CLEC9a binding agents and use thereof

Abstract

To provide CLEC9A binding agents and use thereof.SOLUTION: The present invention relates, in part, to agents that bind Clec9A and their use as diagnostic and therapeutic agents. The present invention further relates to pharmaceutical compositions comprising the Clec9A binding agents and their use in the treatment of various diseases.SELECTED DRAWING: Figure 7B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 542,944, filed August 9, 2017, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates, in part, to binding agents that bind Clec9A and their use as therapeutic and diagnostic agents.

[0003] Explanation of the electronically submitted text file The contents of the following text file, submitted electronically herewith, are incorporated by reference in their entirety into this specification: Computer-readable copy of sequence listing (file name: ORN-033PC_ST25, creation date: August 8, 2018, file size: 808 KB). [Background technology]

[0004] Dendritic cells (DCs) are antigen-presenting cells that process antigens and present them to other cells of the immune system. In particular, dendritic cells can capture antigens and present them on their surface to activate T cells, such as cytotoxic T cells (CTLs). Furthermore, activated dendritic cells can recruit additional immune cells, such as macrophages, eosinophils, natural killer cells, and T cells, such as natural killer T cells.

[0005] Given the important role of dendritic cells in immunity, deviations in dendritic cell function have been linked to diseases such as cancer and autoimmune diseases such as multiple sclerosis. For example, cancer cells can prevent dendritic cell recruitment and activation, rendering dendritic cells ineffective, thereby evading immune detection and destruction. Furthermore, dendritic cells have been found in the brain during central nervous system inflammation and may be involved in the pathogenesis of autoimmune diseases of the brain.

[0006] Thus, there remains a need for improved therapies for diseases, including cancer and multiple sclerosis, through the modification of dendritic cell function. Summary of the Invention [Means for solving the problem]

[0007] In various aspects, the present invention relates to Clec9A-binding agents having at least one targeting moiety that specifically binds to Clec9A. In various embodiments, these Clec9A-binding agents bind to Clec9A but do not functionally modulate (e.g., partially or completely neutralize) Clec9A. Thus, in various embodiments, the Clec9A-binding agents of the present invention are used, for example, to directly or indirectly recruit Clec9A-expressing cells to a site of interest while allowing the cells to signal through Clec9A (i.e., binding of the Clec9A-binding agent does not reduce or eliminate Clec9A signaling at the site of interest). In certain embodiments, the targeting moiety is a single-domain antibody (VHH). In various embodiments, the Clec9A-binding agent further comprises a signaling agent, such as, but not limited to, interferon, interleukin, and tumor necrosis factor, which can be modified to attenuate its activity. In various embodiments, the Clec9A-binding agent comprises an additional targeting moiety that binds to another target of interest (e.g., an antigen, receptor). In one embodiment, the other target of interest (e.g., antigen, receptor) is present on a tumor cell. In another embodiment, the other target of interest (e.g., antigen, receptor) is present on an immune cell. In some embodiments, Clec9A-binding agents of the invention can directly or indirectly recruit immune cells (e.g., dendritic cells) to a site of action (such as, by way of non-limiting example, the tumor microenvironment). In some embodiments, Clec9A-binding agents of the invention promote antigen (e.g., tumor antigen) presentation by dendritic cells.

[0008] In various embodiments, the Clec9A binding agents of the invention are used in the treatment of various diseases or disorders, such as cancer, infectious diseases, immune disorders, and other diseases and disorders, and the invention further encompasses various methods of treatment. [Brief explanation of the drawings]

[0009] [Figure 1-1] Figure 1 shows the nucleotide sequences of 66 different VHHs specific for human Clec9A. Gaps were introduced to align the sequences. The sequences in Figure 1 are assigned sequence identifiers as shown in Example 1. [Figure 1-2] As described for Figure 1-1. [Figure 1-3] As described for Figure 1-1. [Figure 1-4] As described for Figure 1-1. [Figure 1-5] As described for Figure 1-1. [Figure 1-6] As described for Figure 1-1. [Figure 1-7] As described for Figure 1-1. [Figure 1-8] As described for Figure 1-1. [Figure 2-1] Figure 2 shows the amino acid sequences of 66 different VHHs specific to human Clec9A. The complementarity-determining regions (CDR1, CDR2, and CDR3) shown were determined according to Kabat. Gaps were introduced to align the sequences. The 66 different VHHs belong to 25 different CDR3 groups (see Figure 3). VHHs belonging to the same group are highly similar, suggesting that their amino acid sequences arise from somatic hypermutation or from clonally related B cells derived from the same B cell but diversified due to RT and / or PCR errors during library construction. Although VHHs belonging to the same group recognize the same epitope, their other properties (e.g., affinity, potency, stability, expression level, etc.) may differ. The sequences in Figure 2 are assigned sequence identifiers as shown in Example 1. [Figure 2-2]As described for Figure 2-1. [Figure 2-3] As described for Figure 2-1. [Figure 2-4] As described for Figure 2-1. [Figure 3] FIG. 3 is a table showing that 66 different VHHs belong to 25 different CDR3 groups. [Figure 4] FIG. 4 shows the binding of various VHHs to HEK293 T cells transfected with human Clec9A. [Figure 5] Figure 5 shows a human dendritic cell pSTAT1 signaling assay. The chimeras investigated were various anti-human Clec9A VHH / human IFNα R149A. Two doses of substance were investigated: 100 ng / ml and 500 ng / ml. PBS was the control, and data are expressed as fold change in the percentage of pSTAT1+ dendritic cells (data are the average of triplicate data sets). [Figure 6] FIG. 6 shows the purification and production of Clec9A targeting moieties R1CHCL50, 3LEC89, and their variants. [Figure 7A] FIG. 7A is a graph showing the biological activity of a chimeric protein having a Clec9A targeting moiety (R1CHCL50) and a mutated human IFNα2 (R149A) signaling moiety on HL116 and HL116-hCle9A cells. [Figure 7B] FIG. 7B is a graph showing the biological activity of a chimeric protein having a Clec9A targeting moiety (3LEC89) and a mutated human IFNα2 (R149A) signaling moiety on HL116 and HL116-hCle9A cells. [Figure 8] Figure 8 is a graph showing the in vivo antitumor activity of CLEC9A-based AFNs (e.g., 2LEC16-hIFNa2_R149A, 3LEC22-hIFNa2_R149A, 1LEC28-hIFNa2_R149A, 3LEC30-hIFNa2_R149A, and 3LEC89-hIFNa2_R149A) in mice with a humanized immune system bearing RL tumors. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention is based, in part, on the discovery of agents that recognize and bind to Clec9A (e.g., antibodies, such as, by way of non-limiting example, VHHs). In some embodiments, the Clec9A-binding agents of the invention comprise one or more targeting moieties and / or one or more VHHs. They are part of chimeric or fusion proteins with several signal transduction agents. In some embodiments, these Clec9A binding agents bind to Clec9A but do not functionally regulate Clec9A.

[0011] In some embodiments, these Clec9A-binding agents can bind and directly or indirectly recruit immune cells to the site of therapeutic action (e.g., tumor or tumor microenvironment). In some embodiments, Clec9A-binding agents enhance tumor antigen presentation to elicit an effective anti-tumor immune response.

[0012] In some embodiments, the Clec9A-binding agent modulates tumor antigen presentation. In some embodiments, the Clec9A-binding agent modulates the immune response to avoid or reduce autoimmunity. In some embodiments, the Clec9A-binding agent results in immunosuppression. In some embodiments, the Clec9A-binding agent 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 reducing autoreactive T cells in a patient.

[0013] The present invention provides pharmaceutical compositions comprising Clec9A binding agents and their use in the treatment of various diseases, including cancer, autoimmune diseases, and / or neurodegenerative diseases.

[0014] Clec9A binding substance In various embodiments, the Clec9A-binding agents of the present invention are protein-based agents capable of specifically binding to Clec9A. In various embodiments, the Clec9A-binding agents of the present invention are protein-based agents capable of specifically binding to Clec9A without functional modulation (e.g., partial or complete neutralization) of Clec9A. Clec9A is a group 5 C-type lectin-like receptor (CTLR) expressed on the surface of a subset of dendritic cells (i.e., BDCA3+ dendritic cells) specialized in the uptake and processing of materials from dead cells. Clec9A recognizes conserved components in nucleated and anucleated cells that become exposed upon plasma membrane damage. Clec9A is expressed on the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis. Clec9A possesses a cytoplasmic immunoreceptor tyrosine-based activation motif that can recruit Syk kinase and induce pro-inflammatory cytokine production (see Huysamen et al. (2008), JBC, 283:16693-701).

[0015] In various embodiments, the Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that recognizes an epitope present on Clec9A. In certain embodiments, the antigen recognition domain recognizes one or more linear epitopes present on Clec9A. As used herein, a linear epitope refers to any contiguous sequence of amino acids present on Clec9A. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on Clec9A. As used herein, a conformational epitope refers to a portion of one or more amino acids (which may be discontinuous) that forms a three-dimensional surface with characteristics and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0016] In various embodiments, Clec9A binding agents of the invention may bind to full-length and / or mature forms and / or isoforms and / or splice variants and / or fragments and / or any other natural or synthetic analogs, variants, or mutants of human Clec9A. In various embodiments, Clec9A binding agents of the invention may bind to any form of Clec9A, including monomers, dimers, heterodimers, multimers, and associated forms. In one embodiment, the Clec9A binding agent binds to the monomeric form of Clec9A. In another embodiment, the Clec9A binding agent binds to the dimeric form of Clec9A. In a further embodiment, the Clec9A binding agent binds to a glycosylated form of Clec9A, which may be a monomer or a dimer.

[0017] In one embodiment, a Clec9A binding agent of the invention comprises a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on human Clec9A. In one embodiment, human Clec9A comprises the following amino acid sequence: MHEEEIYTSLQWDSPAPDTYQKCLSSNKCSGACCLVMVISCVFCMGLLTASIFLGVKLLQVSTIAMQQQEKLIQQERALLNFTEWKRSCALQMKYCQAFMQNSLSSAHNSSPCPNNWIQNRESCYYVSEIWSIWHTSQENCLKEGSTLLQIESKEEMDFITGSLRKIKGSYDYWVGLSQDGHSGRWLWQDGSSPSPGLLPAERSQSANQVCGYVKSNSLLSSNCSTWKYFICEKYALRSSV (SEQ ID NO: 1)

[0018] In various embodiments, the Clec9A binding agents of the present invention comprise a targeting moiety capable of specific binding. In various embodiments, the Clec9A binding agent comprises a targeting moiety having an antigen recognition domain, such as an antibody or derivative thereof. In certain embodiments, the Clec9A binding agent comprises a targeting moiety that is an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain comprises one variable region (V L ) and one constant region (C L ). The variable regions determine the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity-determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs are called 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.

[0019] In some embodiments, the Clec9A binding agent comprises a targeting moiety that is a specific antibody derivative or format. In some embodiments, Clec9A binding agents of the invention comprise the following targeting moieties: single domain antibodies, recombinant heavy chain antibodies (VHHs), single chain antibodies (scFvs), shark heavy chain antibodies (VNARs), microproteins (cysteine ​​knot proteins, knottins), DARPins; tetranectins; affibodies; transbodies; anticalins; adnectins; affilins; affimers; microbodies; aptamers; alterases; plastic antibodies; phylomers; stradobodies; maxibodies; shrimpbodies; phylomers; armadillo repeat proteins; Kunitz domains, avimers, atrimers, probodies, immunobodies, triomabs, troibodies, pepbodies, vaccibodies, unibodies; duobodies, Fvs, Fabs, Fab's, F(ab')2s, peptidomimetic molecules, or synthetic molecules. These include U.S. Pat. No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Pat. No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Pat. No. 7,250,297, U.S. Pat. No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Pat. No. 7,838,629, U.S. Pat. No. 7,186,524, U.S. Pat. Nos. 6,004,746, 5,475,096, 2004 / 146938, 2004 / 157209, 6,994,982, 6,794,144, 2010 / 239633, 7,803,907, 2010 / 119446, and / or 7,166,697. See also Storz MAbs. 2011 May-Jun;3(3):310-317. I want to be done that.

[0020] In some embodiments, the Clec9A binder comprises a targeting moiety that is a single domain antibody, such as a VHH. The VHH may be derived from organisms that produce VHH antibodies, such as camelids, sharks, or the VHH may be an engineered VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional antibodies from camelids that lack light chains. These heavy chain antibodies contain a single variable domain (VHH). H H) and two constant domains (CH2 and CH3).

[0021] In certain embodiments, the Clec9A binder comprises a VHH. In some embodiments, the VHH is a humanized or camelized VHH.

[0022] In some embodiments, the VHH is a fully human VHH. H In some embodiments, the fully human V domain is H The domain, e.g., HUMABODY, is monovalent, bivalent, or trivalent. In some embodiments, a fully human V H The domains, e.g., HUMABODY, may be monospecific or multispecific, such as monospecific, bispecific, or trispecific. Exemplary fully human V H Domains such as HUMABODIES are described, for example, in WO 2016 / 113555 and WO 2016 / 113557, the entire disclosures of which are incorporated herein by reference.

[0023] In some embodiments, the Clec9A binder comprises a targeting moiety that is a VHH comprising a single amino acid chain having four "framework regions" or FRs and three "complementarity-determining regions" or CDRs. As used herein, a "framework region" or "FR" refers to the region in the variable domain located between the CDRs. As used herein, a "complementarity-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.

[0024] In various embodiments, the Clec9A binder comprises a VHH having a variable domain comprising at least one of a CDR1, CDR2, and / or CDR3 sequence, hi various embodiments, the Clec9A binder comprises a VHH having a variable region comprising at least one of a FR1, FR2, FR3, and / or FR4 sequence.

[0025] In some embodiments, the CDR1 sequence is selected from the following: GRISSINSMG (SEQ ID NO: 2); GSITSINAMG (SEQ ID NO: 3); GRFFRVNAMG (SEQ ID NO: 4); GSSDSINAMG (SEQ ID NO: 5); GSVFSINAWG (SEQ ID NO: 6); GSILSINSMG (SEQ ID NO: 7); VSISSINSMG (SEQ ID NO: 8); GRVFSINAMG (SEQ ID NO: 9); VNIDTLNSMA (SEQ ID NO: 10); GGISSINSMG (SEQ ID NO: 11); GSMHSVNSMA (SEQ ID NO: 12); GDISSINAMG (SEQ ID NO: 13); GSIFSIDAMG (SEQ ID NO: 14); GSIFSINAMG (SEQ ID NO: 15); GSIFSIAAMG (SEQ ID NO: 16); GNIASITAMG (SEQ ID NO: 17); GFTF DDYAIG (SEQ ID NO: 18); GSISSINAMG (SEQ ID NO: 19); VSIFRSYFMG (SEQ ID NO: 20); GSIVSINAIG (SEQ ID NO: 21); RSFSSFNAMG (SEQ ID NO: 22); GSFSSINAMG (SEQ ID NO: 23); GTSFSINGMA (SEQ ID NO: 24); GRTFSTYAMG (SEQ ID NO: 25); GRIFDINAMG (SEQ ID NO: 26); GTLFSINGMA (SEQ ID NO: 27); GSIDSINAMG (SEQ ID NO: 28); GRAFSTNSMG (SEQ ID NO: 29); GSIISINSMG (SEQ ID NO: 30); RNFFSINAMG (SEQ ID NO: 31); GSIVSINSMG (SEQ ID NO: 32); GSIIGINSMG (SEQ ID NO: 33); GRTFPGYVMA (SEQ ID NO: 34); GRTFSINAMG (SEQ ID NO: 35); GRTLSSYTIG (SEQ ID NO: 36); GSFFSINAMG (SEQ ID NO: 37); GSIFSINSMG (SEQ ID NO: 38); GSIFSFNAMG (SEQ ID NO: 39); GRTFSTYAMA (SEQ ID NO: 40); VNIGSLNSMV (SEQ ID NO: 41); GRTLSNYAVG (SEQ ID NO: 42); GSVFSINAMG (SEQ ID NO: 43); GSIFEINSIG (SEQ ID NO: 44); GSIFNINSMG (SEQ ID NO: 45); VNIGTLNSMA (SEQ ID NO: 46); GRIGSINSMG (SEQ ID NO: 47); GRTLSNYAVA (SEQ ID NO: 48); RSFFSFNAMG (SEQ ID NO: 49); GIIFSINAMG (SEQ ID NO: 50); GRIFSVNAMG (SEQ ID NO: 51); GRTFSSYAMA (SEQ ID NO: 52); GSFSSI NVMG (sequence number 53); INSMG (sequence number 54); INAMG (sequence number 55); VNAMG (sequence number 56); INAWG (sequence number 57); LNSMA (sequence number 58); VNSMA (sequence number 59); IDAMG (sequence number 60); IAAMG (sequence number 61); SITAMG (sequence number 62); DYAIG (sequence number 63); SYFMG (sequence number 64); INAIG (sequence number 65); FNAMG (sequence number 66); INGMA (sequence number 67); TYAMG (sequence number 68); TNSMG (sequence number 69); GYVMA (sequence number 70); SYTIG (sequence number 71); TYAMA (sequence number 72); LNSMV (sequence number 73); NYAVG (sequence number 74); INSIG (sequence number 75); NYAVA (sequence number 76); SYAMA (sequence number 77); and INVMG (sequence number 78).

[0026] In some embodiments, the CDR2 sequence is selected from the following: AITNGGAKTYADSVKG (SEQ ID NO: 79); AITSGGRLSYADSVKG (SEQ ID NO: 80); AITNGGQTAYADSVKG (SEQ ID NO: 81); AITSGGRSTYIDSAKG (SEQ ID NO: 82); AITNQGRIAYAPSVNG (SEQ ID NO: 83); AITNDGRTTYVDSVKG (SEQ ID NO: 84); AVTVGGRYAYADSAKN (SEQ ID NO: 85); AITNQGATTYADSVKG (SEQ ID NO: 86); GITGSGQITYANSVRG (SEQ ID NO: 87); AITNGGRTVYGDSVKG (SEQ ID NO: 88) ;AITSGGRLAYAPSVNG (SEQ ID NO: 89); AITNGGRTTYVDSVKG (SEQ ID NO: 90); AITTGGRTTYVDSVKG (SEQ ID NO: 91); AITNQGRLTYADSVKG (SEQ ID NO: 92); AITSGGRRAYADSVKG (SEQ ID NO: 93); AITSASASRTTYADSVKG (SEQ ID NO: 94); CISRSDGSTYYDDSVKG (SEQ ID NO: 95); AITNQGRVTYADSVKG (SEQ ID NO: 96); AITDGGRLAYADSAKG (SEQ ID NO: 97); SITNQGIRNYSTSVMG (SEQ ID NO: 98) 98);AITNQGRTTYADSVKG (SEQ ID NO: 99);AITNGGRIAYGIAVNG (SEQ ID NO: 100);AITNGGRIAYSDSAKG (SEQ ID NO: 101);GITSDGSTGYADSVKG (SEQ ID NO: 102);AISWSGGSTYYADSVKG (SEQ ID NO: 103);AITDQGRLAYADSAKG (SEQ ID NO: 104);AITNGGQTTYADSVKG (SEQ ID NO: 105);AITTGGRTAYVDSVKG (SEQ ID NO: 106);AITSQGRITLADSVKG (SEQ ID NO: 107);AITVDGRLAYA DSAKH (SEQ ID NO: 108); AITNGGRIAYGTSVMG (SEQ ID NO: 109); AITNGGQIAYADSVKG (SEQ ID NO: 110); AITDQGRTTYADSVKG (SEQ ID NO: 111); GITTQGRITYGNSVRG (SEQ ID NO: 112); AITSGGRTTYVDSVKG (SEQ ID NO: 113); AINWRGGDTYYADSVKG (SEQ ID NO: 114); AITDGGAKTYADSVKG (SEQ ID NO: 115); AITNQGRLSYVDSVKG (SEQ ID NO: 116); AITNQGRRTYADSVKG (SEQ ID NO: 117);AITNGGRIAYTDSVKG (SEQ ID NO: 118); AITNGGRTTYADSVKG (SEQ ID NO: 119); AITDGGRLTYADSAKG (SEQ ID NO: 120); AISWSGGSTEYHDSVKG (SEQ ID NO: 121); AITNQGRIAYADSVKG (SEQ ID NO: 122); AINWSSGGISYSNSAKG (SEQ ID NO: 123); AITG; QGRTTYADSVKG (SEQ ID NO: 124); AITNGGQIVYADSVKG (SEQ ID NO: 125); AITTQGRTTYEDSVKG (SEQ ID NO: 126); AITSGGITNYANSVQG (SEQ ID NO: 127); AITVGGRLAYADSAKG (SEQ ID NO: 128); GITGGGQITYANSVRG (SEQ ID NO: 129); AITSQGRSTYADSAKG (SEQ ID NO: 130); AITNGGATVYADSVKG (SEQ ID NO: 131); AITDGGRLTYADSAKN (SEQ ID NO: 132); AINWSSGGISYSNAAK G (SEQ ID NO: 133); AITNXGRTTYADSVKG (SEQ ID NO: 134); AIWWASGGISYANSAKG (SEQ ID NO: 135); AITNQGAPTYADSVKG (SEQ ID NO: 136); RITNLGLPNYADSVTG (SEQ ID NO: 137); RITNLGLPNYADSVKG (SEQ ID NO: 138); AITNGGAKT (SEQ ID NO: 139); AITSGGRLS (SEQ ID NO: 140); AITNGGQTA (SEQ ID NO: 141); AITSGGRST (SEQ ID NO: 142); ITNQGRIA (SEQ ID NO: 143); ITNQGRIAYA PSVNG (SEQ ID NO: 144); AITNDGRTT (SEQ ID NO: 145); AVTVGGRYA (SEQ ID NO: 146); AITNQGATT (SEQ ID NO: 147); GITGSGQIT (SEQ ID NO: 148); AITNGGRTV (SEQ ID NO: 149); AITSGGRLA (SEQ ID NO: 150); AITNGGRTT (SEQ ID NO: 151); AITTGGRTT (SEQ ID NO: 152); AITNQGRLT (SEQ ID NO: 153); AITSGGRRA (SEQ ID NO: 154); AITSASASRTT (SEQ ID NO: 155); CISRSDGSTY (SEQ ID NO: 156); AITNQGRVT (SEQ ID NO: 157); AITDGGRLA (SEQ ID NO: 158); SITNQGIRN (SEQ ID NO: 159); AITNQGRTT (SEQ ID NO: 160); AITNGGRIA (SEQ ID NO: 161); GITSDGSTG (SEQ ID NO: 162); AISWSGGSTY (SEQ ID NO: 163); AITDQGRLA (SEQ ID NO: 164) AITNGGQTT (SEQ ID NO: 165); AITTGGRTA (SEQ ID NO: 166); AITSQGRIT (SEQ ID NO: 167); AITVDGRLA (SEQ ID NO: 168); AITNGGQIA (SEQ ID NO: 169);AITDQGRTT (SEQ ID NO: 170); GITTQGRIT (SEQ ID NO: 171); AITSGGRTT (SEQ ID NO: 172); AINWRGGDTY (SEQ ID NO: 173); AITDGGAKT (SEQ ID NO: 174); AITNQGRLS (SEQ ID NO: 175); AITNQGRRT (SEQ ID NO: 176); AITDGGRLT (SEQ ID NO: 177); AISWSGGSTE (SEQ ID NO: 178); AITNQGRIA (SEQ ID NO: 179); AINWSSGGIS (SEQ ID NO: 180); AITGQGRTT ( SEQ ID NO: 181); AITNGGQIV (SEQ ID NO: 182); AITTQGRTT (SEQ ID NO: 183); AITSGGITN (SEQ ID NO: 184); AITVGGRLA (SEQ ID NO: 185); GITGGGQIT (SEQ ID NO: 186); AITSQGRST (SEQ ID NO: 187); AITNGGATV (SEQ ID NO: 188); AITNXGRTT (SEQ ID NO: 189); AIWWASGGIS (SEQ ID NO: 190); AITNQGAPT (SEQ ID NO: 191); and RITNLGLPN (SEQ ID NO: 192).

[0027] In some embodiments, the CDR3 sequence is selected from the following: FTRRDDY (SEQ ID NO: 193); FQSSGID (SEQ ID NO: 194); WAADYQQY (SEQ ID NO: 195); WNRDRQQY (SEQ ID NO: 196); KPTPVYGSTVGDY (SEQ ID NO: 197); FTRDKDY (SEQ ID NO: 198); WDRDRQQY (SEQ ID NO: 199); FTRTDDY (SEQ ID NO: 200); YDRSSTPY (SEQ ID NO: 201); FTRGDDY (SEQ ID NO: 202); LNSATTY (SEQ ID NO: 203); YTRDEDY (SEQ ID NO: 204); FTRDEDY (SEQ ID NO: 205); KWYDPLVIE YYDN (SEQ ID NO: 206); KADHNDY (SEQ ID NO: 207); FRSGADDY (SEQ ID NO: 208); EVPSTYSCSGFREDY (SEQ ID NO: 209); FAASGMEY (SEQ ID NO: 210); WTTDRQQY (SEQ ID NO: 211); FAGWGKEDY (SEQ ID NO: 212); FSPTGDY (SEQ ID NO: 213); KPTPVYGSTVGDY (SEQ ID NO: 214); KASPVYGSTVEDY (SEQ ID NO: 215); STPRGDSY (SEQ ID NO: 216); EAEGSGREGNFYERS (SEQ ID NO: 217); WDRDR QQY (SEQ ID NO: 218); FTRSDDY (SEQ ID NO: 219); STPRGDSY (SEQ ID NO: 220); FTRTDDY (SEQ ID NO: 221); WTTLGTF (SEQ ID NO: 222); WVRDGQQY (SEQ ID NO: 223); KAIPVYGSTVEDY (SEQ ID NO: 224); KAAATHLSTVADY (SEQ ID NO: 225); FGRFDDY (SEQ ID NO: 226); WGVKTGPESGSGTL (SEQ ID NO: 227); FTRDEDY (SEQ ID NO: 228); RLTTEYDYAY (SEQ ID NO: 229); FTRGNDY (SEQ ID NO: 230); FQSSGID (SEQ ID NO: 231); FSPTDDF (SEQ ID NO: 232); KAIPIYGSTAEDY (SEQ ID NO: 233); FSLTDDY (SEQ ID NO: 234); WTRDRQQY (SEQ ID NO: 235); FTRDEDF (SEQ ID NO: 236); EVEGSGREGNFYGA (SEQ ID NO: 237); PGW DY (SEQ ID NO: 238); YDRSATAY (SEQ ID NO: 239); ASSVLSGTVDY (SEQ ID NO: 240); FAADGMEY (SEQ ID NO: 241); KAAASYVSTVADY (SEQ ID NO: 242); TAKDDY (SEQ ID NO: 243); FTGWGKEDY (SEQ ID NO: 244); WAADYQQY (SEQ ID NO: 245); YDRSATPY (SEQ ID NO: 246); WARDRQQY (SEQ ID NO: 247); WTKDRQQY (SEQ ID NO: 248); FTRTYDY (SEQ ID NO: 249); ASSILSGTVDY (SEQ ID NO: 250); WAADYQQY (SEQ ID NO: 251); KPAPVYGSTVGDY (SEQ ID NO: 252); FAADGMEY (SEQ ID NO: 253); FGSGGG (SEQ ID NO: 254); ASSVLSGTADY (SEQ ID NO: 255); VALKAEY (SEQ ID NO: 256); and EAEGSGREGNFYERS (SEQ ID NO: 257).

