PD-1 and PD-L1 binding agents

Abstract

The present invention relates to PD-1 and PD-L1 binding agents, in particular, the present invention relates in part to agents that bind to PD-1 or PD-L1 and the use of said agents as diagnostic and therapeutic agents. The invention further relates to a pharmaceutical composition comprising said PD-1 or PD-L1 binding agent and to the use of said pharmaceutical composition in the treatment of various diseases.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 2018800657274, application date August 8, 2018, and invention name “PD-1 and PD-L1 binders”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 542,921, filed August 9, 2017, the contents of which are hereby incorporated by reference in their entirety. Technical Field

[0004] The present invention relates, in part, to binding agents that bind to PD-1 or PD-L1 and their use as therapeutic and diagnostic agents.

[0005] Instructions for electronically submitting text documents

[0006] The contents of the text file submitted electronically with this article are incorporated herein by reference in their entirety: Computer-readable copy of the Sequence Listing (File name: ORN-034PC_ST25; Creation date: August 8, 2018; File size: 827 KB). Background Art

[0007] Immunotherapies have been developed to redirect the body's immune system against cancer. Immunotherapies offer cell-specific advantages that other treatment modalities, such as chemotherapy and radiation, lack. Therefore, methods for enhancing the efficacy of immune-based therapies may have clinical benefits. For example, immune checkpoint molecules that provide co-stimulatory or co-inhibitory signals play a central role in regulating T cell immune responses against tumor cells.

[0008] However, despite impressive responses to agents targeting checkpoint molecules, including clinical trials that led to the approval of YERVOY, KEYTRUDA, and OPDIVO, immunotherapies such as checkpoint inhibitors still fail in the vast majority of patients. Furthermore, many immunotherapies are complicated by side effects that significantly narrow the patient's therapeutic window and make them more susceptible to other diseases.

[0009] Therefore, there remains a need for improved immunotherapeutics that can provide targeted therapy against cancer while minimizing side effects. Summary of the Invention

[0010] In various aspects, the present invention relates to binding agents having at least one targeting portion that specifically binds to PD-1 or PD-L1. In various embodiments, these binding agents bind to PD-1 or PD-L1 and functionally regulate (e.g., partially or completely neutralize) PD-1 or PD-L1. In various embodiments, these binding agents bind to PD-1 or PD-L1 but do not functionally regulate (e.g., partially or completely neutralize) PD-1 or PD-L1. Therefore, in various embodiments, the binding agents of the present invention are used, for example, to recruit cells expressing PD-1 or cells expressing PD-L1 directly or indirectly to a target site while still allowing the cells to signal via PD-1 or PD-L1 (i.e., the binding of the PD-1 or PD-L1 binding agent does not reduce or eliminate PD-1 or PD-L1 signaling at the target site). In one embodiment, the targeting portion is a single domain antibody (VHH).

[0011] In various embodiments, the binding agent of the present invention further comprises a signaling agent, such as, but not limited to, interferon, interleukin, and tumor necrosis factor, which may be modified to reduce activity. In various embodiments, the binding agent comprises an additional targeting moiety that binds to other target of interest (e.g., antigen, receptor). In one embodiment, the other target of interest (e.g., antigen, receptor) is present on tumor cells. In another embodiment, the other target of interest (e.g., antigen, receptor) is present on immune cells. In some embodiments, the binding agent of the present invention can recruit immune cells (e.g., dendritic cells) directly or indirectly to the site of action (as a non-limiting example, such as the tumor microenvironment). In some embodiments, the binding agent of the present invention promotes the presentation of antigens (e.g., tumor antigens) by dendritic cells.

[0012] In various embodiments, the binding agents of the invention can be used to treat a variety of diseases or disorders, such as cancer, infection, immune disorders, and other diseases and disorders, and the invention encompasses various methods of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A schematic diagram of the PD-1 or PD-L1 Actaferon cloning strategy described in Example 1 is provided.

[0014] Figure 2 Binding of Actaferon to Hek293T cells transfected with PD-L1 (upper panel) or PD-1 (lower panel) is shown.

[0015] Figure 3PD-1 targeting as assessed by pSTAT1 status in peripheral blood mononuclear cells (PBMCs) is shown. Data are plotted as a percentage of the unstimulated pSTAT1 mean fluorescence intensity (MFI).

[0016] Figure 4 Figure 2 shows PD-1 targeting as assessed by pSTAT1 status in transfected Hek293T cells. The percentage of pSTAT1 cells is plotted.

[0017] Figure 5 Shown is PD-L1 targeting as assessed by pSTAT1 status in MDA-MB-231 cells. Data are plotted as mean fluorescence intensity (MFI).

[0018] Figure 6 Binding of PD-L1 VHH to transfected Hek293T cells is shown. Data are plotted as mean fluorescence intensity (MFI).

[0019] Figures 7A-7G Figure 2 is a graph showing the bioactivity of the PD-L1 VHH Actaferon (AFN). The bioactivity was measured on parental HL116 cells (IFN-responsive cell line stably transfected with the p6-16 luciferase reporter). Luciferase activity was induced by serial dilutions of the PD-L1 VHH AFN in the presence or absence of excess (20 μg / ml) of the corresponding PD-L1 VHH. The PD-L1 VHH tested was 2LIG3 ( Figure 7A )、2LIG27( Figure 7B )、2LIG97( Figure 7C )、2LIG99( Figure 7D )、2LIG176( Figure 7E )、2LIG189( Figure 7F ) and 3LIG8( Figure 7G ).

[0020] Figure 8 Graph showing the bioactivity of Atezolizumab-Actaferon (ate-AFN; i.e., Ate-IFNa2mut) on parental HL116 cells (an IFN-responsive cell line stably transfected with a p6-16 luciferase reporter). Bioactivity was measured by luciferase activity induced by serial dilutions of ate-AFN in the presence or absence of excess (50 μg / ml) Atezolizumab (ate).

[0021] Figure 9 is a graph showing the in vivo anti-tumor activity of 2LIG99-based AFN in the RL tumor model in mice with a humanized immune system.

[0022] Figures 10A-10C The results show that the 102C3 AFN ( Figure 10A )、102C12 AFN( Figure 10B ) or wild-type IFNa2 ( Figure 10C ) Figures of pSTAT1 in Hek293T cells transfected with human PD-1 or empty vector and stimulated by PD-1. The percentage of pSTAT1-positive cells is plotted in these figures. DETAILED DESCRIPTION

[0023] The present invention is based in part on the discovery of binding agents (e.g., antibodies, such as VHHs, as non-limiting examples) that recognize and bind to PD-1 or PD-L1. In some embodiments, the binding agents of the invention are part of a chimeric or fusion protein with one or more targeting moieties and / or one or more signaling agents. In various embodiments, these binding agents bind to PD-1 or PD-L1 and functionally modulate (e.g., partially or completely neutralize) PD-1 or PD-L1. In some embodiments, these binding agents bind to PD-1 or PD-L1 but do not functionally modulate PD-1 or PD-L1.

[0024] The present invention further provides pharmaceutical compositions comprising the binding agent and uses of the pharmaceutical compositions in treating various diseases, including cancer, autoimmune diseases and / or neurodegenerative diseases.

[0025] PD-1 or PD-L1 binders

[0026] In various embodiments, the PD-1 or PD-L1 binding agents of the present invention are protein-based agents that can specifically bind to PD-1 or PD-L1. In various embodiments, the PD-1 or PD-L1 binding agents of the present invention are protein-based agents that can specifically bind to PD-1 or PD-L1 without functionally modulating (e.g., partially or completely neutralizing) PD-1 or PD-L1.

[0027] Programmed cell death protein 1, also known as PD-1 and cluster of differentiation 279 (CD279), is a cell surface receptor expressed primarily on activated T cells, B cells, and macrophages. PD-1 has been shown to negatively regulate antigen receptor signaling when engaged by its ligands, namely PD-L1 and / or PD-L2. PD-1 plays an important role in downregulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 is a type I transmembrane glycoprotein that contains an Ig variable type (V-type) domain responsible for ligand binding and a cytoplasmic tail responsible for binding of signaling molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs, namely ITIM (immunoreceptor tyrosine-based inhibition motif) and ITSM (immunoreceptor tyrosine-based switch motif).

[0028] In various embodiments, the PD-1 binding agent of the present invention comprises a targeting portion having an antigen recognition domain that recognizes an epitope present on PD-1. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on PD-1. As used herein, a linear epitope refers to any continuous amino acid sequence present on PD-1. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on PD-1. As used herein, a conformational epitope refers to one or more amino acid segments (which may be discontinuous) that form a three-dimensional surface having a feature and / or shape and / or tertiary structure that can be recognized by an antigen recognition domain.

[0029] In various embodiments, the PD-1 binding agents of the present invention can bind to full-length and / or mature forms and / or isoforms and / or splice variants and / or fragments and / or any other naturally occurring or synthetic analogs, variants, or mutants of human PD-1. In various embodiments, the PD-1 binding agents of the present invention can bind to any form of human PD-1. In one embodiment, the PD-1 binding agent binds to the phosphorylated form of PD-1.

[0030] In one embodiment, the PD-1 binding agent of the present invention comprises a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on human PD-1. In one embodiment, the human PD-1 comprises the amino acid sequence of SEQ ID NO: 1.

[0031] In another embodiment, the human PD-1 comprises the amino acid sequence of SEQ ID NO: 1 without the amino-terminal signal peptide.

[0032] In various embodiments, the PD-1 binding agent of the present invention comprises a targeting moiety capable of specific binding. In various embodiments, the PD-1 binding agent comprises a targeting moiety having an antigen recognition domain, such as an antibody or a derivative thereof. In one embodiment, the PD-1 binding agent comprises a targeting moiety, which 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 a variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain includes a variable region (V L ) and a constant region (C L). The variable region determines the specificity of the antibody. Each variable region includes three hypervariable regions flanked by four relatively conserved framework regions (FR), also known as complementarity determining regions (CDRs). The three CDRs (referred to as CDR1, CDR2, and CDR3) contribute to the antibody's binding specificity. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0033] In some embodiments, the PD-1 binding agent comprises a targeting moiety, which is an antibody derivative or antibody form. In some embodiments, the PD-1 binding agent of the present invention comprises a targeting moiety, which is a single domain antibody, a heavy chain only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain only shark antibody (VNAR), a microprotein (cysteine ​​knot protein, knottin), a DARPin; a tetranectin; an affibody; a transbody; an anticalin; an adnectin; an affilin; an affimer; a microbody; an aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer; an armadillo repeat protein; a Kunitz domain; a cytochrome c-terminal ... domain), avimer, atrimer, probody, immunobody, triomab, troybody; pepbody; vaccibody, unibody; duobody, Fv, Fab, Fab′, F(ab′)2, peptide mimetic molecule or synthetic molecule as described in the following U.S. Patent Nos. or Patent Publication Nos.: US 7,417,130, US2004 / 132094, US 5,831,012, US2004 / 023334, US 7,250,297, US 6,818,418, US2004 / 209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US2004 / 146938, US2004 / 157209, US 6,994,982, US 6,794,144, US 2010 / 239633, US 7,803,907, US2010 / 119446 and / or US 7,166,697, the contents of which are hereby incorporated by reference in their entirety. See also Storz MAbs. 2011 May-June; 3(3):310-317.

[0034] In some embodiments, the PD-1 binding agent comprises a targeting moiety that is a single domain antibody, such as a VHH. The VHH can be derived from, for example, an organism that produces VHH antibodies, such as a camel, a shark, or a designed VHH. VHHs are therapeutic proteins derived from antibodies that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional antibodies from camels that lack light chains. These heavy chain antibodies contain a single variable domain (V H H) and two constant domains (CH2 and CH3).

[0035] In one embodiment, the PD-1 binding agent comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

[0036] In some embodiments, the VHH comprises a fully human V H domain, such as HUMABODY (Crescendo Biologics, Cambridge, UK). In some embodiments, the fully human V H The domain (e.g., HUMABODY) is monovalent, bivalent, or trivalent. In some embodiments, the fully human V H The domain (eg, HUMABODY) is monospecific or multispecific, such as monospecific, bispecific, or trispecific. H Domains such as HUMABODY are described, for example, in WO2016 / 113555 and WO2016 / 113557, the entire contents of which are incorporated by reference.

[0037] In some embodiments, the PD-1 binding agent 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, "framework regions" or "FRs" refer to the regions of the variable domain located between the CDRs. As used herein, "complementarity determining regions" or "CDRs" refer to the variable region of a VHH that contains an amino acid sequence that is capable of specifically binding to an antigenic target.

[0038] In various embodiments, the PD-1 binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence. In various embodiments, the PD-1 binding agent comprises a VHH having a variable region comprising at least one FR1, FR2, FR3, and FR4 sequence.

[0039] In some embodiments, the PD-1 CDR1 sequence is selected from the group consisting of: GFSMDYYAIA (SEQ ID NO: 2); GFSVDYYAIA (SEQ ID NO: 3); GGFNRVSYMG (SEQ ID NO: 4); GIIKSINFMG (SEQ ID NO: 5); GFILDYYGIG (SEQ ID NO: 6); GLSLDYDGVG (SEQ ID NO: 7); GRTFSSLGMG (SEQ ID NO: 8); GFAFGSYDMG (SEQ ID NO: 9); GFSFGNNDMS (SEQ ID NO: 10); IHAMG (SEQ ID NO: 11); INAMA (SEQ ID NO: 12); SGTMG (SEQ ID NO: 13); GSIASIHAM (SEQ ID NO: 14); GSIASIHAMG (SEQ ID NO: 15); FYGMG (SEQ ID NO: 16); GGTFSFYGMG (SEQ ID NO: 17); YYAIA (SEQ ID NO: 18); NO:18); VSYMG (SEQ ID NO:19); INFMG (SEQ ID NO:20); SLGMG (SEQ ID NO:21); SYDMG (SEQ ID NO:22); and NNDMS (SEQ ID NO:23).

[0040] In some embodiments, the PD-1 CDR2 sequences are selected from: CITGSDFMVDT (SEQ ID NO:24); SVTSGGEI (SEQ ID NO:25); STTSDGRT (SEQ ID NO:26); CISSSDGST (SEQ ID NO:27); AIAWNGAST (SEQ ID NO:28); GINSGGRIT (SEQ ID NO:29); AINSGGGST (SEQ ID NO:30); AITWSGGITYYEDSVKG (SEQ ID NO:31); VITWSGGITYYADSVKG (SEQ ID NO:32); VITVSGGITYYADSVKG (SEQ ID NO:33); AITWSGGITYYADSLKG (SEQ ID NO:34); LISWSGGSTYYEDSVKG (SEQ ID NO:35); SIPWSGGRIYYADSVKG (SEQ ID NO:36); VITWSGGITY (SEQ ID NO:37); VITVSGGITY (SEQ ID NO:38); DIRTSAGRTYYADSVKG (SEQ ID NO:39); DIRTSAGRTY (SEQ ID NO:40); CITGSDFMVDTY (SEQ ID NO:41); CITGSDFMVDTYYVASVKG (SEQID NO:42); SVTSGGEIT (SEQ ID NO:43); SVTSGGEITIADSVKG (SEQ ID NO:44); SVTSGGEITVADSVKG (SEQ ID NO:45); STTSDGRTT (SEQ ID NO:46); STTSDGRTTVADSVKG (SEQID NO:47); CISSSDGSTY (SEQ ID NO:48); AIAWNGASTY (SEQ ID NO:49); AIAWNGASTYYTESVKG (SEQ ID NO:50); GINSGGRITD (SEQ ID NO:51); GINSGGRITDYADSVTG (SEQ ID NO:52); AINSGGGSTY (SEQ ID NO:53); and AINSGGGSTYYADSVKG (SEQ ID NO:54).

[0041] In some embodiments, the PD-1 CDR3 sequence is selected from the group consisting of: AVRSTANTLCPSHYSVMDY (SEQ ID NO: 55); AVRSTANTLCPSHYSIMDY (SEQ ID NO: 56); NADIWVSDARMYNY (SEQ ID NO: 57); NADIWLPSDRMYNY (SEQ ID NO: 58); ATATLCDGGIWGY (SEQ ID NO: 59); AASGLGSVVVTANEYDY (SEQ ID NO: 60); AQGDRSSWHYYGMDY (SEQ ID NO: 61); ATKSDPMTNEYDL (SEQ ID NO: 62); DRAESSWYDY (SEQ ID NO: 63); DKHQSSWYDY (SEQ ID NO: 64); DKHQSSFYDY (SEQ ID NO: 65); DRAQSSWYDY (SEQ ID NO: 66); DRVDSNWYDY (SEQ ID NO: 67). NO:67); KERSTGWDFAS (SEQ ID NO:68); and EMSGISGWDY (SEQ ID NO:69).

[0042] In various exemplary embodiments, the PD-1 binding agent comprises an amino acid sequence selected from the group consisting of:

[0043] 2PD23

[0044] QVQLQESGGGLVQPGGSLRLSCAASGFSMDYYAIAWFRQAPGKEREEISCITGSDFMVDTYYVASVKGRFTISRDNAENTAYLQMNNLKPEDTGVYFCAVRSTANTLCPSHYSVMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO:70);

[0045] 2PD26

[0046] QVQLQESGGGLVQAGGSLRLSCAASGFSMDYYAIAWFRQAPGKEREEISCITGSDFMVDTYYVASVKGRFTISRDNAENTAYLQMNNLKPEDTGVYFCAVRSTANTLCPSHYSVMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:71);

[0047] 2PD90

[0048] QVQLQESGGGLVQPGGSLRLSCSASGFSVDYYAIAWFRQAPGKEREEISCITGSDFMVDTYYVASVKGRFTISRDNAKNTAYLQMNSLKPEDTGVYFCAVRSTANTLCPSHYSIMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:72);

[0049] 2PD-106

[0050] QVQLQESGGGLVQPGGSLRLSCSASGFSMDYYAIAWFRQAPGKEREEISCITGSDFMVDTYYVASVKGRFTISRDNAKNTAHLQMNSLKPEDTGVYFCAVRSTANTLCPSHYSVMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:73);

[0051] 2PD-16

[0052] QVQLQESGGGLVQAGGSLRLSCAASGGFNRVSYMGWYRQAPGTKRELVASVTSGGEITIADSVKGRFTVSRDNSKNTLYLQMNGLKPEDGATYWCNADIWVSDARMYNYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:74);

[0053] 2PD71

[0054] QVQLQESGGGLVQTGESLRLSCAASGGFNRVSYMGWYRQAPGSKRELVASVTSGGEITVADSVKGRFTVSRDNNKNTLYLQMNGLKPEDGATYWCNADIWVSDARMYNYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:75);

[0055] 2PD-152

[0056] QVQLQESGGGLVQTGESLRLSCAASGIIKSINFMGWYRQPPGTKRELVASTTSDGRTTVADSVKGRFTISRDNAKNTIYLEMSSLKPEDTATYWCNADIWLPS DRMYNYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:76);

[0057] 2PD-12

[0058] QVQLQESGGGLVQAGGSLRLSCAVSGFILDYYGIGWFRQAPGKEREAVSCISSSDGSTYYADSVKGRFTISRDNALNTLYLQMNSLKPEDTAVYHCATATLCDGGIWGYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:77);

[0059] 3PD55

[0060] QVQLQESGGGLAQAGGSLRLSCEGSGLSLDYDGVGWFRQAPGKEREAVSCISSSDGSTYYADSVKGRFTISRGNALNTLYLQMNSLKPEDTAVYYCATATLCDGGIWGYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:78);

[0061] 3PD82

[0062] QVQLQESGGGSVQPGGSLRLSCAVSGFILDYYGIGWFRQAPGKEREAVSCISSSDGSTYYADSVKGRFTISRDNALNTLYLQMNSLKPEDTAVYYCATATLCDGGIWGYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:79);

[0063] 2PD8

[0064] QVQLQESGGGSVQAGDSLRLSCTASGRTFSSLGMGWFRQAPGKEREFVSAIAWNGASTYYTESVKGRFTISRDDAKNTVYLQMNSLKPTDTAVYFCAASGLGSVVVTANEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:80);

[0065] 2PD27

[0066] QVQLQESGGGSVQPGKSLRLSCAASGRTFSSLGMGWFRQAPGKEREFVSAIAWNGASTYYTESVKGRFTISRDDAKNTVYLQMNSLKPTDTAVYFCAASGLGSVVVTANEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 81);

[0067] 2PD82

[0068] OR

[0069] 3PD36

[0070] QVQLQESGGGLVQPGGSLRLSCAASGFSFGNNDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATKSDPMTNEYDLWGXGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 83).

[0071] In various exemplary embodiments, the PD-1 binding agent comprises an amino acid sequence selected from SEQ ID NO: 70 to SEQ ID NO: 83 without a terminal histidine tag sequence (ie, HHHHHH; SEQ ID NO: 84).

[0072] In some embodiments, the PD-1 binding agent comprises an amino acid sequence selected from SEQ ID Nos: 70-83 (provided above) without the HA tag (ie, YPYDVPDYGS; SEQ ID NO: 85).

[0073] In some embodiments, the PD-1 binding agent comprises an amino acid sequence selected from SEQ ID Nos: 70-83 (provided above) without an AAA linker.

[0074] In some embodiments, the PD-1-binding agent comprises an amino acid sequence selected from SEQ ID Nos: 70-83 (provided above) without the AAA linker, HA tag, and terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 86).

[0075] In various exemplary embodiments, the PD-1 binding agent comprises an amino acid sequence selected from the group consisting of:

[0076] 102C3:

[0077] OR

[0078] 102C12:

[0079] QVQLQESGGGLVQAGKSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTQVTVSS (SEQ ID NO: 1247).

