formation and / or knob-in-hole mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation.

[0319] In the trans orientation, the targeting moiety and the signaling agent are, in some embodiments, not found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention. In the trans orientation, the targeting moiety and the signaling agent are, in some embodiments, found on separate polypeptide chains in the Fc-based chimeric protein complex of the present invention. In the cis orientation, the targeting moiety and the signaling agent are, in some embodiments, found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention.

[0320] In some embodiments where two or more targeting moieties are present in the heterodimeric protein complex described herein, one targeting moiety may be present in a trans orientation (with respect to the signaling substance), while another targeting moiety may be present in a cis orientation (with respect to the signaling substance). In some embodiments, the signaling substance and the targeting moiety are present on the same end / side (N-terminus or C-terminus) of the Fc domain. In some embodiments, the signaling substance and the targeting moiety are present on different sides / ends (N-terminus or C-terminus) of the Fc domain.

[0321] In some embodiments where two or more targeting moieties are present in the heterodimeric protein complex described herein, the targeting moieties may be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties should be in a trans configuration with respect to each other since they are present on different Fc chains). In some embodiments where two or more targeting moieties are present on the same Fc chain, the targeting moieties may be present on the same or different sides / ends (N-terminus or / C-terminus) of the Fc chain.

[0322] In some embodiments where two or more targeting moieties are present in the heterodimeric protein complex described herein, the targeting moieties may be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties should be in a trans configuration with respect to each other since they are present on different Fc chains). In some embodiments where two or more signaling substances are present on the same Fc chain, the signaling substances may be present on the same or different sides / ends (N-terminus or / C-terminus) of the Fc chain.

[0323] In some embodiments where two or more signaling substances are present in the heterodimeric protein complex described herein, one signaling substance may be present in a trans orientation (relative to the targeting moiety), while another signaling substance may be present in a cis orientation (relative to the targeting moiety).

[0324] In some embodiments, the Fc domain comprises or begins with the core hinge region of wild-type human IgG1, which region comprises the sequence Cys-Pro-Pro-Cys. In some embodiments, the Fc domain also comprises the upper hinge, or a portion thereof (e.g., DKTHTCPPC (see WO 2009053368), EPKSCDKTHTCPPC, or EPKSSDKTHTCPPC (see Lo et al., Protein Engineering vol. 11 no. 6 pp. 495-500, 1998)).

[0325] Fc-based chimeric protein complex The Fc-based chimeric protein complex of the present technology comprises at least one Fc domain disclosed herein, at least one signaling substance (SA) disclosed herein, and at least one targeting moiety (TM) disclosed herein.

[0326] The Fc-based chimeric protein complex of the present invention may include a complex of two fusion proteins each comprising an Fc domain. In some embodiments, the Fc-based chimeric protein complex is a homodimer.

[0327] In some embodiments, the Fc-based chimeric protein complex is a heterodimer. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation / structure. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a cis orientation / structure. In some embodiments, the heterodimeric Fc-based chimeric protein complex does not include a signaling substance and a targeting moiety on a single polypeptide.

[0328] In some embodiments, the Fc-based chimeric protein has an improved in vivo half-life compared to a chimeric protein lacking Fc or a chimeric protein that is not a heterodimer complex. In some embodiments, the Fc-based chimeric protein has improved solubility, stability and other pharmacological properties compared to a chimeric protein lacking Fc or a chimeric protein that is not a heterodimer complex.

[0329] The heterodimeric Fc-based chimeric protein complex is composed of two different polypeptides. In some embodiments described herein, the targeting domain is present on a polypeptide different from the signaling molecule, and thus, a protein can be made that contains only one copy of the targeting domain and, similarly, only one type of signaling molecule (which can control possible interference with desirable properties). Further, in some embodiments, cross-linking of antigens on the cell surface (which may induce undesirable effects) can be avoided by only one targeting domain (e.g., VHH). Further, in some embodiments, one signaling molecule can alleviate possible interference with the binding force mediated by the restoration of effector function, depending on the "crowding" of the molecule and the targeting domain. Further, in some embodiments, the heterodimeric Fc-based chimeric protein complex can have two targeting moieties, which can be arranged on two different polypeptides. For example, in some embodiments, the C-termini of both targeting moieties (e.g., VHH) can be masked to avoid potential autoantibodies or existing antibodies (e.g., VHH autoantibodies or existing antibodies). Further, in some embodiments, a heterodimeric Fc-based chimeric protein complex having a targeting domain on a polypeptide different from, for example, the signaling molecule (e.g., wild-type signaling molecule) can preferentially "bridge" two cell types (e.g., tumor cells and immune cells). Further, in some embodiments, the heterodimeric Fc-based chimeric protein complex has two signaling molecules on different polypeptides, respectively, enabling a more complex effector response.

[0330] Furthermore, in some embodiments, for example, a heterodimeric Fc-based chimeric protein complex having a targeting domain on a polypeptide different from a signaling agent and having a diversity of combinations of targeting moieties and signaling agents is provided in a practical manner. For example, in some embodiments, a polypeptide having any of the targeting moieties described herein may be a "ready-to-use product" combined with a polypeptide having any of the signaling agents described herein to enable rapid generation of various combinations of targeting moieties and signaling agents in a single Fc-based chimeric protein complex.

[0331] In some embodiments, the Fc-based chimeric protein complex includes one or more linkers. In some embodiments, the Fc-based chimeric protein complex includes a linker that connects the Fc domain, the signaling agent, and the targeting moiety. In some embodiments, the Fc-based chimeric protein complex includes a linker that connects the signaling agent and the targeting moiety respectively (or in the case of two or more targeting moieties, connects the signaling agent to one of the targeting moieties). In some embodiments, the Fc-based chimeric protein complex includes a linker that connects each signaling agent 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 to another signaling agent.

[0332] In some embodiments, the Fc-based chimeric protein complex includes two or more targeting moieties. In such embodiments, the targeting moieties may be the same targeting moiety or different targeting moieties.

[0333] In some embodiments, the Fc-based chimeric protein complex comprises two or more signaling agents. In such embodiments, the signaling agents may be the same targeting moiety or different targeting moieties.

[0334] For example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, at least two signaling agents (SA), and at least two targeting moieties (TM), wherein the Fc domain, signaling agent, and targeting moiety are each selected from any of the Fc domains, signaling agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is a homodimer.

[0335] In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 5A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 6A-H. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 7A-H. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 8A-D. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 9A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 10A-J. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 11A-D. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 12A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 13A-J. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 14A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 15A-L. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 16A-L. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 17A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 18A-L. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 19A-L. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 20A-J. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 21A-J. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 22A-F. In various embodiments, the Fc-based chimeric protein complex takes the form of a schematic diagram of any of FIGS. 23A-F.

[0336] In some embodiments, the signaling molecule is linked to the targeting moiety, and the targeting moiety is linked to the Fc domain on the same end (see FIGS. 5A - F). In some embodiments, the Fc domain is a homodimer.

[0337] In some embodiments, the signaling molecule and the targeting moiety are linked to the Fc domain, and the targeting moiety and the signaling molecule are linked on the same end (see FIGS. 5A - F). In some embodiments, the Fc domain is a homodimer.

[0338] In some embodiments, the targeting moiety is linked to the signaling molecule, and the signaling molecule is linked to the Fc domain on the same end (see FIGS. 5A - F). In some embodiments, the Fc domain is a homodimer.

[0339] In some embodiments, the homodimeric Fc-based chimeric protein complex comprises two or more targeting moieties. In some embodiments, there are four targeting moieties and two signaling agents, the targeting moieties are linked to the Fc domain, and the signaling agents are linked to the targeting moieties on the same end (see FIGS. 6A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four targeting moieties and two signaling agents, two of the targeting moieties are linked to the Fc domain, two of the targeting moieties are linked to the signaling agent, and these are linked to the Fc domain on the same end (see FIGS. 6A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four targeting moieties and two signaling agents, two of the targeting moieties are linked to each other, one targeting moiety from each pair is linked to the Fc domain on the same end, and the signaling agent is linked to the Fc domain on the same end (see FIGS. 6A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four targeting moieties and two signaling agents, two of the targeting moieties are linked to each other, one targeting moiety from each pair is linked to the signaling agent, the other targeting moiety of the pair is linked to the Fc domain, and the targeting moieties linked to the Fc domain are linked on the same end (see FIGS. 6A-H). In some embodiments, the Fc domain is a homodimer.