[0028] In various exemplary embodiments, the Clec9A binding agent comprises an amino acid sequence selected from the following sequences: 1LEC7 (SEQ ID NO: 258) QVQLQESGGGLVQPGGSLRLSCAASGRISSINSMGWYRQAPGNQRELVAAITNGGAKTYADSVKGRFTISTDNAGNTVYLQMDSLRPEDTAVYYCKAFTRRDDYWGQGTQITVSSAAAYPYDVPDYGSHHHHHH; 1LEC9 (SEQ ID NO: 259) QVQLQESGGGLVQAGGSLRLLSCAASGSITSINAMGWYRQAPGKQRELVAAITSGGRLSYADSVKGRFTISRDNAESTVALQMNSLKPEDTAVYSCAAFQSSGIDWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC26 (SEQ ID NO: 260) QVQLQESGGGLVQPGGSLRLSCAASGRFFRVNAMGWYRQAPGKQRELVAAITNGGQTAYADSVKGRFTISKESARNTVHLQMSSLKPEDTAVYYCTIWAADYQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC27 (SEQ ID NO: 261) QVQLQESGGGLVQAGESLRLSCAASGSSDSINAMGWYRQAPGKQRELVAAITSGGRSTYIDSAKGRATISRDNARNTAYLQMSSLKAEDTAVYYCTIWNRDRQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC28 (SEQ ID NO: 262) QVQLQESGGGLVQSGGSLRLSCAASGSVFSINAWGWYRQAPGKQRELVAAITNQGRIAYAPSVNGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCNAKPTPVYGSTVGDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC30 (SEQ ID NO: 263) QVQLQESGGGLVQAGGSLRLSCAASGSILSINSMGWYRPALGNQRELVAAITNDGRTTYVDSVKGRFTISRDNAKNTVYL QMNSLKPEDTAVYWCKAFTRDKDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC38 (SEQ ID NO: 264) QVQLQESGGGLVQTGGSLRLSCAASVSISSINSMGWYRQAPGKERELVAAVTVGGRYAYADSAKNRFTISRDDAQNTVHLQMSSLRAEDTAVYYCTIWDRDRQQYWGXGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC42 (SEQ ID NO: 265) QVQLQESGGGLVQPGGSLRLSCAASGRVFSINAMGWYRQAPGKQRELVAAITNQGATTYADSVKGRFTISRDTAGNTVYLQMNSLRPEDTAVHYCKAFTRTDDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC51 (SEQ ID NO: 266) QVQLQESGGGLVQAGGSLLSCAASVNIDTLNSMAWYRQAPGKQRELVAGITGSGQITYANSVRGRFTVSRDNAKSTVYLQMNTLQPEDTAVYYCAAYDRSSTPYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC61 (SEQ ID NO: 267) QVQLQESGGGLVQPGGSLRLSCAASGGISSINSMGWYRQAPGNQRELVAAITNGGRTVYGDSVKGRFTISRDSAGNTVHLQMDSLRPEDTGVYYCKAFTRGDDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC62 (SEQ ID NO: 268) QVQLQESGGGLVQPGGFLSLSCAASGSMHSVNSMAWYRQVPGKQRELVAAITSGGRLAYAPSVNGRFTISRDYAKNTIHLQMNSLEPEDTAVYYCAALNSATTYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC63 (SEQ ID NO: 269) QVQLQESGGGLVQAGGSLRLSCAATGDISSINAMGWHRPARGNERELVAAITNGGRTTYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCKAYTRDEDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC64 (SEQ ID NO: 270) QVQLQESGGGLVRAGGSLRLSCAASGSIFSIDAMGWYRPAHGEQRELVAAITTGGRTTYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCKAFTRDEDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC70 (SEQ ID NO: 271) QVQLQESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAAITNQGRLTYADSVKGRFTISRDNAKNTVFLQMDSLKPEDTAVYYCNAKWYDPLVIEYYDNWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC84 (SEQ ID NO: 272) QVQLQESGGGLVQPGGSLRLSCAASGSIFSIAAMGWYRQAPGKQRELVAAITSGGRRAYADSVKGRFTISRDNDENTVALQMNSLKPEDTDVYYCNAKADHNDYWGQGTQITVSSAAAYPYDVPDYGSHHHHHH; 1LEC88 (SEQ ID NO: 273) QVQLQESGGGLVQPGGSLRLSCAAIGNIASITAMGWYRQAPGKQRELVAAITSASASRTTYADSVKGRFTISRDNAKNTV YLQMNSLQPEDTAVYYCKGFRSGADDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC91 (SEQ ID NO: 274) QVQLQESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKEHEGVSCISRSDGSTYYDDSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEVPSTYSCSGFREDYKGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC92 (SEQ ID NO: 275) QVQLQESGGGLVQPGGSLRLSCAASGSISSINAMGWYRQAPGNQRELVAAITNQGRVTYADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCKVFAASGMEYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1LEC94 (SEQ ID NO: 276) QVQLQESGGGLVQAGESLRLSCAASVSIFRSYFMGWYRQAPGKQRELVAAITDGGRLAYADSAKGRFTISREDTRNTVHLQMSSLKAEDTAVYYCTIWTTDRQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC6 (SEQ ID NO: 277) QVQLQESGGGWVQPGGSLRLSCAATGSIVSINAIGWYRQAPGKQRELVASITNQGIRNYSTSVMGRFTISRDDVKNTVSLQMNSLKPEDSAVYYCKGFAGWGKEDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC13 (SEQ ID NO: 278) QVQLQESGGGLVQAGASLRLLSCAASGSIFSINAMGWYRQAPGKQRELVAAITNQGRTTYADSVKGRFTISRDNAKNTVYLQMDSLEPEDTAIYYCKGFSPTGDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC16 (SEQ ID NO: 279) QVQLQESGGGLVQPGGSLRLSCLASRSFSSFNAMGWYRQAPGKERELVAAITNGGRIAYGIAVNGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAKPTPVYGSTVGDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC20 (SEQ ID NO: 280) QVQLQESGGGLVQAGGSLTLSCAASGSFSSINAMGYYRQAPGKQRELVAAITNGGRIAYSDSAKGRFTISRDSAKNTMYLQMNSLKPEDTDVYYCNAKASPVYGSTVEDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC23 (SEQ ID NO: 281) QVQLQESGGGLVQPGGSLRLSCAASGTSFSINGMAWYRQAPGGQRELVGGITSDGSTGYADSVKGRFTVSRDNAKNTVYLQMNRLKPEDTAVYYCGTSTPRGDSYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC24 (SEQ ID NO: 282) QVQLQESGGGLVQAGGSLLRLSCAASGRTFSTYAMGWFRQAPGKERGLVAAISWSGGSTYYADSVKGRFTIFRDNAENTVYLQMNSLKPEDTAVYYCAAEAEGSGREGNFYERSWYQGQGTQVTVSSAAAYPYDVPDYGSHHHHH; 2LEC26 (SEQ ID NO: 283) QVQLQESGGGLVETGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAAITDQGRLAYADSAKGRFTISRENARNTLHL QMSSLKAEDTAVYYCTIWDRDRQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC38 (SEQ ID NO: 284) QVQLQESGGGLVQPGGSLRLSCAASGRIFDINAMGWYRQAPGKQRELVAAITNGGQTTYADSVKGRFTISRDNAGNTVYLQMNSLRPEDTAVYYCKAFTRSDDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC48 (SEQ ID NO: 285) QVQLQESGGGLVQAGGSLRLSCAASGTLFSINGMAWYRQAPGKRRELVGGITSDGSTGYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCGTSTPRGDSYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC53 (SEQ ID NO: 286) QVQLQESGGGLVQAGGSLRLLSCAASGSIDSINAMGWYRPALGEQRELVAAITTGGRTAYVDSVKGRFTISRDAAKNTVYLQMNSLKPEDTAVYSCKAFTRDTDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC54 (SEQ ID NO: 287) QVQLQESGGGLAQPGGSLQLSCAASGRAFSTNSMGWYRQASGKQRELVAAITSQGRITLADSVKGRFTISSDNTKNTVFLQMNSLKPEDTAVYYCNAWTTLGTFGGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC55 (SEQ ID NO: 288) QVQLQESGGGLVQTGESLSLSCAVASGSIISINSMGWYRQAPEKQRELVAAITVDGRLAYADSAKHRFTISSKESARNTVHLHMSSLKPEDTAVYYCTIWVRDGQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC59 (SEQ ID NO: 289) QVQLQESGGGLVQPGGSLRLSCAVSRNFFSINAMGWYRQAPGKQRELVAAITNGGRIAYGTSVMGRFTISRDDAKNTVDLQMNSLRPEDTAVYYCNAKAIPVYGSTVEDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC60 (SEQ ID NO: 290) QVQLQESGGGLVQPGGSLRLSCAASGRFFRVNAMGWYRQVPGKQRELVAAITNGGQIAYADSVKGRFTISRDSAKNTVYLQMNSLKSEDTDVYYCNAKAAATHLSTVADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC61 (SEQ ID NO: 291) QVQLQESGGGLVQPGGSLRLSCAASGSIVSINSMGWYRQAPGKQRELVAAITDQGRTTYADSVKGRFTISRDDAKNKNTVYLQMNSLKAEDTAVYACKAFGRFDDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC62 (SEQ ID NO: 292) QVQLQESGGGLVQPGGSLRLSCAAYGSIFSINAMGWYRQAPGKERELVAGITTQGRITYGNSVRGRFTISGDNAKNTVYLQMKSLKPEDTAVYYCSAWGVKTGPESGSGTLEGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC63 (SEQ ID NO: 293) QVQLQESGGGLVQAGGSLRLSCAASGSIIGINSMGYYRTAPGKQRELVAAITSGGRTTYVDSVKGRFTISRDNAKNTVYL QMNSLKPEDTAVYFCKAFTRDEDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC67 (SEQ ID NO: 294) QVQLQESGGGLVQAGGSLLRLSCAASGRTFPGYVMAWFRQSPGQEREFAAAINWRGGDTYYADSVKGRFTISRDNVKNTVFLQMNSLKPEDTAVYFCAARLTTEYDYAYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC68 (SEQ ID NO: 295) QVQLQESGGGLVQPGESLRLSCAASGSIFSINAMGWYRQAPGKQRELVAAITDGGAKTYADSVKGRFTISTDNAGNTVYLQMDSLRPEDTAVYYCKAFTRGNDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC76 (SEQ ID NO: 296) QVQLQESGGGLVQAGESLRLSCVVSGRTFSINAMGWYRQAPGKQRELVAAITNQGRLSYVDSVKGRFTISRDNAANTVYLQMNSLKPEDTAVYYCAAFQSSGIDWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC83 (SEQ ID NO: 297) QVQLQESGGGLVQAGGSLLSCAASGRTLSSYTIGWYRQAPGKQRELVAAITNQGRRTYADSVKGRFTISRDNAKNTVYLQMDSLKSEDTAVYYCKGFSPTDDFWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC88 (SEQ ID NO: 298) QVQLQESGGGLVQPGGSLRLSCTASGSFFSINAMGWYRQAPGNQRELVAAITNGGRIAYTDSVKGRFTISNDNAKNTVYLQMNSLKPEDTDVYYCNAKAIPIYGSTAEDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC89 (SEQ ID NO: 299) QVQLQESGGGLVQAGGSLLSCAASGSIFSINSMGWYRQAPGKQRELVAAITNGGRTTYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCKGFSLTDDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC90 (SEQ ID NO: 300) QVQLQESGGGLVQTGGSLRLSCAASGSIFSFNAMGWYRQAPGKQRELVAAITDGGRLTYADSAKGRFTISRENTRNTVHLQMSSLKAEDTADYYCTIWTRDRQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC93 (SEQ ID NO: 301) QVQLQESGGGLVQAGGSLLSCAASGSIFSINAMGWYRPALGEQRELVAAITTGGRTTYVDSVKGRFSISRDNAKNTVYLQMNSLKPEDTAVYFCKAFTRDEDFWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2LEC95 (SEQ ID NO: 302) QVQLQESGGGLVQAGGSLRLSCEASGRTFSTYAMAWFRQAPGKERDLVAAISWSGGSTEYHDSVKGRFTISRDNTKNTVYLQMNSLKAEDTAVYYCAAEVEGSGREGNFYGASWYPGQGTQVTVSSAAAYPYDVPDYGSHHHHH; 3LEC4 (SEQ ID NO: 303) QVQLQESGGGLVQPGGSLRLSCAASGSFFSINAMGWYRQAPGKQRELVAAITNQGRIAYADSVKGRFTISRDNAKNTVYL QMNSLKPEDTAVYYCGRPGWDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC6 (SEQ ID NO: 304) QVQLQESGGGLVQAGGSLRLSCVASVNIGSLNSMVWYRQSPGKQRELVAGITGSGQITYANSVRGRFTVSRDIAKSTAYLQMNTLKPEDTAVYYCAAYDRSATAYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC9 (SEQ ID NO: 305) QVQLQESGGGLVQAGGSLRVSCAASGRTLSNYAVGWWRQAPGKQREFVAAINWSSGGISYSNSAKGRFALSRDNAKNTVYLQMDSLKPEDTAVYYCAAASSVLSGTVDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC11 (SEQ ID NO: 306) QVQLQESGGGLVQPGGSLRLSCAASGSISSINAMGWYRQAPGKQRELVAAITGQGRTTYADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCKVFAADGMEYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC13 (SEQ ID NO: 307) QVQLQESGGGLVQPGGSLRLSCAASGRFFRVNAMGWYRQAPGKQRELVAAITNGGQIVYADSVKGRFTISRDSAKNTVYLQMNSLKSEDTAVYYCNAKAAASYVSTVADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC15 (SEQ ID NO: 308) QVQLQESGGGLVQAGGSLLSCAASGSVFSINAMGWYRQAPEKQRELVAAITTQGRTTYEDSVKGRFTISRDGAQNTVYLQMDSLKPEDTAVYYCKAWTAKDDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC22 (SEQ ID NO: 309) QVQLQESGGGRVQPGGSLRLSCAAIGSIFEINSIGWYRQAPGKQRELVAAITSGGITNYANSVQGRSTISRDNVNNTVYLQMNSLKPEDSAVYYCKGFTGWGKEDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC23 (SEQ ID NO: 310) QVQLQESGGGLVQTGGSLRLSCAASGSIFNINSMGWYRQAPGKQRELVAAITVGGRLAYADSAKGRFTISKESARNTVHLQMSSLKPEDTAVYYCTIWAADYQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC27 (SEQ ID NO: 311) QVQLQESGGGLVQAGGSLLSCAASVNIGTLNSMAWYREAPGKQRELVAGITGGGQITYANSVRGRFTVSRDIAKSTAYLQMNTLKPEDTAVYYCAAYDRSATPYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC30 (SEQ ID NO: 312) QVQLQESGGGLVQTGGSLRLSCAASGSIFSINSMGWYRQAPGKQRELVAAITSQGRSTYADSAKGRFTISLGNARNTVNLQMSSLKTEDTAVYYCTIWARDRQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC36 (SEQ ID NO: 313) QVQLQESGGGLVQPGGSLRLSCAASGRIGSINSMGWYRQAPGKQREMVAAITNGGATVYADSVKGRFTISRDNAGNTVDL HMNSLRPEDSAVYYCKAFTRGDDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC55 (SEQ ID NO: 314) QVQLQESGGGLVQPGGSLKLSCAASGSIFSFNAMGWYRQAPGKQRELVAAITDGGRLTYADSAKNRFTISRENTRNTVHLQMSSLKAEDTAVYYCTIWTKDRQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC57 (SEQ ID NO: 315) QVQLQESGGGLVQPGGSLRLSCAASGRISSINSMGWYRQAPGKQRELVAAITNGGAKTYADSVKGRFTISRDGAGNTVYLQMDNLRPEDTAVYYCKAFTRTYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC61 (SEQ ID NO: 316) QVQLQESGGGLVQAGGSLRVSCAASGRTLSNYAVAWFRQAPGKQREFVAAINWSSGGISYSNAAKGRFALSRDNAKNTVYLQMDSLKPEDTAVYYCAAASSILSGTVDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC62 (SEQ ID NO: 317) QVQLQESGGGLVQPGGSLRLSCAASGRIGSINSMGWYRQAPGKQREMVAAITNGGATVYADSVKGRFTISRDNAGNTVDLHMNSLRPEDSAVYYCTIWAADYQQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC66 (SEQ ID NO: 318) QVQLQESGGGLVQPGGSLRLSCAASRSFFSFNAMGWYRQAPGKQRELVAAITNGGRIAYGTSVMGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNAKPAPVYGSTVGDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC69 (SEQ ID NO: 319) QVQLQESGGGLVQPGGSPRLSCAASGRFFRVNAMGWYRQAPGKQRELVAAITNGGQTAYADSVKGRFTISRDSAKNTVYLQMNSLKSEDTAVYYCKVFAADGMEYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC76 (SEQ ID NO: 320) QVQLQESGGGLVQPGESLRLSCAASGIIFSINAMGWYRQAPGKQRELVAAITNXGRTTYADSVKGRFTISRDNAKNTVTLQMNSLKPEDTAVYYCNAFGSGGGVGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC82 (SEQ ID NO: 321) QVQLQESGGGLVQAGGSLRLLSCAASGRTLSNYAVAWFRQAPGKQRELVAAIWWASGGISYANSAKGRFVLSRDNAKNTVYLQMDSLKPEDTAVYYCAAASSVLSGTADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3LEC89 (SEQ ID NO: 322) QVQLQESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAGNTVYLQMNSLRPEDTAVYYCKAFTRGDDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; or 3LEC94 (SEQ ID NO: 323) QVQLQESGGGSVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGMERELVAAISWSGGSTYYADSVKGRFTISRDNAENTVY LQMNSLKPEDTAVYYCAAEAEGSGREGNFYERSWYQGQGTQVTVSSAAAYPYDVPDYGSHHHHHH.

[0029] In various exemplary embodiments, the Clec9A binding agent comprises an amino acid sequence selected from any one of the sequences provided above, minus the terminal histidine tag sequence (ie, HHHHHH: SEQ ID NO: 324).

[0030] In some embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 258-323 without the HA tag (ie, YPYDVPDYGS; SEQ ID NO: 325).

[0031] In some embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 258-323 (provided above) without the AAA linker (ie, AAA).

[0032] In some embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 258-323 (provided above) without the AAA linker, the HA tag, and the terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 326).

[0033] In various exemplary embodiments, the Clec9A binding agent comprises an amino acid sequence selected from the following sequences: R1CHCL50: QVQLVESGGGLVHPGGSLRLSCAASGSFSSINVMGWYRQAPGKERELVARITNLGLPNYADSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYLVALKAEYWGQGTQVTVSS (SEQ ID NO: 327); R1CHCL50_opt1(E1D-A74S-K83R-Q108L): DVQLVESGGGLVHPGGSLRLSCAASGSFSSINVMGWYRQAPGKERELVARITNLGLPNYADSVTGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCYLVALKAEYWGQGTLVTVSS (SEQ ID NO: 328); R1CHCL50_opt2(E1D-A74S-K83R-Q108L-H13Q): DVQLVESGGGLVQPGGSLRLSCAASGSFSSINVMGWYRQAPGKERELVARITNLGLPNYADSVTGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCYLVALKAEYWGQGTLVTVSS (SEQ ID NO: 329); R1CHCL50_opt3(E1D-A74S-K83R-Q108L-T64K): DVQLVESGGGLVHPGGSLRLSCAASGSFSSINVMGWYRQAPGKERELVARITNLGLPNYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCYLVALKAEYWGQGTLVTVSS (SEQ ID NO: 330); R1CHCL50_opt4(E1D-A74S-K83R-Q108L-H13Q-T64K): DVQLVESGGGLVQPGGSLRLSCAASGSFSSINVMGWYRQAPGKERELVARITNLGLPNYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCYLVALKAEYWGQGTLVTVSS (SEQ ID NO: 331); 3LEC_89(wild type); QVQLQESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAGNTVYLQMNSLRPEDTAVYYCKAFTRGDDYWGQGTQVTVSS (SEQ ID NO: 332); 3LEC_89_opt1(E1D-Q5V-Q108L): DVQLVESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAGNTVYLQMNSLRPEDTAVYYCKAFTRGDDYWGQGTLVTVSS (SEQ ID NO: 333); 3LEC_89_opt2(E1D-Q5V-Q108L-A74S): DVQLVESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNSGNTVYLQMNSLRPEDTAVYYCKAFTRGDDYWGQGTLVTVSS (SEQ ID NO: 334); 3LEC_89_opt3(E1D-Q5V-Q108L-G75K): DVQLVESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCKAFTRGDDYWGQGTLVTVSS (SEQ ID NO: 335); and 3LEC_89_opt4(E1D-Q5V-Q108L-A74S-G75K): DVQLVESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCKAFTRGDDYWGQGTLVTVSS (SEQ ID NO: 336).

[0034] In one embodiment, the targeting moiety comprises an anti-Clec9A antibody as disclosed in Tullett et al., JCI Insight. 2016;1(7):e87102, the entire disclosure of which is incorporated herein by reference.

[0035] In various embodiments, the present invention contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologues (collectively referred to herein as "analogs") of the Clec9A binding agents of the invention described herein. In various embodiments, the amino acid sequence of the Clec9A binding agent further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0036] In various embodiments, the Clec9A binding agent comprises a targeting moiety comprising a sequence that is at least 60% identical to any one of the sequences disclosed herein. For example, the Clec9A binding agent may comprise a targeting moiety that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, or at least about 81% identical to any of the sequences disclosed herein. %, 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., at least about 6% identical to any one of the sequences disclosed herein, e.g., SEQ ID NOs: 327-336). 0%, 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 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).

[0037] In various embodiments, the Clec9A binding agent 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 various embodiments, the Clec9A binding agent comprises a targeting moiety comprising an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acid mutations relative to any one of the sequences disclosed herein. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0038] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0039] "Conservative substitutions" can be made, for example, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0040] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid in the same group of the six standard amino acid groups. For example, replacing Asp with Glu maintains one negative charge in the modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0041] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid from a different group of the six standard amino acid groups (1) to (6) above.

[0042] In various embodiments, substitutions may also include non-classical amino acids. Representative 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, fluoroamino acids, designer amino acids such as β-methylamino acids, C These include α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general. do.

[0043] In various embodiments, the amino acid mutation may be in a CDR (e.g., the CDR1, CDR2, or CDR3 region) of the targeting moiety. In another embodiment, the amino acid change may be in a framework region (FR) (e.g., the FR1, FR2, FR3, or FR4 region) of the targeting moiety.

[0044] Amino acid sequence modification can be achieved by any well-known technique in the art, for example, 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, NY, 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0045] In various embodiments, the mutations do not substantially reduce the ability of the Clec9A binding agent of the invention to specifically bind to Clec9A. In various embodiments, the mutations do not substantially reduce the ability of the Clec9A binding agent of the invention to specifically bind to Clec9A and do not functionally modulate (e.g., partially or fully neutralize) Clec9A.

[0046] In various embodiments, the binding affinity of the Clec9A binding agents of the invention for full-length and / or mature forms and / or isoforms and / or splice variants and / or fragments and / or monomeric and / or dimeric forms and / or any other naturally occurring or synthetic analogs, variants, or mutants (including monomeric and dimeric forms) of human Clec9A is determined by the equilibrium dissociation constant (K D In various embodiments, the Clec9A binding agent comprises a targeting moiety that binds to full-length and / or mature and / or isoforms and / or splice variants and / or fragments and / or any other naturally occurring or synthetic analogs, variants, or mutants of human Clec9A (including monomeric and / or dimeric forms) with a KD of less than 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 about 1 nM.

[0047] In various embodiments, a Clec9A-binding agent comprises a targeting moiety that binds but does not functionally modulate (e.g., partially or fully neutralize) the antigen of interest, i.e., Clec9A. For example, in various embodiments, the targeting moiety of a Clec9A-binding agent simply targets the antigen but does not substantially functionally modulate (e.g., partially or fully inhibit or neutralize) the biological effect of the antigen. In various embodiments, the targeting moiety of a Clec9A-binding agent binds an epitope that is physically distant from the antigenic site important for its biological activity (e.g., the active site of the antigen).

[0048] Such binding without significant functional modulation finds use in various embodiments of the invention, including methods in which the Clec9A-binding agents of the invention are used to directly or indirectly recruit active immune cells to required sites via effector antigens. For example, in various embodiments, the Clec9A-binding agents of the invention may be used to recruit dendritic cells directly or indirectly via Clec9A to tumor cells in methods of shrinking or eliminating tumors (e.g., the Clec9A-binding agent may comprise a targeting moiety having an anti-Clec9A antigen recognition domain and a recognition domain (e.g., an antigen recognition domain) for a tumor antigen or receptor). (The present invention may include a targeting moiety having the formula: (I) (II) (III) (III) (IV ...