[0080] In various embodiments, the PD-1 binding agent comprises an amino acid sequence described in U.S. Publication No. 2017 / 0137517, the entire contents of which are incorporated by reference. For example, in some embodiments, the PD-1 binding agent comprises one of the following sequences in U.S. Publication No. 2017 / 0137517:

[0081] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:87);

[0082] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:88);

[0083] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYYCAGDKHQSSWYDYWGQGTLVKVSS(SEQ ID NO:89);

[0084] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVQVSS(SEQ ID NO:90);

[0085] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTLVTVKS(SEQ ID NO:91);

[0086] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTLVTVQS(SEQ ID NO:92);

[0087] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVKVSS(SEQ ID NO:93);

[0088] EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVQVSS(SEQ ID NO:94);

[0089] EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVKS(SEQ ID NO:95);

[0090] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVQS(SEQ ID NO:96);

[0091] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVSS(SEQ ID NO:97);

[0092] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVKVSS(SEQ ID NO:98);

[0093] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVQVSS(SEQ ID NO:99);

[0094] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVKS(SEQ ID NO:100);

[0095] EVQLVESGGGVVQPGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAEMSGISGWDYWGQGTLVTVQS(SEQ ID NO:101);

[0096] EVQLVESGGGVVQPGGSLRLSCAASGGTFSFYGMGWFRQAPGKEREFVADIRTSAGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAEMSGISGWDYWGQGTLVTVSS(SEQ ID NO:102);

[0097] DVQLVESGGGVVQPGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAEMSGISGWDYWGQGTLVTVSS(SEQ ID NO:103);

[0098] DVQLVESGGGVVQPGGSLRLSCAASGGTFSFYGMGWFRQAPGKEREFVADIRTSAGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAEMSGISGWDYWGQGTLVTSSA(SEQ ID NO:104);

[0099] EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYYCAAEMSGISGWDYWGQGTLVKVSSA(SEQ ID NO:105);

[0100] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTLVQVSSA(SEQ ID NO:106);

[0101] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTLVTVKSA(SEQ ID NO:107);

[0102] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTLVTVQSA(SEQ ID NO:108);

[0103] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTLVKVSSA(SEQ ID NO:109);

[0104] EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVQVSSA(SEQ ID NO:110);

[0105] EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVKSA(SEQ ID NO:111);

[0106] EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVQSA(SEQ ID NO:112);

[0107] EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVSSA(SEQ ID NO:113);

[0108] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVKVSSA(SEQ ID NO:114);

[0109] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVQVSSA(SEQ ID NO:115);

[0110] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVKSA(SEQ ID NO:116);

[0111] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVQSA(SEQ ID NO:117);

[0112] EVQLVESGGGVVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAEMSGISGWDYWGQGTLVTVSSA(SEQ ID NO:118);

[0113] EVQLVESGGGVVQPGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAEMSGISGWDYWGQGTLVTVSSA(SEQ ID NO:119);

[0114] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:120);

[0115] EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREEVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:121);

[0116] EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREEVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:122);

[0117] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREEVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA(SEQ ID NO:123);

[0118] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREEVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA(SEQ ID NO:124);

[0119] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREEVAVITVSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS(SEQ ID NO:125);

[0120] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS(SEQ ID NO:126);

[0121] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS(SEQ ID NO:127);

[0122] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS(SEQ ID NO:128);

[0123] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS(SEQ ID NO:129);

[0124] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDSSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS(SEQ ID NO:130);

[0125] EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREEVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREEVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS(SEQ ID NO:131); and

[0126] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ IDNO:132).

[0127] In some embodiments, the PD-1 binding agent comprises an amino acid sequence selected from SEQ ID NOs: 87-132 having one or more substitutions at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110, and 112 (according to Kabat numbering). In some embodiments, the amino acid at position 1 is E or D. In some embodiments, the amino acid at position 11 is L or V. In some embodiments, the amino acid at position 14 is A or P. In some embodiments, the amino acid at position 52a is W or V. In some embodiments, the amino acid at position 73 is N, S, P, or Q. In some embodiments, the amino acid at position 74 is A or S. In some embodiments, the amino acid at position 83 is K or R. In some embodiments, the amino acid at position 89 is T, V, I, or L. In some embodiments, the amino acid at position 100a is W or F. In some embodiments, the amino acid at position 110 is T, K, or Q. In some embodiments, the amino acid at position 112 is S, K, or Q.

[0128] In various embodiments, the PD-1 binding agent comprises an amino acid sequence described in PCT Publication No. WO 2017 / 087587, the entire contents of which are incorporated by reference. For example, in some embodiments, the PD-1 binding agent comprises one of the following sequences in PCT Publication No. WO 2017 / 087587:

[0129] EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSS (SEQ ID NO: 133);

[0130] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:134);

[0131] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTLVKVSS(SEQ ID NO:135);

[0132] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTLVQVSS(SEQ ID NO:136);

[0133] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVKS(SEQ ID NO:137);

[0134] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVQS(SEQ ID NO:138);

[0135] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVKVSS(SEQ ID NO:139);

[0136] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVQVSS(SEQ ID NO:140);

[0137] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVKS(SEQ ID NO:141);

[0138] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVQS(SEQ ID NO:142);

[0139] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:143);

[0140] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVKVSS(SEQ ID NO:144);

[0141] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVQVSS(SEQ ID NO:145);

[0142] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVKS(SEQ ID NO:146);

[0143] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVQS(SEQ ID NO:147);

[0144] EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:148);

[0145] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:149);

[0146] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYYCAGDKHQSSWYDYWGQGTLVTVSSA(SEQ ID NO:150);

[0147] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTLVKVSSA(SEQ ID NO:151);

[0148] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTLVQVSSA(SEQ ID NO:152);

[0149] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVKSA(SEQ ID NO:153);

[0150] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMCWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVQSA(SEQ ID NO:154);

[0151] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMCWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVKVSSA(SEQ ID NO:155);

[0152] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVQVSSA(SEQ ID NO:156);

[0153] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVKSA(SEQ ID NO:157);

[0154] EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVQSA(SEQ ID NO:158);

[0155] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSA(SEQ ID NO:159);

[0156] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVKVSSA(SEQ ID NO:160);

[0157] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVQVSSA(SEQ ID NO:161);

[0158] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVKSA(SEQ ID NO:162);

[0159] EVQLVESGGGVVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGDKHQSSWYDYWGQGTLVTVQSA(SEQ ID NO:163);

[0160] EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSA(SEQ ID NO:164);

[0161] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSA(SEQ ID NO:165);

[0162] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAIITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:166);

[0163] EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:167);

[0164] EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS(SEQ ID NO:168);

[0165] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA(SEQ ID NO:169);

[0166] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA(SEQ ID NO:170);

[0167] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS(SEQ ID NO:171);

[0168] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS(SEQ ID NO:172); and

[0169] DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDYWGQGTLVTVSS(SEQ ID NO:173).

[0170] In some embodiments, the PD-1 binding agent comprises an amino acid sequence selected from SEQ ID NOs: 133-173 having one or more substitutions at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110, and 112 (according to Kabat numbering). In some embodiments, the amino acid at position 1 is E or D. In some embodiments, the amino acid at position 11 is L or V. In some embodiments, the amino acid at position 14 is A or P. In some embodiments, the amino acid at position 52a is W or V. In some embodiments, the amino acid at position 73 is N, S, P, or Q. In some embodiments, the amino acid at position 74 is A or S. In some embodiments, the amino acid at position 83 is K or R. In some embodiments, the amino acid at position 89 is T, V, I, or L. In some embodiments, the amino acid at position 100a is W or F. In some embodiments, the amino acid at position 110 is T, K, or Q. In some embodiments, the amino acid at position 112 is S, K, or Q.

[0171] In various embodiments, the present invention contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs of the PD-1 binding agents of the invention as described herein (collectively referred to herein as "analogs"). In various embodiments, the amino acid sequence of the PD-1 binding agent further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0172] In various embodiments, the present invention also provides PD-L1 binding agents. Programmed death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a type 1 transmembrane protein that is hypothesized to play a major role in suppressing the immune system. PD-L1 is upregulated in macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and is upregulated in T cells and B cells upon TCR and B cell receptor signaling.

[0173] In various embodiments, the PD-L1 binding agent of the present invention comprises a targeting portion having an antigen recognition domain that recognizes an epitope present on PD-L1. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on PD-L1. As used herein, a linear epitope refers to any continuous amino acid sequence present on PD-L1. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on PD-L1. As used herein, a conformational epitope refers to one or more amino acid segments (which may be discontinuous) that form a three-dimensional surface having a feature and / or shape and / or tertiary structure that can be recognized by an antigen recognition domain.

[0174] In various embodiments, the PD-L1 binding agents of the present invention can bind to full-length and / or mature forms and / or isoforms and / or splice variants and / or fragments and / or any other naturally occurring or synthetic analogs, variants, or mutants of human PD-L1. In various embodiments, the PD-L1 binding agents of the present invention can bind to any form of human PD-L1. In one embodiment, the PD-L1 binding agent binds to the phosphorylated form of PD-L1. In one embodiment, the PD-L1 binding agent binds to the acetylated form of PD-L1.

[0175] In one embodiment, the PD-L1 binding agent of the present invention comprises a targeting portion having an antigen recognition domain that recognizes one or more epitopes present on human PD-L1. In one embodiment, the human PD-L1 comprises the following amino acid sequence (signal peptide is underlined):

[0176] Isotype 1:

[0177] MRIFAVFIFMTYWHLLNA FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQ AEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET(SEQ IDNO:174);

[0178] Isotype 2:

[0179] MRIFAVFIFMTYWHLLNA OR

[0180] Isotype 3:

[0181] MRIFAVFIFMTYWHLLNA FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGD (SEQ ID NO: 176).

[0182] In various embodiments, the PD-L1 binding agent of the present invention comprises a targeting portion capable of specific binding. In various embodiments, the PD-L1 binding agent comprises a targeting portion having an antigen recognition domain, such as an antibody or a derivative thereof. In one embodiment, the PD-L1 binding agent comprises a targeting portion, which 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 a variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain includes a variable region (V L ) and a constant region (C L ). The variable region determines the specificity of the antibody. Each variable region includes three hypervariable regions flanked by four relatively conserved framework regions (FR), also known as complementarity determining regions (CDRs). The three CDRs (referred to as CDR1, CDR2, and CDR3) contribute to the antibody's binding specificity. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0183] In some embodiments, the PD-L1 binding agent comprises a targeting moiety, which is an antibody derivative or antibody form. In some embodiments, the PD-L1 binding agent of the present invention comprises a targeting moiety, which is a single domain antibody, a heavy chain only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain only shark antibody (VNAR), a microprotein (cysteine ​​knot protein, knottin), a DARPin; a tetranectin; an affibody; a transbody; an anticalin; an adnectin; an affilin; an affimer; a microbody; an aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer; an armadillo repeat protein; a Kunitz domain; a phylobody; a stradobody; a maxibody; an evibody; a fynomer; a Kunitz domain; a phylobody; a stradobody; a maxibody; an evibody; a fynomer; a phylobody; a stradobody; a maxibody; a evibody; a fynomer; a phylobody; a strado ... domain), avimer, atrimer, probody, immunobody, triomab, troybody; pepbody; vaccibody, unibody; duobody, Fv, Fab, Fab′, F(ab′)2, peptide mimetic molecule or synthetic molecule as described in the following U.S. Patent Nos. or Patent Publication Nos.: US 7,417,130, US2004 / 132094, US 5,831,012, US2004 / 023334, US 7,250,297, US 6,818,418, US2004 / 209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US2004 / 146938, US2004 / 157209, US 6,994,982, US 6,794,144, US 2010 / 239633, US 7,803,907, US2010 / 119446 and / or US 7,166,697, the contents of which are hereby incorporated by reference in their entirety. See also Storz MAbs. 2011 May-June; 3(3):310-317.

[0184] In some embodiments, the PD-L1 binding agent comprises a targeting moiety that is a single domain antibody, such as a VHH. The VHH can be derived from, for example, an organism that produces VHH antibodies, such as a camel, a shark, or a designed VHH. VHHs are therapeutic proteins derived from antibodies that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional antibodies from camels that lack light chains. These heavy chain antibodies contain a single variable domain (V H H) and two constant domains (CH2 and CH3).

[0185] In one embodiment, the PD-L1 binding agent comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

[0186] In some embodiments, the VHH comprises a fully human V H domain, such as HUMABODY (Crescendo Biologics, Cambridge, UK). In some embodiments, the fully human V H The domain (e.g., HUMABODY) is monovalent, bivalent, or trivalent. In some embodiments, the fully human V H The domain (eg, HUMABODY) is monospecific or multispecific, such as monospecific, bispecific, or trispecific. H Domains such as HUMABODY are described, for example, in WO2016 / 113555 and WO2016 / 113557, the entire contents of which are incorporated by reference.

[0187] In some embodiments, the PD-L1 binding agent 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, "framework regions" or "FRs" refer to the regions of the variable domain located between the CDRs. As used herein, "complementarity determining regions" or "CDRs" refer to the variable region of a VHH that contains an amino acid sequence that is capable of specifically binding to an antigenic target.

[0188] In various embodiments, the PD-L1 binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence. In various embodiments, the PD-L1 binding agent comprises a VHH having a variable region comprising at least one FR1, FR2, FR3, and FR4 sequence.

[0189] In some embodiments, the PD-L1 CDR1 sequences are selected from: GFTLDYYAIG (SEQ ID NO:177); GTIFSINHMD (SEQ ID NO:178); GFTFDDYGMS (SEQ ID NO:179); GFTLDYYAIN (SEQ ID NO:180); GTIFSINRMD (SEQ ID NO:181); GFTFSSYGMS (SEQ ID NO:182); GKIFSGNDMG (SEQ IDNO:183); GFTFNDYAMS (SEQ ID NO:184); GFNLDPYAIA (SEQ ID NO:185); GFTFTAYAMS (SEQ IDNO:186); GFTFDYYAIG (SEQ ID NO:187); GFNLDPYAIG (SEQ ID NO:188); ESIFSIEAMG (SEQ IDNO:189); GRTFSISAMG (SEQ ID NO:190); YYAIG (SEQ ID NO:191); YYAKC (SEQ ID NO:192); QYDVG (SEQ ID NO:193); NSAMG (SEQ ID NO:194); DSIVS (SEQ ID NO:195); INHMD (SEQ IDNO:196); DYGMS (SEQ ID NO:197); YYAIN (SEQ ID NO:198); INRMD (SEQ ID NO:199); SYGMS (SEQ ID NO:200); GNDMG (SEQ ID NO:201); DYAMS (SEQ ID NO:202); PYAIA (SEQ ID NO:203); AYAMS (SEQ ID NO:204); PYAIG (SEQ ID NO:205); IEAMG (SEQ ID NO:206); and ISAMG (SEQ ID NO:207).

[0190] In some embodiments, the PD-L1 CDR2 sequences are selected from: ISSSDGSTY (SEQ ID NO:208); ITSDGFPT (SEQ ID NO:209); IRWNGGSTN (SEQ ID NO:210); ITSDGTPT (SEQ ID NO:211); IDSGGGSTS (SEQ ID NO:212); ITSGGITD (SEQ ID NO:213); ITSDGTPT (SEQ ID NO:214); IDSGGGSTS (SEQ ID NO:215); IRSNGGYTN (SEQ ID NO:216); ISSSDVGTY (SEQ ID NO:217); INSSDGSTY (SEQ ID NO:218); ISGSDSSTY (SEQ ID NO:219); ISSSDVGTY (SEQ ID NO:220); ITSDGTPT (SEQ ID NO:221); ITSDGTPA (SEQ ID NO:222); IDSGGGSTS (SEQ ID NO:223); ISSGDGSKY (SEQ ID NO:224); ISSSDVGTY (SEQ ID NO:225); IFGGGFTN (SEQ ID NO:226); ITSGGITD (SEQ ID NO:227); IDSGGGSTS (SEQ ID NO:228); ITSDGTPT (SEQ ID NO:229); IDSGGGSTS (SEQ ID NO:230); ISSSDVGTY (SEQ ID NO:231); ITWSGGSTS (SEQ ID NO:232); IDSGGGSTS (SEQ ID NO:233); IRSNGGYTN (SEQ ID NO:234); SISSSDGSTYYADSVKG (SEQ IDNO:235); CISSSDGSTYYADSVKG (SEQ ID NO:236); CISGGDNSTYYADSVKG (SEQ ID NO:237); FSSSGGRTIYPDSVKG (SEQ ID NO:238); RITGGGLIAYTDSVKG (SEQ ID NO:239); GISNGGTIKYAESVLG (SEQ ID NO:240); LITSDGFPT (SEQ ID NO:241); LITSDGFPTYADSAKG (SEQID NO:242); AIRWNGGSTN (SEQ ID NO:243); AIRWNGGSTNYADSVKG (SEQ ID NO:244);LITSDGTPT (SEQ ID NO:245); LITSDGTPTYADSAKG (SEQ ID NO:246); AIDSGGGSTS (SEQ ID NO:247); AIDSGGGSTSYADSVKG (SEQ ID NO:248); IITSGGITD (SEQ ID NO:249); IITSGGITDYADAVKG (SEQ ID NO:250); GIRSNGGYTN (SEQ ID NO:251); GIRSNGGYTNYADSVKG (SEQ ID NO:252); CISSSDVGTY (SEQ ID NO:253); CISSSDVGTYYADSVKG (SEQ ID NO:254); CINSSDGSTY (SEQ ID NO:255); CINSSDGSTYYADSVKG (SEQ ID NO:256); CISGSDSSTY (SEQ ID NO:257); CISGSDSSTYYADSVKG (SEQ ID NO:258); LITSDGTPA (SEQ ID NO:259); LITSDGTPAYADSAKG (SEQ ID NO:260); CISSGDGSKY (SEQ ID NO:261); CISSGDGSKYYADSVKG (SEQ ID NO:262); AIFGGGFTN (SEQ ID NO:263); AIFGGGFTNYADSVKG (SEQ ID NO:264); AITWSGGSTS (SEQ ID NO:265); and AITWSGGSTSYTDSVKG (SEQ ID NO:266).;

[0191] In some embodiments, the CDR3 sequence is selected from the group consisting of: DGWSSCRHGINEYLYW (SEQ ID NO: 267); SSGVYNYW (SEQ ID NO: 268); QGYYCSGYGCPR (SEQ ID NO: 269); SGWRLCRPTDEYDYSYW (SEQ ID NO: 270); QGYYCSGYGCSDYW (SEQ ID NO: 271); RDRTIWW (SEQ ID NO: 272); QGYYCSGYGCYP (SEQ ID NO: 273); DGYYYCSDYPHPLYW (SEQ ID NO: 274); DGWRDCTWSNEYAYW (SEQ ID NO: 275); TGWRTCRGLNEYDYW (SEQ ID NO: 276); DLVSGSRLYDYW (SEQ ID NO: 277); MGRTNYGVIYDPNMYNYW (SEQ ID NO: 278). NO:278); SGWRLCRPTDEYDYLYW (SEQ ID NO:279); SQAPITIATMMKPFYDY (SEQ ID NO:280); RHGGPLTVEYFFDY (SEQ ID NO:281); GGWKYCSGYDPEYIY (SEQ ID NO:282); DWYLNSY (SEQ ID NO:283); INSRDG (SEQ ID NO:284); and RQY (SEQ ID NO:285).