[0340] In some embodiments, the homodimeric Fc-based chimeric protein complex comprises two or more signaling agents. In some embodiments where there are four signaling agents and two signaling agents, the two signaling agents are linked to each other, one signaling agent from the pair is linked to the Fc domain on the same end, and the targeting moiety is linked to the Fc domain on the same end (see FIGS. 7A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four signaling agents and two signaling agents, the two signaling agents are linked to the Fc domain on the same end, the two signaling agents are each linked to a targeting moiety, and the targeting moiety is linked to the Fc domain on the same end (see FIGS. 7A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four signaling agents and two signaling agents, the two signaling agents are linked to each other, one signaling agent of the pair is linked to the targeting moiety, and the targeting moiety is linked to the Fc domain on the same end (see FIGS. 7A-H). In some embodiments, the Fc domain is a homodimer.

[0341] For example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, the Fc domain comprising an ion pair forming mutation and / or a knob-in-hole mutation, at least one signaling agent, and at least one targeting moiety, the ion pair forming motif and / or the knob-in-hole motif, the signaling agent, and the targeting moiety being selected from any of the ion pair forming motifs and / or knob-in-hole motifs, signaling agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0342] In some embodiments, the signaling agent is linked to a targeting moiety, which is linked to an Fc domain (see FIGS. 14A-F and 17A-F). In some embodiments, the targeting moiety is linked to a signaling agent, which is linked to an Fc domain (see FIGS. 14A-F and 17A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0343] In some embodiments, the signaling agent and the targeting moiety are linked to an Fc domain (see FIGS. 8A-D, 11A-D, 14A-F, and 17A-F). In some embodiments, the targeting moiety and the signaling agent are linked to different Fc chains on the same end (see FIGS. 8A-D and 11A-D). In some embodiments, the targeting moiety and the signaling agent are linked to different Fc chains on different ends (see FIGS. 8A-D and 11A-D). In some embodiments, the targeting moiety and the signaling agent are linked to the same Fc chain (see FIGS. 14A-F and 17A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0344] In some embodiments where there is one signaling substance and two targeting moieties, the signaling substance is linked to the Fc domain, and the two targeting moieties can be linked to each other using one targeting moiety linked to the Fc domain or can each be linked to the Fc domain (see FIGS. 9A-F, 12A-F, 15A-L, 18A-L, 20A-J, and 21A-J). In some embodiments, the targeting moiety is linked to one Fc chain and the signaling substance is linked to the other Fc chain (see FIGS. 9A-F and 12A-F). In some embodiments, the paired targeting moiety and the signaling substance are linked to the same Fc chain (see FIGS. 15A-L and 18A-L). In some embodiments, one targeting moiety is linked to the Fc domain, the other targeting moiety is linked to the signaling substance, and the paired targeting moiety is linked to the Fc domain (see FIGS. 15A-L and 18A-L, 20A-J, and 21A-J). In some embodiments, the non-paired targeting moiety and the paired targeting moiety are linked to the same Fc chain (see FIGS. 15A-L and 18A-L). In some embodiments, the non-paired targeting moiety and the paired targeting moiety are linked to different Fc chains (see FIGS. 20A-J and 21A-J). In some embodiments, the non-paired targeting moiety and the paired targeting moiety are linked at the same terminus (see FIGS. 20A-J and 21A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0345] In some embodiments where there is one signaling substance and two targeting moieties, the targeting moieties are linked together, the signaling substance is linked to one of the paired targeting moieties, and the targeting moiety not linked to the signaling substance is linked to the Fc domain (see FIGS. 15A-L and 18A-L, 20A-J, and 21A-J). In some embodiments, the paired signaling substance and the non-paired targeting moiety are linked to the same Fc chain (see FIGS. 15A-L and 18A-L). In some embodiments, the paired signaling substance and the non-paired targeting moiety are linked to different Fc chains (see FIGS. 20A-J and 21A-J). In some embodiments, the paired signaling substance and the non-paired targeting moiety are linked on the same end (see FIGS. 20A-J and 21A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0346] In some embodiments where there is one signaling substance and two targeting moieties, the targeting moieties are linked together, the signaling substance is linked to one of the paired targeting moieties, and the targeting moiety not linked to the signaling substance is linked to the Fc domain (see FIGS. 15A-L and 18A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0347] In some embodiments where there is one signaling substance and two targeting moieties, the targeting moieties are linked together, the signaling substance is linked to one of the paired targeting moieties, and the signaling substance is linked to the Fc domain (see FIGS. 15A-L and 18A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0348] In some embodiments where there is one signaling agent and two targeting moieties, both targeting moieties are linked to the signaling agent, and one of the targeting moieties is linked to the Fc domain (see FIGS. 15A - L and 18A - L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0349] In some embodiments where there is one signaling agent and two targeting moieties, the targeting moieties and the signaling agent are linked to the Fc domain (see FIGS. 20A - J and 21A - J). In some embodiments, the targeting moieties are linked terminally (see FIGS. 20A - J and 21A - J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0350] 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. 10A - J and 13A - 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. 22A - F and 23A - F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains a mutation that reduces or eliminates its effector function.

[0351] In some embodiments where there are two signaling agents and one targeting moiety, the targeting moiety is linked to a signaling agent which is linked to an Fc domain, and the other signaling agent is linked to the Fc domain (see Figures 10A-J, 13A-J, 16A-L, and 19A-L). In some embodiments, the targeting moiety and the non-pairing signaling agent are linked to different Fc chains (see Figures 10A-J and 13A-J). In some embodiments, the targeting moiety and the non-pairing signaling agent are linked to different Fc chains on the same end (see Figures 10A-J and 13A-J). In some embodiments, the targeting moiety and the non-pairing signaling agent are linked to different Fc chains at different ends (see Figures 10A-J and 13A-J). In some embodiments, the targeting moiety and the non-pairing signaling agent are linked to the same Fc chain (see Figures 16A-L and 19A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0352] In some embodiments where there are two signaling agents and one targeting moiety, the targeting moiety is linked to a signaling agent which is linked to an Fc domain, and the other signaling agent is linked to the Fc domain (see Figures 10A-J and 13A-J). In some embodiments, the paired signaling agent and the non-pairing signaling agent are linked to different Fc chains (see Figures 10A-J and 13A-J). In some embodiments, the paired signaling agent and the non-pairing signaling agent are linked to different Fc chains on the same end (see Figures 10A-J and 13A-J). In some embodiments, the paired signaling agent and the non-pairing signaling agent are linked to different Fc chains at different ends (see Figures 10A-J and 13A-J). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0353] In some embodiments where there are two signaling agents and one targeting moiety, the signaling agents are linked together, the targeting moiety is linked to one of the paired signaling agents, and the targeting moiety is linked to the Fc domain (see FIGS. 16A-L and 19A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0354] In some embodiments where there are two signaling agents and one targeting moiety, the signaling agents are linked together, one of the signaling agents is linked to the Fc domain, and the targeting moiety is linked to the Fc domain (see FIGS. 16A-L, 19A-L, 22A-F, and 23A-F). In some embodiments, the paired signaling agents and the targeting moiety are linked to the same Fc chain (see FIGS. 16A-L and 19A-L). In some embodiments, the paired signaling agents and the targeting moiety are linked to different Fc chains (see FIGS. 22A-F and 23A-F). In some embodiments, the paired signaling agents and the signaling agents are linked to different Fc chains on the same terminus (see FIGS. 22A-F and 23A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0355] In some embodiments where there are two signaling agents and one targeting moiety, both signaling agents are linked to the targeting moiety and one of the signaling agents is linked to the Fc domain (see FIGS. 16A-L and 19A-L). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

[0356] In some embodiments where there are two signaling substances and one targeting moiety, the signaling substances are linked together, one of the signaling substances is linked to the targeting moiety, and the other signaling substance is linked to the Fc domain (see Figures 16A-L and 19A-L).