[0049] In some embodiments, Clec9A-binding agents enhance antigen presentation by dendritic cells. For example, in various embodiments, Clec9A-binding agents of the invention directly or indirectly recruit dendritic cells to tumor cells via Clec9A, where tumor antigens are then taken up and presented on dendritic cells for the induction of potent humoral and cytotoxic T cell responses.

[0050] In other embodiments (e.g., in the context of treating autoimmune or neurodegenerative diseases), the Clec9A-binding agent comprises a targeting moiety that binds and neutralizes the antigen of interest, i.e., Clec9A. For example, in various embodiments, the methods of the invention may inhibit or reduce Clec9A signaling or expression, e.g., to reduce an immune response.

[0051] Therapeutic Agents Comprising the Clec9A-Binding Substances of the Present Invention Chimeras and fusions with signal transduction molecules In various embodiments, the Clec9A-binding agents of the invention are part of a chimera or fusion with one or more signal transduction agents. Thus, for example, the invention provides chimera or fusion proteins comprising a targeting moiety for Clec9A and one or more signal transduction agents.

[0052] In various embodiments, the signaling agent is modified to have reduced affinity or activity for one or more of its receptors, thereby allowing for attenuation of the activity (including agonism or antagonism) of the chimeric or fusion protein and / or preventing nonspecific signaling or undesired sequestration. In various embodiments, the signaling agent is antagonistic in its wild-type form and has one or more mutations that attenuate its antagonist activity. In various embodiments, the signaling agent is antagonistic due to one or more mutations, e.g., an agonist signaling agent is converted to an antagonist signaling agent, and such converted signaling agent optionally also has one or more mutations that attenuate its antagonist activity (e.g., as described in WO 2015 / 007520, the entire contents of which are incorporated herein by reference).

[0053] Thus, in various embodiments, the signaling agent is a modified (mutated) form of the signaling agent having one or more modifications (e.g., mutations). In various embodiments, the mutations enable the modified signaling agent to have one or more attenuated activities, such as one or more of reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to the non-mutated, i.e., wild-type, form of the signaling agent (e.g., comparing the wild-type and modified (e.g., mutant) forms of the same signaling agent). In some embodiments, mutations that weaken or reduce binding or affinity include mutations that substantially reduce or eliminate binding or activity. In some embodiments, mutations that weaken or reduce binding or affinity are different from mutations that substantially reduce or eliminate binding or activity. As a result, in various embodiments, the mutations enable the signaling agent to have improved safety, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the non-mutated, i.e., wild-type, form of the signaling agent (e.g., comparing the wild-type and modified (e.g., mutant) forms of the same signaling agent).

[0054] As described herein, a substance can have improved safety due to one or more modifications, e.g., mutations. In various embodiments, improved safety means that the chimeric protein provides lower toxicity (e.g., systemic toxicity and / or tissue / organ-related toxicity); and / or reduced or substantially eliminated side effects; and / or increased tolerability, reduced or substantially eliminated adverse events; and / or reduced or substantially eliminated; and / or an expanded therapeutic window.

[0055] In various embodiments, the signaling agent is modified to have one or more mutations that reduce binding affinity or activity for one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or eliminate binding affinity or activity for the receptor. In some embodiments, the activity conferred by the wild-type signaling agent is agonism for the receptor (e.g., activation of a cellular effect at the site of treatment). For example, the wild-type signaling agent may activate its receptor. In such embodiments, the mutation results in the signaling agent being modified to reduce or eliminate the activating effect on the receptor. For example, the mutation may result in the signaling agent being modified to send a reduced activation signal to the target cell, or the activation signal may be eliminated. In some embodiments, the effect conferred by the wild-type signaling agent is antagonism for the receptor (e.g., blocking or suppressing a cellular effect at the site of treatment). For example, the wild-type signaling agent may antagonize or inhibit the receptor. In these embodiments, the mutation results in the signaling agent being modified to reduce or eliminate antagonizing activity for the receptor. For example, the mutations can result in the signal transduction agent being altered to send a reduced inhibitory signal to the target cell, or the inhibitory signal can be eliminated. In various embodiments, the signal transduction agent is antagonistic due to one or more mutations, e.g., an agonist signal transduction agent is converted to an antagonist signal transduction agent (e.g., as described in WO 2015 / 007520, the entire contents of which are incorporated herein by reference), and such converted signal transduction agent optionally also has one or more mutations that reduce its binding affinity or activity to one or more of its receptors, or that substantially reduce or eliminate binding affinity or activity to one or more of its receptors.

[0056] In some embodiments, the reduced affinity or activity for the receptor is reversible by binding to one or more targeting moieties described herein (e.g., a targeting moiety for Clec9A). In other embodiments, the reduced affinity or activity for the receptor is not substantially reversible by the activity of one or more targeting moieties.

[0057] In various embodiments, the chimeric proteins of the present invention have reduced off-target effects because the signaling agents have mutations that weaken or eliminate binding affinity or activity for the receptor. In various embodiments, this reduction in side effects is observed, for example, compared to wild-type signaling agents. In various embodiments, the signaling agents are active on target cells because the targeting moiety(s) compensate for missing / insufficient binding (e.g., without limitation, and / or avidity) required for substantial activation. In various embodiments, the modified signaling agents are substantially inactive en route to the site of therapeutic action and exert their effect substantially on the specifically targeted cell type, thereby greatly reducing undesirable side effects.

[0058] In some embodiments, a signaling agent may contain one or more mutations that weaken or reduce binding or affinity to one receptor (i.e., a therapeutic receptor) and one or more mutations that substantially reduce or eliminate binding or activity to a second receptor. In such embodiments, these mutations may be in the same or different positions. 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 has an altered signaling agent that has both mutations that weaken binding and / or activity for a therapeutic receptor, thereby allowing for a more controlled and targeted therapeutic effect (e.g., compared to a wild-type signaling agent), and mutations that substantially reduce or eliminate binding and / or activity for another receptor, thereby reducing side effects (e.g., compared to a wild-type signaling agent).

[0059] In some embodiments, the substantial reduction or elimination of binding or activity is not substantially reversible with a targeting moiety (a targeting moiety against Clec9A or any other targeting moiety described herein). In some embodiments, the substantial reduction or elimination of binding or activity is reversible with a targeting moiety. In various embodiments, the substantial reduction or elimination of binding or activity against a second receptor may also prevent adverse effects mediated by the other receptor. Alternatively, or in addition, the substantial reduction or elimination of binding or activity against the other receptor reduces or eliminates sequestering of the therapeutic chimeric protein away from the therapeutic site of action, thereby improving therapeutic efficacy. For example, in some embodiments, this eliminates the need for high doses of the chimeric protein of the invention to compensate for losses at other receptors. The ability to reduce such doses further reduces the potential for side effects.

[0060] In various embodiments, the modified signaling agent may be modified to increase the affinity, e.g., binding (e.g., K D ) and / or activation (e.g., when the altered signal transduction agent is an agonist of that receptor, e.g., KA and / or EC 50 ) and / or inhibition (e.g., if the altered signal transduction agent is an antagonist of that receptor, e.g., K I and / or IC 50 The immunomodulatory agent may comprise one or more mutations that reduce, substantially reduce, or eliminate affinity (measurable as agonism or antagonism) for the receptor. In various embodiments, the reduced affinity of the immunomodulatory agent for the receptor allows for attenuated activity (including agonism or antagonism). In such embodiments, the modified signaling agent has about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, or about 80%-100% affinity for the receptor compared to the wild-type signaling agent. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower, relative to the wild-type signaling agent.

[0061] In some embodiments where the altered signaling agent has mutations that reduce binding to one receptor and substantially reduce or eliminate binding to a second receptor, the attenuation or reduction in binding affinity of the altered signaling agent to one receptor is less than the substantial reduction or elimination of affinity to the other receptor. In some embodiments, the attenuation or reduction in binding affinity of the altered signaling agent to one receptor is less than the substantial reduction or elimination of affinity to the other receptor. A qualitative reduction or elimination is less than about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In various embodiments, a substantial reduction or elimination refers to a reduction in binding affinity and / or activity that is greater than a weakening or reduction.

[0062] In various embodiments, the modified signaling agent comprises one or more mutations that reduce the intrinsic activity of the signaling agent to, for example, about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1%, relative to the wild-type signaling agent.

[0063] In some embodiments, the modified signal transduction agent contains one or more mutations that cause the signal transduction 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 signal transduction agent / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity allows the signal transduction agent, e.g., the mutant signal transduction agent, to have a localized, on-target effect and minimize off-target effects that underlie the side effects observed with wild-type signal transduction agents. In some embodiments, the binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold lower, or at least about 25-fold, or at least about 50-fold lower, or at least about 100-fold, or at least about 150-fold lower.

[0064] Receptor binding activity can be measured by using known methods in the art.For example, affinity and / or binding activity can be evaluated by Scatchard plot analysis and computer fitting of binding data (for example, Scatchard, 1949) or by reflectance interferometry under flow-through conditions, as described by Brecht et al. (1993).The entire contents of these documents are incorporated herein by reference.

[0065] In various embodiments, the signaling agent is an immunomodulatory agent, for example, one or more of an interleukin, an interferon, and a tumor necrosis factor.

[0066] In some embodiments, the signaling agent is an interleukin or modified interleukin, including, for example, IL-1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, or a fragment, variant, analog, or family member. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulation of the proliferation of immune cells (e.g., helper T cells, B cells, eosinophils, and lymphocytes), chemotaxis of 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, DC 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1,14-20).

[0067] In some embodiments, the signaling agent is an interferon or an interferon. and modified forms of interferon, such as types I, II, and III. Examples of interferons include, for example, interferon alpha-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21, interferon beta and interferon gamma, interferon kappa, interferon epsilon, interferon tau, and interferon omega.

[0068] In some embodiments, the signal transduction agent is 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.

[0069] The amino acid sequences of the wild-type signal transduction factors described herein are well known in the art. Thus, in various embodiments, the modified signaling agent is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 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 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 74%, 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). 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).

[0070] In various embodiments, the modified signaling agent is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75% identical to the amino acid sequence of any of the signaling agents described herein. , 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 or about 97%, 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).

[0071] In various embodiments, the modified signal transduction agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, which may include conservative and / or non-conservative substitutions, as described elsewhere herein. In various embodiments, the substitutions may also include non-classical amino acids, as described elsewhere herein.

[0072] As described herein, the modified signal transduction agent has a mutation that affects affinity and / or activity for one or more receptors. In various embodiments, there is reduced affinity and / or activity for a therapeutic receptor, e.g., a receptor through which a desired therapeutic effect is mediated (e.g., agonism or antagonism). In various embodiments, the modified signal transduction agent has a mutation that substantially reduces or eliminates affinity and / or activity for a receptor, e.g., a receptor through which a desired therapeutic effect is not mediated (e.g., as a result of disrupted binding). Receptors for modified signal transduction agents, e.g., receptors for one of the cytokines, growth factors, and hormones described herein, are known in the art.

[0073] Examples of mutations that result in reduced affinity and / or activity (e.g., agonist activity) for a receptor can be found in WO 2013 / 107791 and International Application Nos. PCT / EP2017 / 061544 (e.g., for interferons), WO 2015 / 007542 (e.g., for interleukins), and WO 2015 / 007903 (e.g., for TNF), the entire contents of each of which are incorporated herein by reference. Examples of mutations that reduce affinity and / or activity (e.g., agonist activity) for a therapeutic receptor can be found in WO 2015 / 007520, the entire contents of which are incorporated herein by reference.

[0074] In some embodiments, the modified signal transducer comprises one or more mutations that reduce the affinity and / or activity of the signal transducer for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a receptor of the tumor necrosis factor receptor (TNFR) superfamily, a TGF beta receptor, a receptor of the immunoglobulin (Ig) superfamily, and / or a receptor of the tyrosine kinase superfamily.

[0075] In various embodiments, the receptor for the signal transduction 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, IL-11, IL-12, GM-CSF, G-CSF, LIF, CNTF, as well as 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. The condition is listed.

[0076] In various embodiments, the receptor for a signal transduction substance is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterogeneous subunits and are primarily receptors for interferons. This receptor family includes, but is not limited to, receptors for interferon-α, interferon-β, and interferon-γ, IL-10, IL22, and tissue factor. Exemplary type II cytokine receptors include, but are not limited to, IFN-α receptors (e.g., IFNAR1 and IFNAR2), IFN-β receptors, IFN-γ receptors (e.g., IFNGR1 and IFNGR2), and type II IL receptors.

[0077] In various embodiments, the receptor for the signal transduction substance is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors that have a seven-transmembrane structure and are coupled to G proteins for signal transduction. Chemokine receptors include, but are not limited to, CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors (XCR1). Representative chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.

[0078] In various embodiments, the receptor for the signal transduction agent is a TNFR family member. Tumor necrosis factor receptor (TFNR) family members share a cysteine-rich domain (CRD) formed from three disulfide bonds surrounding a core motif of CXXCXXC that creates an elongated molecule. Representative tumor necrosis factor receptor family members include CD120a (TNFRSF1A), CD120b (TNFRSF1B), lymphotoxin beta receptor (LTBR, ​​TNFRSF3), CD134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSF10A (TNFRSF10A), TNFRSF10B (TNFRSF10B), and TNFRSF10C (TNFRSF10C). F10C), TNFRSF10D (TNFRSF10D), RANK (TNFRSF11A), osteoclastogenic factor (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.

[0079] In various embodiments, the receptor for signal transduction substance is TGF beta receptor.TGF beta receptor is a single transmembrane serine / threonine kinase receptor.TGF beta receptor includes but is not limited to TGFBR1, TGFBR2 and TGFBR3.

[0080] In various embodiments, the receptor for the signaling agent is an Ig superfamily receptor. Receptors of the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Receptors of the Ig superfamily include, but are not limited to, , interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

[0081] In various embodiments, the receptor for the signal transduction substance is a tyrosine kinase superfamily receptor. Receptors of the tyrosine kinase tyrosine kinase superfamily are well known in the art. There are about 58 receptor tyrosine kinases (RTKs) classified into 20 subfamilies. Receptors of the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0082] In certain embodiments, the modified signal transduction agent is interferon alpha. In such embodiments, the modified IFN alpha agent has reduced affinity and / or activity for the IFN alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFN alpha agent has substantially reduced or eliminated affinity and / or activity for the IFN alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0083] Mutant forms of interferon alpha are known to those of skill in the art. In an exemplary embodiment, the modified signal transducer is an allelic IFNα2a having the amino acid sequence of SEQ ID NO:337.

[0084] In an exemplary embodiment, the altered signal transducer is an allelic form of IFNα2b having the amino acid sequence of SEQ ID NO: 338 (which differs from IFNα2a at amino acid position 23).

[0085] In some embodiments, the IFNα2 mutant (IFNα2a or IFNα2b) has one or more amino acid mutations introduced at positions 144-154, e.g., amino acid positions 148, 149, and / or 153. In some embodiments, the IFNα2 mutant contains one or more mutations selected from L153A, R149A, and M148A. Such mutants are described, for example, in WO 2013 / 107791 and Piehler et al. (2000) J. Biol. Chem. 275:40425-33, the entire contents of which are incorporated herein by reference.

[0086] In some embodiments, the IFNα2 mutant has reduced affinity and / or activity for IFNAR1. In some embodiments, the IFNα2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A, as described in WO 2010 / 030671, the entire contents of which are incorporated herein by reference.

[0087] In some embodiments, the IFNα2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A, as described in WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0088] In some embodiments, the IFNα2 mutant comprises one or more mutations selected from R120E and R120E / K121E, as described in WO 2015 / 007520 and WO 2010 / 030671, the entire contents of which are incorporated herein by reference. In such embodiments, the IFNα2 mutant antagonizes wild-type IFNα2 activity. In such embodiments, the mutant IFNα2 has reduced affinity and / or activity for IFNAR1 but not I. Activity against FNAR2 is retained.

[0089] In some embodiments, the human IFNα2 mutant comprises one or more mutations selected from (1) R120E and R120E / K121E (without wishing to be bound by theory, these produce an antagonistic effect), and (2) one or more mutations selected from K133A, R144A, R149A, and L153A (without wishing to be bound by theory, these enable, for example, an attenuating effect on IFNAR2). In certain embodiments, the human IFNα2 mutant comprises R120E and L153A.

[0090] In some embodiments, the human IFNα2 variant comprises one or more mutations selected from L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, as disclosed in WO 2013 / 059885, the entire contents of which are incorporated herein by reference. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L30A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or R33A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or M148A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 mutant comprises the mutations H57Y, E58N, Q61S, and / or L153A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 variant comprises the mutations N65A, L80A, Y85A, and / or Y89A as disclosed in WO 2013 / 059885. In some embodiments, the human IFNα2 variant comprises the mutations N65A, L80A, Y85A, Y89A, and / or D114A as disclosed in WO 2013 / 059885.

[0091] In certain embodiments, the modified signal transduction agent is interferon beta. In such embodiments, the modified interferon beta agent has reduced affinity and / or activity for the IFN alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFN beta agent has substantially reduced or eliminated affinity and / or activity for the IFN alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0092] In one embodiment, the modified signal transduction element is IFNβ. In various embodiments, IFNβ includes functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of IFNβ. In various embodiments, IFNβ includes IFNβ from any species. In one embodiment, the chimeric protein includes a modified mouse IFNβ. In one embodiment, the chimeric protein includes a modified human IFNβ. Human IFNβ is a polypeptide containing 166 amino acid residues and having a molecular weight of approximately 22 kDa. The amino acid sequence of human IFNβ is SEQ ID NO: 339.

[0093] In some embodiments, the IFNβ is IFNβ1a, a glycosylated form of human IFNβ. In some embodiments, the IFNβ is IFNβ1b, a non-glycosylated form of human IFNβ with a Met-1 deletion and a Cys-17 to Ser mutation.

[0094] In various embodiments, the modified IFNβ is directed against the IFNAR1 subunit of IFNAR. The modified IFNβ has one or more mutations that reduce its binding or affinity to IFNAR1. In one embodiment, the modified IFNβ has reduced affinity and / or activity for IFNAR1. In various embodiments, the modified IFNβ is human IFNβ and has one or more mutations at positions F67, R71, L88, Y92, I95, N96, K123, and R124. In some embodiments, the one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G. In certain embodiments, the modified IFNβ comprises an F67G mutation. In certain embodiments, the modified IFNβ comprises a K123G mutation. In certain embodiments, the modified IFNβ comprises an F67G and an R71A mutation. In certain embodiments, the modified IFNβ comprises an L88G and a Y92G mutation. In some embodiments, the modified IFNβ comprises Y92G, I95A, and N96G mutations. In some embodiments, the modified IFNβ comprises K123G and R124G mutations. In some embodiments, the modified IFNβ comprises F67G, L88G, and Y92G mutations. In some embodiments, the modified IFNβ comprises F67S, L88S, and Y92S mutations.

[0095] In some embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity to the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFNβ has reduced affinity and / or activity for IFNAR2. In various embodiments, the modified IFNβ is human IFNβ and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. In some embodiments, the one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In certain embodiments, the modified IFNβ comprises a W22G mutation. In certain embodiments, the modified IFNβ comprises an L32A mutation. In certain embodiments, the modified IFNβ comprises an L32G mutation. In some embodiments, the modified IFNβ comprises an R35A mutation. In some embodiments, the modified IFNβ comprises an R35G mutation. In some embodiments, the modified IFNβ comprises a V148G mutation. In some embodiments, the modified IFNβ comprises an R152A mutation. In some embodiments, the modified IFNβ comprises an R152G mutation. In some embodiments, the modified IFNβ comprises a Y155G mutation. In some embodiments, the modified IFNβ comprises a W22G and R27G mutation. In some embodiments, the modified IFNβ comprises an L32A and R35A mutation. In some embodiments, the modified IFNβ comprises an L151G and R152A mutation. In some embodiments, the modified IFNβ comprises a V148G and R152A mutation.

[0096] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with C17S or C17A.

[0097] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with other IFNβ mutations described herein.

[0098] The crystal structure of human IFNβ is known and is described in Karpusas et al., (1998) PNAS, 94(22):11813-11818. In particular, the structure of human IFNβ has been shown to contain five α-helices (i.e., A, B, C, D, and E) and four loop regions (i.e., AB, BC, CD, and DE loops) connecting these helices. In various embodiments, modified IFNβ contains IFNAR-like structures in the A, B, C, D, and E helices and / or the AB, BC, CD, and DE loops. The modified IFNβ has one or more mutations that reduce its binding affinity or activity for any therapeutic receptor. Exemplary mutations are described in International Publication No. 2000 / 023114 and U.S. Patent Application Publication No. 20150011732, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In an exemplary embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 28-30, 32, and 33. In an exemplary embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 36, 37, 39, and 42. In an exemplary embodiment, the modified IFNβ is human IFNβ containing alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In exemplary embodiments, the modified IFNβ is human IFNβ comprising alanine substitutions at amino acid positions 71-73. In exemplary embodiments, the modified IFNβ is human IFNβ comprising alanine substitutions at amino acid positions 92, 96, 99, and 100. In exemplary embodiments, the modified IFNβ is human IFNβ comprising alanine substitutions at amino acid positions 128, 130, 131, and 134. In exemplary embodiments, the modified IFNβ is human IFNβ comprising alanine substitutions at amino acid positions 149, 153, 156, and 159. In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at W22, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0099] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at R27, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0100] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at W22, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R27, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0101] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at L32, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0102] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at R35, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0103] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at L32, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R35, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0104] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at F67, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0105] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at R71. The mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0106] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at F67, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R71, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0107] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at L88, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0108] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at Y92, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0109] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at F67, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at L88, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0110] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at L88, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0111] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at I95, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and further comprises a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0112] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at N96, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0113] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at Y92, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a mutation at I95, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V). is an aliphatic hydrophobic residue selected from methionine (M), and valine (V), and includes a mutation at N96, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0114] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at K123, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0115] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at R124, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0116] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at K123, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R124, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0117] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at L151, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0118] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at R152, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0119] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at L151, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0120] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at V148, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0121] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at V148, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0122] In some embodiments, the mutant IFNβ comprises SEQ ID NO: 339 and a mutation at Y155, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0123] In some embodiments, the present invention provides a method for the detection of a compound comprising: (a) the amino acid sequence and positions of SEQ ID NO: 339; and (b) one or more targeting moieties, wherein the targeting moiety comprises a recognition domain that specifically binds to an antigen or receptor of interest (e.g., Clec9A), and the modified IFNβ and the targeting moiety may optionally be linked by one or more linkers. In various embodiments, the mutation at position W22 is an aliphatic hydrophobic residue selected from G, A, L, I, M, and V. In various embodiments, the mutation at position W22 is G.

[0124] Additional representative IFNβ variants are provided in International Application No. PCT / EP2017 / 061544, the entire disclosure of which is incorporated herein by reference.

[0125] In certain embodiments, the modified signal transduction agent is interferon gamma. In such embodiments, the modified interferon gamma agent has reduced affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains. In some embodiments, the modified interferon gamma agent has substantially reduced or eliminated affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains.

[0126] IFNγ is the only member of the type II class of interferons. IFNγ is primarily produced by natural killer (NK) cells and natural killer T (NKT) cells as part of the innate immune response. IFNγ is also produced by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells, macrophages, dendritic cells, and B cells. Activated IFNγ forms dimers and acts through a heterodimeric receptor (i.e., IFNγ or IFNγR) consisting of IFNγ receptor 1 and IFNγ receptor 2 subunits. IFNγ receptor 1 is the primary ligand-binding subunit, while IFNγ receptor 2 is required for signal transduction and enhances the affinity of IFNγ receptor 1 for its ligand. Binding of the IFNγ dimer to the receptor activates the JAK-STAT signaling pathway, inducing various biological effects.

[0127] In various embodiments, the modified signal transduction agent comprises a modified IFNγ as a signal transduction agent. In various embodiments, IFNγ includes functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of IFNγ. In various embodiments, IFNγ includes IFNγ from any species. In one embodiment, the modified signal transduction agent comprises a modified mouse IFNγ. In another embodiment, the modified signal transduction agent comprises a modified human IFNγ.

[0128] Human IFNγ is a polypeptide comprising 166 amino acid residues. In one embodiment, the human IFNγ has the amino acid sequence of SEQ ID NO: 340, in which the signal peptide comprises the first 23 amino acids.

[0129] As used herein, human IFNγ also refers to mature human IFNγ without the N-terminal signal peptide. In this embodiment, mature human IFNγ contains 143 amino acids and has the amino acid sequence of SEQ ID NO: 341.

[0130] In some embodiments, the human IFNγ is glycosylated human IFNγ. In some embodiments, the human IFNγ is non-glycosylated human IFNγ.

[0131] The sequence of IFNγ is known in the art. In various embodiments, the modified IFNγ has at least about 60% similarity to the known wild-type amino acid sequence of IFNγ, or at least about 60% similarity to the known wild-type amino acid sequence of IFNγ. 1%, 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 or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity to the amino acid sequence.

[0132] In some embodiments, the modified IFNγ is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or 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).

[0133] In some embodiments, the modified IFNγ is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65% identical to a human IFNγ having the amino acid sequence of SEQ ID NO: 341. , 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 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).

[0134] In various embodiments, the modified IFNγ comprises an amino acid sequence having one or more amino acid mutations, which in some embodiments may be independently selected from a substitution, an insertion, a deletion, and a truncation.

[0135] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0136] "Conservative substitutions" can be made, for example, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0137] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid in the same group of the six standard amino acid groups. For example, replacing Asp with Glu maintains one negative charge in the modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0138] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid from a different group of the six standard amino acid groups (1) to (6) above.

[0139] In various embodiments, substitutions also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε Also included are designer amino acids such as Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methylamino acids, C α-methylamino acids, N α-methylamino acids, and amino acid analogs in general.

[0140] In various embodiments, the IFNγ is modified to have one or more mutations. In some embodiments, the mutations allow the modified IFNγ to have one or more attenuated activities, such as one or more of reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to a non-mutated, e.g., wild-type, form of IFNγ. For example, the one or more attenuated activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to a non-mutated, e.g., wild-type, IFNγ, can be for a therapeutic receptor, such as an IFNγ receptor. As a result, in various embodiments, the mutations allow the modified soluble substance to have reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to a non-mutated, e.g., wild-type, IFNγ.