[0192] In various exemplary embodiments, the PD-L1 binding agent comprises an amino acid sequence selected from the group consisting of:

[0193] 2LIG2

[0194] QVQLQESGGGLVQAGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREEVS CISSSDGSTYYADSVKGRFTISRDNAKNTVNLQMNSLKPEDTAVYYCATDGW SSCRHGIN-EYLYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 286);

[0195] or

[0196] 2LIG3

[0197] QVQLQESGGGLVQAGGSLRLSCTASGTIFSINHMDWFRQAPGKQRELVA LITSDGFPTYADSAKGRFTISRDNTKKTVSLQMNSLKPEDTAVYYCHVSSGVY NYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ IDNO:287);

[0198] or

[0199] 2LIG16

[0200] QVQLQESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWV SAIRWNGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCA-QGYY-CSGYGCPRGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:288);

[0201] or

[0202] 2LIG22

[0203] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAINWFRQAPGKEREEVS CISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATSGWR LCRPTDEYDYSYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:289);

[0204] or

[0205] 2LIG27

[0206] QVQLQESGGGVVQAGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVA LITSDGTPTYADSAKGRFTISRDNTKKTVSLQMNSLKPEDTAVYYCHVSSGVY NYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ IDNO:290);

[0207] or

[0208] 2LIG29<000​QVQLQESGGGLVQTGGSLRLSCAASGFTFSSYGMSWVRQTPGKGPEWVSAIDSGGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCA-QGYY-CSGYGCSDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:291);

[0210] or

[0211] 2LIG30

[0212] QVQLQESGGGLVQPGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDNAKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:292);

[0213] or

[0214] 2LIG34

[0215] QVQLQESGGGSVQAGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPTYADSAKGRFTISRDNTKKTVSLQMNSLKPEDTAVYYCHVSSGVYNYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:293);

[0216] or

[0217] 2LIG35

[0218] QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQTPGKGPEWVSAIDSGGGSTSYADSVKGRFTTSRDNAKNTLYLQMNSLKPEDTAVYYCA-QGYY-CSGYGCSDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:294);

[0219] or

[0220] 2LIG48

[0221] QVQLQESGGGLVQPGGSLRLSCAASGFTFNDYAMSWVRQAPGKGLEWVSGIRSNGGYTNYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCA-QGYY-CSGYGCYPGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:295);

[0222] or

[0223] 2LIG65

[0224] QVQLQESGGGLVQAGGSLRLSCAASGFNLDPYAIAWFRQAPGKEREEVS CISSSDVGTYYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATDGY YYCSDYPHPLYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:296);

[0225] or

[0226] 2LIG85

[0227] QVQLQESGGGLVQPGGSLRLSCAASGFTFTAYAMSWFRQAPGKEREEVS CINSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYHCATDGW RDCTWSNEYAYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:297);

[0228] or

[0229] 2LIG86

[0230] QVQLQESGGGLVQPGGSLRLSCAASGFTFDYYAIGWFRQAPGKEREEVS CISGSDSSTYYADSVKGRFTIVRDNAQNTVYLQMNSLKPEDTAIYYCAVTGW RTCRGLNEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:298);

[0231] or

[0232] 2LIG89

[0233] QVQLQESGGGLVQPGGSLRLSCAASGFNLDPYAIAWFRQAPGKEREEVS CISSSDVGTYYADSVKGRFTISRDNTKKTVYLQMNSLKPEDTAVYYCATDGY YYCSDYPHPLYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:299);

[0234] or

[0235] 2LIG97

[0236] QVQLQESGGGLVQAGESLRLSCTASGTIFSINRMDWFRQAPGKQRELVAL ITSDGTPTYADSAKGRFTISRDNTKKTVSLQMNSLKPEDTAVYYCHVSSGVYN YWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ IDNO:300);

[0237] or

[0238] 2LIG99

[0239] QVQLQESGGGLVQAGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVAL ITSDGTPAYADSAKGRFTISRDNTKKTVSLQMNSLKPEDTAVYYCHVSSGVYN YWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ IDNO:301);

[0240] or

[0241] 2LIG109

[0242] QVQLQESGGGLVQSGGSLRLSCKTSGFTFSSYGMSWVRQTPGKGPEWVS AIDSGGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAQGYY-CSGYGCSDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:302);

[0243] or

[0244] 2LIG127

[0245] QVQLQESGGGLVQPGGSLRLSCAASGFNLDPYAIGWFRQAPGKEREEVS CISSGDGSKYYADSVKGRFTMSRDNAKKTVYLQMNSLKPEDTAVYYCATDG YYYCSDYPHPLYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:303);

[0246] or

[0247] 2LIG139

[0248] QVQLQESGGGLVQPGGSLRLSCAVSGFNLDPYAIAWFRQAPGKEREEVSC ISSSDVGTYYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATDGYY YCSDYPHPLYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:304);

[0249] or

[0250] 2LIG176

[0251] QVQLQESGGGLVQAGGSLRLSCAASESIFSIEAMGWYRQAPGKQRELVA AIFGGGFTNYADSVKGRFTISRDNANRTVYLQMNSLKPEDTAVYYCNADLVS GSSRLYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:305);[[ID=第十九]] [[ID=第二十]]

[0252] or

[0253] 2LIG189

[0254] QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELV GIITSGGITDYADAVKGRFTISRDNAKNMMYLQMNSLKPEDTAVYYCNMRDR TIWWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ IDNO:306);

[0255] or

[0256] 3LIG3[[ID=第二十四]] [[ID=第二十五]]

[0257] It should be noted that in the above translation, some of the line item numbers seem to be incorrect in the original text (such as "第十九", "第二十", "第二十四", "第二十五"), which may be due to incorrect numbering in the original. But I have translated it exactly according to the content you provided.QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREEVSCISSSDGSTYYADSVKGRFTISRDNAKNTVNLQMNSLKPEDTAVYYCATDGWSSCRHGINEYLYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:307);

[0258] or

[0259] 3LIG7

[0260] QVQLQESGGGLVQAGGSLRLSCAASGFTFSSYGMSWVRQTPGKGPEWVSAIDSGGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAQGYY - CSGYGCSDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:308);

[0261] or

[0262] 3LIG8

[0263] QVQLQESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPTYADSAKGRFTISRDNTKKTVSLQMNSLKPEDTAVYYCHVSSGVYNYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:309);

[0264] or

[0265] 3LIG9

[0266] QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQTPGKGPEWVSAIDSGGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAQGYYCSGYGCSDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:310);

[0267] or

[0268] 3LIG18

[0269] QVQLQESGGGLVQPGGSLRLSCAASGFNLDPYAIAWFRQAPGKEREEVSCISSSDVGTYYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATDGYYYCSDYPHPLYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:311);

[0270] or

[0271] 3LIG20

[0272] QVQLQESGGGLVXAGGSLRLSCAASGRTFSISAMGWFRQAPGKEREFVA AITWSGGSTSYTDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAIYYCAAMGR TNYGVIYDPNMYNYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:312);

[0273] or

[0274] 3LIG{28}

[0275] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAINWFRQAPGKEREEVS CISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATSGWR LCRPTDEYDYLYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:313);

[0276] or

[0277] 3LIG{29}

[0278] QVQLQESGGGLVQAGGSMRLSCAASGFTFSSYGMSWVRQTPGKGPEWV SAIDSGGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAQGY YCSGYGCSDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO:314);

[0279] or

[0280] 3LIG{30}

[0281] QVQLQESGGGTVQAGGSLRLSCAASGFTFNDYAMSWVRQAPGKGLEW VSGIRSNGGYTNYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAQG YYCSGYGCYPGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQID NO:315);

[0282] or

[0283] 3LIG33

[0284] QVQLQESGGGLVQPGTSLRLSCAASGFTLDYYAIGWFRQAPGKEREEVS CISSSDGSTYYADSVKGRFTISRDNAKNTVNLQMNSLKPEDTAVYYCATDGW SSCRHGINEYLYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 316).

[0285] In various exemplary embodiments, the PD-L1-binding agent comprises an amino acid sequence selected from any one of the above sequences without a terminal histidine tag sequence (ie, HHHHHH; SEQ ID NO: 84).

[0286] In some embodiments, the PD-L1 binding agent comprises an amino acid sequence selected from SEQ ID Nos: 286-316 (provided above) without the HA tag (ie, YPYDVPDYGS; SEQ ID NO: 85).

[0287] In some embodiments, the PD-L1 binding agent comprises an amino acid sequence selected from SEQ ID Nos: 286-316 (provided above) without an AAA linker.

[0288] In some embodiments, the PD-L1-binding agent comprises an amino acid sequence selected from SEQ ID Nos: 286-316 (provided above) without the AAA linker, HA tag, and terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 86).

[0289] In various embodiments, the present invention contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs of the PD-L1 binding agents of the invention as described herein (collectively referred to herein as "analogs"). In various embodiments, the amino acid sequence of the PD-L1 binding agent further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0290] In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety comprising a sequence having at least 60% identity to any of the sequences disclosed herein. For example, the PD-1 or PD-L1 binding agent can comprise a targeting moiety comprising a sequence having 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 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 or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100% identical to any of the sequences disclosed herein. or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% sequence identity).

[0291] In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety comprising an amino acid sequence having one or more amino acid mutations relative to any of the sequences disclosed herein. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety comprising an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations relative to any 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.

[0292] In some embodiments, the amino acid mutation is an amino acid substitution, and can include conservative and / or non-conservative substitutions.

[0293] "Conservative substitutions" can be made, for example, based on similarities in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0294] As used herein, a "conservative substitution" is defined as the exchange of one amino acid listed in the six standard amino acid groups shown above for another amino acid within the same group. For example, exchanging Asp for Glu results in a negative charge being retained in the polypeptide thus modified. In addition, glycine and proline can be substituted for each other based on their ability to disrupt alpha helices.

[0295] As used herein, a "non-conservative substitution" is defined as the exchange of one amino acid listed in different of the six standard amino acid groups (1) to (6) shown above for another amino acid.

[0296] In various embodiments, the substitutions can also include non-classical amino acids. Exemplary non-classical amino acids generally 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-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs.

[0297] In various embodiments, the amino acid mutation can be in a CDR (e.g., CDR1, CDR2, or CDR3) of the targeting moiety. In another embodiment, the amino acid change can be in a framework region (FR) (e.g., FR1, FR2, FR3, or FR4) of the targeting moiety.

[0298] Modification of the amino acid sequence can be achieved using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0299] In various embodiments, the mutation does not substantially reduce the ability of the PD-1 or PD-L1 binding agents of the invention to specifically bind to PD-1 or PD-L1. In various embodiments, the mutation does not substantially reduce the ability of the PD-1 or PD-L1 binding agents of the invention to specifically bind to PD-1 or PD-L1 and does not functionally modulate (e.g., partially or fully neutralize) PD-1 or PD-L1.

[0300] In various embodiments, the equilibrium dissociation constant (K D) is used to describe the binding affinity of the PD-1 or PD-L1 binding agents of the present invention to the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or monomer and / or dimeric form and / or any other naturally occurring or synthetic analogs, variants or mutants (including monomers and / or dimeric forms) of human PD-1 or PD-L1. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds to the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other naturally occurring or synthetic analogs, variants or mutants (including monomers and / or dimeric forms) of human PD-1 or PD-L1, wherein K D Less than about 1 uM, 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.

[0301] In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds to the target antigen but does not functionally modulate (e.g., partially or completely neutralize) the target antigen, i.e., PD-1 or PD-L1. For example, in various embodiments, the targeting moiety of the PD-1 or PD-L1 binding agent merely targets the antigen but does not substantially functionally modulate (e.g., partially or completely inhibit, reduce, or neutralize) the biological effect of the antigen. In various embodiments, the targeting moiety of the PD-1 or PD-L1 binding agent binds to an epitope that is physically separated from an antigenic site that is important for the biological activity of the antigen (e.g., the active site of the antigen).

[0302] In various embodiments, these binding agents bind to and functionally modulate (e.g., partially or fully neutralize) PD-1 or PD-L1.

[0303] Therapeutic agents comprising the PD-1 or PD-L1 binding agents of the present invention

[0304] Chimeras and fusions with signaling agents

[0305] In various embodiments, the PD-1 or PD-L1 binding agents of the invention are part of a chimera or fusion with one or more signaling agents. Thus, the invention provides chimeric or fusion proteins comprising, for example, a targeting moiety to PD-1 or PD-L1 and one or more signaling agents.

[0306] In various embodiments, the signal transducer is modified so as to have reduced affinity or activity for one or more of its receptors, thereby allowing for attenuation of activity (including agonism or antagonism) and / or prevention of non-specific signaling or undesirable sequestration of chimeric or fusion proteins. In various embodiments, the signal transducer is antagonistic in its wild-type form and carries one or more mutations that attenuate its antagonistic activity. In various embodiments, the signal transducer is antagonistic due to one or more mutations, e.g., an agonist signal transducer is converted to an antagonist signal transducer, and optionally, such a converted signal transducer also carries one or more mutations that attenuate its antagonistic activity (e.g., as described in WO 2015 / 007520, the entire contents of which are hereby incorporated by reference).

[0307] Thus, in various embodiments, the signaling agent is a modified (e.g., mutant) form of a signaling agent having one or more mutations (e.g., multiple mutations). In various embodiments, the mutation allows the modified signaling agent to have one or more weakened activities relative to the unmutated, i.e., wild-type form of the signaling agent, such as one or more of reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity (e.g., comparing the wild-type form of the same signaling agent versus the modified (e.g., mutant) form). In some embodiments, mutations that weaken or reduce binding or affinity include those that substantially reduce or eliminate binding or activity. In some embodiments, mutations that weaken or reduce binding or affinity are different from those that substantially reduce or eliminate binding or activity. As a result, in various embodiments, the mutation allows the signaling agent to have improved safety relative to the unmutated, i.e., wild-type signaling agent, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects (e.g., comparing the wild-type form of the same signaling agent versus the modified (e.g., mutant) form).

[0308] As described herein, the agent can have improved safety due to one or more modifications (e.g., mutations). In various embodiments, improved safety means that the chimeric protein of the invention 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 off-target effects; and / or an increased therapeutic window.

[0309] In various embodiments, the signal transducer is modified to have one or more mutations that reduce its binding affinity or activity to one or more of its receptors. In some embodiments, the signal transducer is modified to have one or more mutations that substantially reduce or eliminate its binding affinity or activity to the receptor. In some embodiments, the activity provided by the wild-type signal transducer is an agonist effect at the receptor (e.g., activating a cellular effect at the treatment site). For example, the wild-type signal transducer can activate its receptor. In such embodiments, the mutation reduces or eliminates the activation activity of the modified signal transducer at the receptor. For example, the mutation can cause the modified signal transducer to deliver a weakened activation signal to the target cell, or can eliminate the activation signal. In some embodiments, the activity provided by the wild-type signal transducer is an antagonist effect at the receptor (e.g., blocking or suppressing a cellular effect at the treatment site). For example, the wild-type signal transducer can antagonize or inhibit the receptor. In these embodiments, the mutation reduces or eliminates the antagonist activity of the modified signal transducer at the receptor. For example, the mutation can cause the modified signaling agent to deliver a weakened inhibitory signal to the target cell, or can eliminate the inhibitory signal. In various embodiments, the signaling agent is antagonistic due to one or more mutations, such as converting an agonist signaling agent into an antagonist signaling agent (e.g., as described in WO 2015 / 007520, the entire contents of which are hereby incorporated by reference), and optionally, such converted signaling agent also carries one or more mutations that reduce its binding affinity or activity to one or more of its receptors, or substantially reduce or eliminate its binding affinity or activity to one or more of its receptors.

[0310] In some embodiments, attachment of one or more targeting moieties as described herein (e.g., targeting moieties for PD-1 or PD-L1) can restore the reduced affinity or activity at the receptor. In other embodiments, the activity of one or more of the targeting moieties does not substantially restore the reduced affinity or activity at the receptor.

[0311] In various embodiments, the chimeric proteins of the invention have reduced off-target effects because their signaling agents have mutations that weaken or eliminate binding affinity or activity at the receptor. In various embodiments, such reductions in side effects are observed relative to, for example, wild-type signaling agents. In various embodiments, the signaling agents are active on target cells because the one or more targeting moieties compensate for the absence / deficiency of binding (e.g., but not limited to, and / or avidity) required for substantial activation. In various embodiments, the modified signaling agents are substantially inactive on pathways to the therapeutically active site, and their effects are substantially directed to the specifically targeted cell type, thereby greatly reducing undesirable side effects.

[0312] In some embodiments, the signal transducer can include one or more mutations that weaken or reduce the binding or affinity to a receptor (i.e., a therapeutic receptor) and one or more mutations that substantially reduce or eliminate the binding or activity at a second receptor. In such embodiments, these mutations can be in the same or different positions (i.e., the same mutation or multiple mutations). In some embodiments, the one or more mutations that reduce the binding and / or activity at one receptor are different from the one or more mutations that substantially reduce or eliminate the binding and / or activity at another receptor. In some embodiments, the one or more mutations that reduce the binding and / or activity at one receptor are the same as the one or more mutations that substantially reduce or eliminate the binding and / or activity at another receptor. In some embodiments, the chimeric protein of the present invention has a modified signal transducer that has both a mutation that weakens the binding and / or activity at the therapeutic receptor and thus allows a more controlled on-target therapeutic effect (e.g., relative to a wild-type signal transducer) and a mutation that substantially reduces or eliminates the binding and / or activity at another receptor and thus reduces side effects (e.g., relative to a wild-type signal transducer).

[0313] In some embodiments, the substantial reduction or elimination of binding or activity cannot be substantially restored using a targeting moiety (e.g., a targeting moiety for PD-1 or PD-L1 or any other targeting moiety described herein). In some embodiments, the substantial reduction or elimination of binding or activity can be restored using a targeting moiety. In various embodiments, substantially reducing or eliminating binding or activity at a second receptor can also prevent adverse effects mediated by another receptor. Alternatively or additionally, substantially reducing or eliminating binding or activity at another receptor improves the therapeutic effect because the reduced or eliminated sequestration of the therapeutic chimeric protein is away from the site of therapeutic action. For example, in some embodiments, this avoids the need for a high dose of the chimeric protein of the present invention that can compensate for the loss at another receptor. This ability to reduce the dose also provides a lower potential for side effects.

[0314] In various embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced, substantially reduced, or eliminated affinity for one or more of its receptors, e.g., binding (e.g., K D ) and / or activation (e.g., when the modified signaling agent is an agonist of its receptor, which can be measured as, for example, K A and / or EC 50 ) and / or inhibition (e.g., when the modified signaling agent is an antagonist of its receptor, which can be measured as, for example, K I and / or IC 50 ). In various embodiments, the reduced affinity at the immunomodulator receptor allows for reduced activity (including agonism or antagonism). In such embodiments, the modified signaling agent has an affinity for the receptor of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% relative 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.

[0315] In embodiments where the modified signaling agent has a mutation that reduces binding at one receptor and substantially reduces or eliminates binding at a second receptor, the degree of reduction or decrease in the binding affinity of the modified signaling agent for one receptor is less than the substantial reduction or elimination of affinity for another receptor. In some embodiments, the reduction or decrease in the binding affinity of the modified signaling agent for one receptor is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% lower than the substantial reduction or elimination of affinity for another receptor. In various embodiments, substantial reduction or elimination refers to a greater reduction in binding affinity and / or activity than reduction or decrease.

[0316] 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.

[0317] In some embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have a reduced affinity for its receptor, which is lower than the binding affinity of the one or more targeting moieties to its one or more receptors. In some embodiments, this binding affinity difference exists between the signaling agent / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this binding affinity difference allows the signaling agent (e.g., a mutated signaling agent) to have a localized on-target effect and minimizes the off-target effects that form the basis of the side effects observed in the case of wild-type signaling agents. In some embodiments, this binding affinity is at least about 2 times lower, or at least about 5 times, or at least about 10 times, or at least about 15 times, or at least about 25 times lower, or at least about 50 times, or at least about 100 times, or at least about 150 times lower.

[0318] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be assessed by Scatchard plot analysis and computer fitting of the binding data (e.g., Scatchard, 1949) or by reflectance interferometry spectroscopy under flow-through conditions as described in Brecht et al. (1993), all of which are hereby incorporated by reference in their entirety.

[0319] In various embodiments, the signaling agent is an immunomodulator, such as one or more of an interleukin, an interferon, and a tumor necrosis factor.

[0320] In some embodiments, the signaling agent is an interleukin or a modified interleukin, including, for example, IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-14; IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33; IL-35; IL-36 or a fragment, variant, analog or family member thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes and macrophages. Known functions include stimulation of proliferation of immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis of neutrophils and T lymphocytes, and / or inhibition of interferon. Interleukin activity can be determined using assays known in the art: Matthews et al., Lymphokines and Interferens: A Practical Approach, Clemens et al., eds., IRL Press, Washington, DC 1987, pp. 221-225; and Orencole and Dinarello (1989) Cytokine 1, 14-20.

[0321] In some embodiments, the signal transducer is an interferon or a modified form of an interferon such as type I interferon, type II interferon, and type III interferon. Illustrative interferons include, for example, interferon-α-1, interferon-α-2, interferon-α-4, interferon-α-5, interferon-α-6, interferon-α-7, interferon-α-8, interferon-α-10, interferon-α-13, interferon-α-14, interferon-α-16, interferon-α-17, and interferon-α-21, interferon-β, and interferon-γ, interferon-κ, interferon-ε, interferon-τ, and interferon-ω.

[0322] In some embodiments, the signaling agent is tumor necrosis factor (TNF) or a protein in the tumor necrosis factor (TNF) or TNF family, including but not limited to TNF-α, TNF-β, LT-β, ​​CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and modified forms of TRAIL.

[0323] The amino acid sequences of the wild-type signaling agents described herein are well known in the art. Thus, in various embodiments, the modified signaling agents comprise at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 101%, or at least about 102%, or at least about 103%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 110%, or at least about 111%, or at least about 112%, or at least about 113%, or at least about 114%, or at least about 115%, or at least about 116%, or at least about 117%, or at least about 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 more sequence identity). , 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).

[0324] In various embodiments, the modified signaling agent comprises an amino acid sequence that is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 101%, or at least about 102%, or at least about 103%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 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 about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or at least about 94%, or at least about 95%, or about or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0325] In various embodiments, the modified signaling agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions and can include conservative and / or non-conservative substitutions as described elsewhere herein. In various embodiments, the substitutions can also include non-classical amino acids as described elsewhere herein.

[0326] As described herein, the modified signaling agents carry mutations that affect affinity and / or activity at one or more receptors. In various embodiments, there is a reduced affinity and / or activity for a therapeutic receptor, such as a receptor through which the desired therapeutic effect (excitation or antagonism) is mediated. In various embodiments, the modified signaling agents carry mutations that substantially reduce or eliminate affinity and / or activity at a receptor, such as a receptor through which the desired therapeutic effect (e.g., due to the heterogeneity of the combination) is not mediated. Receptors of any modified signaling agents as described herein, such as one of cytokines, growth factors, and hormones are known in the art.

[0327] Illustrative mutations that provide reduced affinity and / or activity (e.g., agonism) at the receptor are found in WO 2013 / 107791 and 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 hereby incorporated by reference. Illustrative mutations that provide reduced affinity and / or activity (e.g., antagonism) at the receptor are found in WO 2015 / 007520, the entire contents of which are hereby incorporated by reference.

[0328] In some embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and / or activity for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a receptor in the tumor necrosis factor receptor (TNFR) superfamily, a TGF-β receptor, a receptor in the immunoglobulin (Ig) superfamily, and / or a receptor in the tyrosine kinase superfamily.

[0329] In various embodiments, the receptor of the signal transducer is a type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to, receptors for IL2 (β subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and receptors for thrombopoietin (TPO), prolactin, and growth hormone. Illustrative type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptor.

[0330] In various embodiments, the receptor of the signal transducer is a type II cytokine receptor. Type II cytokine receptor is a multimeric receptor consisting of heterologous subunits, and is a receptor primarily used for interferon. This receptor family includes but is not limited to receptors for interferon-α, interferon-β and interferon-γ, IL10, IL22 and tissue factor. Illustrative type II cytokine receptors include but are not limited to IFN-α receptors (e.g., IFNAR1 and IFNAR2), IFN-β receptors, IFN-γ receptors (e.g., IFNGR1 and IFNGR2) and type II IL receptors.

[0331] In various embodiments, the receptor of the signal transducer is a G protein coupled receptor. Chemokine receptors are G protein coupled receptors with seven transmembrane structures and coupled to G proteins to carry out signal transduction. Chemokine receptors include but are not limited to CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors and XC chemokine receptors (XCR1). Exemplary 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.