[0357] In some embodiments where there are two signaling substances and one targeting moiety, each signaling substance is linked to the Fc domain and the targeting moiety is linked to one of the signaling substances (see Figures 16A-L and 19A-L). In some embodiments, the signaling substances are linked to the same Fc chain (see Figures 16A-L and 19A-L).

[0358] In some embodiments, the targeting moiety or signaling substance is linked to the Fc domain, including one or both of the CH2 and CH3 domains and optionally the hinge region. For example, such polypeptides can be made using a vector encoding a targeting moiety, a signaling substance, or a combination thereof linked to the Fc domain as a single nucleotide sequence.

[0359] Multispecific chimeras and fusions with signaling substances In some embodiments, the FAP-binding substances of the present technology are part of a chimera or fusion or Fc-based chimeric protein complex with one or more of the signaling substances and / or one or more additional targeting moieties described herein. Accordingly, the present technology provides chimeric or fusion proteins or Fc-based chimeric protein complexes comprising one or more signaling substances and targeting moieties for FAP and / or one or more additional targeting moieties.

[0360] In some embodiments, the FAP-binding substance of the present technology is multispecific, i.e., the FAP-binding substance comprises two or more targeting moieties having recognition domains that recognize and bind to two or more targets, such as antigens, or receptors, or epitopes. In such embodiments, the FAP-binding substance of the present technology may comprise two or more targeting moieties having recognition domains that recognize and bind to two or more epitopes on the same antigen or on different antigens. In some embodiments, such multispecific FAP-binding substances exhibit advantageous properties such as improved binding affinity and / or improved selectivity. In one embodiment, the FAP-binding substance of the present technology comprises two targeting moieties and is bispecific, i.e., binds to and recognizes two epitopes on the same antigen or on different antigens.

[0361] In some embodiments, the multispecific FAP-binding substance of the present technology comprises two or more targeting moieties, each targeting moiety being an antibody or antibody derivative described herein. In one embodiment, the multispecific FAP-binding substance of the present technology comprises at least one VHH comprising an antigen recognition domain for FAP and one antibody or antibody derivative comprising an antigen recognition domain for a tumor antigen.

[0362] In some embodiments, the multispecific FAP-binding substance of the present invention has two or more targeting moieties that target different antigens or receptors, and one targeting moiety may be attenuated with respect to its antigen or receptor, e.g., the targeting moiety binds to its antigen or receptor with low affinity or low binding strength (e.g., including binding with an affinity or binding strength lower than the affinity or binding strength that the other targeting moiety has for its antigen or receptor, e.g., the difference in binding affinity may be about 10-fold, or 25-fold, or 50-fold, or 100-fold, or 300-fold, or 500-fold, or 1000-fold, or 5000-fold; e.g., the targeting moiety with lower affinity or binding strength binds to its antigen or receptor in the range of mid- to high nM or low- to mid-μM K Dcan bind, while the targeting moiety with higher affinity or binding strength has a K in the range of mid-high pM or low-mid nM D can bind). For example, in some embodiments, the multispecific FAP-binding substance of the present invention includes a deattenuated targeting moiety directed to an indiscriminate antigen or receptor, which can improve targeting to the cell of interest (e.g., via other targeting moieties) and prevent effects spanning multiple cell types that are not targeted for therapy (e.g., by binding indiscriminately to an antigen or receptor with higher affinity than that provided in these embodiments).

[0363] The multispecific FAP-binding substances of the present invention can be constructed using methods known in the art. See, for example, U.S. Patent No. 9,067,991, U.S. Patent Application Publication No. 2011 / 0262348, and International Publication No. 2004 / 041862. The entire contents of these patents are incorporated herein by reference. In an exemplary embodiment, the multispecific FAP-binding substances of the present technology that include two or more targeting moieties can be constructed by chemical cross-linking, for example, reacting amino acid residues with an organic derivatizing agent as described in Blattler et al., Biochemistry 24, 1517-1524 and European Patent No. 294703, the entire contents of which are incorporated herein by reference. In another exemplary embodiment, the multispecific FAP-binding substance that includes two or more targeting moieties is constructed by gene fusion, i.e., by constructing a single polypeptide that includes the polypeptides of the individual targeting moieties. For example, a single polypeptide construct can be formed that encodes a first VHH having an antigen recognition domain for FAP and a second antibody or antibody derivative having an antigen recognition domain for a tumor antigen. Methods for producing bivalent or multivalent VHH polypeptide constructs are disclosed in International Publication No. 96 / 34103, the entire contents of which are incorporated herein by reference. In a further exemplary embodiment, the multispecific FAP-binding substances of the present technology can be constructed by using a linker. For example, the carboxy terminus of a first VHH having an antigen recognition domain for FAP can be linked to the amino terminus of a second antibody or antibody derivative having an antigen recognition domain for a tumor antigen (and vice versa). Examples of linkers that can be used are described herein. In some embodiments, the components of the multispecific FAP-binding substances of the present technology are directly linked to each other without using a linker.

[0364] In some embodiments, the multispecific FAP-binding substance recognizes and binds to FAP and one or more antigens found on one or more immune cells. The immune cells can include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer cells, T lymphocytes (e.g., cytotoxic T lymphocytes, helper T cells), B lymphocytes, plasma cells, dendritic cells, or subsets thereof. In some embodiments, the FAP-binding substance specifically binds to the antigen of interest and effectively mobilizes one or more immune cells either directly or indirectly.

[0365] In some embodiments, the multispecific FAP-binding substance of the present technology recognizes and binds to FAP and one or more antigens found on tumor cells. In these embodiments, the FAP-binding substance of the present invention can directly or indirectly mobilize immune cells to tumor cells or the tumor microenvironment. In some embodiments, the FAP-binding substance of the present invention can directly or indirectly mobilize immune cells, such as immune cells (e.g., CTL) that can kill and / or suppress tumors, to the site of action (such as, but not limited to, the tumor microenvironment).

[0366] In some embodiments, the FAP-binding substance of the present invention can change the balance of immune cells in a manner advantageous for the immune attack on tumors, or find use in a method including changing the balance of immune cells in a manner advantageous for the immune attack on tumors. For example, the FAP-binding substance of the present invention can change the ratio of immune cells at clinically important sites in a manner advantageous for cells that can kill and / or suppress tumors (e.g., T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), neutrophils, B cells, dendritic cells or subsets thereof), and in a manner disadvantageous for cells that protect tumors (e.g., myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg); tumor-associated neutrophils (TAN), M2 macrophages, tumor-associated macrophages (TAM), or subsets thereof). In some embodiments, the FAP-binding substance of the present invention can increase the ratio of effector T cells to regulatory T cells.

[0367] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to a tumor cell-associated antigen. In some embodiments, the targeting moiety mobilizes tumor cells directly or indirectly. For example, in some embodiments, the mobilization of tumor cells is towards one or more effector cells (e.g., immune cells described herein) that can kill and / or suppress the tumor cells. In some embodiments, the targeting moiety mobilizes T cells directly or indirectly to tumor cells by two targeting moieties that interact with their respective antigens on tumors and FAP-positive immune cells (e.g., dendritic cells).

[0368] A tumor cell, or cancer cell, refers to an uncontrolled proliferation of cells or tissues and / or an abnormal increase in cell survival and / or an abnormal increase in the suppression of apoptosis that interferes with the normal functioning of the organs and systems of the body. For example, tumor cells include benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Examples of tumor cells include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), melanoma, myeloma, neuroblastoma, oral cancer (lip, glossal, tongue, oral cavity, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland carcinoma, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, Hodgkin lymphoma and non-Hodgkin lymphoma, and lymphomas including B-cell lymphoma (including low-grade / follicular non-Hodgkin 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, large tumor lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, as well as other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with nevus syndrome, edema (e.g., associated with brain tumors), and cells of the MEGS syndrome, but are not limited thereto.