[0141] In various embodiments, the IFNγ is modified to have a mutation that reduces its binding affinity and / or activity for a therapeutic receptor, such as an IFNγ receptor comprising the IFNγ receptor 1 and IFNγ receptor 2 subunits. In some embodiments, the activity conferred by wild-type IFNγ is agonism for the therapeutic receptor (e.g., activation of a cellular effect at the site of treatment). For example, wild-type IFNγ can activate the therapeutic receptor. In such embodiments, the mutation results in an IFNγ that is modified to reduce its activating effect on the therapeutic receptor.

[0142] In some embodiments, the reduced affinity and / or activity for a therapeutic receptor (e.g., an IFNγ receptor) can be restored by binding of a targeting moiety. In other embodiments, the reduced affinity and / or activity for a therapeutic receptor cannot be substantially restored by binding to a targeting moiety. In various embodiments, the therapeutic chimeric proteins of the present invention reduce off-target effects because the IFNγ has a mutation that weakens its binding affinity and / or activity for a therapeutic receptor. In various embodiments, this reduces side effects observed with, for example, wild-type IFNγ. In various embodiments, the modified IFNγ is substantially inactive en route to the site of therapeutic action and substantially retains its effect on the specifically targeted cell type, thereby greatly reducing undesirable side effects.

[0143] In various embodiments, the modified IFNγ has one or more mutations that cause the IFNγ to have weakened or reduced affinity and / or activity, e.g., binding (e.g., KD) and / or activation (e.g., measurable as K A and / or EC50), for one or more therapeutic receptors (e.g., IFNγ receptors). In various embodiments, the reduced affinity and / or activity for the therapeutic receptor allows for attenuated activity and / or signaling from the therapeutic receptor.

[0144] In various embodiments, the modified IFNγ has one or more mutations that reduce its binding or affinity to the IFNγ receptor 1 subunit and / or biological activity. In one embodiment, the modified IFNγ has reduced affinity and / or activity for the IFNγ receptor 1 subunit. In various embodiments, the modified IFNγ is human IFNγ with one or more mutations at amino acid residues involved in binding to the IFNγ receptor 1 subunit. In some embodiments, the modified IFNγ is human IFNγ with one or more mutations at amino acids located at the interface with the IFNγ receptor 1 subunit. In various embodiments, the one or more mutations are present at an amino acid position selected from, but not limited to, Q1, V5, E9, K12, H19, S20, V22, A23, D24, N25, G26, T27, L30, K108, H111, E112, I114, Q115, A118, E119, and K125 (each relative to SEQ ID NO: 341, which is wild-type human IFNγ lacking the N-terminal signal sequence). In some embodiments, the one or more mutations are substitutions selected from V5E, S20E, V22A, A23G, A23F, D24G, G26Q, H111A, H111D, I114A, Q115A, and A118G (each relative to SEQ ID NO: 341). In some embodiments, the one or more mutations are substitutions selected from V22A, A23G, D24G, H111A, H111D, I114A, Q115A, and A118G.

[0145] In one embodiment, the modified IFNγ comprises the mutations A23G and D24G. In another embodiment, the modified IFNγ comprises the mutations I114A and A118G. In a further embodiment, the modified IFNγ comprises the mutations V5E, S20E, A23F, and G26Q.

[0146] In various embodiments, the modified IFNγ has one or more of the following mutations: a deletion of residue A23, a deletion of residue D24, an S20I substitution, an A23V relaxation, a D21K substitution, and a D24A substitution.

[0147] In some embodiments, the modified IFNγ has one or more mutations that reduce its binding or affinity to the IFNγ receptor 2 subunit and / or biological activity.

[0148] In some embodiments, the modified IFNγ has one or more mutations that reduce its binding or affinity and / or biological activity for both the IFNγ receptor 1 and IFNγ receptor 2 subunits.

[0149] In some embodiments, the modified IFNγ has one or more mutations that reduce its binding or affinity and / or biological activity to IFNγ receptor 1 and one or more mutations that substantially reduce or eliminate binding or affinity and / or biological activity to IFNγ receptor 2. In some embodiments, a chimeric protein having such a modified IFNγ can provide target-selective IFNγ receptor 1 activity (e.g., IFNγ receptor 1 activity can be restored by targeting via a targeting moiety).

[0150] In some embodiments, the modified IFNγ has one or more mutations that reduce its binding or affinity and / or biological activity to IFNγ receptor 1, and one or more mutations that reduce its binding or affinity and / or biological activity to IFNγ receptor 1. In some embodiments, a chimeric protein having such a modified IFNγ can provide target-selective IFNγ receptor 1 and / or IFNγ receptor 1 activity (e.g., IFNγ receptor 1 and IFNγ receptor 2 activity can be restored by targeting via a targeting moiety).

[0151] In various embodiments, the modified IFNγ is C-terminally truncated. In some embodiments, the modified IFNγ is a mature IFNγ comprising the amino acid sequence of SEQ ID NO: 341 with a C-terminal deletion. In such embodiments, the mature IFNγ may comprise a C-terminal truncation of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acid residues. In certain embodiments, the modified IFNγ is a mature IFNγ comprising the amino acid sequence of SEQ ID NO: 341 with a C-terminal deletion of 5 amino acids. In certain embodiments, the modified IFNγ is a mature IFNγ comprising the amino acid sequence of SEQ ID NO: 341 with a C-terminal deletion of 7 amino acids. In one embodiment, the modified IFNγ is a mature IFNγ comprising the amino acid sequence of SEQ ID NO: 341 with a C-terminal deletion of 14 amino acids. In one embodiment, the modified IFNγ is a mature IFNγ comprising the amino acid sequence of SEQ ID NO: 341 with a C-terminal deletion of 15 amino acids. In one embodiment, the modified IFNγ is a mature IFNγ comprising the amino acid sequence of SEQ ID NO: 341 with a C-terminal deletion of 16 amino acids. Further modified IFNγ with C-terminal truncations that can be utilized in the present invention are described in Haelewyn et al., Biochem. J. (1997), 324:591-595 and Lundell et al., Protein Eng. (1991) 4:335-341, the entire contents of which are incorporated herein by reference.

[0152] In various embodiments, the modified IFNγ is a single-chain IFNγ, e.g., as described in Randal et al. (2001) Structure 9:155-163 and Randal et al. (1998) Protein Sci. 7:1057-1060, the entire contents of which are incorporated herein by reference. In some embodiments, the single-chain IFNγ comprises a first IFNγ chain linked at its C-terminus to the N-terminus of a second IFNγ chain. In various embodiments, the first and second IFNγ chains are linked by a linker, as described elsewhere herein.

[0153] In some embodiments, the first IFNγ chain comprises a C-terminal truncation of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acid residues. In certain embodiments, the first IFNγ chain comprises a C-terminal truncation of about 24 amino acid residues. In some embodiments, the second IFNγ comprises an N-terminal truncation of at least about 1, about 2, about 3, about 4, or about 5 amino acid residues. In certain embodiments, the second IFNγ chain comprises an N-terminal truncation of about 3 amino acid residues. In some embodiments, the second IFNγ chain comprises a C-terminal truncation of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acid residues. In various embodiments, the first and / or second IFNγ chain has one or more amino acid 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, as described elsewhere herein. In various embodiments, the first and / or second IFNγ chain includes one or more substitutions selected from V5E, S20E, V22A, A23G, A23F, D24G, G26Q, H111A, H111D, I114A, Q115A, and A118G. In various embodiments, the first and / or second IFNγ chain comprises one or more substitutions selected from V22A, A23G, D24G, H111A, H111D, I114A, Q115A, and A118G. In various embodiments, the first and / or second IFNγ chain comprises A23G and D24G substitutions. In various embodiments, the first and / or second IFNγ chain comprises I114A and A118G substitutions. In another embodiment, the mutations are V5E, S20E, A23F, and G26Q.

[0154] In various embodiments, the first and / or second IFNγ chain comprises one or more substitutions disclosed herein, and further, the first and / or second IFNγ chain comprises a C-terminal truncation disclosed herein.

[0155] In various embodiments, the first and / or second IFNγ chain comprises one or more of the substitutions disclosed herein and a C-terminal truncation disclosed herein.

[0156] The crystal structure of human IFNγ is known and is described, for example, in Ealick et al., (1991) Science, 252:698-702. In particular, the structure of human IFNγ has been shown to contain a core of six α-helices and an extended unfolded sequence in the C-terminal region. In various embodiments, the modified IFNγ has one or more mutations in one or more helices that reduce its binding affinity to a therapeutic receptor (e.g., an IFNγ receptor) and / or biological activity.

[0157] In various embodiments, the modified IFNγ has about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% to 20%, about 20% to 40%, about 50%, about 40% to 60%, about 60% to 80%, or about 80% to 100% of the affinity and / or biological activity for a therapeutic receptor (e.g., the IFNγ receptor or any one of its IFNγ receptor 1 and IFNγ receptor 2 subunits) compared to wild-type IFNγ. In some embodiments, the binding affinity and / or biological activity is at least about 2-fold less, about 3-fold less, about 4-fold less, about 5-fold less, about 6-fold less, about 7-fold less, about 8-fold less, about 9-fold less, at least about 10-fold less, at least about 15-fold less, at least about 20-fold less, at least about 25-fold less, at least about 30-fold less, at least about 35-fold less, at least about 40-fold less, at least about 45-fold less, at least about 50-fold less, at least about 100-fold less, at least about 150-fold less, or about 10-50-fold less, about 50-100-fold less, about 100-150-fold less, about 150-200-fold less, or more than 200-fold less, compared to wild-type IFNγ.

[0158] In various embodiments, the modified IFNγ comprises one or more mutations that reduce the intrinsic activity of IFNγ 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%, for example, compared to wild-type IFNγ.

[0159] In some embodiments, the modified IFNγ comprises one or more mutations that cause the IFNγ to have reduced affinity and / or biological activity for its receptor. In some embodiments, the binding affinity and / or biological activity of the modified IFNγ to its receptor is lower than the binding affinity and / or biological activity of the targeting moiety to its receptor. In some embodiments, this difference in binding affinity and / or biological activity exists between the modified IFNγ / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity and / or biological activity enables the modified IFNγ to have a localized on-target effect while minimizing off-target effects that underlie the side effects observed with wild-type IFNγ. In some embodiments, this binding affinity and / or biological activity 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.

[0160] Receptor binding activity can be measured by using known methods in the art.For example, affinity and / or binding activity can be evaluated by Scatchard plot analysis and computer fitting of binding data (for example, Scatchard, 1949) or by reflectance interferometry under flow-through conditions, as described by Brecht et al. (1993).The entire contents of these documents are incorporated herein by reference.

[0161] In some embodiments, the modified signal transduction agent is a consensus interferon, which is generated by scanning the sequences of several human non-allelic IFNα subtypes and assigning the most frequently observed amino acid at each corresponding position. The consensus interferon differs from IFNα2b at 20 of 166 amino acids (88% homology), and comparison with IFNβ shows identity at more than 30% of amino acid positions. In various embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO: 342:

[0162] In some embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO: 343, which differs from the amino acid sequence of SEQ ID NO: 342 by only one amino acid, i.e., SEQ ID NO: 343 lacks the first methionine residue of SEQ ID NO: 342.

[0163] In various embodiments, the consensus interferon comprises a modified consensus interferon, i.e., a consensus interferon variant, as a signaling agent. In various embodiments, the consensus interferon variant includes a functional derivative, analog, precursor, isoform, splice variant, or fragment of consensus interferon.

[0164] In some embodiments, consensus interferon variants are selected from the consensus interferon variants disclosed in U.S. Patent Nos. 4,695,623, 4,897,471, 5,541,293 and 8,496,921.The entire contents of these documents are incorporated herein by reference.For example, consensus interferon variants can comprise the amino acid sequence of IFN-CON2 or IFN-CON3 as disclosed in U.S. Patent Nos. 4,695,623, 4,897,471 and 5,541,293.In some embodiments, consensus interferon variants comprise the amino acid sequence of IFN-CON2, SEQ ID NO:344.

[0165] In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON3, SEQ ID NO:345.

[0166] In some embodiments, the consensus interferon variant comprises the amino acid sequence of any one of the variants disclosed in U.S. Patent No. 8,496,921. For example, the consensus variant may comprise the amino acid sequence of SEQ ID NO:346.

[0167] In another embodiment, the consensus interferon variant may comprise the amino acid sequence of SEQ ID NO:347.

[0168] In some embodiments, the consensus interferon variant may be pegylated, i.e., include a PEG moiety. In certain embodiments, the consensus interferon variant may include a PEG moiety attached at position S156C of SEQ ID NO: 347.

[0169] In some embodiments, the genetically modified interferon is a variant of human IFNα2a, resulting from the insertion of Asp near position 41 of the sequence of SEQ ID NO: 348 to give SEQ ID NO: 349 (which results in a renumbering of the sequence relative to the IFNα2a sequence), and has the following mutations: Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser, Asp86Glu, Ile104Thr, Gly106Glu, Thr110Glu, Lys117Asn, Arg125Lys, and Lys136Thr. All embodiments herein describing consensus interferon similarly apply to this genetically modified interferon.

[0170] In various embodiments, the consensus interferon variant comprises an amino acid sequence having one or more amino acid mutations, which in some embodiments may be independently selected from substitutions, insertions, deletions, and truncations.

[0171] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0172] In various embodiments, substitutions also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, C α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general).

[0173] In various embodiments, the consensus interferon is modified to have one or more mutations. In some embodiments, the mutations allow the consensus interferon variant to have one or more attenuated activities, such as one or more of reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to a non-mutated, e.g., wild-type, consensus interferon (e.g., a consensus interferon having the amino acid sequence of SEQ ID NO: 345 or 346). For example, compared to a non-mutated, e.g., wild-type, consensus interferon, the one or more attenuated activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, may be for a therapeutic receptor such as IFNAR. As a result, in various embodiments, the mutations allow the consensus interferon variant to have reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to a non-mutated, e.g., wild-type, consensus interferon.

[0174] In various embodiments, the consensus interferon is modified to have a mutation that reduces its binding affinity or activity to a therapeutic receptor, such as IFNAR. In some embodiments, the activity conferred by the consensus interferon is agonism of the therapeutic receptor (e.g., activation of a cellular effect at the site of treatment). For example, the consensus interferon may activate the therapeutic receptor. In such embodiments, the mutation results in a consensus interferon variant with reduced activating effect on the therapeutic receptor.

[0175] In some embodiments, the reduced affinity or activity for a therapeutic receptor can be restored by binding of a targeting moiety. In other embodiments, the reduced affinity or activity for a therapeutic receptor cannot be substantially restored by binding of a targeting moiety. In various embodiments, the therapeutic Fc-based chimeric proteins of the present invention reduce off-target effects because the consensus interferon variant has mutations that weaken its binding affinity or activity for a therapeutic receptor. In various embodiments, this reduces side effects observed with, for example, wild-type consensus interferon. In various embodiments, the consensus interferon variant is substantially inactive en route to the site of therapeutic action and retains its effect substantially on the specifically targeted cell type, thereby greatly reducing undesirable side effects.

[0176] In various embodiments, the consensus interferon variant is a variant of the consensus interferon that has attenuated or reduced activity against one or more therapeutic receptors. The therapeutic receptor may have one or more mutations that cause it to have reduced affinity, e.g., binding (e.g., KD) and / or activation (e.g., measurable as K A and / or EC50). In various embodiments, the reduced affinity for the therapeutic receptor allows for attenuated activity and / or signaling from the therapeutic receptor.

[0177] In various embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to the IFNAR1 subunit of IFNAR. In one embodiment, the consensus interferon variant has reduced affinity and / or activity for IFNAR1. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to the IFNAR2 subunit of IFNAR. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to the IFNAR1 and IFNAR2 subunits.

[0178] In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for IFNAR1 and one or more mutations that substantially reduce or eliminate binding or affinity for IFNAR2. In some embodiments, Fc-based chimeric proteins having such consensus interferon variants can provide target-selective IFNAR1 activity (e.g., IFNAR1 activity can be restored by targeting via a targeting moiety, e.g., SIRPα).

[0179] In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for IFNAR2 and one or more mutations that substantially reduce or eliminate binding or affinity for IFNAR1. In some embodiments, Fc-based chimeric proteins having such consensus interferon variants can provide target-selective IFNAR2 activity (e.g., IFNAR2 activity can be restored by targeting via a targeting moiety, e.g., SIRPα).

[0180] In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to IFNAR1 and one or more mutations that reduce its binding or affinity to IFNAR2. In some embodiments, Fc-based chimeric proteins having such consensus interferon variants can provide target-selective IFNAR1 and / or IFNAR2 activity (e.g., IFNAR1 and IFNAR2 activity can be restored by targeting via a targeting moiety, e.g., SIRPα).

[0181] In some embodiments, the consensus interferon is modified to have one or more amino acid mutations at positions 145-155, e.g., amino acid positions 149, 150 and / or 154, relative to SEQ ID NO: 346. In some embodiments, the consensus interferon is modified to have one or more amino acid mutations at positions 145-155, e.g., amino acid positions 149, 150, and / or 154, relative to SEQ ID NO: 346, where the substitutions are optionally hydrophobic and selected from alanine, valine, leucine, and isoleucine. In some embodiments, the consensus interferon variant comprises one or more mutations selected from M149A, R150A, and L154A, relative to SEQ ID NO: 342.

[0182] In some embodiments, the consensus interferon is based on SEQ ID NO: 342. and modified to have a mutation at amino acid position 121 (i.e., K121). In one embodiment, the consensus interferon comprises a K121E mutation relative to SEQ ID NO:342.

[0183] In some embodiments, the altered signal transduction agent is vascular endothelial growth factor (VEGF). VEGF is a potent growth factor that plays an important role in both physiological and pathological angiogenesis, regulating vascular permeability and acting as a growth factor for cells expressing VEGF receptors. Additional functions include stimulating cell migration, particularly of macrophage lineages and endothelial cells. In addition to at least three receptors (VEGFR-1, VEGFR-2, and VEGFR3), several members of the VEGF growth factor family exist. VEGF family members can bind and activate two or more VEGFR types. For example, VEGF-A binds VEGFR1 and VEGFR2, while VEGF-C can bind VEGFR2 and VEGFR3. VEGFR1 and VEGFR2 activation regulates angiogenesis, and VEGFR3 activation is involved in lymphangiogenesis. Most pro-angiogenic signals are generated from VEGFR2 activation. It has been reported that VEGFR1 activation may be associated with a negative role in angiogenesis. VEGFR1 signaling has also been reported to be important for in vivo tumor progression via bone marrow-derived VEGFR1-positive cells (contributing to the formation of a premetastatic microenvironment in bone). Several VEGF-A-based therapeutic approaches, primarily directed against or neutralizing therapeutic antibodies, have been developed for use in the treatment of various human tumors that depend primarily on angiogenesis. However, these are not without side effects. This is not surprising given that these agents act as general, non-cell / tissue-specific inhibitors of VEGF / VEGFR interactions. Therefore, it would be desirable to restrict VEGF (e.g., VEGF-A) / VEGFR2 inhibition to specific target cells (e.g., tumor vasculature endothelial cells).

[0184] In some embodiments, the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E and VEGF 121 , VEGF 121 b, VEGF 145 , VEGF 165 , VEGF 165 b, VEGF 189, and VEGF 206 These isoforms include various VEGF-A isoforms such as those described above. In some embodiments, the altered signaling agent has reduced affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In some embodiments, the altered signaling agent has reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In certain embodiments, the altered signaling agent has reduced affinity and / or activity for VEGFR-2 (KDR / Flk-1) and / or has reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1). Such embodiments are used, for example, in wound healing methods or in the treatment of ischemia-related diseases (mediated, without intending to be bound by theory, by the effects of VEGFR2 on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1 (Flt-1), which is associated with cancer and pro-inflammatory activity, is avoided. In various embodiments, VEGFR-1 (Flt-1) functions as a decoy receptor, thereby substantially reducing or eliminating affinity for this receptor and avoiding sequestration of the therapeutic agent. In certain embodiments, the modified signal transduction agent has reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or reduced or eliminated affinity and / or activity for VEGFR-2 (KDR / Flk-1). In some embodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signal transduction agent has reduced affinity and / or activity for VEGFR3. Alternatively, the modified signal transduction agent has substantially reduced or eliminated affinity and / or activity for VEGFR3.

[0185] Pro-angiogenic therapies are also important in various diseases (e.g., ischemic heart disease, hemorrhage, etc.), and include VEGF-based therapeutics. Activation of VEGFR2 is pro-angiogenic (acting on endothelial cells). VEGFR1 can stimulate the migration of inflammatory cells (including, e.g., macrophages), leading to inflammation associated with vascular hyperpermeability. Activation of VEGFR1 can also activate myeloid cells associated with tumor microenvironment formation. Therefore, VEGF-based therapeutics selective for VEGFR2 activation would be desirable in this case. Furthermore, cells that specifically target, for example, endothelial cells would be desirable.

[0186] In some embodiments, the altered signal transduction agent has reduced affinity and / or activity (e.g., antagonistic) for VEGFR-2 and / or substantially reduced or eliminated affinity and / or activity for VEGFR-1. When targeted to tumor vasculature endothelial cells via a targeting moiety that binds to a tumor endothelial cell marker (e.g., PSMA), such a construct will specifically inhibit VEGFR2 activation on such marker-positive cells but will not activate VEGFR1 en route to or on the target cells (when activity is eliminated), thus, for example, abolishing the induction of an inflammatory response. This would provide a more selective and safer antiangiogenic therapy for many tumor types than VEGF-A neutralizing therapy.

[0187] In some embodiments, the altered signal transduction agent has reduced affinity and / or activity (e.g., agonist activity) for VEGFR-2 and / or substantially reduced or eliminated affinity and / or activity for VEGFR-1. By targeting vascular endothelial cells, in some embodiments, such constructs promote angiogenesis without inducing the inflammatory response associated with VEGFR1. Thus, such constructs will have targeted pro-angiogenic effects with substantially reduced risk of side effects resulting from systemic activation of VEGFR2 and VEGFR1.

[0188] In an exemplary embodiment, the altered signal transduction agent is a VEGF having the amino acid sequence VEGF165 (wild-type) (SEQ ID NO: 350). 165 is.

[0189] In another exemplary embodiment, the altered signal transduction agent is a VEGF having the amino acid sequence VEGF165b (wild-type) (SEQ ID NO: 351). 165b is.

[0190] In these embodiments, the modified signal transduction agent has a mutation at amino acid 183 (e.g., a substitution mutation at 183, e.g., 183K, 183R, or 183H). Without intending to be bound by theory, it is believed that such mutations may result in reduced receptor binding affinity. See, e.g., U.S. Patent No. 9,078,860, the entire contents of which are incorporated herein by reference.

[0191] In one embodiment, the signal-modifying substance is TNFα. TNF is a pleiotropic cytokine with many diverse functions, including regulating cell proliferation, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and trafficking, inflammation, and septic shock. It binds to two distinct membrane receptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits a very broad expression pattern, while TNFR2 is selectively expressed on specific populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. In response to receptor activation, distinct biological pathways are activated, although some overlap exists. As a general rule, and without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), while TNFR2 signaling is associated with the activation of cell survival signals (e.g., activation of the NFκB pathway). Administration of TNF results in systemic toxicity, primarily due to the involvement of TNFR1. However, activation of TNFR2, like TNFR1, also It should be noted that, due to the diverse actions of TNF, control over TNF targeting and activity is important in the development of TNF-based therapeutic agents.

[0192] In some embodiments, the modified signal transduction agent has reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signal transduction agent has substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2. TNFR1 is expressed in most tissues and is involved in cell death signaling; in contrast, TNFR2 is involved in cell survival signaling. Thus, in embodiments relating to cancer therapy, the modified signal transduction agent has reduced affinity and / or activity for TNFR1 and / or substantially reduced or eliminated affinity and / or activity for TNFR2. In these embodiments, the chimeric protein can target cells in which apoptosis is desired, such as tumor cells or tumor vascular endothelial cells. In embodiments relating to methods of promoting cell survival, e.g., in neurogenesis for the treatment of neurodegenerative disorders, the modified signal transduction agent has reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1. In other words, the chimeric protein, in some embodiments, comprises a modified TNFα agent that can prioritize either death or survival signaling.

[0193] In some embodiments, the chimeric protein 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 chimeras are, in some embodiments, more potent inducers of apoptosis than chimeras having only wild-type TNF and / or mutations that result in reduced affinity and / or activity for TNFR1. Such chimeras are, in some embodiments, used to induce tumor cell death or tumor vascular endothelial cell death (e.g., in the treatment of cancer). Also, in some embodiments, these chimeras inhibit TNF via, for example, TNFR2. reg This avoids or reduces cellular activation, thus further supporting TNFR1-mediated anti-tumor activity in vivo.

[0194] In some embodiments, the chimeric protein has a modified TNF with reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1. Such chimeras, in some embodiments, are more potent activators of cell survival in some cell types, which may be of particular therapeutic interest in a variety of diseases, including, but not limited to, stimulation of neurogenesis. Furthermore, such TNFR2-selected chimeras are also useful in the treatment of autoimmune diseases (e.g., Crohn's disease, diabetes, MS, colitis, etc., and many others described herein). In some embodiments, the chimeras target autoreactive T cells. In some embodiments, the chimeras target T cells. reg Promotes cell activation and indirect suppression of cytotoxic T cells.

[0195] In some embodiments, the chimeric protein results in the death of autoreactive T cells, for example, by activating TNFR2 and / or bypassing TNFR1 (e.g., by modified TNF with reduced affinity and / or activity for TNFR2 and / or with substantially reduced or eliminated affinity and / or activity for TNFR1). Without wishing to be bound by theory, these autoreactive T cells have altered apoptosis / survival signaling due to changes in NFκB pathway activity / signaling. In some embodiments, the chimera induces autoreactive T cells with impaired or altered NFκB pathway activity and, optionally, altered sensitivity to certain death-inducing signals (e.g., TNFR2 activation), underlying an imbalance in cell death (apoptosis) / survival signaling profiles. resulting in the death of

[0196] In some embodiments, TNFR2-based chimeras have additional therapeutic applications for a variety of autoimmune diseases, particularly diseases including cardiac disease, demyelinating and neurodegenerative disorders, and infectious diseases.