[0332] In various embodiments, the receptor for the signaling agent is a TNFR family member. Tumor necrosis factor receptor (TNFR) family members share a cysteine-rich domain (CRD) formed by three disulfide bonds surrounding a CXXCXXC core motif, forming an elongated molecule. Exemplary tumor necrosis factor receptor family members include: CD120a (TNFRSF1A), CD120b (TNFRSF1B), lymphotoxin beta receptor (LTBR, ​​TNFRSF3), CD134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSF10A (TNFRSF10A), TNFRSF10B (TNFRSF10B), TNFRSF10C (TNFRSF10C), TNFRSF10D (TNFRSF10D), RANK (TNFRSF11A), osteoprotegerin (TNFRSF11B), TNFRSF11C (TNFRSF11C), TNFRSF11D (TNFRSF11D), RANK (TNFRSF11A), osteoprotegerin (TNFRSF11B), TNFRSF11C (TNFRSF11C), TNFRSF11D (TNFRSF11D), RANK (TNFRSF11A), osteoprotegerin (TNFRSF11 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.

[0333] In various embodiments, the receptor of the signal transduction agent is a TGF-β receptor. The TGF-β receptor is a single-pass serine / threonine kinase receptor. TGF-β receptors include but are not limited to TGFBR1, TGFBR2 and TGFBR3.

[0334] In various embodiments, the receptor for the signaling agent is an Ig superfamily receptor. Receptors in the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Receptors in the Ig superfamily include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

[0335] In various embodiments, the receptor of the signal transducer is a tyrosine kinase superfamily receptor. Receptors in the tyrosine kinase superfamily are well known in the art. There are approximately 58 known receptor tyrosine kinases (RTKs), which are divided into 20 subfamilies. Receptors in the tyrosine kinase superfamily include, but are not limited to, FGF receptors and various isotypes thereof, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0336] In one embodiment, the modified signaling agent is interferon alpha. In such embodiments, the modified IFN-alpha agent has reduced affinity and / or activity for the IFN-alpha / beta receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFN-alpha agent has substantially reduced or eliminated affinity and / or activity for the IFN-alpha / beta receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2 chains.

[0337] Mutated forms of interferon alpha are known to those skilled in the art. In one illustrative embodiment, the modified signaling agent is an allelic form of IFN-alpha 2a having the amino acid sequence of SEQ ID NO: 317.

[0338] In an illustrative embodiment, the modified signaling agent is an allelic form of IFN-α2b having the amino acid sequence of SEQ ID NO: 318 (which differs from IFN-α2a at amino acid position 23).

[0339] In some embodiments, the IFN-α2 mutant (IFN-α2a or IFN-α2b) is mutated at one or more amino acids at positions 144-154, such as amino acid positions 148, 149, and / or 153. In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from L153A, R149A, and M148A. Such mutants are described, for example, in WO2013 / 107791 and Piehler et al., (2000) J. Biol. Chem, 275: 40425-33, the entire contents of all of which are hereby incorporated by reference.

[0340] In some embodiments, the IFN-α2 mutant has reduced affinity and / or activity for IFNAR1. In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A as described in WO2010 / 030671, the entire contents of which are hereby incorporated by reference.

[0341] In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A as described in WO2008 / 124086, the entire contents of which are hereby incorporated by reference.

[0342] In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from R120E and R120E / K121E as described in WO2015 / 007520 and WO2010 / 030671, the entire contents of which are hereby incorporated by reference. In such embodiments, the IFN-α2 mutant antagonizes wild-type IFN-α2 activity. In such embodiments, the mutant IFN-α2 has reduced affinity and / or activity for IFNAR1, while retaining affinity and / or activity for IFNR2.

[0343] In some embodiments, the human IFN-α2 mutant comprises (1) one or more mutations selected from R120E and R120E / K121E, without wishing to be bound by theory, which mutations can produce an antagonistic effect; and (2) one or more mutations selected from K133A, R144A, R149A and L153A, without wishing to be bound by theory, which mutations allow for attenuation of the effect at, for example, IFNAR2. In one embodiment, the human IFN-α2 mutant comprises R120E and L153A.

[0344] In some embodiments, the human IFN-α2 mutant comprises one or more mutations selected from L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A as disclosed in WO 2013 / 059885, the entire contents of which are hereby incorporated by reference. In some embodiments, the human IFN-α2 mutant comprises the mutations H57Y, E58N, Q61S and / or L30A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations H57Y, E58N, Q61S and / or R33A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations H57Y, E58N, Q61S and / or M148A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations H57Y, E58N, Q61S and / or L153A disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations N65A, L80A, Y85A and / or Y89A as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations N65A, L80A, Y85A, Y89A and / or D114A as disclosed in WO 2013 / 059885.

[0345] In one embodiment, the modified signaling agent is interferon beta. In such embodiments, the modified interferon beta agent has reduced affinity and / or activity for IFN-α / β receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified interferon beta agent has substantially reduced or eliminated affinity and / or activity for IFN-α / β receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2 chains.

[0346] In illustrative embodiments, the modified signaling agent is IFN-β. In various embodiments, the IFN-β encompasses functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of IFN-β. In various embodiments, the IFN-β encompasses IFN-β derived from any species. In one embodiment, the chimeric protein comprises a modified form of mouse IFN-β. In another embodiment, the chimeric protein comprises a modified form of human IFN-β. Human IFN-β is a polypeptide having a molecular weight of approximately 22 kDa comprising 166 amino acid residues. The amino acid sequence of human IFN-β is SEQ ID NO: 319.

[0347] In some embodiments, the human IFN-β is IFN-β-1a, which is a glycosylated form of human IFN-β. In some embodiments, the human IFN-β is IFN-β-1b, which is a non-glycosylated form of human IFN-β, having a Met-1 deletion and a Cys-17 to Ser mutation.

[0348] In various embodiments, the modified IFN-β has one or more mutations, and the one or more mutations reduce the binding or affinity of the modified IFN-β to the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFN-β has reduced affinity and / or activity at IFNAR1. In various embodiments, the modified IFN-β is human IFN-β and has one or more mutations at positions F67, R71, L88, Y92, I95, N96, K123 and R124. In some embodiments, the one or more mutations are selected from the substitutions of F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G and R124G. In one embodiment, the modified IFN-β includes F67G mutation. In one embodiment, the modified IFN-β includes K123G mutation. In one embodiment, the modified IFN-β comprises F67G and R71A mutations. In one embodiment, the modified IFN-β comprises L88G and Y92G mutations. In one embodiment, the modified IFN-β comprises Y92G, I95A and N96G mutations. In one embodiment, the modified IFN-β comprises K123G and R124G mutations. In one embodiment, the modified IFN-β comprises F67G, L88G and Y92G mutations. In one embodiment, the modified IFN-β comprises F67S, L88S and Y92S mutations.

[0349] In some embodiments, the modified IFN-β has one or more mutations, and the one or more mutations reduce the binding or affinity of the modified IFN-β to the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFN-β has reduced affinity and / or activity at IFNAR2. In various embodiments, the modified IFN-β is human IFN-β and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152 and Y155. In some embodiments, the one or more mutations are selected from the substitutions of W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G and Y155G. In one embodiment, the modified IFN-β includes a W22G mutation. In one embodiment, the modified IFN-β includes an L32A mutation. In one embodiment, the modified IFN-β comprises an L32G mutation. In one embodiment, the modified IFN-β comprises an R35A mutation. In one embodiment, the modified IFN-β comprises an R35G mutation. In one embodiment, the modified IFN-β comprises a V148G mutation. In one embodiment, the modified IFN-β comprises an R152A mutation. In one embodiment, the modified IFN-β comprises an R152G mutation. In one embodiment, the modified IFN-β comprises a Y155G mutation. In one embodiment, the modified IFN-β comprises a W22G and R27G mutation. In one embodiment, the modified IFN-β comprises an L32A and R35A mutation. In one embodiment, the modified IFN-β comprises an L151G and R152A mutation. In one embodiment, the modified IFN-β comprises a V148G and R152A mutation.

[0350] In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H, in combination with C17S or C17A.

[0351] In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H, in combination with any other IFN-β mutations described herein.

[0352] The crystal structure of human IFN-β is known and is described in Karpusas et al., (1998) PNAS, 94 (22): 11813-11818. Specifically, it has been shown that the structure of human IFN-β includes five α helices (i.e., A, B, C, D, and E) and four loops (i.e., AB, BC, CD, and DE rings) connecting these helices. In various embodiments, the modified IFN-β has one or more mutations in A, B, C, D, E helices and / or AB, BC, CD, and DE rings, and the one or more mutations reduce the binding affinity or activity of the modified IFN-β at treatment receptors such as IFNAR. Exemplary mutations are described in WO2000 / 023114 and US20150011732, the entire contents of which are hereby incorporated by reference. In an exemplary embodiment, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In an exemplary embodiment, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 28-30, 32, and 33. In an exemplary embodiment, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 36, 37, 39, and 42. In an exemplary embodiment, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In an exemplary embodiment, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 71-73. In an exemplary embodiment, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 92, 96, 99, and 100. In an exemplary embodiment, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 128, 130, 131, and 134. In an exemplary embodiment, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 149, 153, 156, and 159. In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at W22 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0353] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at R27 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0354] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at W22 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R27 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0355] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at L32 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0356] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at R35 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0357] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at L32, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at R35, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0358] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at F67 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0359] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at R71 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0360] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at F67, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R71, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0361] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0362] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at Y92 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0363] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at F67, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at L88, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0364] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0365] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at I95 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V); and a mutation at Y92 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0366] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at N96 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0367] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at Y92, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at I95, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), methionine (M), and valine (V); and a mutation at N96, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0368] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at K123 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0369] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at R124 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0370] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 188 and a mutation at K123 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R124 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0371] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at L151 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0372] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at R152 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0373] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at L151, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at R152, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0374] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at V148 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0375] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at V148, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R152, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0376] In some embodiments, the mutant IFN-β comprises SEQ ID NO: 319 and a mutation at Y155 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0377] In some embodiments, the present invention relates to a chimeric protein comprising: (a) a modified IFN-β having an amino acid sequence of SEQ ID NO: 319 and a mutation at position W22, wherein the mutation is an aliphatic hydrophobic residue; and (b) one or more targeting moieties comprising a recognition domain that specifically binds to a target antigen or receptor (e.g., PD-1 or PD-L1), the modified IFN-β and the one or more targeting moieties being optionally connected to one or more linkers. In various embodiments, the mutation at position W22 is an aliphatic hydrophobic residue selected from G, A, L, I, M, and V. In various embodiments, the mutation at position W22 is G.

[0378] Other exemplary IFN-β mutants are provided in PCT / EP2017 / 061544, the entire disclosure of which is incorporated herein by reference.

[0379] In one embodiment, the modified signaling agent is interferon gamma. In such embodiments, the modified interferon gamma agent has reduced affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon gamma agent has substantially reduced or eliminated affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., IFNGR1 and IFNGR2 chains.

[0380] IFN-γ is the only member of the type II interferon class. As part of the innate immune response, IFN-γ is mainly produced by natural killer (NK) and natural killer T (NKT) cells. CD4 Th1 and CD8 cytotoxic T lymphocytes (CTL) effector T cells, macrophages, dendritic cells and B cells also produce IFN-γ. Activated IFN-γ forms dimers, which act through heterodimeric receptors (i.e., IFN-γ receptors or IFN-γRs) composed of IFN-γ receptor 1 and IFN-γ receptor 2 subunits. IFN-γ receptor 1 is the main ligand binding subunit, while IFN-γ receptor 2 is required for signal transduction and also increases the affinity of IFN-γ receptor 1 for its ligand. The binding of IFN-γ dimers to receptors activates the JAK-STAT signaling pathway, thereby triggering various biological effects.

[0381] In various embodiments, the modified signaling agent comprises a modified form of IFN-γ as a signaling agent. In various embodiments, the IFN-γ encompasses functional derivatives, analogs, precursors, isoforms, splice variants or fragments of IFN-γ. In various embodiments, the IFN-γ encompasses IFN-γ derived from any species. In one embodiment, the modified signaling agent comprises a modified form of mouse IFN-γ. In another embodiment, the modified signaling agent comprises a modified form of human IFN-γ.

[0382] Human IFN-γ is a polypeptide comprising 166 amino acid residues. In one embodiment, the human IFN-γ has the amino acid sequence of SEQ ID NO: 320.

[0383] As used herein, human IFN-γ may also refer to mature human IFN-γ without the N-terminal signal peptide. In this embodiment, the mature human IFN-γ comprises 143 amino acids and has the amino acid sequence of SEQ ID NO: 321.

[0384] In some embodiments, the human IFN-γ is a glycosylated form of human IFN-γ. In some embodiments, the human IFN-γ is a non-glycosylated form of human IFN-γ.

[0385] The sequence of IFN-γ is known in the art. In various embodiments, the modified IFN-γ comprises an amino acid sequence that is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 101%, or at least about 102%, or at least about 103%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 110%, or at least about 111%, or at least about 112%, or at least about 113%, or at least about 114%, or at least about 115%, or at least about 116%, or at least about 117%, or at least about 1 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 or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0386] In some embodiments, the modified IFN-γ comprises a polypeptide that has at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 101%, or at least about 102%, or at least about 103%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 110%, or at least about 111%, or at least about 112%, or at least about 113%, or at least about 114%, or at least about 115%, or at least about 116%, or at least about 6%, 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%. At least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0387] In some embodiments, the modified IFN-γ comprises a polypeptide that has at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 101%, or at least about 102%, or at least about 103%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 110%, or at least about 111%, or at least about 112%, or at least about 113%, or at least about 114%, or at least about 115%, or at least about 116%, or at least about 6%, 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%. At least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0388] In various embodiments, the modified IFN-γ comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0389] In some embodiments, the amino acid mutation is an amino acid substitution, and can include conservative and / or non-conservative substitutions.

[0390] "Conservative substitutions" can be made, for example, based on similarities in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0391] As used herein, a "conservative substitution" is defined as the exchange of one amino acid listed in the six standard amino acid groups shown above for another amino acid within the same group. For example, exchanging Asp for Glu results in a negative charge being retained in the polypeptide thus modified. In addition, glycine and proline can be substituted for each other based on their ability to disrupt alpha helices.

[0392] As used herein, a "non-conservative substitution" is defined as the exchange of one amino acid listed in different of the six standard amino acid groups (1) to (6) shown above for another amino acid.

[0393] In various embodiments, the substitutions can also include non-classical amino acids (e.g., generally, selenocysteine, pyrrolysine, N-formylmethionine, beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoroamino acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl amino acids, and amino acid analogs).

[0394] In various embodiments, the IFN-γ is modified to have one or more mutations. In some embodiments, the mutation allows the modified IFN-γ to have one or more weakened activities relative to the unmutated (e.g., wild-type) form of IFN-γ, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity. For example, relative to the unmutated (e.g., wild-type) form of IFN-γ, the one or more weakened activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity can be at a therapeutic receptor such as an IFN-γ receptor. As a result, in various embodiments, the mutation allows the modified soluble agent to have reduced systemic toxicity, reduced side effects, and reduced off-target effects relative to the unmutated (e.g., wild-type) form of IFN-γ.

[0395] In various embodiments, the IFN-γ is modified to have a mutation that reduces its binding affinity and / or activity at a treatment receptor such as an IFN-γ receptor including IFN-γ receptor 1 and IFN-γ receptor 2 subunits. In some embodiments, the activity provided by the wild-type IFN-γ is an agonism at the treatment receptor (e.g., activating the cellular effect of the treatment site). For example, the wild-type IFN-γ can activate a treatment receptor. In such embodiments, the mutation causes the activation activity of the modified IFN-γ at the treatment receptor to be reduced.

[0396] In some embodiments, being connected to a targeting moiety can restore the affinity and / or activity that was reduced at the treatment receptor (e.g., IFN-γ receptor). In other embodiments, being connected to a targeting moiety can substantially not restore the affinity and / or activity that was reduced at the treatment receptor. In various embodiments, the therapeutic chimeric protein of the present invention reduces off-target effects because the IFN-γ has a mutation that weakens the binding affinity and / or activity at the treatment receptor. In various embodiments, it is observed that side effects are reduced relative to, for example, wild-type IFN-γ. In various embodiments, the modified IFN-γ is substantially inactive to the pathway of the therapeutic active site, and its effect is substantially directed to the cell type specifically targeted, thereby greatly reducing undesirable side effects.

[0397] In various embodiments, the modified IFN-γ has one or more mutations that cause the IFN-γ to have attenuated or reduced affinity and / or activity, e.g., binding (e.g., KD) and / or activation (measurable as, e.g., KA and / or EC50), for one or more therapeutic receptors (e.g., IFN-γ receptor). In various embodiments, reduced affinity and / or activity at the therapeutic receptor allows for attenuation of activity and / or signaling from the therapeutic receptor.

[0398] In various embodiments, the modified IFN-γ has one or more mutations that reduce the binding or affinity and / or biological activity of the modified IFN-γ to the IFN-γ receptor 1 subunit. In one embodiment, the modified IFN-γ has reduced affinity and / or activity at the IFN-γ receptor 1 subunit. In various embodiments, the modified IFN-γ is human IFN-γ, and the human IFN-γ has one or more mutations at amino acid residues involved in binding to the IFN-γ receptor 1 subunit. In some embodiments, the modified IFN-γ is human IFN-γ, and the human IFN-γ has one or more mutations at an amino acid residue located at the interface with the IFN-γ receptor 1 subunit. In various embodiments, the one or more mutations are at an amino acid 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: 321, which is wild-type human IFN-γ and lacks its 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: 321). In an embodiment, the one or more mutations are substitutions selected from V22A, A23G, D24G, H111A, H111D, I114A, Q115A, and A118G.

[0399] In one embodiment, the modified IFN-γ comprises mutations A23G and D24G. In another embodiment, the modified IFN-γ comprises mutations I114A and A118G. In another embodiment, the modified IFN-γ comprises mutations V5E, S20E, A23F, and G26Q.

[0400] In various embodiments, the modified IFN-γ has one or more of the following mutations: deletion of residue A23, deletion of residue D24, S20I substitution, A23V substitution, D21K substitution, and D24A substitution.

[0401] In some embodiments, the modified IFN-γ has one or more mutations that reduce the binding or affinity of the modified IFN-γ to the IFN-γ receptor 2 subunit and / or the biological activity.

[0402] In some embodiments, the modified IFN-γ has one or more mutations that reduce the binding or affinity of the modified IFN-γ to IFN-γ receptor 1 and IFN-γ receptor 2 subunits and / or the biological activity of the modified IFN-γ.

[0403] In some embodiments, the modified IFN-γ has one or more mutations that reduce its binding or affinity and / or biological activity for IFN-γ receptor 1 and one or more mutations that substantially reduce or eliminate its binding or affinity and / or biological activity for IFN-γ receptor 2. In some embodiments, a chimeric protein having such a modified IFN-γ can provide target-selective IFN-γ receptor 1 activity (e.g., can restore IFN-γ receptor 1 activity via targeting via a targeting moiety).

[0404] In some embodiments, the modified IFN-γ has one or more mutations that reduce its binding or affinity and / or biological activity for IFN-γ receptor 1 and one or more mutations that reduce its binding or affinity and / or biological activity for IFN-γ receptor 1. In some embodiments, a chimeric protein having such a modified IFN-γ can provide target-selective IFN-γ receptor 1 and / or IFN-γ receptor 1 activity (e.g., can restore IFN-γ receptor 1 and IFN-γ receptor 2 activity via targeting via a targeting moiety).

[0405] In various embodiments, the modified IFN-γ is truncated at the C-terminus. In some embodiments, the modified IFN-γ is mature IFN-γ, and the mature IFN-γ comprises the amino acid sequence SEQ ID NO with C-terminal deletion: 321. In such embodiments, the mature IFN-γ can comprise 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 C-terminal truncation. In one embodiment, the modified IFN-γ is mature IFN-γ, and the mature IFN-γ comprises the amino acid sequence SEQ ID NO with C-terminal deletion of 5 amino acids: 321. In one embodiment, the modified IFN-γ is a mature IFN-γ comprising the amino acid sequence of SEQ ID NO: 321 with a C-terminal deletion of 7 amino acids. In one embodiment, the modified IFN-γ is a mature IFN-γ comprising the amino acid sequence of SEQ ID NO: 321 with a C-terminal deletion of 14 amino acids. In one embodiment, the modified IFN-γ is a mature IFN-γ comprising the amino acid sequence of SEQ ID NO: 321 with a C-terminal deletion of 15 amino acids. In one embodiment, the modified IFN-γ is a mature IFN-γ comprising the amino acid sequence of SEQ ID NO: 321 with a C-terminal deletion of 16 amino acids. Additional modified IFN-γ with C-terminal truncations that can be used 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 hereby incorporated by reference.

[0406] In various embodiments, the modified IFN-γ is a single-chain IFN-γ as described in, for example, Randal et al. (2001) Structure 9:155-163 and Randal et al. (1998) Protein Sci.7:1057-1060, the entire contents of which are hereby incorporated by reference. In some embodiments, the single-chain IFN-γ comprises a first IFN-γ chain, which is connected to the N-terminus of the second IFN-γ chain at its C-terminus. In various embodiments, the first IFN-γ chain and the second IFN-γ chain are connected by a linker as described elsewhere herein.

[0407] In some embodiments, the first IFN-γ chain comprises 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 C-terminal truncation. In one embodiment, the first IFN-γ chain comprises a C-terminal truncation of about 24 amino acid residues. In some embodiments, the second IFN-γ chain comprises an N-terminal truncation of at least about 1, about 2, about 3, about 4 or about 5 amino acid residues. In one embodiment, the second IFN-γ chain comprises an N-terminal truncation of about 3 amino acid residues. In some embodiments, the second IFN-γ 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, as described elsewhere herein, the first IFN-γ chain and / or the second IFN-γ chain comprise one or more amino acid mutations at Q1, V5, E9, K12, H19, S20, V22, A23, D24, N25, G26, T27, L30, K108, H111, E112, I114, Q115, A118, E119, and K125. In various embodiments, the first IFN-γ chain and / or the second IFN-γ chain comprise one or more substitutions selected from the group consisting of V5E, S20E, V22A, A23G, A23F, D24G, G26Q, H111A, H111D, I114A, Q115A, and A118G. In various embodiments, the first IFN-γ chain and / or the second IFN-γ chain comprise one or more substitutions selected from the group consisting of V22A, A23G, D24G, H111A, H111D, I114A, Q115A, and A118G. In various embodiments, the first IFN-γ chain and / or the second IFN-γ chain comprise A23G and D24G substitutions. In various embodiments, the first IFN-γ chain and / or the second IFN-γ chain comprise I114A and A118G substitutions. In another embodiment, the mutations are V5E, S20E, A23F, and G26Q.