[0369] Examples of tumor cells, or cancer cells, include, but are not limited to, carcinomas, such as various subtypes (including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myeloid, acute lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell), lymphomas and myelomas (including, for example, Hodgkin and non-Hodgkin lymphomas, light chain type, non-secretory MGUS, and plasmacytoma), and central nervous system cancers (such as brain tumors (such as gliomas (such as astrocytomas, oligodendrogliomas, and ependymomas), meningiomas, pituitary adenomas, and neuromas), and spinal cord tumors (such as meningiomas and neurofibromas)).

[0370] Examples of tumor antigens include MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, am11, prostate specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, tumor antigens of the MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-Al2, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), tumor antigens of the GAGE family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, 3-catenin and gamma-catenin, p120ctn, gp100 Pme1117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, tumor antigens of the Smad family, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, and BCMA (TNFRSF17), among others, but not limited thereto. In some embodiments, the FAP-binding substance comprises a targeting moiety that binds to one or more of these tumor antigens.

[0371] In some embodiments, the multispecific FAP-binding substance of the invention recognizes and binds to FAP and an antigen on tumor cells. In some embodiments, the multispecific FAP-binding substance directly or indirectly recruits CTLs to tumor cells or to the tumor microenvironment.

[0372] In some embodiments, the multispecific FAP-binding substance of the invention has targeting moieties that target two different cells (e.g., to form a synapse) or the same cell (e.g., to obtain a higher concentration of signaling effector).

[0373] In some embodiments, the multi-specific FAP-binding substance of the present technology comprises a targeting moiety having an antigen recognition domain that specifically binds to a T cell-related target (e.g., an antigen, a receptor). In some embodiments, the targeting moiety recruits T cells directly or indirectly. In certain embodiments, the antigen recognition domain specifically binds to effector T cells. In some embodiments, the antigen recognition domain recruits effector T cells directly or indirectly to, for example, in some embodiments, a treatment site (e.g., a site having one or more diseased cells or cells to be modulated to obtain a therapeutic effect). Examples of effector T cells include cytotoxic T cells (e.g., αβTCR, CD3+, CD8+, CD45RO+); CD4+ effector T cells (e.g., αβTCR, CD3+, CD4+, CCR7+, CD62Lhi, IL7R / CD127+); CD8+ effector T cells (e.g., αβTCR, CD3+, CD8+, CCR7+, CD62Lhi, IL7R / CD127+); effector memory T cells (e.g., CD62Llow, CD44+, TCR, CD3+, IL7R / CD127+, IL15R+, CCR7low); central memory T cells (e.g., CCR7+, CD62L+, CD27+; or CCR7hi, CD44+, CD62Lhi, TCR, CD3+, IL7R / CD127+, IL15R+); CD62L+ effector T cells; CD8+ effector memory T cells (TEM) including early effector memory T cells (CD27+CD62L-) and late effector memory T cells (CD27-CD62L-) (TemE and TemL, respectively); CD127(+)CD25(low / -) effector T cells; CD127(-)CD25(-) effector T cells; CD8+ stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca(+)); TH1 effector T cells (e.g., CXCR3+, CXCR6 + and CCR5+; or αβTCR, CD3+, CD4+, IL12R+, IFNγR+, CXCR3+), TH2 effector T cells (e.g., CCR3 + , CCR4 +and CCR8+; or αβTCR, CD3+, CD4+, IL4R+, IL33R+, CCR4+, IL17RB+, CRTH2+); TH9 effector T cells (e.g., αβTCR, CD3+, CD4+); TH17 effector T cells (e.g., αβTCR, CD3+, CD4+, IL23R+, CCR6+, IL1R+); CD4+CD45RO+CCR7+ effector T cells, ICOS+ effector T cells; CD4+CD45RO+CCR7(−) effector T cells; and effector T cells that secrete IL2, IL4 and / or IFNγ.

[0374] Examples of target T cell antigens include, for example, the following (including the extracellular domain if applicable): CD8, CD3, SLAMF4, IL2Rα, 4-1BB / TNFRSF9, IL2Rβ, ALCAM, B7-1, IL4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL6R, CCR3, IL7Rα, CCR4, CXCRl / ILSRA, CCR5, CCR6, IL10Rα, CCR7, IL10Rβ, CCRS, IL12Rβ1, CCR9, IL12Rβ2, CD2, IL13Rα1, IL13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrinα4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / 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 γ chain / IL2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, thrombopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAILRI / TNFRSFIOA, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL1R1 and TSLPR. In some embodiments, the FAP binding substance comprises a targeting moiety that binds to one or more of these exemplary T cell antigens.

[0375] In some embodiments, the multispecific FAP-binding substance of the present technology comprises a VHH that is a single amino acid chain having 4 "framework regions" or FRs and 3 "complementary determining regions" or CDRs and comprises a targeting moiety for CD8. As used herein, "framework region" or "FR" refers to a region in the variable domain located between CDRs. As used herein, "complementary determining region" or "CDR" refers to a variable region in a VHH that comprises an amino acid sequence capable of specifically binding to an antigenic target.

[0376] In some embodiments, the multispecific FAP-binding substance of the present technology comprises a VHH for CD8 having a variable domain that comprises at least one of the CD8 CDR1, CD8 CDR2, and / or CD8 CDR3 sequences.

[0377] In some embodiments, the CD8 CDR1 sequence is selected from SEQ ID NO: 192 or SEQ ID NO: 193.

[0378] In some embodiments, the CD8 CDR2 sequence is selected from SEQ ID NO: 194 or SEQ ID NO: 195.

[0379] In some embodiments, the CD8 CDR3 sequence is selected from SEQ ID NO: 196 or SEQ ID NO: 197 or SEQ ID NO: 198

[0380] In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from R3HCD27 (SEQ ID NO: 199) or R3HCD129 (SEQ ID NO: 200) or R2HCD26 (SEQ ID NO: 201).

[0381] In some embodiments, the CD8 targeting moiety comprises a VHH having a variable domain that comprises at least one of the CD8 CDR1, CD8 CDR2, and / or CD8 CDR3 sequences described below.

[0382] In some embodiments, the CD8 CDR1 sequence is selected from SEQ ID NO: 202 to SEQ ID NO: 270.

[0383] In some embodiments, the CD8 CDR2 sequence is selected from SEQ ID NO: 271 to SEQ ID NO: 339.

[0384] In some embodiments, the CD8 CDR3 sequence is selected from SEQ ID NO: 340 to SEQ ID NO: 408.