[0197] In one embodiment, the wild-type TNFα has the amino acid sequence of SEQ ID NO:352.

[0198] In such embodiments, the modified TNFα agent has mutations at one or more amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, resulting in a modified TNFα with reduced receptor binding affinity. See, e.g., U.S. Patent No. 7,993,636, the entire contents of which are incorporated herein by reference.

[0199] In some embodiments, the modified human TNFα portion has mutations 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 WO 2015 / 007903, the entire contents of which are incorporated herein by reference (GenBank Accession Number BAG70306, Version BAG70306.1 G1:197692685, numbered according to the human TNF sequence). In some embodiments, the modified human TNFα portion 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 some embodiments, the human TNFα portion has a mutation selected from Y87Q, Y87L, Y87A, and Y87F, and Y87H. In another embodiment, the human TNFα portion has a mutation selected from I97A, I97Q, and I97S. In a further embodiment, the human TNFα portion has a mutation selected from Y115A and Y115G. In some embodiments, the human TNFα portion has an E146K mutation. In some embodiments, the human TNFα portion has Y87H and E146K mutations. In some embodiments, the human TNFα portion has Y87H and A145R mutations. In some embodiments, the human TNFα portion has R32W and S86T mutations. In some embodiments, the human TNFα portion has R32W and E146K mutations. In some embodiments, the human TNFα portion has L29S and R32W mutations. In some embodiments, the human TNFα portion has D143N and A145R mutations. In certain embodiments, the human TNFα portion has D143N and A145R mutations.In certain embodiments, the human TNFα portion has A145T, E146D, and S147D mutations.In one embodiment, the human TNFα portion has A145T and S147D mutations.

[0200] In some embodiments, the modified TNFα agent comprises one or more mutations selected from N39Y, S147Y, and Y87H, as described in WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0201] In some embodiments, the modified human TNFα portion has mutations that result in receptor selectivity as described in International Application No. PCT / IB2016 / 001668, the entire contents of which are incorporated herein by reference. In some embodiments, the mutations to TNF are selective for TNFR1. In some embodiments, the TNFR1-selective TNFR1-selective TNFR1-selective TNNFα portion has mutations that result in receptor selectivity as described in International Application No. PCT / IB2016 / 001668, the entire contents of which are incorporated herein by reference. In some embodiments, the mutations to TNF are selective for TNFR1. The mutation to F is at 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 at 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 mutations to TNF that are TNFR2 selective are one or more of A145R, A145T / S147D, and A145T / E146D / S147D.

[0202] In certain embodiments, the altered signal transduction agent is TNFβ. TNFβ can form homotrimers or heterotrimers with LTβ (LTα1β2). In some embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2 and / or herpesvirus entry mediator (HEVM) and / or LTβR.

[0203] In one embodiment, the wild-type TNFβ has the amino acid sequence of SEQ ID NO:353.

[0204] In such embodiments, the modified soluble agent can include a mutation at one or more amino acid positions 106-113, which results in a modified TNFβ with reduced receptor binding affinity for TNFR2. In certain embodiments, the modified soluble agent has one or more substitution mutations at amino acid positions 106-113. In exemplary 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 agent has an insertion of about 1 to about 3 amino acids at positions 106-113.

[0205] In some embodiments, the modifier is a TNF family member (e.g., TNFα, TNFβ), which may be in a single-chain trimer form, as described in WO 2015 / 007903, the entire contents of which are incorporated herein by reference.

[0206] In some embodiments, the modifier is a TNF family member (e.g., TNFα, TNFβ) that has reduced affinity and / or activity, i.e., antagonist activity, for TNFR1 (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 modifier is a TNF family member (e.g., TNFα, TNFβ) that also, optionally, has substantially reduced or eliminated affinity and / or activity for TNFR2. In some embodiments, the modifier is a TNF family member (e.g., TNFα, TNFβ) that has reduced affinity and / or activity, i.e., antagonist activity, for TNFR2 (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 modifier is a TNF family member (e.g., TNFα, TNFβ) that also optionally has substantially reduced or eliminated affinity and / or activity for TNFR1. Constructs of such embodiments can be 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 in a single chain trimer form, as described in WO 2015 / 007903.

[0207] In certain embodiments, the modified signal transduction agent is TRAIL. In some embodiments, the modified TRAIL agent has 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 agent has reduced affinity and / or activity for DR4 (TRAIL-RI) and / or DR5 (TRAIL-RII) and / or DcR1 and / or DcR2.

[0208] In one embodiment, wild-type TRAIL has the amino acid sequence of SEQ ID NO:354.

[0209] In such embodiments, the modified TRAIL agent may contain mutations at amino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209, T214-L220, K224-A226, W231, E236-L239, E249-K251, T261-H264, and H270-E271 (GenBank accession number NP_003801, version 10NP_003801.1, G1:4507593, numbering based on the human sequence; see above).

[0210] 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. Representative mutations include one or more of the following amino acid positions: Y189, R191, Q193, H264, I266, and D267. For example, the mutations may be one or more of Y189Q, R191K, Q193R, H264R, I266L, and D267Q. In certain embodiments, the modified TRAIL substance contains the mutations Y189Q, R191K, Q193R, H264R, I266L, and D267Q.

[0211] In some embodiments, the modified TRAIL substance may contain 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. Representative 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 contains 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 which are incorporated herein by reference.

[0212] In certain embodiments, the modified signal transduction agent is TGFα. In such embodiments, the modified TGFα agent has reduced affinity and / or activity for the epidermal growth factor receptor (EGFR). In some embodiments, the modified TGFα agent has substantially reduced or eliminated affinity and / or activity for the epidermal growth factor receptor (EGFR).

[0213] In certain embodiments, the altered signal transduction agent is TGFβ. In such embodiments, the altered signal transduction agent is a reduced TGFBR1 and / or TGFBR2 signal transduction agent. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for TGFBR1 and / or TGFBR2. In some embodiments, the modified signaling agent optionally has 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, TGF-beta selects TGFBR1 over TGFBR2 or TGFBR2 over TGFBR1. Similarly, without intending to be bound by theory, LAP may act as a reservoir of ligand for the TGF-beta receptor. In some embodiments, the modified signaling agent has reduced affinity and / or activity for TGFBR1 and / or TGFBR2 and / or substantially reduced or eliminated affinity and / or activity for latency-associated peptide (LAP). In some embodiments, such chimeras are used in Kamuracchi-Engelman disease or other diseases associated with inappropriate TGF-beta signaling.

[0214] In some embodiments, the modified substance is a TGF family member (e.g., TGFα, TGFβ) that has reduced affinity and / or activity, i.e., antagonist activity, for one or more of TGFBR1, TGFBR2, and TGFBR3 (e.g., natural antagonist activity or antagonist activity that is the 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 modified substance is a TGF family member (e.g., TGFα, TGFβ) that also optionally has substantially reduced or eliminated affinity and / or activity for one or more of TGFBR1, TGFBR2, and TGFBR3.

[0215] In some embodiments, the modified substance is a TGF family member (e.g., TGFα, TGFβ) that has reduced affinity and / or activity, i.e., antagonist activity, for TGFBR1 and / or TGFBR2 (e.g., natural antagonist activity or antagonist activity that is the 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 modified substance is a TGF family member (e.g., TGFα, TGFβ) that also, optionally, has substantially reduced or eliminated affinity and / or activity for TGFBR3.

[0216] In some embodiments, the modified signal transduction agent is an interleukin. In some embodiments, the modified signal transduction agent is IL-1. In some embodiments, the modified signal transduction agent is IL-1α or IL-1β. In some embodiments, the modified signal transduction agent has reduced affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signal transduction agent has substantially reduced or eliminated affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signal transduction agent has reduced affinity and / or activity for IL-1R2. In some embodiments, the modified signal transduction agent has substantially reduced or eliminated affinity and / or activity for IL-1R2. For example, in some embodiments, the modified IL-1 agent avoids interaction with IL-1R2, thus substantially reducing its function as a decoy and / or sink for therapeutic agents.

[0217] In one embodiment, wild-type IL-1β has the amino acid sequence of IL-1 beta (mature, wild-type) (SEQ ID NO: 355).

[0218] IL-1 is a pro-inflammatory cytokine and an important immune system regulator. IL-1 is a potent activator of CD8 T cell responses, increasing the proportion of Th17 cells and enhancing the proliferation of IFNγ- and IL-4-producing cells. + A potent regulator of T cells, antigen-specific CD8 + Enhances T cell proliferation, differentiation, peripheral migration, and memory. IL-1 receptors include IL-1R1 and IL-1R2. Binding to and signaling through IL-1R1 constitutes the mechanism by which IL-1 mediates many of its biological (and pathological) effects. IL-1R2 can function as a decoy receptor, thereby reducing the availability of IL-1 for interaction and signaling through IL-1R1.

[0219] In some embodiments, the modified IL-1 has reduced affinity and / or activity (e.g., agonist activity) for IL-1R1. In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and / or activity for IL-1R2. Such embodiments result in the prevention of restorable IL-1 / IL-1R1 signaling and loss of therapeutic chimeras to ILR2 and consequently reduced IL-1 dosage (e.g., compared to wild-type or chimeras having only attenuating mutations to ILR1). Such constructs are used, e.g., in methods for treating cancer, including, e.g., stimulating the immune system to mount an anti-cancer response.

[0220] In some embodiments, the modified IL-1 has reduced affinity and / or activity for IL-1R1 (e.g., antagonist activity, e.g., naturally occurring 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 some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and / or activity for IL-1R2. In such embodiments, IL-1 / IL-1R1 signaling is not restorable, and loss of therapeutic chimeras to ILR2 and consequently reduced IL-1 dosages are required (e.g., compared to wild-type or chimeras having only attenuating mutations to ILR1). Such constructs are used in methods for treating, e.g., autoimmune diseases, including, e.g., suppressing the immune system.

[0221] In such embodiments, the modified signal transducer has a deletion of amino acids 52-54, which produces a modified human IL-1β with reduced binding affinity for type I IL-1R and reduced biological activity. See, e.g., WO 1994 / 000491, the entire contents of which are incorporated herein by reference. In some embodiments, the modified human IL-1β is selected from the group consisting of 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, E and one or more substitution mutations selected from 221S / N224A, N224S / K225S, E244K, N245Q (where X can be any amino acid change, e.g., a non-conservative change), which exhibit reduced binding to IL-1R, as described, for example, in WO 2015 / 007542 and WO 2015 / 007536, the entire contents of which are incorporated herein by reference (GenBank Accession No. NP_000567, version NP-000567.1, G1:10835145, numbered based on the human IL-1β sequence). In some embodiments, the modified human IL-1β may 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 certain embodiments, the modified human IL-1β comprises the mutations Q131G and Q148G. In certain embodiments, the modified human IL-1β comprises the mutations Q148G and K208E. In some embodiments, the modified human IL-1β comprises the mutations R120G and Q131G. In some embodiments, the modified human IL-1β comprises the mutations R120G and H146A. In some embodiments, the modified human IL-1β comprises the mutations R120G and H146N. In some embodiments, the modified human IL-1β comprises the mutations R120G and H146R. In some embodiments, the modified human IL-1β comprises the mutations R120G and H146E. In some embodiments, the modified human IL-1β comprises the mutations R120G and H146G. In some embodiments, the modified human IL-1β comprises the mutations R120G and K208E. In some embodiments, the modified human IL-1β comprises the mutations R120G, F162A, and Q164E.

[0222] In certain embodiments, the modified signal transduction agent is IL2. In such embodiments, the modified signal transduction agent has reduced affinity and / or activity for IL2Rα and / or IL2Rβ and / or IL2Rγ. In some embodiments, the modified signal transduction agent has reduced affinity and / or activity for IL2Rβ and / or IL2Rγ. In some embodiments, the modified signal transduction agent has 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 antagonistic to IL2Rβ and / or IL2Rγ. For example, the constructs of the present invention can be used to target CD8 receptors bearing IL2 receptors β and γ. + It favors attenuated activation of T cells (which can confer antitumor effects) and T cells bearing IL2 receptors α, β, and γ. reg(which can have immunosuppressive, tumor-promoting effects). Furthermore, in some embodiments, selectivity for IL2Rβ and / or IL2Rγ over IL2Rα avoids IL2 side effects such as pulmonary edema. IL2-based chimeras are also useful for treating autoimmune diseases, for example, when the modified IL2 is antagonistic to IL2Rβ and / or IL2Rγ (e.g., naturally antagonistic activity or antagonistic 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). For example, the constructs of the present invention can be used to target CD8 IL2 receptors β and γ. + It favors attenuated T cell suppression (and thus suppresses immune responses) and T cells with IL2 receptors α, β, and γ. reg Alternatively, in some embodiments, chimeras with IL2 are not preferred. reg activation, and therefore immunosuppression, of CD8 + For example, these constructs are used to treat diseases or conditions that would benefit from immunosuppression, such as autoimmune disorders, without a preference for T cell activation.

[0223] In some embodiments, the chimeric protein is Clec9A + In some embodiments, these constructs comprise a targeting moiety described herein that targets dendritic cells, and the modified IL2 agent has reduced affinity and / or activity for IL2Rβ and / or IL2Rγ and / or substantially reduced or eliminated affinity and / or activity for IL2Rα. + Targets dendritic cell activity, typically T reg In some embodiments, such constructs have an enhanced immunostimulatory effect compared to wild-type IL2 (by not stimulating Tregs, without wishing to be bound by theory), while eliminating or reducing the systemic toxicity associated with IL2.

[0224] In one embodiment, wild-type IL2 has the amino acid sequence of IL2 (mature, wild-type) (SEQ ID NO: 356).

[0225] In such embodiments, the modified human IL2 substance contains one or more mutations at amino acid positions L72 (L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K), F43 (F43A, F43G, F43S, F43T, F43Q, F43E, F43N, F43D, F43R, or F43K), F44 (F44A, F44G, F44S, F44T, F44Q, F44E, F44N, F44D, F44R, or F44K), F45 (F45A, F45B, F45C, F45D, F45E, F45F, F45G, F45H, F45I ... and at position Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K). Without wishing to be bound by theory, it is believed that these modified IL2 agents have reduced affinity for the high-affinity IL2 receptor and maintain affinity for the intermediate-affinity IL2 receptor compared to wild-type IL2. See, e.g., U.S. Patent Application Publication No. 2012 / 0244112, the entire contents of which are incorporated herein by reference.

[0226] In some embodiments, the modified IL2 agent has one or more mutations at amino acid positions R38, F42, Y45, and E62. For example, the modified IL2 agent may include one or more of R38A, F42A, Y45A, and E62A. In some embodiments, the modified IL2 agent may include a mutation at C125, such as C125S. In such embodiments, the modified IL2 agent 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 which is incorporated herein by reference. In some embodiments, the modified IL2 agent having mutations 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, modified IL2 agents having mutations at R38, F42, Y45, and / or E62 are less toxic than wild-type IL2 agents. Chimeric proteins comprising modified IL2 agents with substantially reduced affinity and / or activity for IL2Rα may find use, for example, in oncology. In other embodiments, modified IL2 agents may have substantially reduced affinity and / or activity for IL2Rβ, for example, as described in WO 2016 / 025385, the entire disclosure of which is incorporated herein by reference. In such embodiments, the modified IL2 agents may induce proliferation of Treg cells but not CD8+ The chimeric proteins containing modified IL2 substances with substantially reduced affinity and / or activity for IL2Rβ may find use, for example, in the treatment of autoimmune diseases. In some embodiments, the modified IL2 substances may have one or more mutations at amino acid positions N88, D20, and / or A126. For example, the modified IL2 substances may include one or more of N88R, N88I, N88G, D20H, Q126L, and Q126F.

[0227] In various embodiments, the modified IL2 agent may include a mutation at D109 or C125. For example, the mutation may be D109C or C125S. In some embodiments, a modified IL2 having a mutation at D109 or C125 may be utilized for conjugation to a PEG moiety.

[0228] In certain embodiments, the altered signal transduction agent is IL3. In some embodiments, the altered signal transduction agent has reduced affinity and / or activity for the IL3 receptor, which is a heterodimer having a unique alpha chain paired with a common beta (beta c or CD131) subunit. In some embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for the IL3 receptor, which is a heterodimer having a unique alpha chain paired with a common beta (beta c or CD131) subunit.

[0229] In certain embodiments, the altered signal transduction agent is IL4. In such embodiments, the altered signal transduction agent has reduced affinity and / or activity for type 1 and / or type 2 IL4 receptors. In such embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for type 1 and / or type 2 IL4 receptors. Type 1 IL4 receptors are composed of IL4Rα subunits with a common γ chain. The type 2 IL4 receptor is composed of an IL4Rα subunit bound to a different subunit known as IL-13Rα1 and specifically binds IL4. In some embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for the type 2 IL4 receptor.

[0230] In one embodiment, wild-type IL4 has the amino acid sequence of IL2 (mature, wild-type) (SEQ ID NO: 357).

[0231] In such embodiments, the modified IL4 agents have 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 agents maintain activity mediated by the type I receptor but significantly reduce biological activities mediated by other receptors. See, e.g., U.S. Pat. No. 6,433,157, the entire contents of which are incorporated herein by reference.

[0232] In certain embodiments, the modified signal transduction agent is IL6. IL6 signals through a cell surface type I cytokine receptor complex comprising the ligand-binding IL6R chain (CD126) and the signal transduction component gp130. IL6 can also bind to a soluble form of IL6R (sIL6R), which is the extracellular portion of IL6R. The sIL6R / IL6 complex is involved in neurite outgrowth and neuronal survival and may therefore be important for nerve regeneration through remyelination. Thus, in some embodiments, the modified signal transduction agent has reduced affinity and / or activity for IL6R / gp130 and / or sIL6R. In some embodiments, the modified signal transduction agent has substantially reduced or eliminated affinity and / or activity for IL6R / gp130 and / or sIL6R.

[0233] In one embodiment, wild-type IL6 has the amino acid sequence of IL6 (mature, wild-type) (SEQ ID NO: 358).

[0234] In such embodiments, the modified signal transduction agent has one or more mutations at amino acids 58, 160, 163, 171, or 177. Without wishing to be bound by theory, it is believed that these modified IL6 agents exhibit reduced binding affinity for IL6Rα and reduced biological activity. See, e.g., WO 97 / 10338, the entire contents of which are incorporated herein by reference.

[0235] In certain embodiments, the altered signaling agent is IL-10. In such embodiments, the altered signaling agent has reduced affinity and / or activity for IL-10 receptor 1 and IL-10 receptor 2. In some embodiments, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for IL-10 receptor 1 and IL-10 receptor 2.

[0236] In certain embodiments, the altered signal transduction agent is IL-11. In such embodiments, the altered signal transduction agent has reduced affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130. In such embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130.

[0237] In some embodiments, the altered signal transduction agent is IL-12. The altered signaling agent has reduced affinity and / or activity for IL-12Rβ1 and / or IL-12Rβ2. In such embodiments, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for IL-12Rβ1 and / or IL-12Rβ2.

[0238] In certain embodiments, the altered signal transduction agent is IL-13. In such embodiments, the altered signal transduction agent has reduced affinity and / or activity for the IL4 receptor (IL4Rα) and IL-13Rα1. In some embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for the IL4 receptor (IL4Rα) or IL-13Rα1.

[0239] In certain embodiments, wild-type IL-13 has the amino acid sequence of IL-13 (mature, wild-type) (SEQ ID NO: 359).

[0240] In such embodiments, the modified IL-13 material 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, it is believed that these modified IL-13 materials exhibit reduced biological activity. See, e.g., International Publication No. WO 2002 / 018422, the entire contents of which are incorporated herein by reference.

[0241] In certain embodiments, the altered signal transduction agent is IL-18. In some embodiments, the altered signal transduction agent has reduced affinity and / or activity for IL-18Rα and / or IL-18Rβ. In some embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for IL-18Rα and / or IL-18Rβ. In some embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for IL-18Rα2, an isoform of IL-18Rα that lacks the TIR domain required for signal transduction.

[0242] In certain embodiments, wild-type IL-18 has the amino acid sequence of IL-18 (wild-type) (SEQ ID NO: 360).

[0243] In such embodiments, the modified IL-18 agent may comprise one or more mutations in amino acids or amino acid regions selected from Y37 to K44, R49 to Q54, D59 to R63, E67 to C74, R80, M87 to A97, N127 to K129, Q139 to M149, K165 to K171, R183 and Q190 to N191 (GenBank Accession Number AAV38697, version AAV38697.1, GI:54696650, numbering based on the human IL-18 sequence), as described in WO 2015 / 007542, the entire contents of which are incorporated herein by reference.

[0244] In certain embodiments, the altered signal transduction agent is IL33. In such embodiments, the altered signal transduction agent has reduced affinity and / or activity for ST2 receptor 1 and IL-1RAcP. In some embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for ST2 receptor and IL-1RAcP.

[0245] In one embodiment, the wild-type IL33 has the amino acid sequence of SEQ ID NO:361.

[0246] In such embodiments, the modified IL33 material may comprise I113-Y122, S, as described in WO 2015 / 007542, the entire contents of which are incorporated herein by reference. The sequence may contain one or more mutations in amino acids or amino acid regions selected from E127 to E139, E144 to D157, Y163 to M183, E200, Q215, L220 to C227, and T260 to E269 (GenBank accession number NP_254274, version 254274.1, Gl:15559209, numbering based on the human sequence).

[0247] In one embodiment, the altered signal transduction agent is epidermal growth factor (EGF). EGF is a family of potent growth factors. Members include EGF, HB-EGF, and others, such as TGFα, amphiregulin, neuregulin, epiregulin, and betacellulin. EGF family receptors include EGFR (ErbB1), ErbB2, ErbB3, and ErbB4, which can function as homodimeric and / or heterodimeric receptor subtypes. Different EGF family members exhibit distinct selectivity for various receptor subtypes. For example, EGF binds to ErbB1 / ErbB1, ErbB1 / ErbB2, ErbB4 / ErbB2, and several other heterodimeric subtypes. HB-EGF has a similar pattern, but binds to ErbB4 / 4. Positive or negative modulation of EGF (EGF-like) growth factor signaling is of great therapeutic interest. For example, inhibition of EGFR signaling is of interest in the treatment of various cancers in which EGFR signaling constitutes a major growth-promoting signal, or stimulation of EGFR signaling is of therapeutic interest in, for example, wound healing (acute and chronic) and oral mucositis (a major side effect of various cancer therapies, including but not limited to radiation therapy).

[0248] In some embodiments, the altered signal transduction agent has reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4. Such embodiments are used, for example, in methods for treating wounds. In some embodiments, the altered signal transduction agent binds to one or more of ErbB1, ErbB2, ErbB3, and ErbB4 and antagonizes the activity of the receptor. In such embodiments, the altered signal transduction agent has reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4, thereby antagonizing the activity of the receptor in a weakened manner. Such embodiments are used, for example, in the treatment of cancer. In certain embodiments, the altered signal transduction agent has reduced affinity and / or activity for ErbB1. ErbB1 is a therapeutic target for kinase inhibitors—most of which have side effects because they are not very selective (e.g., gefitinib, erlotinib, afatinib, brigatinib, and icotinib). In some embodiments, attenuated antagonistic ErbB1 signaling is more on-target and has fewer side effects than other agents that target the EGF receptor.

[0249] In some embodiments, the altered signal transduction agent has reduced affinity and / or activity for ErbB1 (e.g., antagonist activity, e.g., naturally occurring 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) and / or substantially reduced or eliminated affinity and / or activity for ErbB4 or other subtypes with which it may interact. Specific targeting via the targeting moiety will achieve cell-selective inhibition of ErbB1 / ErbB1 receptor activation (antagonism, e.g., naturally occurring 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) without involving other receptor subtypes that may be associated with inhibition-related side effects. Thus, in contrast to EGFR kinase inhibitors, which inhibit EGFR activity in all cell types in the body, such constructs may be used to inhibit activated EGFR in a cell-selective manner (e.g., by receptor amplification, overexpression, etc.) with reduced side effects. This would provide anti-EGFR (ErbB1) drug action (tumor cells with EGFR signaling).

[0250] In some embodiments, the altered signal transduction agent has reduced affinity and / or activity (e.g., antagonist activity) for ErbB4 and / or other subtypes with which it interacts. Selective activation of ErbB1 signaling is achieved by targeting specific target cells via a targeting moiety (e.g., epithelial cells). Such constructs are used in some embodiments for wound treatment (promoting wound healing) with reduced side effects, particularly for the treatment of chronic conditions and for applications other than local administration of therapeutic agents (e.g., systemic wound healing).

[0251] In certain embodiments, the modified signal transduction substance is insulin or an insulin analog. In some embodiments, the modified insulin or insulin analog has 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 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 allows for the control of diabetes, obesity, metabolic disorders, etc., while avoiding cancer-promoting effects by shifting away from the IGF1 or IGF2 receptor.

[0252] In some embodiments, the altered signal transduction substance is insulin-like growth factor-I or insulin-like growth factor-II (IGF1 or IGF2). In some embodiments, the altered signal transduction substance is IGF1. In such embodiments, the altered signal transduction substance has reduced affinity and / or activity for the insulin receptor and / or IGF1 receptor. In some embodiments, the altered signal transduction substance binds to the IGF1 receptor and antagonizes the activity of the receptor. In such embodiments, the altered signal transduction substance has reduced affinity and / or activity for the IGF1 receptor, thereby allowing the activity of the receptor to be antagonized in an attenuated manner. In some embodiments, the altered signal transduction substance has substantially reduced or eliminated affinity and / or activity for the IGF1 receptor. In some embodiments, the altered signal transduction substance has reduced affinity and / or activity for the IGF2 receptor, thereby allowing the activity of the receptor to be antagonized in an attenuated manner. In some embodiments, the altered signal transduction substance has substantially reduced or eliminated affinity and / or activity for the insulin receptor, thus not interfering with insulin signaling. In various embodiments, this is applicable to cancer treatment. In various embodiments, the agent may prevent IR isoform A from creating resistance to cancer treatment.

[0253] In one embodiment, the chimeric protein has (i) a targeting moiety to Clec9A and (ii) a targeting moiety directed to tumor cells, along with any modified or mutated signal transduction agent described herein. In one embodiment, the chimeric protein has a targeting moiety directed to Clec9A on dendritic cells and a second targeting moiety directed to PD-L1 or PD-L2 on tumor cells.