[0408] In various embodiments, the first IFN-γ chain and / or the second IFN-γ chain comprises one or more substitutions as disclosed herein, and the first IFN-γ chain and / or the second IFN-γ chain comprises a C-terminal truncation as disclosed herein.

[0409] In various embodiments, the first IFN-γ chain and / or the second IFN-γ chain comprises one or more substitutions as disclosed herein and a C-terminal truncation as disclosed herein.

[0410] The crystal structure of human IFN-γ is known and is described in, for example, Ealick et al., (1991) Science, 252:698-702. Specifically, it has been shown that the structure of human IFN-γ includes a core of six α helices and an unfolded sequence extending in the C-terminal region. In various embodiments, the modified IFN-γ has one or more mutations in the one or more helices, and the one or more mutations reduce the binding affinity and / or biological activity of the modified IFN-γ at a therapeutic receptor (e.g., an IFN-γ receptor).

[0411] In various embodiments, the modified IFN-γ has about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% of the affinity and / or biological activity of wild-type IFN-γ for a therapeutic receptor (e.g., IFN-γ receptor or any of its IFN-γ receptor 1 and IFN-γ receptor 2 subunits) relative to wild-type IFN-γ. In some embodiments, the binding affinity and / or biological activity 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 IFN-γ.

[0412] In various embodiments, the modified IFN-γ comprises one or more mutations that reduce the intrinsic activity of the IFN-γ, e.g., to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1%, relative to the wild-type IFN-γ.

[0413] In some embodiments, the modified IFN-γ comprises one or more mutations that cause the modified IFN-γ to have a reduced affinity and / or biological activity for the receptor. In some embodiments, the binding affinity and / or biological activity of the modified IFN-γ to the receptor is lower than the binding affinity and / or biological activity of the targeting portion to its receptor. In some embodiments, this binding affinity and / or biological activity difference exists between the modified IFN-γ / receptor and the targeting portion / receptor on the same cell. In some embodiments, this binding affinity and / or biological activity difference allows the modified IFN-γ to have a localized on-target effect and minimizes the off-target effect that constitutes the basis of the side effects observed in the case of wild-type IFN-γ. In some embodiments, the binding affinity and / or biological activity is at least about 2 times lower, or at least about 5 times, or at least about 10 times, or at least about 15 times, or at least about 25 times lower, or at least about 50 times, or at least about 100 times, or at least about 150 times lower.

[0414] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be assessed by Scatchard plot analysis and computer fitting of the binding data (e.g., Scatchard, 1949) or by reflectance interferometry spectroscopy under flow-through conditions as described in Brecht et al. (1993), all of which are hereby incorporated by reference in their entirety.

[0415] In some embodiments, the modified signal transduction agent is a consensus interferon. By scanning the sequences of several human non-allelic IFN-α subtypes and assigning the most commonly observed amino acids at each corresponding position, a consensus interferon can be generated. The consensus interferon has 20 amino acids different from IFN-α2b in 166 amino acids (88% homology) and is shown to have identity at more than 30% of amino acid positions compared to IFN-β. In various embodiments, the consensus interferon comprises the amino acid sequence SEQ ID NO: 322.

[0416] In some embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO:323, which differs from the amino acid sequence of SEQ ID NO:322 by one amino acid, ie, SEQ ID NO:323 lacks the initial methionine residue of SEQ ID NO:322.

[0417] In various embodiments, the consensus interferon includes a modified form of the consensus interferon, i.e., a consensus interferon variant, as a signaling agent. In various embodiments, the consensus interferon encompasses a functional derivative, analog, precursor, isoform, splice variant, or fragment of the consensus interferon.

[0418] In one embodiment, the consensus interferon variant is selected from the consensus interferon variants disclosed in U.S. Patent Nos. 4,695,623, 4,897,471, 5,541,293 and 8,496,921, all of which are hereby incorporated by reference in their entirety. For example, the consensus interferon variant may comprise the amino acid sequence of IFN-CON2 or IFN-CON3 as disclosed in U.S. Patent Nos. 4,695,623, 4,897,471 and 5,541,293. In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON2, SEQ ID NO: 324.

[0419] In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON3, SEQ ID NO:325.

[0420] In one embodiment, the consensus interferon variant comprises the amino acid sequence of any variant disclosed in U.S. Patent No. 8,496,921. For example, the consensus variant may comprise the amino acid sequence of SEQ ID NO: 326.

[0421] In another embodiment, the consensus interferon variant may comprise the amino acid sequence of SEQ ID NO:327.

[0422] In some embodiments, the consensus interferon variant may be PEGylated, ie, comprise a PEG moiety. In one embodiment, the consensus interferon variant may comprise a PEG moiety attached to position S156C of SEQ ID NO: 327.

[0423] In some embodiments, the engineered interferon is a variant of human IFN-α2a, with an insertion of Asp at approximately position 41 of SEQ ID NO: 328 to produce SEQ ID NO: 329 (resulting in renumbering of the sequence relative to the IFN-α2a sequence) and the following mutations: Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser, Asp86Glu, Ile104Thr, Gly106Glu, Thr110Glu, Lys117Asn, Arg125Lys, and Lys136Thr. All embodiments describing the consensus interferon herein are equally applicable to the engineered interferon.

[0424] In various embodiments, the consensus interferon variant comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0425] In some embodiments, the amino acid mutation is an amino acid substitution, and can include conservative and / or non-conservative substitutions.

[0426] In various embodiments, the substitutions can also include non-classical amino acids (e.g., generally, selenocysteine, pyrrolysine, N-formylmethionine, beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoroamino acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl amino acids, and amino acid analogs).

[0427] In various embodiments, the consensus interferon is modified to have one or more mutations. In some embodiments, the mutation allows the consensus interferon variants to have one or more weakened activities relative to the consensus interferon (e.g., with an amino acid sequence SEQ ID NO: 325 or 326 consensus interferon) unmutated (e.g., wild-type) form, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity. For example, relative to the consensus interferon unmutated (e.g., wild-type) form, the one or more weakened activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity can be at a therapeutic receptor such as IFNAR. As a result, in various embodiments, the mutation allows the consensus interferon variants to have reduced systemic toxicity, reduced side effects, and reduced off-target effects relative to the consensus interferon unmutated (e.g., wild-type) form.

[0428] In various embodiments, the shared interferon is modified to have a mutation that reduces its binding affinity or activity for treating receptors such as IFNAR. In some embodiments, the activity provided by the shared interferon is the agonism at the treatment receptor (for example, activating the cellular effect at the treatment site). For example, the shared interferon can activate treatment receptors. In such embodiments, the mutation causes the activation activity of the shared interferon variants at the treatment receptor to be reduced.

[0429] In some embodiments, being connected to a targeting moiety can restore the affinity or activity that has been reduced at the treatment receptor. In other embodiments, being connected to a targeting moiety can not restore the affinity or activity that has been reduced at the treatment receptor substantially. In various embodiments, the Fc-based therapeutic chimeric protein of the present invention reduces off-target effects because the consensus interferon variants have mutations that weaken the binding affinity or activity at the treatment receptor. In various embodiments, it is observed that side effects exist relative to, for example, wild-type consensus interferon. In various embodiments, the consensus interferon variants are substantially inactive to the pathway of the treatment active site, and their effects are substantially directed to the cell type of specific targeting, thereby greatly reducing undesirable side effects.

[0430] In various embodiments, the consensus interferon variants have one or more mutations that cause the consensus interferon variants to have weakened or reduced affinity for one or more therapeutic receptors, e.g., binding (e.g., KD) and / or activation (measurable as, e.g., KA and / or EC50). In various embodiments, reduced affinity at the therapeutic receptor allows for attenuation of activity and / or signaling from the therapeutic receptor.

[0431] In various embodiments, the total interferon variants have one or more mutations, and the one or more mutations reduce the combination or avidity of the total interferon variants to the IFNAR1 subunit of IFNAR. In one embodiment, the total interferon variants have the affinity and / or activity of reduction at IFNAR1. In some embodiments, the total interferon variants have one or more mutations, and the one or more mutations reduce the combination or avidity of the total interferon variants to the IFNAR2 subunit of IFNAR. In some embodiments, the total interferon variants have one or more mutations, and the one or more mutations reduce the combination or avidity of the total interferon variants to IFNAR1 and IFNAR2 subunits.

[0432] 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 its binding or affinity for IFNAR2. In some embodiments, an Fc-based chimeric protein with such a consensus interferon variant can provide target-selective IFNAR1 activity (e.g., IFNAR1 activity can be restored via targeting by a targeting moiety (e.g., SIRPα)).

[0433] 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 its binding or affinity for IFNAR1. In some embodiments, an Fc-based chimeric protein with such a consensus interferon variant can provide target-selective IFNAR2 activity (e.g., IFNAR2 activity can be restored via targeting by a targeting moiety (e.g., SIRPα).

[0434] 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 substantially reduce its binding or affinity to IFNAR2. In some embodiments, an Fc-based chimeric protein with such a consensus interferon variant can provide target-selective IFNAR1 and / or IFNAR2 activity (e.g., IFNAR1 and / or IFNAR2 activity can be restored via targeting by a targeting moiety (e.g., SIRPα).

[0435] In some embodiments, relative to SEQ ID NO: 326, the consensus interferon is modified to have a mutation at one or more amino acids at positions 145-155 (such as amino acid positions 149, 150 and / or 154). In some embodiments, relative to SEQ ID NO: 326, the consensus interferon is modified to have a mutation at one or more amino acids at positions 145-155 (such as amino acid positions 149, 150 and / or 154), the substitution being optionally hydrophobic and selected from alanine, valine, leucine and isoleucine. In some embodiments, the consensus interferon mutant comprises one or more mutations selected from M149A, R150A and L154A and relative to SEQ ID NO: 323.

[0436] In one embodiment, the consensus interferon is modified to have a mutation at amino acid position 121 (ie, K121) relative to SEQ ID NO: 323. In one embodiment, the consensus interferon comprises a K121E mutation relative to SEQ ID NO: 323.

[0437] In some embodiments, the modified signal transducer is vascular endothelial growth factor (VEGF). VEGF is a powerful growth factor that plays a major role in physiological and pathological angiogenesis, regulates vascular permeability, and can serve as a growth factor on cells expressing VEGF receptors. Among other functions, additional functions also include stimulating cell migration in macrophage lineages and endothelial cells. There are several members in the VEGF growth factor family, as well as at least three receptors (VEGFR-1, VEGFR-2, and VEGFR-3). Members of the VEGF family can combine with and activate more than one VEGFR type. For example, VEGF-A combines VEGFR-1 and VEGFR-2, while VEGF-C combines VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 activate and regulate angiogenesis, while VEGFR-3 activation is associated with lymphangiogenesis. The main pro-angiogenic signal is generated by VEGFR-2 activation. It is reported that VEGFR-1 activation may be associated with the negative effects in angiogenesis. It has also been reported that VEGFR-1 signal transduction is important for the in vivo tumor progression of VEGFR-1 positive cells (contributing to forming a metastasis niche in bone) via bone marrow origin. Several therapeutic antibodies based on VEGF-A guidance / neutralization have been developed, and these therapies are mainly used in the treatment of various human tumors that rely on angiogenesis. But these therapies are not without side effects. Considering that these therapies work as general non-cell / tissue-specific VEGF / VEGFR interaction inhibitors, this may not be surprising. Therefore, it is desirable that VEGF (e.g., VEGF-A) / VEGFR-2 inhibition is limited to specific target cells (e.g., tumor vascular endothelial cells).

[0438] In some embodiments, the VEGF is VEGF-A, VEGF-B, VEFG-C, VEGF-D, or VEGF-E, and isoforms thereof, including various isoforms of VEGF-A, such as VEGF 121 , VEGF 121 b. VEGF 145 , VEGF 165 , VEGF 165 b. VEGF 189 and VEGF 206. In some embodiments, the modified signaling agent has a reduced affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In one embodiment, the modified signaling agent has a reduced affinity and / or activity for VEGFR-2 (KDR / Flk-1) and / or has a substantially reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1). Such embodiments can be used, for example, in wound healing methods or treatments of diseases associated with ischemia (without wishing to be bound by theory, these diseases are mediated by the effects of VEGFR-2 on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1 (Flt-1) associated with cancer and proinflammatory activity is avoided. In various embodiments, VEGFR-1 (Flt-1) acts as a decoy receptor, thereby significantly reducing or eliminating affinity at this receptor, thereby avoiding sequestration of the therapeutic agent. In one embodiment, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1), and / or has a substantially reduced or eliminated affinity and / or activity for VEGFR-2 (KDR / Flk-1). In some embodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for VEGFR-3. Alternatively, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for VEGFR-3.

[0439] Pro-angiogenesis therapy is also very important in various diseases (such as ischemic heart disease, hemorrhage, etc.), and includes VEGF-based therapy. The activation of VEGFR-2 has a pro-angiogenic effect (acting on endothelial cells). The activation of VEFGR-1 can stimulate inflammatory cells (including, for example, macrophages) to migrate and cause the high vascular permeability associated with inflammation. The activation of VEFGR-1 can also promote the formation of tumor microhabitats associated with bone marrow. Therefore, in this case, it will be desirable to select a therapeutic agent based on VEGF for VEGFR-2 activation. In addition, it is desirable that cell-specific targeting, for example, endothelial cells.

[0440] In some embodiments, the modified signaling agent has a reduced affinity and / or activity (e.g., antagonism) for VEGFR-2, and / or has a substantially reduced or eliminated affinity and / or activity for VEGFR-1. For example, when targeting tumor vascular endothelial cells via a targeting portion bound to a tumor endothelial cell marker (e.g., PSMA, etc.), such a construct will specifically inhibit VEGFR-2 activation on such marker-positive cells without activating VEGFR-1 on the way and on the target cells (if the activity is eliminated), thereby eliminating the induction of an inflammatory response. Compared to VEGF-A neutralization therapy, this will provide a more selective and safer anti-angiogenic therapy for many tumor types.

[0441] In some embodiments, the modified signal transducer has a reduced affinity and / or activity (e.g., agonism) to VEGFR-2, and / or has a substantially reduced or eliminated affinity and / or activity to VEGFR-1. In some embodiments, by targeting vascular endothelial cells, such constructs promote angiogenesis without inducing an inflammatory response associated with VEGFR-1. Therefore, such constructs will have a targeted pro-angiogenic effect and substantially reduce the risk of side effects caused by systemic activation of VEGFR-2 and VEGR-1.

[0442] In one illustrative embodiment, the modified signaling agent is VEGF 165 , the VEGF 165 It has the amino acid sequence SEQ ID NO:330.

[0443] In another illustrative embodiment, the modified signaling agent is VEGF 165b , the VEGF 165b It has the amino acid sequence SEQ ID NO:331.

[0444] In these embodiments, the modified signaling agent has a mutation at amino acid I83 (e.g., a substitution mutation at I83, such as I83K, I83R, or I83H). Without wishing to be bound by theory, it is believed that such mutations can result in reduced receptor binding affinity. See, for example, U.S. Patent No. 9,078,860, the entire contents of which are hereby incorporated by reference.

[0445] In one embodiment, the modified signaling agent is TNF-α. TNF is a pleiotropic cytokine with many different functions, including regulation of cell growth, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and migration, inflammation, and septic shock. It binds to two unique membrane receptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits a very broad expression pattern, while TNFR2 is preferentially expressed on certain populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. Very distinct biological pathways are activated in response to receptor activation, but there is also some overlap. In general, without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), while TNFR2 signaling is associated with the activation of cell survival signals (e.g., activation of the NFkB pathway). Administration of TNF is systemic toxic, and this is primarily due to TNFR1 binding. However, it should be noted that activation of TNFR2 is also associated with a variety of activities, and like TNFRl, control of TNF targeting and activity is important in the context of development of TNF-based therapeutics.

[0446] In some embodiments, the modified signaling agent has reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2. TNFR1 is expressed in most tissues and is involved in cell death signaling, while TNFR2, in contrast, is involved in cell survival signaling. Thus, in embodiments directed to methods of treating cancer, the modified signaling agent has reduced affinity and / or activity for TNFR1 and / or substantially reduced or eliminated affinity and / or activity for TNFR2. In these embodiments, the chimeric protein can be targeted to cells in need of apoptosis, such as tumor cells or tumor vascular endothelial cells. In embodiments directed to methods of promoting cell survival, for example, in neurogenesis for the treatment of neurodegenerative disorders, the modified signaling agent has reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1. In other words, in some embodiments, the chimeric protein of the invention comprises a modified TNF-α agent that allows for favorable death or survival signaling.

[0447] In some embodiments, the chimeric protein comprises a modified TNF having reduced affinity and / or activity for TNFR1 and / or substantially reduced or eliminated affinity and / or activity for TNFR2. In some embodiments, such chimeras are more effective inducers of apoptosis than wild-type TNF and / or chimeras carrying only one or more mutations that contribute to reduced affinity and / or activity for TNFR1. In some embodiments, such chimeras can be used to induce tumor cell death or tumor vascular endothelial cell death (e.g., for the treatment of cancer). In addition, in some embodiments, for example, such chimeras avoid or reduce activation of T cells via TNFR2. reg cells, thus further supporting TNFR1-mediated antitumor activity in vivo.

[0448] In some embodiments, the chimeric protein has a modified TNF that has reduced affinity and / or activity for TNFR2 and / or has substantially reduced or eliminated affinity and / or activity for TNFR1. In some embodiments, such chimeras are more effective cell survival activators in some cell types that may be specific therapeutic targets in various disease settings, including but not limited to stimulating neurogenesis. In addition, such chimeras that favor TNFR2 can also be used to treat autoimmune diseases (e.g., Crohn's disease, diabetes, MS, colitis, etc. and numerous other autoimmune diseases described herein). In some embodiments, the chimeras target autoreactive T cells. In some embodiments, the chimeras promote T reg Cell activation and indirect suppression of cytotoxic T cells.

[0449] In some embodiments, the chimera causes death of autoreactive T cells (e.g., modified TNF with reduced affinity and / or activity for TNFR2 and / or substantially reduced or abolished affinity and / or activity for TNFR1), e.g., by activating TNFR2 and / or avoiding TNFR1. Without wishing to be bound by theory, these autoreactive T cells have their apoptosis / survival signaling altered, e.g., by changes in NFkB pathway activity / signaling. In some embodiments, the chimera causes death of autoreactive T cells that have lesions or modifications to the NFkB pathway that underlie an imbalance in the cell death (apoptosis) / survival signaling properties of the autoreactive T cells and optionally alter their susceptibility to certain death-inducing signals (e.g., TNFR2 activation).

[0450] In some embodiments, TNFR-2 based chimeras have additional therapeutic applications in a wide range of diseases, including various autoimmune diseases, cardiac diseases, demyelinating and neurodegenerative disorders, and infectious diseases, among others.

[0451] In one embodiment, the wild-type TNF-α has the amino acid sequence of SEQ ID NO:332.

[0452] In such embodiments, the modified TNF-α agent has a mutation at one or more amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, thereby generating a modified TNF-α with reduced receptor binding affinity. See, e.g., U.S. Patent No. 7,993,636, the entire contents of which are hereby incorporated by reference.

[0453] In some embodiments, the modified human TNF-α portion has a mutation at one or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91, 197, T105, P106, A109, P113, Y115, E127, N137, D143, A145, and E146 as described, e.g., in WO / 2015 / 007903, the entire contents of which are hereby incorporated by reference (numbering is based on the human TNF sequence, Genbank Accession No. BAG70306, version BAG70306.1, G1: 197692685). In some embodiments, the modified human TNF-α portion has a substitution mutation selected from the group consisting of L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145R, A145T, E146D, E146K, and S147D. In one embodiment, the human TNF-α portion has a mutation selected from the group consisting of Y87Q, Y87L, Y87A, Y87F, and Y87H. In another embodiment, the human TNF-α portion has a mutation selected from the group consisting of I97A, I97Q, and I97S. In another embodiment, the human TNF-α portion has a mutation selected from the group consisting of Y115A and Y115G. In one embodiment, the human TNF-α portion has an E146K mutation. In one embodiment, the human TNF-α portion has a Y87H and E146K mutation. In one embodiment, the human TNF-α portion has a Y87H and A145R mutation. In one embodiment, the human TNF-α portion has an R32W and S86T mutation. In one embodiment, the human TNF-α portion has an R32W and E146K mutation. In one embodiment, the human TNF-α portion has an L29S and R32W mutation. In one embodiment, the human TNF-α portion has a D143N and A145R mutation. In one embodiment, the human TNF-α portion has D143N and A145R mutations. In one embodiment, the human TNF-α portion has A145T, E146D, and S147D mutations. In one embodiment, the human TNF-α portion has A145T and S147D mutations.

[0454] In some embodiments, the modified TNF-α agent has one or more mutations selected from N39Y, S147Y, and Y87H as described in WO2008 / 124086, the entire contents of which are hereby incorporated by reference.