[0385] In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from the following sequences: 1CDA7 (SEQ ID NO: 409); or 1CDA12 (SEQ ID NO: 410); or 1CDA14 (SEQ ID NO: 411); or 1CDA15 (SEQ ID NO: 412); or 1CDA17 (SEQ ID NO: 413); or 1CDA18 (SEQ ID NO: 414); or 1CDA19 (SEQ ID NO: 415); or 1CDA24 (SEQ ID NO: 416); or 1CDA26 (SEQ ID NO: 417); or 1CDA28 (SEQ ID NO: 418); or 1CDA37 (SEQ ID NO: 419); or 1CDA43 (SEQ ID NO: 420); or 1CDA45 (SEQ ID NO: 421); or 1CDA47 (SEQ ID NO: 422); or 1CDA48 (SEQ ID NO: 423); or 1CDA58 (SEQ ID NO: 424); or 1CDA65 (SEQ ID NO: 425); or 1CDA68 (SEQ ID NO: 426); or 1CDA73 (SEQ ID NO: 427); or 1CDA75 (SEQ ID NO: 428); or 1CDA86 (SEQ ID NO: 429); or 1CDA87 (SEQ ID NO: 430); or 1CDA88 (SEQ ID NO: 431); or 1CDA89 (SEQ ID NO: 432); or 1CDA92 (SEQ ID NO: 433); or 1CDA93 (SEQ ID NO: 434); or 2CDA1 (SEQ ID NO: 435); or 2CDA5 (SEQ ID NO: 436); or 2CDA22 (SEQ ID NO: 437); or 2CDA28 (SEQ ID NO: 438); or 2CDA62 (SEQ ID NO: 439); or 2CDA68 (SEQ ID NO: 440); or 2CDA73 (SEQ ID NO: 441); or 2CDA74 (SEQ ID NO: 442); or 2CDA75 (SEQ ID NO: 443); or 2CDA77 (SEQ ID NO: 444); or 2CDA81 (SEQ ID NO: 445); or 2CDA87 (SEQ ID NO: 446); or 2CDA88 (SEQ ID NO: 447); or 2CDA89 (SEQ ID NO: 448); or 2CDA91 (SEQ ID NO: 449); or 2CDA92 (SEQ ID NO: 450); or 2CDA93 (SEQ ID NO: 451); or 2CDA94 (SEQ ID NO: 452); or 2CDA95 (SEQ ID NO: 453); or 3CDA3 (SEQ ID NO: 454); or 3CDA8 (SEQ ID NO: 455); or 3CDA11 (SEQ ID NO: 456); or 3CDA18 (SEQ ID NO: 457); or 3CDA19 (SEQ ID NO: 458);or 3CDA21 (SEQ ID NO: 459); or 3CDA24 (SEQ ID NO: 460); or 3CDA28 (SEQ ID NO: 461); or 3CDA29 (SEQ ID NO: 462); or 3CDA31 (SEQ ID NO: 463); or 3CDA32 (SEQ ID NO: 464); or 3CDA33 (SEQ ID NO: 465); or 3CDA37 (SEQ ID NO: 466); or 3CDA40 (SEQ ID NO: 467); or 3CDA41 (SEQ ID NO: 468); or 3CDA48 (SEQ ID NO: 469); or 3CDA57 (SEQ ID NO: 470); or 3CDA65 (SEQ ID NO: 471); or 3CDA70 (SEQ ID NO: 472); or 3CDA73 (SEQ ID NO: 473); or 3CDA83 (SEQ ID NO: 474); or 3CDA86 (SEQ ID NO: 475); or 3CDA88 (SEQ ID NO: 476); or 3CDA90 (SEQ ID NO: 477).;

[0386] In various exemplary embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include a terminal histidine tag sequence (i.e., HHHHHH: SEQ ID NO: 43).

[0387] In some embodiments, the CD8 targeting moiety comprises the amino acid sequence described in U.S. Patent Application Publication No. 2014 / 0271462. The entire contents of this patent are incorporated herein by reference. In some embodiments, the CD8 targeting moiety comprises the amino acid sequence described in Table 0.1, Table 0.2, Table 0.3, and / or FIGS. 1A - 121 of U.S. Patent Application Publication No. 2014 / 0271462. The entire contents of this patent are incorporated herein by reference. In some embodiments, the CD8 targeting moiety comprises HCDR1 of SEQ ID NO: 478 or 479 and / or HCDR2 of SEQ ID NO: 478 or 479 and / or HCDR3 of SEQ ID NO: 478 or 479 and / or LCDR1 of SEQ ID NO: 480 and / or LCDR2 of SEQ ID NO: 480 and / or LCDR3 of SEQ ID NO: 480.

[0388] In some embodiments, the technology contemplates the use of any natural or synthetic analogs, variants, mutants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the CD8-targeting moieties of the invention described herein. In some embodiments, the amino acid sequence of the CD8-targeting moiety further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0389] In some embodiments, the multispecific FAP-binding agent of the technology comprises a targeting moiety having an antigen recognition domain that specifically binds to a B cell-related target (e.g., an antigen, a receptor). In some embodiments, the targeting moiety mobilizes B cells directly or indirectly to, for example, a therapeutic agent site (e.g., a site having one or more diseased cells or cells to be modulated to obtain a therapeutic effect). By way of non-limiting example, in some embodiments, B cell antigens include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, CDw150, and B cell maturation antigen (BCMA). In some embodiments, the FAP-binding agent comprises a targeting moiety that binds one or more of the above B cell antigens.

[0390] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to a natural killer cell-related target (e.g., an antigen, a receptor). In some embodiments, the targeting moiety recruits natural killer cells directly or indirectly to, for example, a therapeutic agent site (e.g., a site having one or more diseased cells or cells to be regulated to obtain a therapeutic effect). In some embodiments, by way of non-limiting example, examples of natural killer cell antigens include, for example, TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, Kidalpha, DNAM-1, LMIR5 / CD300LB, Fc-epsilonRII, LMIR6 / CD300LE, Fc-gammaRI / CD64, MICA, Fc-gammaRIIB / CD32b, MICB, Fc-gammaRIIC / CD32c, MULT-1, Fc-gammaRIIA / CD32a, Nectin-2 / CD112, Fc-gammaRIII / CD16, NKG2A, FcRH1 / IRTA5, NKG2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1p, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, and ULBP-3. In some embodiments, the FAP-binding substance includes a targeting moiety that binds one or more of the above NK cell antigens.

[0391] In some embodiments, the targeting moiety recognizes and / or binds to FMS-like tyrosine kinase 3 (Flt3), or is a natural ligand for Flt3, such as FMS-like tyrosine kinase 3 ligand (Flt3L) or a truncated region thereof (e.g., a region capable of binding Flt3). In some embodiments, the targeting moiety is the extracellular domain of Flt3L. In some embodiments, the targeting moiety comprises an Flt3L domain, the Flt3L domain is a single-chain dimer, and optionally one Flt3L domain is linked to another Flt3L domain via one or more linkers, and the linker is a flexible linker.

[0392] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to macrophage / monocyte-related targets (e.g., antigens, receptors). In some embodiments, the targeting moiety mobilizes macrophages / monocytes directly or indirectly to, for example, a therapeutic agent site (e.g., a site having one or more diseased cells or cells to be regulated to obtain a therapeutic effect). In some embodiments, non-limiting examples of macrophage / monocyte antigens include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common gamma chain, integrin alpha4 / CD49d, BLAME / SLAMF8, integrin alphaX / CD11c, CCL6 / C10, integrin beta2 / CD18, CD155 / PVR, integrin beta3 / CD61, CD31 / PECAM-1, Latexin, CD36 / SR-B3, leukotriene B4R1, CD40 / TNFRSF5, LIMPII / SR-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-gammaRI / CD64, osteopontin, Fc-gammaRIIB / CD32b, PD-L2, Fc-gammaRIIC / CD32c, siglec-3 / CD33, Fc-gammaRIIA / CD32a, SIGNR1 / CD209, Fc-gammaRIII / CD16, SLAM, GM-CSFRalpha, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-gammaRI, TLR4, IFN-gammaR2, TREM-1, ILIRII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4, IL10Ralpha, ALCAM, IL10Rbeta, aminopeptidase N / ANPEP, ILT2 / CD85j, common gamma chain, ILT3 / CD85k, ClqR1 / CD93, ILT4 / CD85d,CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / CDIIb, CCR8, integrin αX / CDIIc, CD155 / PVR, integrin 32 / CD18, CD14, integrin 133 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMFS, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD105, NCAM-L1, Fc-γRI / CD64, PSGL-1, Fc-γRIIIICD16, RP105, G-CSF R, L-selectin, GM-CSFRα, siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL6R, TREM-2, CXCRI / IL8RA, TREM-3 and TREMLITTLT-1. In some embodiments, the FAP binding substance comprises a targeting moiety that binds to one or more of the above macrophage / monocyte cell antigens.,

[0393] In some embodiments, the multi-specific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to dendritic cell-related targets (e.g., antigens, receptors). In some embodiments, the targeting moiety mobilizes dendritic cells directly or indirectly to, for example, a therapeutic agent site (e.g., a site having one or more diseased cells or cells to be regulated to obtain a therapeutic effect). In some embodiments, non-limiting examples of dendritic cell (DC) antigens include, but are not limited to, for example, FAP, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-EI, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-Pl / C0LEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL12 / IL23p40, 4-Amino-1,8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin α4 / CD49d, Aag, integrin p2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, ClqR1 / 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, Chern23, PD-L2, CLEC-1, RP105, CLEC-2, CLEC-8, siglec-2 / CD22, CRACC / SLAMF7, siglec-3 / CD33, DC-SIGN, DEC205, siglec-5, DC-SIGNR / CD299, siglec-6, DCAR, siglec-7, DCIR / CLEC4A, siglec-9, DEC-205, siglec-10, Dectin-1 / CLEC7A, siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, DEP-1 / CD148, SIGNR4, DLEC, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, DEC205, and vanilloid R1. In some embodiments, the FAP binding substance comprises a targeting moiety that binds to one or more of the above DC antigens.