[0254] In one embodiment, the chimeric protein has (i) a targeting moiety directed to Clec9A and (ii) a targeting moiety directed to a checkpoint inhibitor marker, along with any modified or mutated interferon described herein. In an embodiment, the chimeric protein has a targeting moiety directed to Clec9A on dendritic cells and a second targeting moiety directed to PD-1.

[0255] In various embodiments, the signaling agent is a toxin or a toxic enzyme. In some embodiments, the toxins or toxic enzymes are 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 which is incorporated herein by reference. In such embodiments, the chimeric proteins of the present invention may be used to induce cell death in a cell-type-specific manner. In such embodiments, the toxins may be modified, e.g., mutated, to reduce the affinity and / or activity of the toxin to reduce its effectiveness, as described herein for other signaling molecules.

[0256] Multispecific chimeras and fusions with signal transduction agents In various embodiments, the Clec9A-binding agents of the invention are part of a chimera or fusion with one or more signal transduction agents and / or one or more additional targeting moieties described herein. Thus, for example, the invention provides chimera or fusion proteins comprising one or more signal transduction agents and a targeting moiety for Clec9A and / or one or more additional targeting moieties.

[0257] In various embodiments, Clec9A-binding agents of the invention are multispecific, i.e., they comprise two or more targeting moieties having recognition domains that recognize and bind to two or more targets, e.g., antigens, receptors, or epitopes. In such embodiments, Clec9A-binding agents of the invention 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 or different receptors. In various embodiments, such multispecific Clec9A-binding agents exhibit advantageous properties, such as improved avidity and / or selectivity. In certain embodiments, Clec9A-binding agents of the invention comprise two targeting moieties and are bispecific, i.e., they bind to and recognize two epitopes on the same antigen or on different antigens.

[0258] In various embodiments, the multispecific Clec9A-binding agents of the invention comprise two or more targeting moieties, each of which is an antibody or antibody derivative described herein. In one embodiment, the multispecific Clec9A-binding agents of the invention comprise at least one VHH comprising an antigen-recognition domain against Clec9A and one antibody or antibody derivative comprising an antigen-recognition domain against a tumor antigen.

[0259] In various embodiments, a multispecific Clec9A binding agent of the invention has two or more targeting moieties that target different antigens or receptors, and one targeting moiety can be attenuated for that antigen or receptor, e.g., a targeting moiety that binds the antigen or receptor with low affinity or avidity (e.g., including binding with a lower affinity or avidity than the other targeting moieties have for that antigen or receptor, e.g., the difference between binding affinities can 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., a targeting moiety with a lower affinity or avidity can bind to the antigen or receptor with a K in the mid-to-high nM or low-to-mid μM range). D While higher affinity or avidity targeting moieties may bind with K in the mid-to-high pM or low-to-mid nM range. D For example, in some embodiments, the present multispecific Clec9A binding agents comprise attenuated targeting moieties directed against promiscuous antigens or receptors, which may improve targeting to cells of interest (e.g., via other targeting moieties) and prevent cross-cell effects, including those not targeted therapeutically (e.g., by binding to promiscuous antigens or receptors with higher affinity than provided in these embodiments).

[0260] The multispecific Clec9A binding agents 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. 20110262348, and International Publication No. WO 2004 / 041862, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the multispecific Clec9A binding agents of the present invention comprising two or more targeting moieties can be constructed by chemically crosslinking, for example, amino acid residues, using methods such as those described in Blattler, the entire contents of which are incorporated herein by reference. These may be constructed by reacting organic derivatization reagents such as those described in [End Page 110] et al., Biochemistry 24, 1517-1524 and European Patent No. 294703. In another exemplary embodiment, multispecific Clec9A binding agents containing two or more targeting moieties are constructed by gene fusion, i.e., constructing a single polypeptide containing individual targeting moiety polypeptides. For example, a single polypeptide construct may be formed encoding a first VHH having an antigen-recognition domain against Clec9A and a second antibody or antibody derivative having an antigen-recognition domain against a tumor antigen. Methods for producing bivalent or multivalent VHH polypeptide constructs are disclosed in International Publication No. WO 96 / 34103, the entire contents of which are incorporated herein by reference. In a further exemplary embodiment, the multispecific Clec9A binding agents of the present invention may be constructed by using a linker. For example, the carboxy terminus of a first VHH having an antigen-recognition domain against Clec9A may be linked to the amino terminus of a second antibody or antibody derivative having an antigen-recognition domain against a tumor antigen (or vice versa). Exemplary linkers that can be used are described herein. In some embodiments, the components of the multispecific Clec9A binding agents of the invention are directly linked to one another without the use of a linker.

[0261] In various embodiments, the multispecific Clec9A-binding agents of the invention recognize and bind to Clec9A and one or more antigens found on one or more immune cells, which may 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, natural killer T cells), B lymphocytes, plasma cells, dendritic cells, or subsets thereof. In some embodiments, the Clec9A-binding agents specifically bind to an antigen of interest and effectively recruit, directly or indirectly, one or more immune cells.

[0262] In various embodiments, the multispecific Clec9A-binding agents of the invention recognize and bind to Clec9A and one or more antigens found on tumor cells. In these embodiments, the Clec9A-binding agents of the invention can directly or indirectly recruit immune cells to tumor cells or to the tumor microenvironment. In some embodiments, the Clec9A-binding agents of the invention can directly or indirectly recruit immune cells, e.g., immune cells (e.g., CTLs) capable of killing and / or suppressing tumors, to a site of action (such as, but not limited to, the tumor microenvironment).

[0263] In some embodiments, Clec9A-binding agents of the invention can alter the balance of immune cells in favor of tumor immune attack, or are used in methods that involve altering the balance of immune cells. For example, Clec9A-binding agents of the invention can alter the ratio of immune cells at clinically important sites in favor of cells capable of killing and / or suppressing tumors (e.g., T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), neutrophils, B cells, dendritic cells, or subsets thereof) and against cells that protect tumors (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs); tumor-associated neutrophils (TANs), M2 macrophages, tumor-associated macrophages (TAMs), or subsets thereof). In some embodiments, Clec9A-binding agents of the invention can increase the ratio of effector T cells to regulatory T cells.

[0264] In some embodiments, the multispecific Clec9A-binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a tumor cell-associated antigen. In some embodiments, the targeting moiety directly or indirectly recruits tumor cells. For example, in some embodiments, tumor cell recruitment is to 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 directly or indirectly recruits T cells to tumor cells by virtue of the two targeting moieties interacting with their respective antigens on the tumor and Clec9A-positive immune cells (e.g., dendritic cells).

[0265] Tumor cells, or cancer cells, refer to uncontrolled proliferation of cells or tissues and / or abnormally increased cell survival and / or abnormally increased inhibition of apoptosis that interferes with the normal functioning of bodily organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. Examples of tumor cells include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney or renal cancer, and thyroid cancer. cancer), laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lip, tongue, tonsil, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland carcinoma, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulvar cancer, lymphomas including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)), small lymphocytic (SL) NHL, intermediate-grade / follicular NH These include, but are not limited to, L, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and cells of abnormal blood vessel proliferation associated with Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and phacomatosis, edema (e.g., associated with brain tumors), and Meigs syndrome.

[0266] Tumor cells or cancer cells also include, but are not limited to, carcinomas, such as various subtypes (including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myeloid, acute lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell), lymphomas and myelomas (including, for example, Hodgkin's and non-Hodgkin's lymphoma, light chain, non-secretory MGUS, and plasmacytoma), and central nervous system cancers (e.g., brain tumors (e.g., gliomas (e.g., astrocytoma, oligodendroglioma, and ependymoma), meningioma, pituitary adenoma, and neuroma), and spinal cord tumors (e.g., meningioma and neurofibroma)).

[0267] Examples of tumor antigens include MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), and T-cell receptor / CD3. Zeta chain, tumor antigens of the MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5) , GAGE ​​family tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, tumor antigens of the Smad family, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 These include, but are not limited to, CT-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). In various embodiments, the Clec9A-binding agent comprises a targeting moiety that binds one or more of these tumor antigens.

[0268] In some embodiments, the multispecific Clec9A-binding agents recognize and bind to Clec9A as well as antigens on tumor cells, hi some embodiments, the multispecific Clec9A-binding agents directly or indirectly recruit CTLs to tumor cells or the tumor microenvironment.

[0269] In various embodiments, the multispecific Clec9A binding agents have targeting moieties that target two different cells (e.g., to create a synapse) or the same cell (e.g., to achieve a higher concentration of a signaling agent effect).

[0270] In some embodiments, the multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a T cell-associated target (e.g., antigen, receptor). In some embodiments, the targeting moiety directly or indirectly recruits T cells. In certain embodiments, the antigen recognition domain specifically binds to effector T cells. In some embodiments, the antigen recognition domain directly or indirectly recruits effector T cells, for example, in some embodiments, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of 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 +, IL-15R + , CCR7low); central memory T cells (e.g., CCR7 + , CD62L + , CD27 + or CCR7hi, CD44 + , CD62Lhi, TCR, CD3 + , IL7R / CD127 + , IL-15R + );CD62L + Effector T cells; early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L - ) (TemE and TemL, respectively) containing CD8 + Effector Memory T cells (TEM);CD127( + )CD25(low / - ) effector T cells; CD127( - )CD25( - ) Effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca( + )); TH1 effector T cells (e.g., CXCR3 + , CXCR6 + and CCR5 + ; or αβTCR, CD3 + , CD4 + , IL-12R + , IFNγR + , CXCR3 + ), TH2 effector T cells (e.g., CCR3 + , CCR4 + and CCR8 + ; or αβTCR, CD3 + , CD4 + , IL4R + , IL33R + , CCR4 + , IL-17RB + , CRTH2 +); TH9 effector T cells (e.g., αβTCR, CD3 + , CD4 + ); TH17 effector T cells (e.g., αβTCR, CD3 + , CD4 + , IL23R + , CCR6 + , IL-1R + );CD4 + CD45RO + CCR7 + Effector T cells, ICOS + Effector T cells; CD4 + CD45RO + CCR7( - ) effector T cells; and IL2, IL4 and / or IFN-γ secreting effector T cells.

[0271] Examples of T cell antigens of interest include, for example, the following (including extracellular domains, if applicable): CD8, CD3, SLAMF4, IL2Rα, 4-1BB / TNFRSF9, IL2Rβ, ALCAM, B7-1, IL4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL6R, CCR3, IL7Rα, CCR4, CXCR1 / ILSRA, CCR5, CCR6, IL-10Rα, CCR7, IL-10Rβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, and lutegrin. α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15 S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40Ligand / 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 gamma chain / IL2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCC R / WSX-1, DNAM-1, thymopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas Ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAILR1 / TNFRSF1OA, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, and TSLPR. In various embodiments, the Clec9A-binding agent comprises a targeting moiety that binds one or more of these exemplary T cell antigens.

[0272] In some embodiments, a multispecific Clec9A binder of the invention comprises a targeting moiety for CD8 that is a VHH comprising a single amino acid chain having four "framework regions" or FRs and three "complementarity-determining regions" or CDRs. As used herein, a "framework region" or "FR" refers to the region in the variable domain located between the CDRs. As used herein, a "complementarity-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.

[0273] In various embodiments, the multispecific Clec9A binding agents of the invention comprise a VHH against CD8 having a variable domain comprising at least one of CDR1, CDR2, and / or CDR3 sequences.

[0274] In some embodiments, the CDR1 sequence is selected from SEQ ID NO:362 or SEQ ID NO:18.

[0275] In some embodiments, the CDR2 sequence is selected from SEQ ID NO:363 or SEQ ID NO:364.

[0276] In some embodiments, the CDR3 sequence is selected from SEQ ID NO: 365 or SEQ ID NO: 366 or SEQ ID NO: 367

[0277] In various embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from the following sequences: R3HCD27 (SEQ ID NO: 368) or R3HCD129 (SEQ ID NO: 369) or R2HCD26 (SEQ ID NO: 370).

[0278] In various embodiments, the CD8 targeting moiety comprises a VHH having a variable domain comprising at least one of the CDR1, CDR2, and / or CDR3 sequences as described below.

[0279] In some embodiments, the CDR1 sequence is selected from SEQ ID NO:371 to SEQ ID NO:438 or SEQ ID NO:52.

[0280] In some embodiments, the CDR2 sequence is selected from SEQ ID NO:439 to SEQ ID NO:507.

[0281] In some embodiments, the CDR3 sequence is selected from SEQ ID NO:508 to SEQ ID NO:576.

[0282] In various embodiments, the CD8 targeting moiety is 1CDA7 (SEQ ID NO: 577) or 1CDA12 (SEQ ID NO: 578) or 1CDA14 (SEQ ID NO: 579) or 1CDA15 (SEQ ID NO: 580) or 1CDA17 (SEQ ID NO: 581) or 1CDA18 (SEQ ID NO: 582) or 1CDA19 (SEQ ID NO: 583) or 1CDA24 (SEQ ID NO: 584) or 1CDA26 (SEQ ID NO: 585) or 1CDA28 (SEQ ID NO: 586) or 1CDA37 (SEQ ID NO: 587) or 1CDA43 (SEQ ID NO: 588) or 1CDA45 (SEQ ID NO: 589) or 1CDA47 (SEQ ID NO: 590) or 1CDA48 (SEQ ID NO: 591) or 1CDA58 (SEQ ID NO: 592) or 1CDA65 (SEQ ID NO: 593) or 1CDA68 (SEQ ID NO: 594) or 1CDA73 (SEQ ID NO: 595) or 1CDA75 (SEQ ID NO: 596) or 1CDA86 (SEQ ID NO: 597) or 1C DA87 (SEQ ID NO: 598) or 1CDA88 (SEQ ID NO: 599) or 1CDA89 (SEQ ID NO: 600) or 1CDA92 (SEQ ID NO: 601) or 1CDA93 (SEQ ID NO: 602) or 2CDA1 (SEQ ID NO: 603) or 2CDA5 (SEQ ID NO: 604) or 2CDA22 (SEQ ID NO: 605) or 2CDA28 (SEQ ID NO: 606) or 2CDA62 (SEQ ID NO: 607) or 2CDA68 (SEQ ID NO: 608) or 2CDA73 (SEQ ID NO: 609) or 2CDA74 (SEQ ID NO: 610) or 2CDA75 (SEQ ID NO: 611) or 2CDA77 (SEQ ID NO: 612) or 2CDA81 (SEQ ID NO: 613) or 2CDA87 (SEQ ID NO: 614) or 2CDA88 (SEQ ID NO: 615) or 2CDA89 (SEQ ID NO: 616) or 2CDA91 (SEQ ID NO: 617) or 2CDA92 (SEQ ID NO: 618) or 2CDA93 (SEQ ID NO: 619) or 2CDA94 (SEQ ID NO: 620) Sequence number 620) or 2CDA95 (SEQ ID NO: 621) or 3CDA3 (SEQ ID NO: 622) or 3CDA8 (SEQ ID NO: 623) or 3CDA11 (SEQ ID NO: 624) or 3CDA18 (SEQ ID NO: 625) or 3CDA19 (SEQ ID NO: 626) or 3CDA21 (SEQ ID NO: 627) or 3CDA24 (SEQ ID NO: 628) or 3CDA28 (SEQ ID NO: 629) or 3CDA29 (SEQ ID NO: 630) or 3CDA31 (SEQ ID NO: 631) or 3CDA32 (SEQ ID NO: 632) or 3CDA33 (SEQ ID NO: 633) 33) or 3CDA37 (SEQ ID NO:634) or 3CDA40 (SEQ ID NO:635) or 3CDA41 (SEQ ID NO:636) or 3CDA48 (SEQ ID NO:637) or 3CDA57 (SEQ ID NO:638) or 3CDA65 (SEQ ID NO:639) or 3CDA70 (SEQ ID NO:640) or 3CDA73 (SEQ ID NO:641) or 3CDA83 (SEQ ID NO:642) or 3CDA86 (SEQ ID NO:643) or 3CDA88 (SEQ ID NO:644) or 3CDA90 (SEQ ID NO:645). In various representative 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:324).

[0283] In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 577-645, which does not include the HA tag (ie, YPYDVPDYGS; SEQ ID NO: 325).

[0284] In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 577-645 (provided above) without the AAA linker (ie, AAA).

[0285] In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 577-645 (provided above), excluding the AAA linker, HA tag, and terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 326). In various embodiments, the CD8 targeting moiety comprises an amino acid sequence set forth in U.S. Patent Application Publication No. 2014 / 0271462, the entire contents of which are incorporated herein by reference. In various embodiments, the CD8 targeting moiety comprises an amino acid sequence set forth in Table 0.1, Table 0.2, Table 0.3, and / or Figures 1A-12I of U.S. Patent Application Publication No. 2014 / 0271462, the entire contents of which are incorporated herein by reference. In various embodiments, the CD8 targeting moiety comprises HCDR1 of SEQ ID NO: 646 or 647 and / or HCDR2 of HCDR1 of SEQ ID NO: 646 or 647 and / or HCDR3 of HCDR1 of SEQ ID NO: 646 or 647 and / or LCDR1 of LCDR1 of SEQ ID NO: 648 and / or LCDR2 of LCDR1 of SEQ ID NO: 648 and / or LCDR3 of LCDR1 of SEQ ID NO: 648, as provided below.

[0286] In various embodiments, the present invention contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologues (collectively referred to herein as "analogs") of the CD8-directed targeting moieties of the present invention described herein. In various embodiments, the amino acid sequence of a CD8-directed targeting moiety further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0287] In some embodiments, the multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a B cell-associated target (e.g., antigen, receptor). In some embodiments, the targeting moiety directly or indirectly recruits B cells to a therapeutic site (e.g., a site containing one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of B cell antigens of interest include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD10, CD11, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120, CD121, CD Examples of Clec9A-binding proteins include CD4, 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 various embodiments, the Clec9A-binding agent comprises a targeting moiety that binds one or more of these exemplary B-cell antigens.

[0288] In some embodiments, multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a natural killer cell-associated target (e.g., antigen, receptor). In some embodiments, the targeting moiety directly or indirectly recruits, for example, natural killer cells to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of natural killer cell antigens of interest include, for example, TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, Kir1alpha, DNAM-1, LMIR5 / CD300LB, Fc-epsilon RII, LMIR6 / CD300LE, Fc-γRl / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, and FcRH1 / IRT. A5, NKG2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, and ULBP-3. In various embodiments, the Clec9A binding agent comprises a targeting moiety that binds one or more of these exemplary NK cell antigens.

[0289] In some embodiments, multispecific Clec9A-binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a macrophage / monocyte-associated target (e.g., antigen, receptor). In some embodiments, the targeting moiety directly or indirectly recruits macrophages / monocytes to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of macrophage / monocyte antigens of interest include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin α4 / CD49d, BLAME / SLAMF8, integrin αX / CDllc, CCL6 / C10, integrin β2 / CD18, CD155 / PVR, integrin β3 / CD61, CD31 / PECAM-1, latexin, CD36 / SR-B3, leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLA MF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, Endoglin / CD105, Osteoactivin / GPNMB , Fc-γRI / CD64, osteopontin, Fc-γRIIB / CD32b, PD-L2, Fc-γRIIC / CD32c, Siglec-3 / CD33, Fc-γRIIA / CD32a, SIGNR1 / CD209, Fc-γRIII / CD16, SLAM, GM-CSFRα, TCCR / WSX-1, ICAM-2 / CD10 2, TLR3, IFN-γRl, TLR4, IFN-gannaR2, TREM-l, IL-lRII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF 4, IL-10Rα, ALCAM, IL-10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, ClqR1 / C D93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / CDll b, CCR8, integrin αX / CDllc, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / 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, CXCRl / IL8RA, TREM-3, and TREMLl / TLT-1. In various embodiments, the CLEC9A binding agent comprises a targeting moiety that binds one or more of these exemplary macrophage / monocyte antigens.

[0290] In some embodiments, multispecific Clec9A-binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a dendritic cell-associated target (e.g., antigen, receptor). In some embodiments, the targeting moiety directly or indirectly recruits dendritic cells to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect).Examples of dendritic cell antigens of interest include, for example, CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL23p40, 4-Amino-1, 8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, and lutegrin. α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4 Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, ClqR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBCCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLl, CD2F-10 / SLAMF9, Osteoactivin GPNMB, Chern23, PD-L2, CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, DEC-205, 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, T. These include CCR / 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 various embodiments, the CLEC9A binding agent comprises a targeting moiety that binds one or more of these exemplary DC antigens.

[0291] In various embodiments, the chimeric proteins of the present invention comprise a targeting moiety comprising an amino acid sequence that is at least 60% identical to any one of the sequences disclosed herein. For example, a chimeric protein can have at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 12 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 The targeting moiety may comprise an amino acid sequence that is at least 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 the targeting moiety.

[0292] In some embodiments, multispecific Clec9A-binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds a target (e.g., antigen, receptor) on an immune cell selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, and eosinophils. In some embodiments, the antigen recognition domain directly or indirectly recruits megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, and eosinophils, for example, in some embodiments, to a treatment site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect).

[0293] In some embodiments, multispecific Clec9A-binding agents of the invention comprise a targeting moiety having an antigen-recognition domain that specifically binds to a megakaryocyte- and / or platelet-associated target (e.g., antigen, receptor). Examples of megakaryocyte and / or platelet antigens include, for example, GPIIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, Clec9A-binding agents comprise a targeting moiety that binds one or more of these exemplary megakaryocyte and / or platelet antigens.

[0294] In some embodiments, the multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to an erythrocyte-associated target (e.g., antigen, receptor). Examples of erythrocyte antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb / IIIa), CD41b (GPIIb), CD71 ( transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2 (MHC-II), and Rh antigens. In various embodiments, the Clec9A binding agent comprises a targeting moiety that binds one or more of these exemplary erythrocyte antigens.

[0295] In some embodiments, the multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a mast cell-associated target (e.g., antigen, receptor). Examples of mast cell antigens of interest include, for example, SCFR / CD117, Fc ε RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMA1, FCERL1A, FCER2, TPSABl. In various embodiments, the Clec9A binding agent comprises a targeting moiety that binds one or more of these mast cell antigens.

[0296] In some embodiments, the multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a basophil-associated target (e.g., antigen, receptor). Examples of basophil antigens of interest include, for example, Fc ε These include RI, CD203c, CD123, CD13, CD107a, CD107b, and CD164. In various embodiments, the Clec9A binding agent comprises a targeting moiety that binds one or more of these basophil antigens.

[0297] In some embodiments, the multispecific Clec9A-binding agents of the invention comprise a targeting moiety having an antigen-recognition domain that specifically binds to a neutrophil-associated target (e.g., antigen, receptor). Examples of neutrophil antigens of interest include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRIII), CD18 protein (LFA-1, CR3, and p150, 95), CD45, CD67, and CD177. In various embodiments, the Clec9A-binding agents comprise a targeting moiety that binds one or more of these neutrophil antigens.

[0298] In some embodiments, the multispecific Clec9A-binding agents of the invention comprise a targeting moiety having an antigen-recognition domain that specifically binds to an eosinophil-associated target (e.g., antigen, receptor). Examples of eosinophil antigens of interest include, for example, CD35, CD44, and CD69. In various embodiments, the Clec9A-binding agents comprise a targeting moiety that binds one or more of these eosinophil antigens.

[0299] In various embodiments, the multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to any suitable antigen or receptor or cell surface marker known to those of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Examples of tissue-specific markers include endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAMl, PROCR, SELE, SELP, TEK, THBD, VCAMl, and VWF; smooth muscle cell surface markers such as ACTA2, MYHlO, MYHl1, MYH9, and MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COLlAl, COL1A2, COL3A1, FAP, and PH-4; epithelial cell surface markers such as CDlD, K6IRS2, KRTlO, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCl, and TACSTDl; and CD13, TFNA, and alpha-v beta-3 (α). V β3), angiogenic markers such as E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN. In various embodiments, the Clec9A binding agent comprises a targeting moiety that binds one or more of these antigens.

[0300] In various embodiments, the multispecific Clec9A binding agents of the invention are expressed on T cells. The targeting moiety includes an antigen recognition domain that specifically binds to one or more of the following checkpoint markers: PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

[0301] In various embodiments, the multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to one or more of checkpoint markers, e.g., PD-1 / PD-L1 or PD-L2, CD28 / CD80 or CD86, CTLA4 / CD80 or CD86, ICOS / ICOSL or B7RP1, BTLA / HVEM, KIR, LAG3, CD137 / CD137L, OX40 / OX40L, CD27, CD40L, TIM3 / Gal9, and A2aR.

[0302] By way of non-limiting example, in various embodiments, a multispecific Clec9A-binding agent of the invention comprises a targeting moiety directed to (i) CD8; (ii) one or more of checkpoint markers expressed on T cells, e.g., PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, Cd27, CD40L, TIM3, and A2aR; and / or (iii) the targeting moiety is directed to tumor cells in addition to any modified (e.g., mutant) signal transduction agent described herein.

[0303] In various embodiments, the multispecific Clec9A-binding agents have one or more targeting moieties directed against PD-1. In some embodiments, the Clec9A-binding agents have one or more targeting moieties that selectively bind a PD-1 polypeptide. In some embodiments, the Clec9A-binding agents comprise one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a PD-1 polypeptide.

[0304] In various embodiments, the multispecific Clec9A binders of the invention comprise a VHH against PD1 having a variable domain comprising at least one of the CDR1, CDR2, and / or CDR3 sequences.

[0305] In some embodiments, the PD1 CDR1 sequence is selected from SEQ ID NO:649 to SEQ ID NO:662.

[0306] In some embodiments, the PD1 CDR2 sequence is selected from SEQ ID NO: 663 to SEQ ID NO: 676.

[0307] In some embodiments, the PD1 CDR3 sequence is selected from SEQ ID NO: 677 to SEQ ID NO: 689.

[0308] In various exemplary embodiments, the PD1 targeting moiety comprises an amino acid sequence selected from the following sequences: 2PD23 (SEQ ID NO: 690) or 2PD26 (SEQ ID NO: 691) or 2PD90 (SEQ ID NO: 692) or 2PD106 (SEQ ID NO: 693) or 2PD16 (SEQ ID NO: 694) or 2PD71 (SEQ ID NO: 695) or 2PD152 (SEQ ID NO: 696) or 2PD12 (SEQ ID NO: 697) or 3PD55 (SEQ ID NO: 698) or 3PD82 (SEQ ID NO: 699) or 2PD8 (SEQ ID NO: 700) or 2PD27 (SEQ ID NO: 701) or 2PD82 (SEQ ID NO: 702) or 3PD36 (SEQ ID NO: 703).