[0455] In some embodiments, the modified human TNF-α portion has a mutation that provides receptor selectivity as described in PCT / IB2016 / 001668, the entire contents of which are hereby incorporated by reference. In some embodiments, the TNF mutation is selective for TNF-R1. In some embodiments, the selective TNF mutation for TNF-R1 is at one or more of positions R32, S86, and E146. In some embodiments, the selective TNF mutation for TNF-R1 is one or more of R32W, S86T, and E146K. In some embodiments, the selective TNF mutation for TNF-R1 is one or more of R32W, R32W / S86T, R32W / E146K, and E146K. In some embodiments, the TNF mutation is selective for TNF-R2. In some embodiments, the selective TNF mutation for TNF-R2 is at one or more of positions A145, E146, and S147. In some embodiments, the TNF-R2 selective TNF mutation is one or more of A145T, A145R, E146D, and S147D. In some embodiments, the TNF-R2 selective TNF mutation is one or more of A145R, A145T / S147D, and A145T / E146D / S147D.

[0456] In one embodiment, the modified signaling agent is TNF-β. TNF-β can form a homotrimer or heterotrimer with LT-β (LT-α1β2). In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2 and / or herpes virus entry mediator (HEVM) and / or LT-βR.

[0457] In one embodiment, the wild-type TNF-β has the amino acid sequence of SEQ ID NO:333.

[0458] In such embodiments, the modified soluble agent may comprise a mutation at one or more amino acids at positions 106-113, thereby generating a modified TNF-β with reduced receptor binding affinity for TNFR2. In one embodiment, the modified soluble agent has one or more substitution mutations at amino acid positions 106-113. In illustrative embodiments, the substitution mutations are selected from the group consisting of 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.

[0459] In some embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β), which may be in the form of a single-chain trimer as described in WO 2015 / 007903, the entire contents of which are incorporated by reference.

[0460] In some embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β) that has reduced affinity and / or activity, i.e., antagonistic activity, at TNFR1 (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are hereby incorporated by reference). In these embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β) that optionally also has substantially reduced or abolished affinity and / or activity for TNFR2. In some embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β) that has reduced affinity and / or activity, i.e., antagonistic activity, at TNFR2 (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are hereby incorporated by reference). In these embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β), which optionally also has substantially reduced or eliminated affinity and / or activity for TNFR1. The constructs of such embodiments can be used, for example, in methods for suppressing TNF responses in a cell-specific manner. In some embodiments, the antagonist TNF family member (e.g., TNF-α, TNF-β) is in the form of a single chain trimer as described in WO 2015 / 007903.

[0461] In one embodiment, the modified signaling 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 substantially reduced or eliminated affinity and / or activity for DR4 (TRAIL-RI) and / or DR5 (TRAIL-RII) and / or DcR1 and / or DcR2.

[0462] In one embodiment, the wild-type TRAIL has the amino acid sequence of SEQ ID NO:334.

[0463] In such embodiments, the modified TRAIL agent may comprise mutations at amino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209, T214-I220, K224-A226, W231, E236-L239, E249-K251, T261-H264, and H270-E271 (numbering is according to the human sequence, Genbank accession number NP_003801, version 10NP_003801.1, Gl:4507593; see above).

[0464] In some embodiments, the modified TRAIL agent may comprise one or more mutations that substantially reduce the affinity and / or activity of the modified TRAIL agent to TRAIL-R1. In such embodiments, the modified TRAIL agent may specifically bind to TRIL-R2. Exemplary mutations include mutations at one or more amino acid positions Y189, R191, Q193, H264, I266, and D267. For example, the mutation may be one or more of Y189Q, R191K, Q193R, H264R, I266L, and D267Q. In one embodiment, the modified TRAIL agent comprises mutations Y189Q, R191K, Q193R, H264R, I266L, and D267Q.

[0465] In some embodiments, the modified TRAIL agent may comprise one or more mutations that substantially reduce the affinity and / or activity of the modified TRAIL agent for TRAIL-R2. In such embodiments, the modified TRAIL agent may specifically bind to TRIL-R1. Exemplary mutations include mutations at one or more amino acid positions G131, R149, S159, N199, K201, and S215. For example, the mutation may be one or more of G131R, R149I, S159R, N199R, K201H, and S215D. In one embodiment, the modified TRAIL agent comprises 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:el035, the entire disclosure of which is hereby incorporated by reference.

[0466] In one embodiment, the modified signaling agent is TGFα. In such embodiments, the modified TGFα agent has reduced affinity and / or activity for epidermal growth factor receptor (EGFR). In some embodiments, the modified TGFα agent has substantially reduced or eliminated affinity and / or activity for epidermal growth factor receptor (EGFR).

[0467] In one embodiment, the modified signaling agent is TGFβ. In such embodiments, the modified signaling agent has reduced affinity and / or activity for TGFBR1 and / or TGFBR2. 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 reduced or substantially reduced or eliminated affinity and / or activity for TGFBR3, without wishing to be bound by theory, and the TGFBR3 may serve as a reservoir of ligands for the TGF-β receptor. In some embodiments, TGFβ may be more inclined to TGFBR1 relative to TGFBR2, or TGFβ may be more inclined to TGFBR2 relative to TGFBR1. Similarly, without wishing to be bound by theory, LAP may serve as a reservoir of ligands for the TGF-β receptor. In some embodiments, the modified signaling agent has reduced affinity and / or activity for TGFBR1 and / or TGFBR2 and / or has substantially reduced or eliminated affinity and / or activity for latent associated peptide (LAP). In some embodiments, such chimeras can be used for Camurati-Engelmann disease or other diseases associated with inappropriate TGFβ signaling.

[0468] In some embodiments, the modified agent is a TGF family member (e.g., TGFα, TGFβ) that has reduced affinity and / or activity, i.e., antagonistic activity (e.g., natural antagonistic activity or antagonistic activity caused by one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are hereby incorporated by reference herein). In these embodiments, the modified agent is a TGF family member (e.g., TGFα, TGFβ) that also optionally has substantially reduced or eliminated affinity and / or activity at one or more of TGFBR1, TGFBR2, TGFBR3.

[0469] In some embodiments, the modified agent is a TGF family member (e.g., TGFα, TGFβ) that has reduced affinity and / or activity, i.e., antagonistic activity, at TGFBR1 and / or TGFBR2 (e.g., natural antagonistic activity or antagonistic activity resulting from one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are hereby incorporated by reference herein). In these embodiments, the modified agent is a TGF family member (e.g., TGFα, TGFβ) that also optionally has substantially reduced or eliminated affinity and / or activity at TGFBR3.

[0470] In one embodiment, the modified signaling agent is an interleukin. In one embodiment, the modified signaling agent is IL-1. In one embodiment, the modified signaling agent is IL-1α or IL-1β. In some embodiments, the modified signaling agent has reduced affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signaling agent has reduced affinity and / or activity for IL-1R2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-1R2. For example, in some embodiments, the modified IL-1 agent of the present invention avoids interaction at IL-1R2 and thus substantially reduces its function as an attractant and / or receptor for therapeutic agents.

[0471] In one embodiment, the wild-type IL-1β has the amino acid sequence of SEQ ID NO: 335.

[0472] IL1 is a pro-inflammatory cytokine and an important regulator of the immune system. It is a potent activator of CD4 T cell responses, increasing the proportion of Th17 cells and the expansion of cells producing IFNγ and IL-4. + A potent regulator of T cells, thereby enhancing antigen-specific CD8 + T cell expansion, differentiation, migration to the periphery and memory. IL-1 receptors include IL-1R1 and IL-1R2. Binding to IL-1R1 and signaling via IL-1R1 constitute the mechanism by which IL-1 mediates many of its biological (and pathological) activities. IL1-R2 can act as a decoy receptor, thereby reducing the availability of IL-1 for interaction and signaling via IL-1R1.

[0473] In some embodiments, the modified IL-1 has a reduced affinity and / or activity (e.g., agonistic activity) for IL-1R1. In some embodiments, the modified IL-1 has a substantially reduced or eliminated affinity and / or activity for IL-1R2. In such embodiments, there is recoverable IL-1 / IL-1R1 signaling, and the loss of therapeutic chimeras at IL-R2 is prevented, and thus the dose of required IL-1 is reduced (e.g., relative to wild-type or chimeras carrying only attenuated mutations for IL-R1). Such constructs can be used, for example, to treat cancer, including, for example, methods of stimulating the immune system to establish an anti-cancer response.

[0474] In some embodiments, the modified IL-1 has reduced affinity and / or activity for IL-1R1 (e.g., antagonistic activity, such as natural antagonistic activity or antagonistic activity due to one or more mutations, see, for example, WO2015 / 007520, the entire contents of which are hereby incorporated by reference). In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and / or activity for IL-1R2. In such embodiments, there is irreversible IL-1 / IL-1R1 signaling, and the loss of therapeutic chimeras at IL-R2 is prevented, and thus the required dose of IL-1 is reduced (e.g., relative to wild-type or chimeras carrying only attenuated mutations for IL-R1). Such constructs can be used, for example, to treat autoimmune diseases, including, for example, methods of suppressing the immune system.

[0475] In such embodiments, the modified signaling agent has a deletion of amino acids 52-54, which results in 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 hereby incorporated by reference. In some embodiments, the modified human IL-1β has an amino acid sequence 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, E221 One or more substitution mutations of S / N224A, N224S / K225S, E244K, N245Q (wherein X can be any amino acid change, e.g., a non-conservative change), thereby exhibiting reduced binding to IL-1R, as described, e.g., in WO 2015 / 007542 and WO / 2015 / 007536, the entire contents of which are hereby incorporated by reference (numbering is based on the human IL-1β sequence, Genbank accession number NP_000567, version NP-000567.1, G1: 10835145). 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 one embodiment, the modified human IL-1β comprises mutations Q131G and Q148G. In one embodiment, the modified human IL-1β comprises mutations Q148G and K208E. In one embodiment, the modified human IL-1β comprises mutations R120G and Q131G. In one embodiment, the modified human IL-1β comprises mutations R120G and H146A. In one embodiment, the modified human IL-1β comprises the mutations R120G and H146N. In one embodiment, the modified human IL-1β comprises the mutations R120G and H146R. In one embodiment, the modified human IL-1β comprises the mutations R120G and H146E.In one embodiment, the modified human IL-1β comprises the mutations R120G and H146G. In one embodiment, the modified human IL-1β comprises the mutations R120G and K208E. In one embodiment, the modified human IL-1β comprises the mutations R120G, F162A, and Q164E.

[0476] In one embodiment, the modified signaling agent is IL-2. In such embodiments, the modified signaling agent has reduced affinity and / or activity for IL-2Rα and / or IL-2Rβ and / or IL-2Rγ. In some embodiments, the modified signaling agent has reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-2Rα. Such embodiments may be relevant to the treatment of cancer, for example, when the modified IL-2 is agonistic at IL-2Rβ and / or IL-2Rγ. For example, the constructs of the present invention may be beneficial in attenuating CD8 T cells with IL2 receptors β and γ. + Activation of T cells (which can provide antitumor effects) is detrimental to T cells with IL-2 receptors α, β, and γ. reg (This can provide an immunosuppressive, tumor-promoting effect.) Furthermore, in some embodiments, preference for IL-2Rβ and / or IL-2Rγ over IL-2Rα avoids many IL-2 side effects, such as pulmonary edema. Moreover, IL-2-based chimeras can be used to treat autoimmune diseases, for example, when the modified IL-2 has antagonism at IL-2Rβ and / or IL-2Rγ (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, for example, WO 2015 / 007520, the entire contents of which are hereby incorporated by reference). For example, the constructs of the present invention may be beneficial in attenuating the effects of IL-2 on CD8 T cells with IL2 receptors β and γ. + The inhibition of T cells (and thus the suppression of the immune response) is to the detriment of T cells with IL2 receptors α, β and γ. reg Alternatively, in some embodiments, chimeras carrying IL-2 may be beneficial for T reg activation, and thus favors immune suppression over CD8 + Activation of T cells. For example, these constructs can be used to treat diseases that would benefit from immune suppression, such as autoimmune diseases.

[0477] In some embodiments, the chimeric protein has a CD8 specific antigen binding protein as described herein. +T cell targeting moieties and modified IL-2 agents with reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ and / or substantially reduced or abolished affinity and / or activity for IL-2Rα. In some embodiments, these constructs provide for targeting of CD8 + T cell activity, and generally T reg In some embodiments, such constructs have an enhanced immunostimulatory effect compared to wild-type IL-2 (e.g., without wishing to be bound by theory, by not stimulating Tregs) while eliminating or reducing the systemic toxicity associated with IL-2.

[0478] In one embodiment, the wild-type IL-2 has the amino acid sequence of SEQ ID NO:336.

[0479] In such embodiments, the modified IL-2 agent has one or more mutations at amino acids L72 (L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K), and 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 IL-2 agents have reduced affinity for the high-affinity IL-2 receptor and retain affinity for the medium-affinity IL-2 receptor compared to the wild-type IL-2. See, for example, US Patent Publication No. 2012 / 0244112, the entire contents of which are hereby incorporated by reference.

[0480] In some embodiments, the modified IL-2 agent has one or more mutations at amino acids R38, F42, Y45, and E62. For example, the modified IL-2 agent may include one or more of R38A, F42A, Y45A, and E62A. In some embodiments, the modified IL-2 agent may include a mutation at C125. For example, the mutation may be C125S. In such embodiments, the modified IL-2 agent may have a substantially reduced affinity and / or activity for IL-2Rα, as described, for example, in Carmenate et al. (2013) The Journal of Immunology, 190: 6230-6238, the entire disclosure of which is hereby incorporated by reference. In some embodiments, the modified IL-2 agent having a mutation at R38, F42, Y45, and / or E62 is capable of inducing the expansion of effector cells, including CD8+ T cells and NK cells rather than Treg cells. In some embodiments, the modified IL-2 agent having a mutation at R38, F42, Y45 and / or E62 is less toxic than a wild-type IL-2 agent. Chimeric proteins comprising the modified IL-2 agent having substantially reduced affinity and / or activity for IL-2Rα can be used, for example, in oncology.

[0481] In other embodiments, the modified IL-2 agent can have a substantially reduced affinity and / or activity for IL-2R β, as described in, for example, WO2016 / 025385, the entire disclosure of which is hereby incorporated by reference. In such embodiments, the modified IL-2 agent can induce the expansion of Treg cells rather than effector cells (such as CD8+ T cells and NK cells). The chimeric protein comprising the modified IL-2 agent having a substantially reduced affinity and / or activity for IL-2R β can be used, for example, to treat autoimmune diseases. In some embodiments, the modified IL-2 agent can include one or more mutations at amino acids N88, D20 and / or A126. For example, the modified IL-2 agent can include one or more of N88R, N88I, N88G, D20H, Q126L and Q126F.

[0482] In various embodiments, the modified IL-2 agent can comprise a mutation at D109 or C125. For example, the mutation can be D109C or C125S. In some embodiments, a modified IL-2 having a mutation at D109 or C125 can be used for attachment to a PEG moiety.

[0483] In one embodiment, the modified signaling agent is IL-3. In some embodiments, the modified signaling agent has reduced affinity and / or activity for the IL-3 receptor, which is a heterodimer with a unique α chain paired with a common β (βc or CD131) subunit. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for the IL-3 receptor, which is a heterodimer with a unique α chain paired with a common β (βc or CD131) subunit.

[0484] In one embodiment, the modified signaling agent is IL-4. In such embodiments, the modified signaling agent has reduced affinity and / or activity for type 1 and / or type 2 IL-4 receptors. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for type 1 and / or type 2 IL-4 receptors. The type 1 IL-4 receptor is composed of IL-4Rα subunits with a common γ chain and specifically binds to IL-4. The type 2 IL-4 receptor includes an IL-4Rα subunit that is bound to a different subunit called IL-13Rα1. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for the type 2 IL-4 receptor.

[0485] In one embodiment, the wild-type IL-4 has the amino acid sequence of SEQ ID NO:337.

[0486] In such embodiments, the modified IL-4 agent has one or more mutations at amino acids R121 (R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W), E122 (E122F), Y124 (Y124A, Y124Q, Y124R, Y124S, Y124T), and S125 (S125A). Without wishing to be bound by theory, it is believed that these modified IL-4 agents maintain type I receptor-mediated activity but significantly reduce other receptor-mediated biological activities. See, for example, U.S. Patent No. 6,433,157, the entire contents of which are hereby incorporated by reference.

[0487] In one embodiment, the modified signal transducer is IL-6. IL-6 carries out signal transduction through the cell surface type I cytokine receptor complex, and the complex includes ligand binding IL-6R chain (CD126) and signal transduction component gp130. IL-6 can also be bound to the soluble form of IL-6R (sIL-6R), which is the extracellular part of IL-6R. The sIL-6R / IL-6 complex may be involved in neurite outgrowth and neuronal survival, and therefore may be very important in the nerve regeneration performed by remyelination. Therefore, in some embodiments, the modified signal transducer has a reduced affinity and / or activity for IL-6R / gp130 and / or sIL-6R. In some embodiments, the modified signal transducer has a substantially reduced or eliminated affinity and / or activity for IL-6R / gp130 and / or sIL-6R.

[0488] In one embodiment, the wild-type IL-6 has the amino acid sequence of IL-6 (mature form, wild-type) (SEQ ID NO: 338).

[0489] In such embodiments, the modified signaling 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 IL-6 agents exhibit reduced binding affinity to IL-6Rα and reduced biological activity. See, for example, WO 97 / 10338, the entire contents of which are hereby incorporated by reference.

[0490] In one embodiment, the modified signaling agent is IL-10. In such embodiments, the modified signaling agent has reduced affinity and / or activity for IL-10 receptor 1 and IL-10 receptor 2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-10 receptor 1 and IL-10 receptor 2.

[0491] In one embodiment, the modified signaling agent is IL-11. In such embodiments, the modified signaling agent has reduced affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130. In this embodiment, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130.

[0492] In one embodiment, the modified signaling agent is IL-12. In this embodiment, the modified signaling agent has reduced affinity and / or activity for IL-12Rβ1 and / or IL-12Rβ2. In this embodiment, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-12Rβ1 and / or IL-12Rβ2.

[0493] In one embodiment, the modified signaling agent is IL-13. In this embodiment, the modified signaling agent has reduced affinity and / or activity for the IL-4 receptor (IL-4Rα) and IL-13Rα1. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for the IL-4 receptor (IL-4Rα) or IL-13Rα1.

[0494] In one embodiment, the wild-type IL-13 has the amino acid sequence of IL-13 (mature form, wild-type) (SEQ ID NO: 339).

[0495] In such embodiments, the modified IL-13 agent 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 agents exhibit reduced biological activity. See, for example, WO 2002 / 018422, the entire contents of which are hereby incorporated by reference.

[0496] In one embodiment, the modified signaling agent is IL-18. In some embodiments, the modified signaling agent has reduced affinity and / or activity for IL-18Rα and / or IL-18Rβ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-18Rα and / or IL-18Rβ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for type II IL-18Rα, i.e., an IL-18Rα isoform that lacks the TIR domain required for signaling.

[0497] In one embodiment, the wild-type IL-18 has the amino acid sequence of IL-18 (wild-type) (SEQ ID NO: 340).

[0498] In such embodiments, the modified IL-18 agent may comprise one or more mutations at an amino acid or amino acid region selected from Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N127-K129, Q139-M149, K165-K171, R183, and Q190-N191 as described in WO / 2015 / 007542, the entire contents of which are hereby incorporated by reference (numbering is based on the human IL-18 sequence, Genbank Accession No. AAV38697, Version AAV38697.1, G1: 54696650).

[0499] In one embodiment, the modified signaling agent is IL-33. In this embodiment, the modified signaling agent has reduced affinity and / or activity for the ST-2 receptor and IL-1RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for the ST-2 receptor and IL-1RAcP.

[0500] In one embodiment, the wild-type IL-33 has the amino acid sequence of SEQ ID NO: 341.

[0501] In such embodiments, the modified IL-33 agent may comprise one or more mutations at an amino acid or amino acid region selected from I113-Y122, S127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227, and T260-E269 as described in WO / 2015 / 007542, the entire contents of which are hereby incorporated by reference (numbering is based on the human sequence, Genbank Accession No. NP_254274, Version NP_254274.1, G1: 15559209).

[0502] In one embodiment, the modified signaling agent is epidermal growth factor (EGF). EGF is a member of a family of potent growth factors. Members include EGF, HB-EGF, and other members such as TGFα, amphiregulin, neuregulin, epiregulin, and beta cell protein. EGF family receptors include EGFR (ErbB1), ErbB2, ErbB3, and ErbB4. These receptors can function as homodimers and / or heterodimers. Different EGF family members exhibit different selectivity for various receptor subtypes. For example, EGF is associated with ErbB1 / ErbB1, ErbB1 / ErbB2, ErbB4 / ErbB2, and several other heterodimer subtypes. HB-EGF has a similar pattern, but it is also associated with ErbB4 / 4. Actively or negatively modulating EGF (EGF-like) growth factor signaling has considerable therapeutic significance. For example, inhibiting EGFR signaling is of interest in the treatment of various cancers in which EGFR signaling is the primary growth-promoting signal. Alternatively, stimulation of EGFR signaling may have therapeutic implications, for example, in promoting wound healing (acute and chronic), and in treating oral mucositis, a major side effect of various cancer therapies, including but not limited to radiation therapy.

[0503] In some embodiments, the modified signaling agent has a reduced affinity and / or activity for ErbB1, ErbB2, ErbB3 and / or ErbB4. Such embodiments can be used, for example, in methods for treating wounds. In some embodiments, the modified signaling agent binds to one or more ErbB1, ErbB2, ErbB3 and ErbB4 and antagonizes the activity of these receptors. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for ErbB1, ErbB2, ErbB3 and / or ErbB4, which allows the activity of these receptors to be antagonized in a weakened manner. Such embodiments can be used, for example, in the treatment of cancer. In one embodiment, the modified signaling agent has a 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 targeted and has fewer side effects than other agents targeting the EGF receptor.

[0504] In some embodiments, the modified signaling agent has reduced affinity and / or activity (e.g., antagonism, e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are hereby incorporated by reference) for ErbBl and / or has substantially reduced or abolished affinity and / or activity for ErbB4 or other subtypes that may interact therewith. By specific targeting via a targeting moiety, cell-selective inhibition of ErbBl / ErbBl receptor activation (antagonism, e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are hereby incorporated by reference) will be achieved without involving other receptor subtypes that may be associated with inhibition-related side effects. Thus, compared to EGFR kinase inhibitors that inhibit EGFR activity in all cell types in vivo, this construct would provide cell-selective (e.g., tumor cells with activated EGFR signaling due to receptor amplification, overexpression, etc.) anti-EGFR (ErbBl) drug effects with reduced side effects.