[0394] In some embodiments, the chimeric protein or Fc-based chimeric protein complex of the invention comprises a targeting moiety comprising an amino acid sequence that is at least 60% identical to any one of the sequences disclosed herein. For example, in some embodiments, the chimeric protein or Fc-based chimeric protein complex 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 (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) to any one of the sequences disclosed herein and may comprise a targeting moiety.

[0395] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to a target (e.g., an antigen, a receptor) on immune cells selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, and eosinophils. In some embodiments, the antigen recognition domain recruits megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, and eosinophils, for example, directly or indirectly to a treatment site (e.g., a site having one or more diseased cells or cells to be modulated to obtain a therapeutic effect).

[0396] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to a megakaryocyte and / or platelet-related target (e.g., an antigen, a receptor). By way of non-limiting example, in some embodiments, megakaryocyte and / or platelet antigens include, for example, GP11b / 111a, GP1b, vWF, PF4, and TSP. In some embodiments, the FAP-binding substance includes a targeting moiety that binds one or more of the above megakaryocyte and / or platelet antigens.

[0397] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to an erythrocyte-related target (e.g., an antigen, a receptor). In some embodiments, by way of non-limiting example, examples of erythrocyte antigens include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein 11b / 111a), CD41b (GPllb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2 (MHC-I1), and rhesus antigens. In some embodiments, the FAP-binding substance includes a targeting moiety that binds one or more of the above erythrocyte antigens.

[0398] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to mast cell-related targets (e.g., antigens, receptors). In some embodiments, by way of non-limiting example, mast cell antigens include, for example, SCFR / CD117, Fca, CD2, CD25, CD35, CD88, CD203c, C5R1, CMAI, FCERIA, FCER2, TPSABI. In some embodiments, the FAP-binding substance includes a targeting moiety that binds one or more of the above mast cell antigens.

[0399] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to basophil-related targets (e.g., antigens, receptors). In some embodiments, by way of non-limiting example, basophil antigens include, for example, Fca, CD203c, CD123, CD13, CD107a, CD107b, and CD164. In some embodiments, the FAP-binding substance includes a targeting moiety that binds one or more of the above basophil antigens.

[0400] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to neutrophil-related targets (e.g., antigens, receptors). In some embodiments, by way of non-limiting example, neutrophil antigens include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRlll), CD18 protein (LFA-1, CR3, and p150,95), CD45, CD67, and CD177. In some embodiments, the FAP-binding substance includes a targeting moiety that binds one or more of the above neutrophil antigens.

[0401] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to an eosinophil-related target (e.g., antigen, receptor). In some embodiments, non-limiting examples of basophil antigens include, for example, CD35, CD44, and CD69. In some embodiments, the FAP-binding substance includes a targeting moiety that binds one or more of the above eosinophil antigens.

[0402] In some embodiments, the multispecific FAP-binding substance of the present technology includes a targeting moiety having an antigen recognition domain that specifically binds to any suitable antigen or receptor or cell surface marker known to those skilled in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. In some embodiments, non-limiting examples of tissue-specific markers include endothelial cell markers (e.g., ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAM1, PROCR, SELE, SELP, TEK, THBD, VCAM1, VWF, etc.); smooth muscle cell surface markers (e.g., ACTA2, MYH10, MYH11, MYH9, MYOCD, etc.); fibroblast (stromal) cell surface markers (e.g., ALCAM, CD34, COL1A1, COL1A2, COL3A1, FAP, PH-4, etc.); epithelial cell surface markers (e.g., CD1D, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUC1, TACSTD1, etc.); neovascular markers (e.g., CD13, TFNA, alpha-v beta-3 (αvβ3), E-selectin, etc.); and adipocyte surface markers (e.g., ADIPOQ, FABP4, and RETN, etc.), but are not limited thereto. In some embodiments, the FAP-binding substance includes a targeting moiety that binds one or more of the above antigens.

[0403] In some embodiments, the multispecific FAP-binding substance of the present technology comprises a targeting moiety having an antigen recognition domain that specifically binds to a checkpoint marker expressed on T cells. In some embodiments, the checkpoint marker is one or more checkpoint markers selected from PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD4OL, TIM3, and A2aR.

[0404] In some embodiments, the multispecific FAP-binding substance of the present technology comprises a targeting moiety having an antigen recognition domain that specifically binds to a checkpoint marker. In some embodiments, the checkpoint marker is one or more checkpoint markers selected from 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 / OX4OL, CD27, CD4OL, TIM3 / Ga19, and A2aR.

[0405] By way of non-limiting example, in some embodiments, the multispecific FAP-binding substance of the present invention comprises (i) CD8; (ii) a targeting moiety directed to one or more of the checkpoint markers expressed on T cells, such as PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, 0X40, Cd27, CD4OL, TIM3, and A2aR, and / or (iii) the targeting moiety is directed to tumor cells together with any of the modified (e.g., mutant) signaling substances described herein.

[0406] In some embodiments, the present multi-specific FAP-binding substance has one or more targeting moieties directed to PD-1. In some embodiments, the FAP-binding substance has one or more targeting moieties that selectively bind to the PD-1 polypeptide. In some embodiments, the FAP-binding substance comprises one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds to the PD-1 polypeptide.

[0407] In some embodiments, the multi-specific FAP-binding substance of the present technology comprises a VHH against PD-1 having a variable domain comprising at least one of the PD-1 CDR1, PD-1 CDR2, and / or PD-1 CDR3 sequences.

[0408] In some embodiments, the PD-1 CDR1 sequence is selected from SEQ ID NOs: 481 to 494.

[0409] In some embodiments, the PD-1 CDR2 sequence is selected from SEQ ID NOs: 495 to 508.

[0410] In some embodiments, the PD-1 CDR3 sequence is selected from SEQ ID NOs: 509 to 521.

[0411] In various exemplary embodiments, the PD-1 targeting moiety comprises an amino acid sequence selected from the following sequences: 2PD23: (SEQ ID NO: 522); or 2PD26: (SEQ ID NO: 523); or 2PD90: (SEQ ID NO: 524); or 2PD-106: (SEQ ID NO: 525); or 2PD-16: (SEQ ID NO: 526); or 2PD71: (SEQ ID NO: 527); or 2PD-152: (SEQ ID NO: 528); or 2PD-12: (SEQ ID NO: 529); or 3PD55: (SEQ ID NO: 530); or 3PD82: (SEQ ID NO: 531); or 2PD8: (SEQ ID NO: 532); or 2PD27: (SEQ ID NO: 533); or 2PD82: (SEQ ID NO: 534); or 3PD36: (SEQ ID NO: 535).

[0412] In various exemplary embodiments, the PD-1 targeting moiety comprises an amino acid sequence selected from any one of the above that does not include a terminal histidine tag sequence (i.e., HHHHHH: SEQ ID NO: 43).