[0309] In various exemplary embodiments, the PD1 targeting moiety includes a terminal histidine tag sequence (i.e. , HHHHHH: SEQ ID NO: 324).

[0310] In some embodiments, the PD1 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 690-703 (provided above) that does not include the HA tag (i.e., YPYDVPDYGS; SEQ ID NO: 325).

[0311] In some embodiments, the PD1 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 690-703 (provided above) without the AAA linker (i.e., AAA).

[0312] In some embodiments, the PD1 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 690-703 (provided above), excluding the AAA linker, HA tag, and terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 326). In certain embodiments, the targeting moiety comprises the anti-PD-1 antibody pembrolizumab (also known as MK-3475, Keytruda), or a fragment thereof. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, et al. (2013) New England Journal of Medicine 369(2):134-44, US 8,354,509, and WO 2009 / 114335, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, pembrolizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 704 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 705.

[0313] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody, nivolvam (also known as BMS-936558, MDX-1106, ONO-4538, Opdivo), or a fragment thereof. Nivolvam (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449 and WO 2006 / 121168, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, nivolvam or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:706 and / or a light chain comprising the amino acid sequence of SEQ ID NO:707.

[0314] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody pidilizumab (also known as CT-011, hBAT, or hBAT-1), or a fragment thereof. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in U.S. Patent Application Publication No. 2008 / 0025980 and WO 2009 / 101611, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a light chain polypeptide comprising an amino acid sequence selected from SEQ ID NOS:15-18 of U.S. Patent Application Publication No. 2008 / 0025980: SEQ ID NO:15 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:708); SEQ ID NO:16 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:709); SEQ ID NO:17 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:710); SEQ ID NO:18 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:711). and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 20-24 of U.S. Patent Application Publication No. 2008 / 0025980: SEQ ID NO: 20 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 712); SEQ ID NO: 21 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 713); SEQ ID NO: 22 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 714); SEQ ID NO: 23 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 715); SEQ ID NO: 24 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 716).

[0315] In one embodiment, the targeting moiety comprises a light chain comprising SEQ ID NO: 18 of US Patent Application Publication No. 2008 / 0025980 and a heavy chain comprising SEQ ID NO: 22 of US Patent Application Publication No. 2008 / 0025980.

[0316] In certain embodiments, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0317] In some embodiments, the targeting moiety comprises the pd-L2-Fc fusion protein AMP-224, which is disclosed in International Publication Nos. 2010 / 027827 and 2011 / 066342, the entire disclosures of which are incorporated herein by reference. In such embodiments, the targeting moiety may comprise a targeting domain comprising SEQ ID NO:4 (SEQ ID NO:717) of International Publication No. 2010 / 027827 and / or a B7-DC fusion protein comprising SEQ ID NO:83 (SEQ ID NO:718) of International Publication No. 2010 / 027827.

[0318] In some embodiments, the targeting moiety comprises the peptide AUNP12 or any other peptide disclosed in U.S. Patent Application Publication No. 2011 / 0318373 or U.S. Patent No. 8,907,053. For example, the targeting moiety can comprise AUNP12 having the following sequence of SEQ ID NO:719 (i.e., Compound 8 or SEQ ID NO:49 of U.S. Patent Application Publication No. 2011 / 0318373): SNTSESFK(SNTSESF)FRVTQLAPKAQIKE-NH2 [ka]

[0319] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E3 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:720; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:721.

[0320] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E8 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:722; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:723.

[0321] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1H3 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738. The entire disclosure of this patent. is incorporated herein by reference. In an exemplary embodiment, 1H3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 724; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 725.

[0322] In certain embodiments, the targeting moiety comprises a VHH directed against PD-1, e.g., as disclosed in U.S. Patent No. 8,907,065 and International Publication No. WO 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In exemplary embodiments, the VHH against PD-1 comprises SEQ ID NOS: 347-351 of U.S. Patent No. 8,907,065: SEQ ID NOS: 347 (SEQ ID NOS: 726) of U.S. Patent No. 8,907,065; SEQ ID NOS: 348 (SEQ ID NOS: 727) of U.S. Patent No. 8,907,065; SEQ ID NOS: 349 (SEQ ID NOS: 728) of U.S. Patent No. 8,907,065; SEQ ID NOS: 350 (SEQ ID NOS: 729) of U.S. Patent No. 8,907,065; and SEQ ID NOS: 351 (SEQ ID NOS: 730) of U.S. Patent No. 8,907,065.

[0323] In certain embodiments, the targeting moiety comprises any one of the anti-PD-1 antibodies or fragments thereof, as disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and International Publication No. WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS:25-29 of U.S. Patent Application Publication No. 2011 / 0271358: SEQ ID NO:25 (SEQ ID NO:731) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO:26 (SEQ ID NO:732) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO:27 (SEQ ID NO:733) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO:28 (SEQ ID NO:734) of U.S. Patent Application Publication No. 2011 / 0271358; and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 30-33 of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 30 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 736); SEQ ID NO: 31 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 737); SEQ ID NO: 32 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 738); SEQ ID NO: 33 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 739).

[0324] In various embodiments, the multispecific Clec9A binding agents of the invention comprise one or more anti-PD-1 antibodies, or antibody fragments thereof, selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene Ltd.).

[0325] In various embodiments, the multispecific Clec9A-binding agents of the invention have one or more targeting moieties directed against PD-L1. In some embodiments, the Clec9A-binding agents have one or more targeting moieties that selectively bind to a PD-L1 polypeptide. In some embodiments, the Clec9A-binding agents comprise one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds to a PD-L1 polypeptide.

[0326] In various embodiments, the multispecific Clec9A binders of the invention comprise a VHH against PDL1 having a variable domain comprising at least one of the CDR1, CDR2, and / or CDR3 sequences.

[0327] In some embodiments, the PD-L1 CDR1 sequence is SEQ ID NO: 740 to SEQ ID NO: 7 There are 70 to choose from.

[0328] In some embodiments, the PD-L1 CDR2 sequence is selected from SEQ ID NO:771 to SEQ ID NO:801.

[0329] In some embodiments, the PD-L1 CDR3 sequence is selected from SEQ ID NO:802 to SEQ ID NO:832.

[0330] In various exemplary embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from the following: 2LIG2 (SEQ ID NO: 833) or 2LIG3 (SEQ ID NO: 834) or 2LIG16 (SEQ ID NO: 835) or 2LIG22 (SEQ ID NO: 836) or 2LIG27 (SEQ ID NO: 837) or 2LIG29 (SEQ ID NO: 838) or 2LIG30 (SEQ ID NO: 839) or 2LIG34 (SEQ ID NO: 840) or 2LIG35 (SEQ ID NO: 841) or 2LIG48 (SEQ ID NO: 842) or 2LIG65 (SEQ ID NO: 843) or 2LIG85 (SEQ ID NO: 844) or 2LIG86 (SEQ ID NO: 845) or 2LIG89 (SEQ ID NO: 846) or 2LIG 97 (SEQ ID NO: 847) or 2LIG99 (SEQ ID NO: 848) or 2LIG109 (SEQ ID NO: 849) or 2LIG127 (SEQ ID NO: 850) or 2LIG139 (SEQ ID NO: 851) or 2LIG176 (SEQ ID NO: 852) or 2LIG189 (SEQ ID NO: 853) or 3LIG3 (SEQ ID NO: 854) or 3LIG7 (SEQ ID NO: 855) or 3LIG8 (SEQ ID NO: 856) or 3LIG9 (SEQ ID NO: 857) or 3LIG18 (SEQ ID NO: 858) or 3LIG20 (SEQ ID NO: 859) or 3LIG28 (SEQ ID NO: 860) or 3LIG29 (SEQ ID NO: 861) or 3LIG30 (SEQ ID NO: 862) or 3LIG33 (SEQ ID NO: 863).

[0331] In various exemplary embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include the terminal histidine tag sequence (i.e., HHHHHH: SEQ ID NO: 324).

[0332] In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 833-863 (provided above) that does not include the HA tag (i.e., YPYDVPDYGS; SEQ ID NO: 325).

[0333] In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 833-863 (provided above) without the AAA linker (i.e., AAA).

[0334] In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 833-863 (provided above), excluding the AAA linker, HA tag, and terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 326). In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab), or a fragment thereof. MEDI4736 is selective for PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80 receptors. MEDI4736 and antigen-binding fragments thereof for use in the methods provided herein comprise heavy and light chains or heavy and light chain variable regions. The sequence of MEDI4736 is disclosed in WO 2016 / 06272, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:864 and / or a light chain comprising the amino acid sequence of SEQ ID NO:865.

[0335] In exemplary embodiments, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 (SEQ ID NO:866) of WO 2016 / 06272; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 (SEQ ID NO:867) of WO 2016 / 06272.

[0336] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (also known as MPDL3280A, RG7446), or a fragment thereof. In an exemplary embodiment, atezolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 868; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 869.

[0337] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody avelumab (also known as MSB0010718C), or a fragment thereof. In an exemplary embodiment, atezolizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 870; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 871.

[0338] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105), or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, BMS-936559 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:872; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:873.

[0339] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3G10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 3G10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:874; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:875.

[0340] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10A5, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 10A5, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:876; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:877.

[0341] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 5F8, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 5F8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:878; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:879.

[0342] In one embodiment, the targeting moiety is the anti-PD-L1 antibody 10H10, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In exemplary embodiments, 10H10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:880; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:881.

[0343] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1B12, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 1B12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:882; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:883.

[0344] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 7H1, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 7H1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:884; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:885.

[0345] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 11E6, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 11E6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:886; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:887.

[0346] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 12B7, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 12B7 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:888; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:889.

[0347] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 13G4, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 13G4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:890; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:891.

[0348] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1E12, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E12, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:892; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:893.

[0349] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1F4 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1F4 or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. 1F4 or an antigen-binding fragment thereof for use in the method comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:894; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:895.

[0350] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2G11, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 2G11, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:896; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:897.

[0351] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3B6, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 3B6, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:898; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:899.

[0352] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3D10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738 and WO 2012 / 145493. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3D10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:900; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:901.

[0353] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS: 34-38 of U.S. Patent Application Publication No. 2011 / 0271358: SEQ ID NO: 34 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 902); SEQ ID NO: 35 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 903); SEQ ID NO: 36 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 904); SEQ ID NO: 37 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 905); U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 906); 358 (SEQ ID NO: 38 (SEQ ID NO: 906); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 39-42 of U.S. Patent Application Publication No. 2011 / 0271358: SEQ ID NO: 39 (SEQ ID NO: 907) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 40 (SEQ ID NO: 908) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 41 (SEQ ID NO: 909) of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 42 (SEQ ID NO: 910) of U.S. Patent Application Publication No. 2011 / 0271358.

[0354] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.7A4, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 (SEQ ID NO:911) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:7 (SEQ ID NO:912) of WO 2011 / 066389.

[0355] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.9D10, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.9D10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (SEQ ID NO: 913) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (SEQ ID NO: 914) of WO 2011 / 066389.

[0356] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.14H9, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:22 (SEQ ID NO:915) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 (SEQ ID NO:916) of WO 2011 / 066389.

[0357] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.20A8, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.20A8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:32 (SEQ ID NO:917) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:37 (SEQ ID NO:918) of WO 2011 / 066389.

[0358] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.15G8, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3.15G8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:42 (SEQ ID NO:919) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:47 (SEQ ID NO:920) of WO 2011 / 066389.

[0359] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.18G1, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 3.18G1, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:52 (SEQ ID NO:921) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:57 (SEQ ID NO:922) of WO 2011 / 066389.

[0360] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4OPT, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Pat. No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4OPT, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Pat. No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the entire disclosures of which are incorporated herein by reference. 2.7A4OPT or an antigen-binding fragment thereof for use in the provided methods comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62 (SEQ ID NO: 923) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 67 (SEQ ID NO: 924) of WO 2011 / 066389.

[0361] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9OPT, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.14H9OPT, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:72 (SEQ ID NO:925) of WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:77 (SEQ ID NO:926) of WO 2011 / 066389.

[0362] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 061142, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein are selected from the group consisting of SEQ ID NOs: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of WO 2016 / 061142: SEQ ID NO: 18 of WO 2016 / 061142 (SEQ ID NO: 927); SEQ ID NO: 30 of WO 2016 / 061142 (SEQ ID NO: 928); SEQ ID NO: 38 of WO 2016 / 061142 (SEQ ID NO: 929); SEQ ID NO: 46 from WO 2016 / 061142 (SEQ ID NO: 930); SEQ ID NO: 50 from WO 2016 / 061142 (SEQ ID NO: 931); SEQ ID NO: 54 from WO 2016 / 061142 (SEQ ID NO: 932); SEQ ID NO: 62 from WO 2016 / 061142 (SEQ ID NO: 933); SEQ ID NO: 70 from WO 2016 / 061142 (SEQ ID NO: 934); SEQ ID NO: 78 from WO 2016 / 061142 (SEQ ID NO: 935) and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO 2016 / 061142: SEQ ID NO: 22 of WO 2016 / 061142 (SEQ ID NO: 936); SEQ ID NO: 26 of WO 2016 / 061142 (SEQ ID NO: 937); SEQ ID NO: 34 of WO 2016 / 061142 (SEQ ID NO: 938); SEQ ID NO: 34 of WO 2016 / 061142 (SEQ ID NO: 939); 42 (SEQ ID NO:939); SEQ ID NO:58 of WO 2016 / 061142 (SEQ ID NO:940); SEQ ID NO:66 of WO 2016 / 061142 (SEQ ID NO:941); SEQ ID NO:74 of WO 2016 / 061142 (SEQ ID NO:942); SEQ ID NO:82 of WO 2016 / 061142 (SEQ ID NO:943); SEQ ID NO:86 of WO 2016 / 061142 (SEQ ID NO:944).

[0363] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 022630, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein are selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WO 2016 / 022630: SEQ ID NO: 2 of WO 2016 / 022630 (SEQ ID NO: 945); SEQ ID NO: 6 of WO 2016 / 022630 (SEQ ID NO: 946); SEQ ID NO: 1 of WO 2016 / 022630 (SEQ ID NO: 1046); SEQ ID NO: 14 of WO 2016 / 022630 (SEQ ID NO: 948); SEQ ID NO: 18 of WO 2016 / 022630 (SEQ ID NO: 949); SEQ ID NO: 22 of WO 2016 / 022630 (SEQ ID NO: 950); SEQ ID NO: 26 of WO 2016 / 022630 (SEQ ID NO: 951); SEQ ID NO: 30 of WO 2016 / 022630 (SEQ ID NO: 952); a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 34 of WO 2016 / 022630 (SEQ ID NO: 953); SEQ ID NO: 38 of WO 2016 / 022630 (SEQ ID NO: 954); SEQ ID NO: 42 of WO 2016 / 022630 (SEQ ID NO: 955); SEQ ID NO: 46 of WO 2016 / 022630 (SEQ ID NO: 956); and / or SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 of WO 2016 / 022630: SEQ ID NO: 4 of WO 2016 / 022630 (SEQ ID NO: 957); SEQ ID NO: 8 of WO 2016 / 022630 (SEQ ID NO: 958); SEQ ID NO: 12 of WO 2016 / 022630 (SEQ ID NO: 959); SEQ ID NO: 16 of WO 2016 / 022630 (SEQ ID NO: 960); SEQ ID NO: 20 of WO 2016 / 022630 (SEQ ID NO: 961); SEQ ID NO: 24 of WO 2016 / 022630 (SEQ ID NO: 962); SEQ ID NO: 28 of WO 2016 / 022630 (SEQ ID NO: 963); SEQ ID NO: 32 of WO 2016 / 022630 (SEQ ID NO: 964); SEQ ID NO: 36 of WO 2016 / 022630 (SEQ ID NO: 965); SEQ ID NO: 40 of WO 2016 / 022630 (SEQ ID NO: 966); SEQ ID NO: 44 of WO 2016 / 022630 (SEQ ID NO: 967); SEQ ID NO: 48 of WO 2016 / 022630 (SEQ ID NO: 968).

[0364] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2015 / 112900, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein has a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 38, 50, 82, and 86 of WO 2015 / 112900: SEQ ID NO: 38 of WO 2015 / 112900 (SEQ ID NO: 969); SEQ ID NO: 50 of WO 2015 / 112900 (SEQ ID NO: 970); SEQ ID NO: 82 of WO 2015 / 112900 (SEQ ID NO: 971); SEQ ID NO: 86 of WO 2015 / 112900 (SEQ ID NO: 972); and / or SEQ ID NOs: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of WO 2015 / 112900: SEQ ID NO: 42 of WO 2015 / 112900 (SEQ ID NO: 972). SEQ ID NO: 42 of WO 2015 / 112900 (SEQ ID NO: 973); SEQ ID NO: 46 of WO 2015 / 112900 (SEQ ID NO: 974); SEQ ID NO: 54 of WO 2015 / 112900 (SEQ ID NO: 975); SEQ ID NO: 58 of WO 2015 / 112900 (SEQ ID NO: 976); SEQ ID NO: 62 of WO 2015 / 112900 (SEQ ID NO: 977); SEQ ID NO: 66 of WO 2015 / 112900 (SEQ ID NO: 978); SEQ ID NO: 70 of WO 2015 / 112900 (SEQ ID NO: 979); SEQ ID NO: 74 of WO 2015 / 112900 (SEQ ID NO: 980); SEQ ID NO: 78 of WO 2015 / 112900 (SEQ ID NO: 981).

[0365] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2010 / 077634 and U.S. Patent No. 8,217,149. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, an anti-PD-L1 or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:20 (SEQ ID NO:982) of WO 2010 / 077634; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:21 (SEQ ID NO:983) of WO 2010 / 077634.

[0366] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies obtainable from the hybridomas available under CNCM Accession Nos. CNCM I-4122, CNCM I-4080, and CNCM I-4081, as disclosed in U.S. Patent Application Publication No. 20120039906, the entire disclosures of which are incorporated herein by reference.

[0367] In some embodiments, the targeting moiety comprises a VHH directed against a PD-L1 antibody, e.g., as disclosed in U.S. Patent No. 8,907,065 and WO 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In exemplary embodiments, the VHH directed against PD-L1 comprises SEQ ID NOS: 394-399 of U.S. Patent No. 8,907,065: SEQ ID NOS: 394 of U.S. Patent No. 8,907,065 (SEQ ID NOS: 984); 395 of U.S. Patent No. 8,907,065 (SEQ ID NOS: 985); 396 of U.S. Patent No. 8,907,065 (SEQ ID NOS: 986); 397 of U.S. Patent No. 8,907,065 (SEQ ID NOS: 987); 398 of U.S. Patent No. 8,907,065 (SEQ ID NOS: 988); and 399 of U.S. Patent No. 8,907,065 (SEQ ID NOS: 989).

[0368] In various embodiments, the multispecific Clec9A-binding agents have one or more targeting moieties directed against PD-L2. In some embodiments, the Clec9A-binding agents have one or more targeting moieties that selectively bind to a PD-L2 polypeptide. In some embodiments, the Clec9A-binding agents comprise one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds a PD-L2 polypeptide.

[0369] In one embodiment, the targeting moiety comprises a VHH directed against PD-L2, for example, as disclosed in U.S. Patent No. 8,907,065 and WO 2008 / 071447, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the VHH against PD-1 comprises SEQ ID NOs:449-455 of U.S. Patent No. 8,907,065: SEQ ID NO:449 of U.S. Patent No. 8,907,065 (SEQ ID NO:990); SEQ ID NO:450 of U.S. Patent No. 8,907,065 (SEQ ID NO:991); SEQ ID NO:451 of U.S. Patent No. 8,907,065 (SEQ ID NO:992); SEQ ID NO:452 of U.S. Patent No. 8,907,065 (SEQ ID NO:993); SEQ ID NO:453 of U.S. Patent No. 8,907,065 (SEQ ID NO:994); SEQ ID NO:454 of U.S. Patent No. 8,907,065 (SEQ ID NO:995); SEQ ID NO:455 of U.S. Patent No. 8,907,065 (SEQ ID NO:996).

[0370] In some embodiments, the targeting moiety comprises any one of the anti-PD-L2 antibodies disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS:43-47 of U.S. Patent Application Publication No. 2011 / 0271358: SEQ ID NO:43 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:997); SEQ ID NO:44 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:998); SEQ ID NO:45 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:999); SEQ ID NO:46 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:1000); U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:1001); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 48-51 of U.S. Patent Application Publication No. 2011 / 0271358; SEQ ID NO: 48 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1002); SEQ ID NO: 49 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1003); SEQ ID NO: 50 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1004); SEQ ID NO: 51 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1005).

[0371] In various embodiments, the targeting moieties of the present invention are 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% of any of the sequences disclosed herein. , 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% identical to any of the sequences disclosed herein). %, 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 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).

[0372] In various embodiments, targeting moieties of the invention can comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences that target PD-1, PD-L1, and / or PD-L2 as disclosed herein.

[0373] Additional antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind or target PD-1, PD-L1, and / or PD-L2 are described in WO 2011 / 066389, U.S. Patent Application Publication No. 2008 / 0025980, U.S. Patent Application Publication No. 2013 / 0034559, U.S. Patent No. 8,779,108 ... No. 8,779,108, U.S. Patent No. 8,779,108, U.S. Patent No. 8,779,108, U.S. Patent No. 8,779,108, U.S. Patent No. 8,779,108, U.S. Patent No. 8,779,108, U.S. Patent No. 8,779,108, U.S. Patent No. 8,779 No. 2014 / 0356353, U.S. Patent No. 8,609,089, U.S. Patent Application Publication No. 2010 / 028330, U.S. Patent Application Publication No. 2012 / 0114649, WO 2010 / 027827, WO 2011 / 066342, U.S. Patent No. 8,907,065, WO 2016 / 062722, WO 2009 / 101611, WO 2010 / 027 827, WO 2011 / 066342, WO 2007 / 005874, WO 2001 / 014556, U.S. Patent Application Publication No. 2011 / 0271358, WO 2010 / 036959, WO 2010 / 077634, U.S. Patent No. 8,217,149, U.S. Patent Application Publication No. 2012 / 0039906, WO 2012 / 145493, U.S. and WO 2015 / 112900, the entire disclosures of which are incorporated herein by reference.

[0374] In various embodiments, the multispecific Clec9A binding agents of the present technology comprise a targeting moiety directed against signal-regulatory protein alpha-1 (SIRP1α). SIRP1α (also known as SIRPα) belongs to a family of cellular immune receptors that includes inhibitory (SIRPα), activating (SIRPβ), non-signaling (SIRPγ), and soluble (SIRPδ) members. SIRP1α is primarily expressed in macrophages, granulocytes, myeloid cells (DCs), mast cells, and their precursors, including hematopoietic stem cells. SIRP1α is a widespread It interacts with the widely expressed transmembrane glycoprotein CD47 and acts as an inhibitory receptor that regulates phagocytosis. In particular, the binding of SIRP1α on macrophages by CD47 expressed on target cells generates an inhibitory signal that negatively regulates phagocytosis of target cells.

[0375] In various embodiments, the SIRP1α targeting moiety is a targeting moiety that specifically recognizes and binds to SIRP1α on macrophages.

[0376] In various embodiments, the SIRP1α targeting moiety is a targeting moiety that specifically recognizes and binds to SIRP1α on monocytes.

[0377] In various embodiments, the SIRP1α targeting moiety is a targeting moiety that specifically recognizes and binds to SIRP1α on a TAM.

[0378] In various embodiments, the SIRP1α targeting moiety is a targeting moiety that specifically recognizes and binds to SIRP1α on dendritic cells, including but not limited to, cDC2s and pDCs.

[0379] In various embodiments, the SIRP1α targeting moiety comprises a targeting moiety having an antigen recognition domain that recognizes SIRP1α. In certain embodiments, the antigen recognition domain recognizes one or more linear epitopes present on SIRP1α. As used herein, a linear epitope refers to any continuous sequence of amino acids present on SIRP1α. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on SIRP1α. As used herein, a conformational epitope refers to a portion of one or more amino acids (which may be discontinuous) that forms a three-dimensional surface with characteristics and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0380] In some embodiments, the SIRP1α targeting moiety can bind to the full-length form and / or mature form and / or isoforms and / or splice variants and / or fragments and / or any other natural or synthetic analogs, variants, or mutants of SIRP1α. In certain embodiments, the SIRP1α targeting moiety is human SIRP1α. In various embodiments, the SIRP1α targeting moiety can bind to any form of human SIRP1α, including monomers, dimers, heterodimers, multimers, and associated forms. In certain embodiments, the SIRP1α targeting moiety binds to the monomeric form of SIRP1α. In other embodiments, the SIRP1α targeting moiety binds to the dimeric form of SIRP1α.

[0381] In some embodiments, the SIRP1α targeting moiety comprises a recognition domain that recognizes one or more epitopes present on human SIRP1α. In some embodiments, the SIRP1α targeting moiety comprises a recognition domain that recognizes human SIRP1α having a signal peptide sequence. A representative human SIRP1α polypeptide is SEQ ID NO: 1006.

[0382] In certain embodiments, the SIRP1α targeting moiety comprises a recognition domain that recognizes human SIRP1α without the signal peptide sequence. A representative human SIRP1α polypeptide without the signal peptide sequence is SEQ ID NO:1007.

[0383] In certain embodiments, the SIRP1α targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP1α isoform 2 (SEQ ID NO: 1008).

[0384] In certain embodiments, the SIRP1α targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP1α isoform 4 (SEQ ID NO: 1009).

[0385] In various embodiments, the SIRP1α targeting moiety can be any protein-based substance capable of specific binding, such as an antibody or a derivative thereof. In certain embodiments, the SIRP1α targeting moiety comprises an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain comprises one variable region (V L ) and one constant region (C L ). The variable regions determine the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity-determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs are called 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.