[0505] In some embodiments, the modified signaling agent has reduced affinity and / or activity (e.g., agonism) for ErbB4 and / or other isoforms that may interact with it. By targeting specific target cells via the targeting moiety, selective activation of ErbB1 signaling (e.g., epithelial cells) is achieved. In some embodiments, such constructs can be used to treat wounds (promote healing) with reduced side effects, and are particularly useful for treating chronic conditions and applications other than topical administration of therapeutic agents (e.g., systemic wound healing).

[0506] In one embodiment, the modified signaling agent is insulin or an insulin analog. In some embodiments, the modified insulin or insulin analog has a reduced affinity and / or activity for the insulin receptor and / or IGF1 or IGF2 receptor. In some embodiments, the modified insulin or insulin analog has a substantially reduced or eliminated affinity and / or activity for the insulin receptor and / or IGF1 or IGF2 receptor. The weakened response at the insulin receptor allows control of diabetes, obesity, metabolic disorders, etc., while directly away from the IGF1 or IGF2 receptor avoids the effects of pro-cancer.

[0507] In one embodiment, the modified signaling agent is insulin-like growth factor I or insulin-like growth factor II (IGF-1 or IGF-2). In one embodiment, the modified signaling agent is IGF-1. In this embodiment, the modified signaling agent has a reduced affinity and / or activity for the insulin receptor and / or IGF1 receptor. In one embodiment, the modified signaling agent can bind to the IGF1 receptor and antagonize the activity of the receptor. In this embodiment, the modified signaling agent has a reduced affinity and / or activity for the IGF1 receptor, which allows the activity of the receptor to be antagonized in a weakened manner. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for the insulin receptor and / or IGF1 receptor. In some embodiments, the modified signaling agent has a reduced affinity and / or activity for the IGF2 receptor, which allows the activity of the receptor to be antagonized in a weakened manner. In one embodiment, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for the insulin receptor and / or IGF2 receptor, and therefore does not interfere with insulin signaling. In various embodiments, this applies to cancer treatment.In various embodiments, the agents of the invention can prevent IR isoform A from causing resistance to cancer treatment.

[0508] In one embodiment, a chimeric protein of the invention has (i) a targeting moiety to PD-1 or PD-L1 and (ii) a targeting moiety to tumor cells, and any of the modified or mutated signaling agents described herein.

[0509] In one embodiment, a chimeric protein of the invention has (i) a targeting moiety to PD-1 or PD-L1 and (ii) a targeting moiety to a checkpoint inhibitor marker, and any of the modified or mutated interferons described herein.

[0510] In various embodiments, the signal transducer is a toxin or a toxic enzyme. In some embodiments, the toxin or toxic enzyme is derived from plants and bacteria. Illustrative 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, cucurbitacin, abrin, gelonin and pokeweed antiviral proteins. Additional toxins include those disclosed in Mathew et al., (2009) Cancer Sci 100 (8): 1359-65, all of which are hereby incorporated by reference. In such embodiments, the chimeric protein of the present invention can be used to induce cell death in a cell type-specific manner. In such embodiments, the toxin can be modified, for example, mutated to reduce the affinity and / or activity of the toxin in order to weaken the effect, as described for other signal transducers herein.

[0511] Multispecific chimeras and fusions with signaling agents

[0512] In various embodiments, the PD-1 or PD-L1 binding agents of the invention are part of a chimera or fusion with one or more signaling agents and / or one or more additional targeting moieties as described herein. Thus, the invention provides chimeric or fusion proteins comprising one or more signaling agents and a targeting moiety to PD-1 or PD-L1 and / or one or more additional targeting moieties.

[0513] In various embodiments, the PD-1 or PD-L1 binding agent of the present invention is multispecific, that is, the PD-1 or PD-L1 binding agent comprises two or more targeting moieties having a recognition domain that recognizes and binds to two or more targets (e.g., antigens or receptors or epitopes). In such embodiments, the PD-1 or PD-L1 binding agent of the present invention may comprise two or more targeting moieties having a recognition domain that recognizes and binds to two or more epitopes on the same antigen or on different antigens. In various embodiments, such multispecific PD-1 or PD-L1 binding agents exhibit many advantageous properties, such as increased affinity and / or improved selectivity. In one embodiment, the PD-1 or PD-L1 binding agent of the present invention comprises two targeting moieties and is bispecific, i.e., binds to and recognizes two epitopes on the same antigen or on different antigens.

[0514] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention comprise two or more targeting moieties, each of which is an antibody or antibody derivative as described herein. In one embodiment, the multispecific PD-1 or PD-L1 binding agents of the invention comprise at least one VHH comprising an antigen recognition domain against PD-1 or PD-L1 and one antibody or antibody derivative comprising an antigen recognition domain against a tumor antigen.

[0515] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention have two or more targeting moieties that target different antigens or receptors, and one targeting moiety can attenuate its antigen or receptor, for example, the targeting moiety binds its antigen or receptor with low affinity or avidity (including, for example, with an affinity or avidity that is lower than the affinity or avidity that the other targeting moiety has for its antigen or receptor, for example, the difference in binding affinity 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; for example, the lower affinity or avidity targeting moiety can bind with a K in the mid to high nM or low to mid μM range). D Binds to its antigen or receptor, while the higher affinity or avidity targeting moiety can bind with a K in the mid to high pM or low to mid nM range. D For example, in some embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention comprise attenuated targeting moieties directed against promiscuous antigens or receptors, thereby improving targeting to target cells (e.g., via other targeting moieties) and preventing effects on multiple types of cells, including those not targeted by the therapy (e.g., by binding to the promiscuous antigen or receptor with higher affinity than provided in these embodiments).

[0516] The multispecific PD-1 or PD-L1 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 Publication No. 20110262348, and WO 2004 / 041862, the entire contents of which are hereby incorporated by reference. In one illustrative embodiment, the multispecific PD-1 or PD-L1 binding agents of the present invention comprising two or more targeting moieties can be constructed by chemical cross-linking, for example, by reacting amino acid residues with an organic derivatizing agent as described in Blattler et al., Biochemistry 24, 1517-1524 and EP294703, the entire contents of which are hereby incorporated by reference. In another illustrative embodiment, the multispecific PD-1 or PD-L1 binding agents comprising two or more targeting moieties are constructed by genetic fusion, i.e., constructing a single polypeptide comprising the polypeptides of the individual targeting moieties. For example, a single polypeptide construct can be formed that encodes a first VHH having an antigen recognition domain for PD-1 or PD-L1 and a second antibody or antibody derivative having an antigen recognition domain for a tumor antigen. A method for producing bivalent or multivalent VHH polypeptide constructs is disclosed in PCT patent application WO 96 / 34103, the entire contents of which are hereby incorporated by reference. In another illustrative embodiment, the multispecific PD-1 or PD-L1 binding agent of the present invention can be constructed by using a linker. For example, the carboxyl terminus of a first VHH having an antigen recognition domain for PD-1 or PD-L1 can be linked to the amino terminus of a second antibody or antibody derivative having an antigen recognition domain for a tumor antigen (or vice versa). Exemplary linkers that can be used are described herein. In some embodiments, the components of the multispecific PD-1 or PD-L1 binding agent of the present invention are directly linked to each other without the use of a linker.

[0517] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the present invention recognize and bind to PD-1 or PD-L1 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, T helper cells, natural killer T cells), B lymphocytes, plasma cells, dendritic cells, or subsets thereof. In some embodiments, the PD-1 or PD-L1 binding agent specifically binds to the target antigen and effectively recruits one or more immune cells directly or indirectly.

[0518] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention recognize and bind to PD-1 or PD-L1 and one or more antigens found on tumor cells. In these embodiments, the PD-1 or PD-L1 binding agents of the invention can directly or indirectly recruit immune cells to tumor cells or the tumor microenvironment. In some embodiments, the PD-1 or PD-L1 binding agents of the invention can directly or indirectly recruit immune cells, such as immune cells (e.g., CTLs) that can kill and / or suppress tumor cells, to the site of action (as a non-limiting example, such as the tumor microenvironment).

[0519] In some embodiments, the PD-1 or PD-L1 binding agents of the present invention can be or can be used in methods involving altering the balance of immune cells that favors immune attack of tumors. For example, the PD-1 or PD-L1 binding agents of the present invention can alter the ratio of immune cells at clinically important sites, thereby favoring cells that can kill and / or suppress tumors (e.g., T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), 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, the PD-1 or PD-L1 binding agents of the present invention can increase the ratio of effector T cells to regulatory T cells.

[0520] In some embodiments, the multispecific PD-1 or PD-L1 binding agent of the present invention comprises a targeting portion having a recognition domain that specifically binds to an antigen associated with a tumor cell. In some embodiments, the targeting portion directly or indirectly recruits tumor cells. For example, in some embodiments, tumor cells are recruited to one or more effector cells (e.g., immune cells described herein) that can kill and / or suppress tumor cells. In some embodiments, the targeting portion directly or indirectly recruits T cells to tumor cells, for example, by two targeting portions that interact with their respective antigens on tumors and CD8 positive immune cells (e.g., T cells).

[0521] Tumor cells or cancer cells refer to cells or tissues with uncontrolled growth and / or an abnormal increase in cell survival time and / or inhibition of apoptosis that interferes with the normal function of body organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. Illustrative tumor cells include, but are not limited to, the following: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal 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, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; vulvar cancer; lymphomas, including Hodgkin's and non-Hodgkin's lymphomas, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-mitotic cell NHL; bulky NHL; and disease NHL); mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus hamartomas, edema (e.g., edema associated with brain tumors), and Meigs' syndrome.

[0522] Tumor cells or cancer cells also include, but are not limited to, carcinomas, such as various subtypes including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myeloid, acute lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell), lymphomas and myelomas (including, for example, Hodgkin's and non-Hodgkin's lymphomas, light chain, non-secretory, MGUS, and plasmacytoma), and central nervous system cancers (including, for example, brain (e.g., gliomas (e.g., astrocytomas, oligodendrogliomas, and ependymomas), meningiomas, pituitary adenomas and neuromas), and spinal cord tumors (e.g., meningiomas and neurofibromas).

[0523] Illustrative tumor antigens include, but are not limited to, MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal-related antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-ζ chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A 11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE ​​family tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, Smad family tumor antigens, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, 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 PD-1 or PD-L1 binding agents of the invention comprise a targeting moiety that binds one or more of these tumor antigens.

[0524] In some embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention recognize and bind to PD-1 or PD-L1 and an antigen on a tumor cell.

[0525] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention have targeting moieties that target two different cells (e.g., to form a synapse) or the same cell (e.g., to obtain a more concentrated signaling agent effect).

[0526] In some embodiments, the multispecific PD-1 or PD-L1 binding agent of the present invention comprises a targeting portion having an antigen recognition domain that specifically binds to a target associated with a T cell (e.g., an antigen, a receptor). In some embodiments, the targeting portion directly or indirectly recruits T cells. In one embodiment, 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 treatment site (e.g., a location with one or more disease cells or cells that are regulated for therapeutic effect). Illustrative effector T cells include cytotoxic T cells (e.g., αβTCR, CD3 + 、CD8 + 、CD45RO + ); CD4 + Effector T cells (e.g., αβTCR, CD3 + 、CD4 + 、CCR7 + 、CD62L high、IL - 7R / CD127 + );CD8 + Effector T cells (e.g., αβTCR, CD3 + 、CD8 + 、CCR7 + 、CD62L high、IL - 7R / CD127 + ); effector memory T cells (e.g., CD62L low, CD44 + , TCR, CD3 + IL - 7R / CD127 + IL-15R + , CCR7 low); central memory T cells (e.g., CCR7 + 、CD62L + 、CD27 + ; or CCR7 high, CD44 + 、CD62L high、TCR、CD3 +IL-7R / CD127 + IL-15R + );CD62L + Effector T cells; CD8 + Effector memory T cells (TEMs), including early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L - ) (TemE and TemL, respectively); CD127 ( + )CD25(low / -) effector T cells; CD127( - )CD25( - ) effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g. CD44(low)CD62L(high)CD122(high)sca( + )); TH1 effector T cells (e.g. CXCR3 + 、CXCR6 + and CCR5 + ; or αβTCR, CD3 + 、CD4 + IL-12R + IFNγR + 、CXCR3 + ), TH2 effector T cells (e.g. CCR3 + 、CCR4 + and CCR8 + ; or αβTCR, CD3 + 、CD4 + , IL-4R + IL-33R + 、CCR4 + IL-17RB + , CRTH2 + ); TH9 effector T cells (e.g., αβTCR, CD3 + 、CD4 + ); TH17 effector T cells (e.g., αβTCR, CD3 + 、CD4 + IL-23R + 、CCR6 + , IL-1R + ); CD4 + CD45RO + CCR7 + Effector T cells, ICOS + Effector T cells; CD4 +CD45RO + CCR7( - ) effector T cells; and effector T cells that secrete IL-2, IL-4 and / or IFN-γ.

[0527] Illustrative T cell antigens of interest include, for example (and where appropriate, the extracellular domains): CD8, CD3, SLAMF4, IL-2Rα, 4-1BB / TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL-4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL-6R, CCR3, IL-7Rα, CCR4, CXCR1 / IL-S RA, CCR5, CCR6, IL-10Rα, CCR 7, IL-1 ORβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrin α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD 11b, CDS, integrin αX / CD11c, integrin β2 / CD1S, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common γ chain / IL-2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, thymopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNF RIII / TNFRSF1B, TRAIL R1 / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, and TSLP R. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds one or more of these illustrative T cell antigens.

[0528] In some embodiments, the multispecific PD-1 or PD-L1 binding agents of the present invention comprise a targeting moiety directed against CD8, which 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, "framework regions" or "FRs" refer to the regions of the variable domain located between the CDRs. As used herein, "complementarity determining regions" or "CDRs" refer to the variable regions of a VHH that contain amino acid sequences that are capable of specifically binding to an antigenic target.

[0529] In various embodiments, a multispecific PD-1 or PD-L1 binding agent of the invention comprises a VHH directed against CD8 having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence.

[0530] In some embodiments, the CDR1 sequence is selected from SEQ ID NO:342 or SEQ ID NO:343.

[0531] In some embodiments, the CDR2 sequence is selected from SEQ ID NO:344 or SEQ ID NO:345.

[0532] In some embodiments, the CDR3 sequence is selected from SEQ ID NO:346 or SEQ ID NO:347 or SEQ ID NO:348.

[0533] In various embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from the group consisting of: R3HCD27 (SEQ ID NO: 349) or R3HCD129 (SEQ ID NO: 350) or R2HCD26 (SEQ ID NO: 351).

[0534] In various embodiments, the CD8 targeting moiety comprises a VHH having a variable domain comprising at least one CDR1, CDR2 and / or CDR3 sequence as described below.

[0535] In some embodiments, the CDR1 sequence is selected from SEQ ID NO:352 to SEQ ID NO:420.

[0536] In some embodiments, the CDR2 sequence is selected from SEQ ID NO:421 to SEQ ID NO:489.

[0537] In some embodiments, the CDR3 sequence is selected from SEQ ID NO:490 to SEQ ID NO:558.

[0538] In various embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from the group consisting of 1CDA 7 (SEQ ID NO: 559) or 1CDA 12 (SEQ ID NO: 560) or 1CDA 14 (SEQ ID NO: 561) or 1CDA 15 (SEQ ID NO: 562) or 1CDA 17 (SEQ ID NO: 563) or 1CDA 18 (SEQ ID NO: 564) or 1CDA 19 (SEQ ID NO: 565) or 1CDA 24 (SEQ ID NO: 566) or 1CDA 26 (SEQ ID NO: 567) or 1CDA 28 (SEQ ID NO: 568) or 1CDA 37 (SEQ ID NO: 569) or 1CDA 43 (SEQ ID NO: 570) or 1CDA 45 (SEQ ID NO: 571) or 1CDA 47 (SEQ ID NO: 572) or 1CDA 48 (SEQ ID NO: 573) or 1CDA 58 (SEQ ID NO: 574) or 1CDA 65 (SEQ ID NO: 575) or 1CDA 68 (SEQ ID NO: 576) or 1CDA 73 (SEQ ID NO: 577) or 1CDA75 (SEQ ID NO: 578) or 1CDA 86 (SEQ ID NO: 579) or 1CDA 87 (SEQ ID NO: 580) or 1CDA 88 (SEQ ID NO: 581) or 1CDA 89 (SEQ ID NO: 582) or 1CDA 92 (SEQ ID NO: 583) or 1CDA 93 (SEQ ID NO: 584) or 2CDA 1 (SEQ ID NO: 585) or 2CDA 5 (SEQ ID NO: 586) or 2CDA 22 (SEQ ID NO: 587) or 2CDA 28 (SEQ ID NO: 588) NO:588) or 2CDA 62 (SEQ ID NO:589) or 2CDA 68 (SEQ ID NO:590) or 2CDA 73 (SEQ ID NO:591) or 2CDA 74 (SEQ ID NO:592) or 2CDA 75 (SEQ ID NO:593) or 2CDA 77 (SEQ ID NO:594) or 2CDA 81 (SEQ ID NO:595) or 2CDA 87 (SEQ ID NO:596) or 2CDA88 (SEQ ID NO:597) or 2CDA 89 (SEQ ID NO:598) or 2CDA 91 (SEQ ID NO:599) or 2CDA 92 (SEQ ID NO:600) or 2CDA93 (SEQ ID NO: 601) or 2CDA 94 (SEQ ID NO: 602) or 2CDA 95 (SEQ ID NO: 603) or 3CDA 3 (SEQ ID NO: 604) or 3CDA 8 (SEQ ID NO: 605) or 3CDA 11 (SEQ ID NO: 606) or 3CDA 18 (SEQ ID NO: 607) or 3CDA 19 (SEQ ID NO: 608) or 3CDA 21 (SEQ ID NO: 609) or 3CDA 24 (SEQ ID NO: 610) or 3CDA 28 (SEQ ID NO: 611) or 3CDA 29 (SEQ ID NO: 612) or 3CDA 31 (SEQ ID NO: 613) or 3CDA 32 (SEQ ID NO: 614) or 3CDA 33 (SEQ ID NO: 615) or 3CDA37 (SEQ ID NO: 616) NO: 616) or 3CDA 40 (SEQ ID NO: 617) or 3CDA 41 (SEQ ID NO: 618) or 3CDA 48 (SEQ ID NO: 619) or 3CDA 57 (SEQ ID NO: 620) or 3CDA 65 (SEQ ID NO: 621) or 3CDA 70 (SEQ ID NO: 622) or 3CDA 73 (SEQ ID NO: 623) or 3CDA 83 (SEQ ID NO: 624) or 3CDA 86 (SEQ ID NO: 625) or 3CDA 88 (SEQ ID NO: 626) or 3CDA 90 (SEQ ID NO: 627).

[0539] In various exemplary embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from any one of the above sequences without a terminal histidine tag sequence (ie, HHHHHH; SEQ ID NO: 84).

[0540] In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from SEQ ID Nos: 559-627 (provided above) without the HA tag (ie, YPYDVPDYGS; SEQ ID NO: 85).

[0541] In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from SEQ ID Nos: 559-627 (provided above) without an AAA linker.

[0542] In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from SEQ ID Nos: 559-627 (provided above) without the AAA linker, HA tag, and terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 86). In various embodiments, the CD8 targeting moiety comprises an amino acid sequence described in U.S. Patent Publication No. 2014 / 0271462, the entire contents of which are incorporated by reference. In various embodiments, the CD8 targeting moiety comprises an amino acid sequence described in Table 0.1, Table 0.2, Table 0.3, and / or Table 0.4 of U.S. Patent Publication No. 2014 / 0271462. Figure 1 A - Amino acid sequences depicted in Figure 12I, the entire contents of which are incorporated by reference. In various embodiments, the CD8 targeting moiety comprises a HCDR1 of HCDR1 of SEQ ID NO: 22 or 23, and / or a HCDR2 of HCDR1 of SEQ ID NO: 22 or 23, and / or a HCDR3 of HCDR1 of SEQ ID NO: 22 or 23, and / or a LCDR1 of LCDR1 of SEQ ID NO: 24, and / or a LCDR2 of LCDR1 of SEQ ID NO: 24, and / or a LCDR3 of LCDR1 of SEQ ID NO: 24, as provided in SEQ ID NO: 628, SEQ ID NO: 629, or SEQ ID NO: 630.

[0543] In various embodiments, the present invention contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs of the targeting moieties for CD8 as described herein (collectively referred to herein as "analogs"). In various embodiments, the amino acid sequence of the targeting moiety for CD8 also includes amino acid analogs, amino acid derivatives or other non-classical amino acids.

[0544] In some embodiments, a multispecific PD-1 or PD-L1 binding agent of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a target (e.g., an antigen, a receptor) associated with a B cell. In some embodiments, the targeting moiety directly or indirectly recruits B cells, for example, in some embodiments, to a treatment site (e.g., a location with one or more diseased cells or cells to be modulated for therapeutic effect). Illustrative target B cell antigens include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDwl30, CD138, CDwl50, and B cell maturation antigen (BCMA). In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds to one or more of these illustrative B cell antigens.

[0545] In some embodiments, a multispecific PD-1 or PD-L1 binding agent of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a target (e.g., an antigen, a receptor) associated with natural killer cells. In some embodiments, the targeting moiety directly or indirectly recruits natural killer cells, for example, in some embodiments, to a treatment site (e.g., a location with one or more diseased cells or cells to be modulated for therapeutic effect). Illustrative natural killer cell antigens of interest include, for example, TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, Kir1α, DNAM-1, LMIR5 / CD300LB, Fc-εRII, LMIR6 / CD300LE, Fc-γR1 / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Integrin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NKG 2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc receptor-like protein 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, and ULBP-3. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds one or more of these illustrative NK cell antigens.