[0413] In some embodiments, the targeting moiety comprises the anti-PD-1 antibody pembrolizumab (also known as MK-3475, Keytruda®), or a fragment thereof. In some embodiments, the targeting moiety is one or more of pembrolizumab and other humanized anti-PD-1 antibodies disclosed in Hamid, et al. (2013) New England Journal of Medicine 369(2):134-44, U.S. Patent No. 8,354,509, and International Publication No. 2009 / 114335. The entire disclosures of these are incorporated herein by reference. In some embodiments, by way of non-limiting example, the pembrolizumab or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO: 536); and / or a light chain comprising the amino acid sequence of SEQ ID NO: 537.

[0414] In some embodiments, the targeting moiety comprises the anti-PD-1 antibody nivolumab (also known as BMS-936558, MDX-1106, ONO-4538, Opdivo®), or a fragment thereof. In some embodiments, the targeting moiety is one or more of nivolumab (clone 5C4) that specifically binds to PD-1 and other human monoclonal antibodies disclosed in U.S. Patent No. 8,008,449 and International Publication No. 2006 / 121168. The entire disclosures of these patents are incorporated herein by reference. In some embodiments, by way of non-limiting example, the nivolumab or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO: 538); and / or a light chain comprising the amino acid sequence of (SEQ ID NO: 539).

[0415] In some embodiments, the targeting moiety comprises the anti-PD-1 antibody pidilizumab (also known as CT-011, hBAT or hBAT-1), or a fragment thereof. In some embodiments, pidilizumab and other humanized anti-PD-I monoclonal antibodies are selected from the pidilizumab and other humanized anti-PD-1 monoclonal antibodies disclosed in US Patent Application Publication No. 2008 / 0025980 and International Publication No. 2009 / 101611. The entire disclosures of these patents are incorporated herein by reference. In some embodiments, by way of non-limiting example, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises one or more light chain variable regions comprising an amino acid sequence selected from the sequences disclosed in US Patent Application Publication No. 2008 / 0025980: (SEQ ID NO: 540); (SEQ ID NO: 541); (SEQ ID NO: 542); and (SEQ ID NO: 543); and / or a heavy chain variable region comprising an amino acid sequence selected from the sequences disclosed in US Patent Application Publication No. 2008 / 0025980: (SEQ ID NO: 544); (SEQ ID NO: 545); (SEQ ID NO: 546); (SEQ ID NO: 547) and (SEQ ID NO: 548).

[0416] In some embodiments, the targeting moiety comprises a light chain comprising (SEQ ID NO: 549); and a heavy chain comprising (SEQ ID NO: 550).

[0417] In some embodiments, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0418] In some embodiments, the targeting moiety comprises the PD-L2-Fc fusion protein AMP-224 or a fragment thereof, which is disclosed in International Publication Nos. 2010 / 027827 and 2011 / 066342. The entire disclosures of these patents are incorporated herein by reference. In some embodiments, the targeting moiety comprises a B7-DC fusion protein comprising (SEQ ID NO: 551) and / or (SEQ ID NO: 552).

[0419] In some embodiments, the targeting moiety comprises the peptide AUNP12 or any other peptide disclosed in U.S. Patent Application Publication No. 2011 / 0318373 or U.S. Patent No. 8,907,053. In some embodiments, by way of non-limiting example, the targeting moiety is: SNTSESFK (SNTSESF) FRVTQLAPKAQIKE-NH 2 (SEQ ID NO: 553) [Chemical Formula] and comprises the AUNP12 sequence of (i.e., Compound 8 of U.S. Patent Application Publication No. 2011 / 0318373).

[0420] In some embodiments, the targeting moiety comprises the anti-PD-1 antibody 1E3, or a fragment thereof, disclosed in U.S. Patent Application Publication No. 2014 / 0044738. The entire disclosure of this patent is incorporated herein by reference. In some embodiments, by way of non-limiting example, 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 554); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 555).

[0421] In certain embodiments, the targeting moiety comprises the anti-PD-1 antibody 1E8, or a fragment thereof, disclosed in U.S. Patent Application Publication No. 2014 / 0044738. The entire disclosure of this patent is incorporated herein by reference. In some embodiments, by way of non-limiting example, 1E8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 556); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 557).

[0422] In some embodiments, the targeting moiety comprises the anti-PD-1 antibody 1H3, or a fragment thereof, disclosed in U.S. Patent Application Publication No. 2014 / 0044738. The entire disclosure of this patent is incorporated herein by reference. In some embodiments, by way of non-limiting example, the 1H3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 558); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 559).

[0423] In some embodiments, the targeting moiety comprises a VHH directed to PD-1, disclosed in U.S. Patent No. 8,907,065 and International Publication No. 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In some embodiments, by way of non-limiting example, the VHH against PD-1 comprises one or more of the sequences disclosed in U.S. Patent No. 8,907,065: (SEQ ID NO: 560); (SEQ ID NO: 561); (SEQ ID NO: 562); (SEQ ID NO: 563); or (SEQ ID NO: 564).

[0424] In some embodiments, the targeting moiety comprises any one of the anti-PD-1 antibodies or fragments thereof disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and International Publication No. 2010 / 036959. The entire contents of these patents are incorporated herein by reference. In some embodiments, by way of non-limiting example, the antibody or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising one or more amino acid sequences selected from the sequences disclosed in U.S. Patent Application Publication No. 2011 / 0271358: (SEQ ID NO: 565); (SEQ ID NO: 566); (SEQ ID NO: 567); (SEQ ID NO: 568); or (SEQ ID NO: 569); and / or a light chain comprising one or more amino acid sequences selected from the sequences disclosed in U.S. Patent Application Publication No. 2011 / 0271358: (SEQ ID NO: 570); (SEQ ID NO: 571); (SEQ ID NO: 572); or (SEQ ID NO: 573).

[0425] In some embodiments, the multi-specific FAP-binding substance of the present invention comprises one or more antibodies directed to PD-1 selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene Ltd.), or antibody fragments thereof.

[0426] In some embodiments, the multi-specific FAP-binding substance has one or more targeting moieties directed to PD-L1. In some embodiments, the FAP-binding substance has one or more targeting moieties that selectively bind to a PD-L1 polypeptide. In some embodiments, the FAP-binding substance comprises one or more of an antibody, antibody derivative or format, peptide or polypeptide, or fusion protein that selectively binds to a PD-L1 polypeptide.

[0427] In some embodiments, the multi-specific FAP-binding substance of the present technology comprises a VHH against PD-L1 having a variable domain comprising at least one of the PD-L1 CDR1, PD-L1 CDR2, and / or PD-L1 CDR3 sequences.

[0428] In some embodiments, the PD-L1 CDR1 sequence is selected from SEQ ID NOs: 574 to 604.

[0429] In some embodiments, the PD-L1 CDR2 sequence is selected from SEQ ID NOs: 605 to 635.

[0430] In some embodiments, the PD-L1 CDR3 sequence is selected from SEQ ID NOs: 636 to 666.

[0431] In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from the following sequences: 2LIG2: (SEQ ID NO: 667); or 2LIG3: (SEQ ID NO: 668); or 2LIG16: (SEQ ID NO: 669); or 2LIG22: (SEQ ID NO: 670); or 2LIG27: (SEQ ID NO: 671); or 2LIG29: (SEQ ID NO: 672); or 2LIG30: (SEQ ID NO: 673); or 2LIG34: (SEQ ID NO: 674); or 2LIG35: (SEQ ID NO: 675); or 2LIG48: (SEQ ID NO: 676); or 2LIG65: (SEQ ID NO: 677); or 2LIG85: (SEQ ID NO: 678); or 2LIG86: (SEQ ID NO: 679); or 2LIG89: (SEQ ID NO: 680); or 2LIG97: (SEQ ID NO: 681); or 2LIG99: (SEQ ID NO: 682); or 2LIG109: (SEQ ID NO: 683); or 2LIG127: (SEQ ID NO: 684); or 2LIG139: (SEQ ID NO: 685); or 2LIG176: (SEQ ID NO: 686); or 2LIG189: (SEQ ID NO: 687); or 3LIG3: (SEQ ID NO: 688); or 3LIG7: (SEQ ID NO: 689); or 3LIG8: (SEQ ID NO: 690); or 3LIG9: (SEQ ID NO: 691); or 3LIG18: (SEQ ID NO: 692); or 3LIG20: (SEQ ID NO: 693); or 3LIG28: (SEQ ID NO: 694); or 3LIG29: (SEQ ID NO: 695); or 3LIG30: (SEQ ID NO: 696); or 3LIG33: (SEQ ID NO: 697).