[0386] In some embodiments, the SIRP1α targeting moiety comprises an antibody derivative or format. In some embodiments, the SIRP1α targeting moiety of the present invention is a single domain antibody, a recombinant heavy chain antibody (VHH), a single chain antibody (scFv), a shark heavy chain antibody (VNAR), a microprotein (cysteine ​​knot protein, knottin), a DARPin, a tetranectin, an affibody, a transbody, an anticalin, an adnectin, an affilin, a microbody, a peptide aptamer, an alterase, a plastic antibody, a phylomer, a stradobody, a maxibody, a shrimpbody, a phylomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troibody, a pepbody, a vaccibody, a unibody, an affimer, a duobody, an Fv, a Fab, a Fab', a F(ab')2, a peptidomimetic molecule, or a synthetic molecule. These include U.S. Pat. No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Pat. No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Pat. No. 7,250,297, U.S. Pat. No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Pat. No. 7,838,629, U.S. Pat. No. 7,186,524, U.S. Pat. Nos. 6,004,746, 5,475,096, 2004 / 146938, 2004 / 157209, 6,994,982, 6,794,144, 2010 / 239633, 7,803,907, 2010 / 119446, and / or 7,166,697. See also May-Jun;3(3):310-317.

[0387] In one embodiment, the SIRP1α targeting moiety comprises a VHH derived from an organism that produces VHH antibodies, such as camelids or sharks, or a single-domain antibody, such as a designed VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies derived from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3).

[0388] In certain embodiments, the SIRP1α targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

[0389] In some embodiments, the VHH is a fully human VHH. H In some embodiments, the fully human V domain is H The domain, e.g., HUMABODY, is monovalent, bivalent, or trivalent. In some embodiments, a fully human V H The domains, e.g., HUMABODY, may be monospecific or multispecific, such as monospecific, bispecific, or trispecific. Exemplary fully human V H Domains such as HUMABODIES are described, for example, in WO 2016 / 113555 and WO 2016 / 113557, the entire disclosures of which are incorporated herein by reference.

[0390] For example, in some embodiments, the SIRP1α targeting moiety comprises one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, a VHH, or a fusion protein that selectively binds SIRP1α. In some embodiments, the SIRP1α targeting moiety comprises an antibody or a derivative thereof that specifically binds SIRP1α. In some embodiments, the SIRP1α targeting moiety is a camelid heavy chain antibody (VHH) that specifically binds SIRP1α.

[0391] In various embodiments, the SIRP1α targeting moiety may include any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences known to recognize and bind to SIRP1α.

[0392] In various embodiments, the present technology contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the SIRP1α targeting moieties described herein. In various embodiments, the amino acid sequence of the SIRP1α targeting moiety further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0393] In various embodiments, the SIRP1α targeting moiety comprises an amino acid sequence having one or more amino acid mutations relative to any targeting moiety sequence known to recognize and bind to SIRP1α. In various embodiments, the SIRP1α targeting moiety comprises an amino acid sequence having 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 15, 20, 30, 40, or 50 amino acid mutations relative to any targeting moiety sequence known to recognize and bind to SIRP1α. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0394] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0395] "Conservative substitutions" can be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0396] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid in the same group of the six standard amino acid groups. For example, replacing Asp with Glu maintains one negative charge in the modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0397] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid from a different group of the six standard amino acid groups (1) to (6) above.

[0398] In various embodiments, substitutions may also include non-classical amino acids. Representative 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, fluoroamino acids, designer amino acids such as β-methylamino acids, C These include α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general.

[0399] In various embodiments, the amino acid mutation may be in a CDR (e.g., the CDR1, CDR2, or CDR3 region) of the targeting moiety. In another embodiment, the amino acid change may be in a framework region (FR) (e.g., the FR1, FR2, FR3, or FR4 region) of the targeting moiety.

[0400] Amino acid sequence modification can be achieved by any well-known technique in the art, for example, 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, NY, 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0401] In various embodiments, the mutations do not substantially reduce the ability of the SIRP1α targeting moiety to specifically recognize and bind to SIRP1α. In various embodiments, the mutations do not substantially reduce the ability of the SIRP1α targeting moiety to specifically bind to SIRP1α and do not functionally modulate (e.g., partially or fully neutralize) SIRP1α.

[0402] In various embodiments, the SIRP1α targeting moiety binds but does not functionally modulate the antigen of interest, i.e., SIRP1α. For example, in various embodiments, the SIRP1α targeting moiety only targets the antigen but does not substantially functionally modulate (e.g., partially or completely inhibit or neutralize) the biological effect of the antigen. In various embodiments, the SIRP1α targeting moiety binds an epitope that is physically distant from the antigenic site important for its biological activity (e.g., the active site of the antigen).

[0403] In other embodiments, the SIRP1α targeting moiety binds and functionally modulates an antigen of interest, i.e., SIRP1α. For example, in various embodiments, the SIRP1α targeting moiety targets an antigen, i.e., SIRP1α, and functionally modulates (e.g., partially or completely inhibits or neutralizes) the biological effect of the antigen. Such binding, along with functional modulation, is used in various embodiments of the present invention, including methods in which the chimeric proteins of the present invention are used to directly or indirectly recruit active immune cells to a required site via an effector antigen.

[0404] In various embodiments, the multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to XCR1, e.g., on DCs. In one embodiment, the multispecific Clec9A binding agents of the invention comprise a targeting moiety having an antigen recognition domain comprising all or part of XCL1.

[0405] In various embodiments, the multispecific Clec9A-binding agent has a targeting moiety with a recognition domain that specifically binds to a target (e.g., an antigen, a receptor) that is part of a non-cellular structure. In some embodiments, the antigen or receptor is not an integral component of an intact cell or cellular structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-proteinaceous non-cellular marker, including, but not limited to, extracellular deposits such as nucleic acids or cholesterol, including DNA or RNA, e.g., DNA released from necrotic tumor cells.

[0406] In some embodiments, the target of interest (e.g., antigen, receptor) is part of the noncellular components of the stroma or extracellular matrix (ECM) or a marker associated therewith. As used herein, stroma refers to the connective and supportive framework of a tissue or organ. The stroma may comprise a collection of cells, such as fibroblasts / myofibroblasts / glial cells, epithelial, adipose, immune, vascular, smooth muscle, and immune cells, along with the extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target of interest (e.g., antigen, receptor) is part of the noncellular components of the stroma, such as the extracellular matrix and extracellular molecules. As used herein, ECM refers to the noncellular components present in all tissues and organs. The ECM consists of a large collection of biochemically distinct components, including, but not limited to, proteins, glycoproteins, proteoglycans, and polysaccharides. These ECM components are typically produced by neighboring cells and secreted into the ECM by exocytosis. Once secreted, ECM components often assemble to form a complex network of macromolecules. In various embodiments, the chimeric proteins of the present invention comprise a targeting moiety that recognizes a target (e.g., an antigen or receptor or non-protein molecule) located on any component of the ECM. Examples of components of the ECM include, but are not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers, and other ECM proteins or ECM non-proteins, e.g., polysaccharides and / or lipids, or ECM-associated molecules (e.g., proteins or non-proteins, e.g., polysaccharides, nucleic acids, and / or lipids).

[0407] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on an ECM proteoglycan. Proteoglycans are glycosylated proteins. A basic proteoglycan unit comprises a core protein with one or more covalently attached glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+), which attract water via osmosis and keep the ECM and resident cells hydrated. Proteoglycans can also capture and store growth factors within the ECM. Examples of proteoglycans that can be targeted by the chimeric proteins of the present invention include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In certain embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on a non-proteoglycan polysaccharide, such as hyaluronic acid.

[0408] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on ECM fibers. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety recognizes one or more epitopes on collagen or collagen fibers. Collagen is the most abundant protein in the ECM. Collagen exists as a fibrous protein in the ECM and provides structural support to resident cells. In one or more embodiments, the targeting moiety recognizes various types of collagen present in the ECM, including, but not limited to, fibrillar collagens (types I, II, III, V, XI), facit collagens (types IX, XII, XIV), short-chain collagens (types VIII, X), basement membrane collagens (type IV), and / or type VI, VII, or XIII collagen. Elastin fibers provide tissue with elasticity, allowing the tissue to stretch as needed and then return to its original state. In some embodiments, the targeting moiety recognizes one or more epitopes on elastin or elastin fibers.

[0409] In some embodiments, the targeting moiety recognizes one or more ECM proteins, including, but not limited to, tenascin, fibronectin, fibrin, laminin, or nidogen / entactin.

[0410] In certain embodiments, the targeting moiety recognizes and binds to tenascin. The tenascin (TN) family of glycoproteins includes at least four members: tenascin-C, tenascin-R, tenascin-X, and tenascin-W. The primary structure of tenascin proteins contains several common motifs ordered in the same contiguous sequence: amino-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain. Each protein member is associated with typical variations in the number and nature of the EGF-like and fibronectin type III repeats. Isoform variants also exist, particularly with respect to tenascin-C. More than 27 splice variants and / or isoforms of tenascin-C are known. In certain embodiments, the targeting moiety recognizes and binds to tenascin-CA1. Similarly, tenascin-R also has various splice variants and isoforms. Tenascin-R typically exists as a dimer or trimer. Tenascin-X is the largest member of the tenascin family and is known to exist as a trimer. Tenascin-W exists as a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on the tenascin protein. In some embodiments, the targeting moiety recognizes the monomeric, dimeric, trimeric, and / or hexameric forms of the tenascin protein.

[0411] In certain embodiments, the targeting moiety recognizes and binds to fibronectin. Fibronectin is a glycoprotein that connects cells to collagen fibers in the ECM, allowing cells to migrate through the ECM. Upon binding to integrins, fibronectin unfolds to form functional dimers. In some embodiments, the targeting moiety recognizes monomeric and / or dimeric forms of fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. In exemplary embodiments, the targeting moiety recognizes fibronectin extracellular domain A (EDA) or fibronectin extracellular domain B (EDB). Elevated EDA levels are associated with various diseases and disorders, including psoriasis, rheumatoid arthritis, diabetes, and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing the EDA isoform and can be used to target the chimeric protein to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing the EDB isoform. In various embodiments, such targeting moieties can be used to target the chimeric protein to tumor cells, including tumor neovasculature.

[0412] In certain embodiments, the targeting moiety recognizes and binds to fibrin, another protein substance often found in the matrix network of the ECM. Fibrin is formed by the action of the protease thrombin on fibrinogen, which polymerizes it. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes both monomeric and polymerized forms of fibrin.

[0413] In certain embodiments, the targeting moiety recognizes and binds to laminin. It is a major component of basement membranes and the protein network foundation for cells and organs. Laminin is a heterotrimeric protein comprising an α chain, a β chain, and a γ chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes the monomeric, dimeric, and trimeric forms of laminin.

[0414] In certain embodiments, the targeting moiety recognizes and binds to nidogen or entactin. Nidogen / entactin are a family of highly conserved sulfated glycoproteins. They are major structural components of basement membranes and function to connect laminin with the collagen IV network in the basement membrane. Members of this family include nidogen-1 and nidogen-2. In various embodiments, the targeting moiety recognizes an epitope on nidogen-1 and / or nidogen-2.

[0415] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an epitope present on any of the targets described herein (e.g., an ECM protein). In certain embodiments, the antigen recognition domain recognizes one or more linear epitopes present on the protein. As used herein, a linear epitope refers to any continuous sequence of amino acids present on a protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on the protein. As used herein, a conformational epitope refers to a portion of one or more amino acids (which may be discontinuous) that forms a three-dimensional surface with characteristics and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0416] In various embodiments, the targeting moiety may bind to the full-length and / or mature and / or isoforms and / or splice variants and / or fragments and / or any other natural or synthetic analogs, variants, or mutants of any of the targets (e.g., ECM proteins) described herein. In various embodiments, the targeting moiety may bind to any form of protein described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers, and associated forms. In various embodiments, the targeting moiety may bind to any post-translationally modified form of protein described herein, such as glycosylated and / or phosphorylated forms.

[0417] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an extracellular molecule such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In certain embodiments, DNA is shed into the extracellular space from necrotic or apoptotic tumor cells or other diseased cells.

[0418] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more noncellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaques are known: Fibro-lipid (fibro-fatty) plaques are characterized by the accumulation of lipid-laden cells beneath the intima of an artery. Beneath the endothelium lies a fibrous cap that covers the atherosclerotic plaque core. The core contains lipid-laden cells (macrophages and smooth muscle cells) with increased tissue cholesterol and cholesterol ester content, fibrin, proteoglycans, collagen, elastin, and necrotic cellular debris. In advanced plaques, the central core of the plaque typically contains extracellular cholesterol deposits (released from dead cells), which form areas of cholesterol crystals with empty needle-like spaces. The periphery of the plaque contains younger foam cells and capillaries. Fibrous plaques are also localized subintima within the arterial wall, resulting in wall thickening and proliferation, and sometimes, discrete localized narrowing of the lumen with slight atrophy of the muscularis. Fibrous plaques are composed of collagen fibers (eosinophils) These plaques contain plaques containing phospholipids (hematoxylinophils), calcium deposits (hematoxylinophils), and lipid-loaded cells. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular components of these plaques, such as fibrin, proteoglycans, collagen, elastin, necrotic cell debris, and calcium or other mineral deposits or precipitates. In some embodiments, the necrotic cell debris is nucleic acid, e.g., DNA or RNA released from dying cells.

[0419] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures found in brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures localized in amyloid plaques found in the brains of patients with Alzheimer's disease. For example, the targeting moiety recognizes and binds to the peptide amyloid beta. The peptide amyloid beta is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in brain plaques found in patients with Huntington's disease. In various embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in plaques associated with other neurodegenerative or musculoskeletal diseases, such as Lewy body dementia and inclusion body myositis.

[0420] Linkers and Functional Groups In various embodiments, Clec9A-binding agents may contain one or more functional groups, residues, or moieties. In various embodiments, the one or more functional groups, residues, or moieties are attached to or genetically fused to any of the signal transduction or targeting moieties described herein. In some embodiments, such functional groups, residues, or moieties impart one or more desirable properties or functionalities to the Clec9A-binding agents of the invention. Examples of such functional groups and techniques for introducing them into Clec9A-binding agents are known in the art; see, for example, Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

[0421] In various embodiments, the Clec9A binding agent may be conjugated and / or fused to another agent to extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties. In some embodiments, the Clec9A binding agent may be fused or conjugated to one or more of PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, etc. In some embodiments, the Clec9A binding agent may be fused or conjugated to an antibody or antibody fragment, such as an Fc fragment. For example, the chimeric protein may be fused to the N-terminus or C-terminus of the Fc domain of human immunoglobulin (Ig) G. In various embodiments, each individual chimeric protein is fused to one or more agents described in BioDrugs (2015) 29:215-239, the entire contents of which are incorporated herein by reference.

[0422] In some embodiments, the functional group, residue, or moiety comprises a suitable pharmaceutically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly(ethylene glycol) or mPEG). In some embodiments, the attachment of a PEG moiety increases the half-life and / or reduces the immunogenicity of the Clec9A-binding protein. Any suitable form of PEGylation is generally used, such as PEGylation used in the art for antibodies and antibody fragments (including, but not limited to, single domain antibodies such as VHHs); see, e.g., Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, See Adv. Drug Deliv. Rev. 54, 453-456 (2003), Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003), and WO 04 / 060965, the entire contents of which are incorporated herein by reference. Various reagents for protein PEGylation are also commercially available, for example, from Nektar Therapeutics, USA. In some embodiments, site-specific PEGylation, particularly via cysteine ​​residues, is used (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003), the entire contents of which are incorporated herein by reference). For example, for this purpose, PEG can be attached to a natural cysteine ​​residue in the Clec9A binding agent of the present invention. In some embodiments, the Clec9A binding agents of the present invention are modified to appropriately introduce one or more cysteine ​​residues for PEG attachment, or an amino acid sequence containing one or more cysteine ​​residues for PEG attachment may be fused to the amino and / or carboxy termini of the Clec9A binding agent using techniques known in the art.

[0423] In some embodiments, the functional group, residue, or moiety comprises N-linked or O-linked glycosylation, hi some embodiments, the N-linked or O-linked glycosylation is introduced as part of a co-translational and / or post-translational modification.

[0424] In some embodiments, the functional group, residue, or moiety comprises one or more detectable labels or other signal-generating groups or moieties. Suitable labels and techniques for their attachment, use, and detection are known in the art and include, but are not limited to, fluorescent labels (e.g., fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine and fluorescent metals, e.g., Eu or other metals of the lanthanide series), phosphorescent labels, chemiluminescent labels, or bioluminescent labels (e.g., luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, dioxetane, or GFP and its analogs), radioisotopes, metals, metal chelates, or metal cations. or other metals or metal cations that are particularly suitable for use in in vivo, in vitro, or in situ diagnostics and imaging, as well as chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase). Other suitable labels include moieties that can be detected using NMR or ESR spectroscopy. The VHHs and polypeptides of the invention so labelled may, depending on the choice of the particular label, be used, for example, for in vitro, in vivo or in situ assays (immunoassays known per se such as ELISAs, RIAs and EIAs and other "sandwich" methods) and for in vivo diagnostic and imaging purposes.

[0425] In some embodiments, the functional group, residue, or moiety comprises a tag that is attached to or genetically fused to the Clec9A-binding agent. In some embodiments, the Clec9A-binding agent can comprise a single tag or multiple tags. The tag can be, for example, a peptide, sugar, or DNA molecule that does not inhibit or interfere with binding of the Clec9A-binding agent to Clec9A or any other antigen of interest, such as a tumor antigen. In various embodiments, the tag is at least about: 3-5 amino acids in length, 5-8 amino acids in length, 8-12 amino acids in length, 12-15 amino acids in length, or 15-20 amino acids in length. Examples of tags are described, for example, in U.S. Patent Application Publication No. 2013 / 0058962. In some embodiments, The tag is an affinity tag, such as glutathione-S-transferase (GST) and histidine (His) tags. In certain embodiments, the Clec9A binder comprises a His tag.

[0426] In some embodiments, the functional group, residue, or moiety comprises a chelating group, for example, for chelating one of a metal or metal cation. Suitable chelating groups include, for example, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0427] In some embodiments, the functional group, residue, or moiety comprises a functional group that is one half of a specific binding pair, such as a biotin-(streptavidin) binding pair. Such functional groups can be used to link Clec9A-binding agents of the invention to another protein, polypeptide, or chemical compound that is bound to the other half of the binding pair, i.e., through the formation of a binding pair. For example, a Clec9A-binding agent of the invention can be conjugated to biotin and then bound to another protein, polypeptide, compound, or carrier that is conjugated to avidin or streptavidin. Such a conjugated Clec9A-binding agent can be used, for example, as a reporter in a diagnostic system in which a detectable signal-generating agent is conjugated to avidin or streptavidin. Such binding pairs can also be used to bind Clec9A-binding agents to carriers, including carriers suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described in Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs may also be used to link therapeutically active agents to the Clec9A binding agents of the invention.

[0428] In some embodiments, Clec9A binding agents of the invention optionally comprise one or more linkers. In some embodiments, the Clec9A binding agent comprises a linker connecting each binding domain and / or targeting moiety. In some embodiments, the Clec9A binding agent comprises a linker connecting each signaling agent and targeting moiety (or, in the case of two or more targeting moieties, connecting the signaling agent to one of the targeting moieties). In some embodiments, linkers may be used to connect various functional groups, residues, or moieties described herein to the Clec9A binding agent. In some embodiments, the linker is a single amino acid or multiple amino acids that do not affect or reduce the stability, orientation, binding, neutralization, and / or efflux properties of the binding domain and binding protein. In various embodiments, the linker is selected from a peptide, protein, sugar, or nucleic acid.

[0429] In some embodiments, Clec9A binding agents of the invention comprise a linker connecting the targeting moiety and the signaling agent, hi some embodiments, chimeric proteins of the invention comprise a linker within the signaling agent (e.g., in the case of single-chain TNF, may comprise two linkers resulting in a trimer).

[0430] The present invention contemplates the use of various linker sequences. In various embodiments, the linker may be derived from a natural multidomain protein or may be an empirical linker, as described, for example, in Chichili et al., (2013), Protein Sci. 22(2):153-167; Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker may be designed using a linker design database and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369 and Crasto et al., (2000), Protein Eng. 13(5):309-312. In various embodiments, the linker may be derived from a natural multidomain protein or may be an empirical linker, as described, for example, in Chichili et al., (2013), Protein Sci. 22(2):153-167; Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369; Alternatively, the linker may be functional. For example, but not limited to, the linker may function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve biological activity of the Clec9A binding agents of the invention.

[0431] In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids in length. For example, the linker can be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is a polypeptide. In some embodiments, the linker is more than about 100 amino acids in length. For example, the linker can be greater than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is flexible. In other embodiments, the linker is rigid.

[0432] In some embodiments, the linker length allows for effective binding of the targeting moiety and signal transduction agent to their receptors. For example, in some embodiments, the linker length allows for effective binding of one targeting moiety and signal transduction agent to a receptor on the same cell, and effective binding of the other targeting moiety to a different cell. Examples of cell pairs are provided elsewhere herein.

[0433] In some embodiments, the length of the linker is at least equal to the shortest distance between the binding sites of a targeting moiety and a signal transduction agent to a receptor on the same cell. In some embodiments, the length of the linker is at least 2, 3, 4, 5, 10, 20, 25, 50, 100 times longer than the shortest distance between the binding sites of a targeting moiety and a signal transduction agent to a receptor on the same cell.

[0434] In some embodiments, a linker connects two targeting moieties to each other, and the linker has a short length; and a linker connects a targeting moiety and a signal transduction agent, and the linker is longer than the linker connecting the two targeting moieties. For example, the difference in amino acid length between the linker connecting the two targeting moieties and the linker connecting the targeting moiety and the signal transduction agent can be about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids. In some embodiments, the linker is flexible. In other embodiments, the linker is rigid.

[0435] In various embodiments, the linker is substantially composed of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine). For example, in some embodiments, the linker is (Gly4Ser) n wherein n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NOs: 1010 to 1017). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 1018). Examples of additional linkers include, but are not limited to, linkers having the following sequences: LE, GGGGS (SEQ ID NO: 1010), ( GGGGS) n (n = 1 to 4) (SEQ ID NOs: 1010 to 1013), (Gly) (SEQ ID NO: 1019), (Gly) (SEQ ID NO: 1020), (EAAAK) n (n = 1 to 3) (SEQ ID NOs: 1021 to 1023), A(EAAAK) nA (n = 2 to 5) (SEQ ID NOs: 1024 to 1027), AEAAAKEAAAKA (SEQ ID NO: 1024), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 1028), PAPAP (SEQ ID NO: 1029), KESGSVSSEQLAQFRSLD (SEQ ID NO: 1030), EGKSSGSGSESKST (SEQ ID NO: 1031), GSAGSAAGSGEF (SEQ ID NO: 1032), and (XP) n , X represents any amino acid, e.g., Ala, Lys, or Glu. In various embodiments, the linker is (GGS) n (n=1 to 20) (SEQ ID NO: 1033 to SEQ ID NO: 1052). In some embodiments, the linker is G. In some embodiments, the linker is AAA. In some embodiments, the linker is (GGGG...

Claims

1. 1. A Clec9A binding agent comprising a recombinant heavy chain antibody (VHH) comprising three complementarity determining regions (CDR1, CDR2, and CDR3), CDR1 is the amino acid sequence of SEQ ID NO: 53; CDR2 is the amino acid sequence of SEQ ID NO: 138; CDR3 is the amino acid sequence of SEQ ID NO: 256; comprising an amino acid sequence having at least 98% identity to one of SEQ ID NOs: 330 and 331; Clec9A binding substance.

2. 2. The Clec9A binding agent of claim 1, comprising the amino acid sequence of SEQ ID NO:

330.

3. 2. The Clec9A binding agent of claim 1, comprising the amino acid sequence of SEQ ID NO:

331.

4. The Clec9A binding substance of claim 1 , wherein the VHH is a humanized VHH.

5. 2. The Clec9A binding agent of claim 1, comprising a signaling agent comprising modified human IFNα2 comprising an R149A mutation.

6. 10. The Clec9A binding agent of claim 1, comprising one or more additional targeting moieties.

7. 7. The Clec9A binding agent of claim 6, wherein the one or more additional targeting moieties recognize and optionally functionally modulate a tumor antigen.

8. 7. The Clec9A binding agent of claim 6, wherein the one or more additional targeting moieties recognize an antigen on an immune cell and optionally functionally modulate the antigen on the immune cell.

9. 9. The Clec9A-binding agent of claim 8, wherein the immune cell is selected from a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, and an NK cell.

10. 10. The Clec9A binding agent of claim 1, which recruits cytotoxic T cells to tumor cells or to the tumor environment.

11. 2. The Clec9A binding agent of claim 1, which recognizes and binds to Clec9A without substantially functionally modulating its activity.

12. A recombinant nucleic acid composition encoding the Clec9A-binding agent of any one of claims 1 to 11.

13. A host cell comprising the nucleic acid of claim 12.

14. 12. Use of a Clec9A binding agent according to any one of claims 1 to 11 in the manufacture of a medicament for the treatment of lymphoma.

15. (a) a Clec9A binding agent according to any one of claims 1 to 11; and (b) modified human IFNα2 having one or more mutations that confer improved safety compared to wild-type IFNα2; Including, A chimeric protein, wherein the Clec9A binding agent and the modified human IFNα2 are optionally linked by one or more linkers.

16. The chimeric protein of claim 15, wherein the modified human IFNα2 comprises one or more mutations at positions R120, M148, R149, and L153.

17. The chimeric protein of claim 16, wherein the modified human IFNα2 comprises one or more mutations selected from R120E, R149A, and L153A.

18. (a) a Clec9A binding agent according to any one of claims 1 to 11; and (b) modified human IFNβ having one or more mutations that confer improved safety compared to wild-type IFNβ; Including, A chimeric protein, wherein the Clec9A binding agent and the modified human IFNβ are optionally linked by one or more linkers.

19. 19. The chimeric protein of claim 18, wherein the modified human IFNβ comprises one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155.

20. 20. The chimeric protein of claim 19, wherein the modified human IFN beta comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, and R152G.

21. A pharmaceutical composition for treating lymphoma, comprising the Clec9A binding agent of any one of claims 1 to 11.

Images