[0432] In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include a terminal histidine tag sequence (i.e., HHHHHH: SEQ ID NO: 43).

[0433] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab), or a fragmen...

Claims

Claim 1: A fibroblast activation protein (FAP) binding agent comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 1087 and comprising a targeting moiety having three complementarity determining regions (CDR1, CDR2, and CDR3), CDR1 consists of the amino acid sequence of SEQ ID NO: 1108, CDR2 consists of the amino acid sequence of SEQ ID NO: 128, and CDR3 consists of the amino acid sequence of SEQ ID NO: 159; Fibroblast activation protein (FAP) binding agent.

2. The FAP-binding substance of claim 1, which is a full-length antibody, a single domain antibody, an antibody composed only of recombinant heavy chains (VHH), a single-chain antibody (scFv), an antibody composed only of shark heavy chains (VNAR), Fv, Fab, Fab', or F(ab')2.

3. The FAP binding substance of claim 1, further comprising one or more signal transduction substances, optionally the targeting moiety and the signal transduction substances being linked with one or more linkers, and optionally the signal transduction substances being selected from one or more of wild-type interferon, wild-type interleukin, wild-type tumor necrosis factor, or modified forms thereof. Claim 4: The signal transduction agent is a modified interferon alpha 2 (IFNα2) comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 176 or 177, wherein the modified IFNα2 has one or more mutations that confer improved safety compared to wild-type IFNα2 having the amino acid sequence of SEQ ID NO: 176 or 177; Optionally, said modified IFNα2 is: (a) one or more mutations at positions 144-154 relative to SEQ ID NO:176 or SEQ ID NO:177; (b) one or more mutations at positions R149, L15, A19, R22, R23, L26, F27, L30, K31, D32, R33, H34, D35, Q40, H57, E58, Q61, F64, N65, T69, L80, Y85, Y89, T106, D114, L117, R120, R125, K133, K134, R144, A145, M148, S152, L153, ​​and N156 relative to SEQ ID NO: 176 or 177; and Optionally, the mutation is: (a) R149A, L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57Y, E58N, Q61S, F64A, N65A, T69A based on SEQ ID NO: 176 or 177 , L80A, Y85A, Y89A, D114R, L117A, R120A, R125A, K133A, K134A, R144A, A145G, A145M, M148A, S152A, L153A, and N156A, optionally M148A, R149A, or L153A; or (b) one or more mutations selected from R149A, R33A, T106X 3 , R120E, R144X 1 , A145X 2 , M148A, and L153A based on the amino acid sequence of SEQ ID NO: 176 or 177, wherein X 1 is selected from A, S, T, Y, L, and I, X 2 is selected from G, H, Y, K, and D, and X 3 is selected from A and E; The FAP-binding substance according to claim 3 .

5. The signal transduction agent is a modified interferon IFNα1 having mutations at one or more amino acids at positions C86, L15, A19, R23, S25, L30, D32, R33, H34, Q40, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157 relative to SEQ ID NO: 1042, and optionally the mutations are C86S, L15A, A19W, R23A, S25A, L30A, L30V, D32A, R33K, R33A, R33Q, H34A, Q40A, C86A, D115R, L1 18A, K121A, K121E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E, R145G, R145H, R145I, R145K, R145L, R145N, R145Q, R145S, R145T, R145V, R145Y, A146D, A146E 4. The FAP-binding substance of claim 3, wherein the amino acid sequence is selected from the group consisting of A146G, A146H, A146I, A146K, A146L, A146M, A146N, A146Q, A146R, A146S, A146T, A146V, A146Y, M149A, M149V, R150A, S153A, L154A, and N157A.

6. The FAP binding substance of claim 3, wherein the signal transduction substance is a modified interferon beta (IFNβ) comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 178, the modified IFNβ having one or more mutations that confer improved safety compared to wild-type IFNβ having the amino acid sequence of SEQ ID NO: 178, optionally the modified IFNβ comprising one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155 of SEQ ID NO: 178, and optionally the modified IFNβ comprising one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G based on SEQ ID NO:

178.

7. The signal transduction agent is a modified tumor necrosis factor alpha (TNFα) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:182, wherein the modified TNFα has one or more mutations that confer reduced receptor binding affinity relative to wild-type TNFα having the amino acid sequence of SEQ ID NO:182, and optionally the modified TNFα has one or more mutations at positions 87, 97, 115, 29, 31, 32, 84, 85, 86, 88, 89, 145, 146, and 147 relative to SEQ ID NO:182, and optionally the mutations are Y87Q, Y87L, Y87A, Y87F, Y87H, I97A, I97Q, I97S, Y115G ... A, L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, V91G, V91A, T105G, P106G, A109Y, P113G, E127G, N137G, D143N, A145G, A145R, A145T, E146D, E146K, and S14 7D, and optionally the mutation at position Y87 is selected from Y87Q, Y87L, Y87A, and Y87F, and / or the mutation at position I97 is selected from I97A, I97Q, and I97S, and / or the mutation at position Y115 is selected from Y115A and Y115G.

8. A FAP-binding substance according to any one of claims 1 to 7, wherein the FAP-binding substance comprises one or more additional targeting moieties, optionally the one or more additional targeting moieties recognize or functionally modulate a tumor antigen and / or an antigen on an immune cell, optionally the immune cell is selected from a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, and an NK cell, optionally the one or more additional targeting moieties recognize or functionally modulate PD-1, PD-L1, PD-L2, CTLA4, OX40L, OX40, CD20, XCR1, Flt3, or Clec9A, and optionally the FAP-binding substance recognizes or binds to FAP without substantially functionally modulating its activity.

9. A recombinant nucleic acid encoding a FAP-binding substance described in any one of claims 1 to 8.

10. A host cell containing the recombinant nucleic acid described in claim 9.

11. Use of a FAP binding substance according to any one of claims 1 to 8 in the manufacture of a medicament for treating or preventing cancer.

12. The use of claim 11, further comprising a chemotherapeutic agent that is a DNA intercalating agent such as doxorubicin, cisplatin, daunorubicin, or epirubicin, and optionally wherein the FAP-binding agent directly or indirectly recruits immune cells to the tumor or tumor microenvironment.

13. Use of a FAP-binding substance according to any one of claims 1 to 8 in the manufacture of a medicament for treating or preventing an autoimmune disease and / or a neurodegenerative disease, comprising: optionally, said autoimmune and / or neurodegenerative disease is multiple sclerosis; Optionally, the FAP binding agent results in immunosuppression in the patient. use.

14. Use of a FAP-binding substance according to any one of claims 1 to 8 in the manufacture of a medicament for treating or preventing a fibrotic disease, comprising: Optionally, the fibrotic disease is selected from liver fibrosis, pulmonary fibrosis, primary sclerosing cholangitis (PSC), chronic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis C infection, alcoholic liver disease, liver injury, cirrhosis, and myelodysplastic syndrome; use.

15. A chimeric protein comprising a FAP-binding substance according to any one of claims 1 to 8 and a wild-type or modified signal transduction substance.

16. A recombinant nucleic acid encoding the chimeric protein described in claim 15.

17. A host cell containing the recombinant nucleic acid described in claim 16.

18. The FAP-binding substance according to any one of claims 1 to 8. an Fc domain, optionally with one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in said Fc domain, and / or stabilize the hinge region in said Fc domain; and Wild-type or modified signal transduction An Fc-based chimeric protein complex comprising:

19. A recombinant nucleic acid encoding the Fc-based chimeric protein described in claim 18.

20. A host cell containing the recombinant nucleic acid described in claim 19.

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