[0546] In some embodiments, the multispecific PD-1 or PD-L1 binding agent of the present invention comprises a targeting portion having an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with a macrophage / monocyte. In some embodiments, the targeting portion directly or indirectly recruits macrophages / monocytes, for example, in some embodiments, to a treatment site (e.g., a location with one or more disease cells or cells that are regulated for therapeutic effect). Illustrative target macrophage / monocyte antigens include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common β chain, integrin α4 / CD49d, BLAME / SLAMF8, integrin αX / CDllc, CCL6 / C10, integrin β2 / CD18, CD155 / PVR, integrin β3 / CD61, CD31 / PECAM-1, latex, CD36 / SR-B3, leukotriene B4 R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD8 4 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, endothelin / CD105, osteoactivin / GPNMB, Fc-γRI / CD64, osteopontin, Fc-γRIIB / CD32b, PD-L2, Fc-γRIIC / CD32c, Siglec-3 / CD33, Fc-γRIIA / CD32a, SIGNR1 / CD209, Fc-γRIII / CD16, SLAM, GM-CSF Rα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γR1, TLR4, IFN-γR2, TREM-1, IL-1 RII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF 4, IL-10Rα, ALCAM, IL-10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, C1q R1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / CD11b, CCR8, integrin αX / CD11c, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSF In some embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds to one or more of these illustrative macrophage / monocyte antigens.

[0547] In some embodiments, a multispecific PD-1 or PD-L1 binding agent of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a target (e.g., an antigen, a receptor) associated with a dendritic cell. In some embodiments, the targeting moiety directly or indirectly recruits dendritic cells, for example, in some embodiments, to a treatment site (e.g., a location with one or more diseased cells or cells to be modulated for therapeutic effect). Illustrative dendritic cell antigens of interest include, for example, Clec9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23p40, 4-amino-1,8-naphthalene dicarboxamide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, rutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerhans protein, B7-2 / CD86, leukotriene B4 R1, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, Clq R1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LB CCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAML1, CD2F-10 / SLAMF9, osteoactivin GPNMB, Chern 23. PD-L2, CLEC-1, RP105, CLEC-2, 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, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, DEC205, and capsaicin R1. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds one or more of these illustrative DC antigens.

[0548] In some embodiments, the multispecific PD-1 or PD-L1 binding agent of the present invention comprises a targeting moiety against Clec9A, which 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, "framework regions" or "FRs" refer to the regions of the variable domain located between the CDRs. As used herein, "complementarity determining regions" or "CDRs" refer to the variable regions of a VHH that contain amino acid sequences that are capable of specifically binding to an antigenic target.

[0549] In various embodiments, a multispecific PD-1 or PD-L1 binding agent of the invention comprises a VHH directed against Clec9A having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence.

[0550] In an exemplary embodiment, the CDR1 sequence is selected from SEQ ID NO:631 to SEQ ID NO:650.

[0551] In an exemplary embodiment, the CDR2 sequence is selected from SEQ ID NO:651 to SEQ ID NO:672.

[0552] In exemplary embodiments, the CDR3 sequence is selected from SEQ ID NO: 673 to SEQ ID NO: 687; or LGR; or VIK.

[0553] In various embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from the group consisting of: R2CHCL8 (SEQ ID NO: 688); R1CHCL50 (SEQ ID NO: 689); R1CHCL21 (SEQ ID NO: 690); R2CHCL87 (SEQ ID NO: 691); R2CHCL24 (SEQ ID NO: 692); R2CHCL38 (SEQ ID NO: 693); R1CHCL16 (SEQ ID NO: 694); R2CHCL10 (SEQ ID NO: 695); R1CHCL34 (SEQ ID NO: 696); R1CHCL82 (SEQ ID NO: 697); R2CHCL3 (SEQ ID NO: 698); R2CHCL69 (SEQ ID NO: 699); R1CHCL56 (SEQ ID NO: 700); R2CHCL32 (SEQ ID NO: 701). NO:701); R2CHCL49 (SEQ ID NO:702); R2CHCL53 (SEQ ID NO:703); R2CHCL22 (SEQ ID NO:704); R2CHCL25 (SEQ ID NO:705); R2CHCL18 (SEQ ID NO:706); R1CHCL23 (SEQ ID NO:707); R1CHCL27 (SEQ ID NO:708); R2CHCL13 (SEQ ID NO:709); R2CHCL14 (SEQ ID NO:710); R2CHCL42 (SEQ ID NO:711); R2CHCL41 (SEQ ID NO:712); R2CHCL94 (SEQ ID NO:713); or R2CHCL27 (SEQ ID NO:714).

[0554] In various embodiments, the Clec9A targeting moiety comprises a VHH having a variable domain comprising at least one CDR1, CDR2 and / or CDR3 sequence as described below.

[0555] In some embodiments, the CDR1 sequence is selected from SEQ ID NO:715 to SEQ ID NO:780.

[0556] In some embodiments, the CDR2 sequence is selected from SEQ ID NO:781 to SEQ ID NO:846.

[0557] In some embodiments, the CDR3 sequence is selected from SEQ ID NO:847 to SEQ ID NO:912.

[0558] In various exemplary embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from the group consisting of: 1LEC 7 (SEQ ID NO: 913).

[0559] In various exemplary embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from any one of the above sequences without a terminal histidine tag sequence (ie, HHHHHH; SEQ ID NO: 84).

[0560] In some embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from SEQ ID Nos: 913-978 (provided above) without the HA tag (ie, YPYDVPDYGS; SEQ ID NO: 85).

[0561] In some embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from SEQ ID Nos: 913-978 (provided above) without an AAA linker.

[0562] In some embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from SEQ ID Nos: 913-978 (provided above) without the AAA linker, HA tag, and terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 86).

[0563] 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 hereby incorporated by reference.

[0564] In various embodiments, the present invention contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs of the targeting moieties for Clec9A as described herein (collectively referred to herein as "analogs"). In various embodiments, the amino acid sequence of the targeting moiety for Clec9A further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0565] In various embodiments, a chimeric protein of the invention comprises a targeting moiety comprising an amino acid sequence that is at least 60% identical to any of the sequences disclosed herein. For example, the chimeric protein can comprise a targeting moiety comprising an amino acid sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, 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 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 or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100% identical to any of the sequences disclosed herein. or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% sequence identity).

[0566] In some embodiments, the multispecific PD-1 or PD-L1 binding agent of the present invention comprises a targeting portion having an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) on an immune cell selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, 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 location with one or more disease cells or cells that are regulated for therapeutic effect).

[0567] In some embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with megakaryocytes and / or platelets. Illustrative target megakaryocyte and / or platelet antigens include, for example, GP IIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds to one or more of these illustrative megakaryocyte and / or platelet antigens.

[0568] In some embodiments, the multispecific PD-1 or PD-L1 binding agent of the present invention comprises a targeting portion having an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with red blood cells. Illustrative target red blood cell antigens include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb / IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2 (MHC-II), and rhesus monkey antigens. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting portion that binds to one or more of these illustrative red blood cell antigens.

[0569] In some embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention comprise a targeting portion having an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with mast cells. Illustrative mast cell antigens of interest include, for example, SCFR / CD117, Fc ε RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMA1, FCER1A, FCER2, TPSAB1. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds one or more of these illustrative mast cell antigens.

[0570] In some embodiments, a multispecific PD-1 or PD-L1 binding agent of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with basophils. Illustrative target basophil antigens include, for example, Fc ε RI, CD203c, CD123, CD13, CD107a, CD107b, and CD 164. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds one or more of these illustrative basophil antigens.

[0571] In some embodiments, the multispecific PD-1 or PD-L1 binding agent of the present invention comprises a targeting portion having an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with neutrophils. Illustrative target neutrophil antigens include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRIII), CD18 protein (LFA-1, CR3 and p150, 95), CD45, CD67, and CD177. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting portion that binds to one or more of these illustrative neutrophil antigens.

[0572] In some embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with eosinophils. Illustrative target eosinophil antigens include, for example, CD35, CD44, and CD69. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds to one or more of these illustrative eosinophil antigens.

[0573] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to any appropriate antigen or receptor or cell surface marker known to those skilled in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Illustrative tissue-specific markers include, but are not limited to, endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAM1, PROCR, SELE, SELP, TEK, THBD, VCAM1, VWF; smooth muscle cell surface markers such as ACTA2, MYH10, MYH11, MYH9, MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COL1A1, COL1A2, COL3A1, FAP, PH-4; epithelial cell surface markers such as CD1D, K61RS2, KRT10, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUC1, TACSTD1; neovascularization markers such as CD13, TFNA, α-vβ-3 (α V β3), E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN. In various embodiments, the PD-1 or PD-L1 binding agent comprises a targeting moiety that binds to one or more of these illustrative antigens.

[0574] In various embodiments, a multispecific PD-1 or PD-L1 binding agent of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a checkpoint marker expressed on T cells, such as one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

[0575] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to a checkpoint marker, e.g., one or more of PD-1 / PD-L1 or PD-L2, CD28 / CD80 or CD86, CTLA4 / CD80 or CD86, ICOS / ICOSL or B7RP1, BTLA / HVEM, KIR, LAG3, CD137 / CD137L, OX40 / OX40L, CD27, CD40L, TIM3 / Gal9, and A2aR.

[0576] As non-limiting examples, in various embodiments, a multispecific PD-1 or PD-L1 binding agent of the invention comprises (i) a targeting moiety to CD8; (ii) a targeting moiety to a checkpoint marker expressed on T cells, such as one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, Cd27, CD40L, TIM3, and A2aR; and / or (iii) a targeting moiety to tumor cells, and any of the modified (e.g., mutated) signaling agents described herein.

[0577] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the present invention comprise one or more targeting moieties directed against PD-1 and a PD-1-binding VHH as disclosed elsewhere herein. In some embodiments, the PD-1 or PD-L1 binding agents have one or more targeting moieties that selectively bind to a PD-1 polypeptide. In some embodiments, the PD-1 or PD-L1 binding agents comprise one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-1 polypeptide. In some embodiments, the PD-1 binding agents comprise one or more PD-1 binding agents disclosed below in combination with one or more signaling agents disclosed herein.

[0578] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody pembrolizumab (also known as MK-3475, KEYTRUDA) or a fragment thereof. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid et al. (2013) New England Journal of Medicine 369(2):134-44, US 8,354,509, and WO 2009 / 114335, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the pembrolizumab or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 979 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 980.

[0579] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody Nivolumab (also known as BMS-936558, MDX-1106, ONO-4538, OPDIVO) or a fragment thereof. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US 8,008,449 and WO 2006 / 121168, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, Nivolumab or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 981 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 982.

[0580] 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 US2008 / 0025980 and WO 2009 / 101611, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 15-18 of US2008 / 0025980: SEQ ID No: 15 of US2008 / 0025980 (SEQ ID NO: 983); SEQ ID No: 16 of US2008 / 0025980 (SEQ ID NO: 984); SEQ ID No: 17 of US2008 / 0025980 (SEQ ID NO: 985); SEQ ID No: 18 of US2008 / 0025980 (SEQ ID NO: 986); and / or a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 20-24 of US2008 / 0025980: SEQ ID No: 20 of US2008 / 0025980 (SEQ ID NO: NO: 987); SEQ ID No: 21 (SEQ ID NO: 988) of US2008 / 0025980; SEQ ID No: 22 (SEQ ID NO: 989) of US2008 / 0025980; SEQ ID No: 23 (SEQ ID NO. NO:990); SEQ ID No:24 (SEQ ID NO:991) of US2008 / 0025980.

[0581] In one embodiment, the targeting moiety comprises a light chain comprising SEQ ID NO: 18 of US2008 / 0025980 and a heavy chain comprising SEQ ID NO: 22 of US2008 / 0025980.

[0582] In one embodiment, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0583] In one embodiment, the targeting moiety comprises the PD-L2-Fc fusion protein AMP-224 disclosed in WO2010 / 027827 and WO 2011 / 066342, the entire disclosures of which are hereby incorporated by reference. In this embodiment, the targeting moiety may include a targeting domain comprising SEQ ID NO: 4 (SEQ ID NO: 992) of WO2010 / 027827 and / or a B7-DC fusion protein comprising SEQ ID NO: 83 (SEQ ID NO: 993) of WO2010 / 027827.

[0584] In one embodiment, the targeting moiety comprises the peptide AUNP 12 or any other peptide disclosed in US2011 / 0318373 or 8,907,053. For example, the targeting moiety may comprise AUNP12 having the sequence of SEQ ID NO: 994 (i.e., compound 8 of US2011 / 0318373 or SEQ ID NO: 49):

[0585] SNTSESFK(SNTSESF)FRVTQLAPKAQIKE-NH2

[0586]

[0587] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E3 or a fragment thereof as disclosed in US2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 995 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 996.

[0588] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E8 or a fragment thereof as disclosed in US2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 1E8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 997 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 998.

[0589] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1H3 or a fragment thereof as disclosed in US2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 1H3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 999 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1000.

[0590] In one embodiment, the targeting moiety comprises a VHH directed against PD-1 as disclosed in, for example, US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the VHH against PD-1 comprises SEQ ID NOs: 347-351 of US 8,907,065: SEQ ID No: 347 of US 8,907,065 (SEQ ID NO: 1001); SEQ ID No: 348 of US 8,907,065 (SEQ ID NO: 1002); SEQ ID No: 349 of US 8,907,065 (SEQ ID NO: 1003); SEQ ID No: 350 of US 8,907,065 (SEQ ID NO: 1004); or SEQ ID No: 351 of US 8,907,065 (SEQ ID NO: 1005).

[0591] In some embodiments, the PD-1 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 1001-1005 having one or more substitutions at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 (according to Kabat numbering). In some embodiments, the amino acid at position 11 is L, M, S, V, or W. In some embodiments, the amino acid at position 37 is F, Y, H, I, L, or V. In some embodiments, the amino acid at position 44 is G, E, A, D, Q, R, S, or L. In some embodiments, the amino acid at position 45 is L, R, C, I, L, P, Q, or V. In some embodiments, the amino acid at position 47 is W, L, F, A, G, I, M, R, S, V, or Y. In some embodiments, the amino acid at position 83 is R, K, N, E, G, I, M, Q, or T. In some embodiments, the amino acid at position 84 is P, A, L, R, S, T, D, or V. In some embodiments, amino acid position 103 is W, P, R, or S; 104 is G or D. In some embodiments, the amino acid at position 108 is Q, L, or R.

[0592] In one embodiment, the targeting moiety comprises any one of the anti-PD-1 antibodies or fragments thereof as disclosed in US2011 / 0271358 and WO2010 / 036959, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 25-29 of US2011 / 0271358: SEQ ID No: 25 (SEQ ID NO: 1006) of US2011 / 0271358; SEQ ID No: 26 (SEQ ID NO: 1007) of US2011 / 0271358; SEQ ID No: 27 (SEQ ID NO: 1008) of US2011 / 0271358; SEQ ID No: 28 (SEQ ID NO: 1009) of US2011 / 0271358; SEQ ID No: 29 (SEQ ID NO: 1010) of US2011 / 0271358; and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 25-29 of US2011 / 0271358. Amino acid sequences of Nos. 30-33: SEQ ID No. 30 (SEQ ID NO: 1011) of US2011 / 0271358; SEQ ID No. 31 (SEQ ID NO: 1012) of US2011 / 0271358; SEQ ID No. 32 (SEQ ID NO: 1013) of US2011 / 0271358; or SEQ ID No. 33 (SEQ ID NO: 1014) of US2011 / 0271358.

[0593] In various embodiments, the multispecific PD-1 or PD-L1 binding agent of the invention comprises one or more antibodies or antibody fragments thereof against PD-1 selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene Ltd.).

[0594] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the present invention have one or more targeting moieties directed against PD-L1. In some embodiments, the PD-1 or PD-L1 binding agents have one or more targeting moieties that selectively bind to a PD-L1 polypeptide. In some embodiments, the PD-1 or PD-L1 binding agents comprise one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L1 polypeptide. In some embodiments, the PD-L1 binding agents comprise one or more PD-1 binding agents disclosed below in combination with one or more signaling agents disclosed herein.

[0595] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab) or a fragment thereof. MEDI4736 is selective for PD-L1 and blocks the binding of PD-L1 to PD-1 and CD80 receptors. MEDI4736 and its antigen-binding fragments used in the methods provided herein comprise a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. The sequence of MEDI4736 is disclosed in WO / 2016 / 06272, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, MEDI4736 or its antigen-binding fragments used in the methods provided herein comprise a heavy chain comprising the amino acid sequence SEQ ID NO: 1015 and / or a light chain comprising the amino acid sequence SEQ ID NO: 1016.

[0596] In illustrative embodiments, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (SEQ ID NO: 1017) of WO / 2016 / 06272 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 (SEQ ID NO: 1018) of WO / 2016 / 06272.

[0597] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (also known as MPDL3280A, RG7446) or a fragment thereof. In illustrative embodiments, atezolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1019 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 1020.

[0598] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody avelumab (also known as MSB0010718C) or a fragment thereof. In illustrative embodiments, avelumab 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: 1021 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 1022.

[0599] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105) or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, BMS-936559 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1023 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1024.

[0600] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3G10 or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 3G10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1025 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1026.

[0601] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10A5 or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 10A5 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1027 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1028.

[0602] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 5F8 or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 5F8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1029 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1030.

[0603] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10H10 or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 10H10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1031 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1032.

[0604] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 1B12 or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 1B12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1033 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1034.

[0605] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 7H1 or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 7H1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1035 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1036.

[0606] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 11E6 or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 11E6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1037 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1038.

[0607] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 12B7 or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 12B7 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1039 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1040.

[0608] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 13G4 or a fragment thereof as disclosed in US2013 / 0309250 and WO2007 / 005874, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 13G4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1041 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1042.

[0609] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1E12 or a fragment thereof as disclosed in US2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 1E12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1043 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1044.

[0610] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1F4 or a fragment thereof as disclosed in US2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 1F4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1045 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1046.

[0611] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2G11 or a fragment thereof as disclosed in US2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 2G11 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1047 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1048.

[0612] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3B6 or a fragment thereof as disclosed in US2014 / 0044738, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 3B6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1049 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1050.

[0613] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3D10 or a fragment thereof as disclosed in US2014 / 0044738 and WO2012 / 145493, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 3D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1051 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1052.

[0614] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies as disclosed in US2011 / 0271358 and WO2010 / 036959, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 34-38 of US2011 / 0271358: SEQ ID No: 34 (SEQ ID NO: 1053) of US2011 / 0271358; SEQ ID No: 35 (SEQ ID NO: 1054) of US2011 / 0271358; SEQ ID No: 36 (SEQ ID NO: 1055) of US2011 / 0271358; SEQ ID No: 37 (SEQ ID NO: 1056) of US2011 / 0271358; SEQ ID No: 38 (SEQ ID NO: 1057) of US2011 / 0271358; and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 34-38 of US2011 / 0271358: SEQ ID No: 34 (SEQ ID NO: 1053) of US2011 / 0271358; SEQ ID No: 35 (SEQ ID NO: 1054) of US2011 / 0271358; Amino acid sequences of Nos. 39-42: SEQ ID No. 39 (SEQ ID NO: 1058) of US2011 / 0271358; SEQ ID No. 40 (SEQ ID NO: 1059) of US2011 / 0271358; SEQ ID No. 41 (SEQ ID NO: 1060) of US2011 / 0271358; or SEQ ID No. 42 (SEQ ID NO: 1061) of US2011 / 0271358.

[0615] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4 or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US Pat. No. 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.7A4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 2 (SEQ ID NO: 1062) of WO 2011 / 066389 and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 1063.

[0616] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 2.9D10 or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US Pat. No. 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.9D10 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: 1064) of WO 2011 / 066389 and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 1065.

[0617] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9 or a fragment thereof as disclosed in WO 2011 / 066389, US 8,779,108, and US 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.14H9 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 22 (SEQ ID NO: 1066) of WO 2011 / 066389 and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 27 (SEQ ID NO: 1067) of WO 2011 / 066389.

[0618] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.20A8 or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US Pat. No. 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.20A8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 32 (SEQ ID NO: 1068) of WO 2011 / 066389 and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 37 (SEQ ID NO: 1069) of WO 2011 / 066389.

[0619] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3.15G8 or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US Pat. No. 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 3.15G8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 42 (SEQ ID NO: 1070) of WO 2011 / 066389 and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 47 (SEQ ID NO: 1071) of WO 2011 / 066389.

[0620] In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3.18G1 or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US Pat. No. 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 3.18G1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 52 (SEQ ID NO: 1072) of WO 2011 / 066389 and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 57 (SEQ ID NO: 1073) of WO 2011 / 066389.

[0621] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A40PT or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US 2014 / 0356353, and US 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, 2.7A40PT or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 62 (SEQ ID NO: 1074) of WO 2011 / 066389 and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 67 (SEQ ID NO: 1075) of WO 2011 / 066389.

[0622] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H90PT or a fragment thereof as disclosed in WO 2011 / 066389, US Pat. No. 8,779,108, and US Pat. No. 2014 / 0356353, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the 2.14H90PT or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No: 72 (SEQ ID NO: 1076) of WO 2011 / 066389 and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 77 (SEQ ID NO: 1077) of WO 2011 / 066389.

[0623] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2016 / 061142, the entire contents of which are hereby incorporated by reference.In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of WO2016 / 061142: SEQ ID No: 18 of WO2016 / 061142 (SEQ ID NO: 1078); SEQ ID No: 30 of WO2016 / 061142 (SEQ ID NO: 1079); SEQ ID No: 38 of WO2016 / 061142 (SEQ ID NO: 1080); SEQ ID No: 46 of WO2016 / 061142 (SEQ ID NO: 1081); SEQ ID No: 50 of WO2016 / 061142 (SEQ ID NO: 1082); SEQ ID No: 54 of WO2016 / 061142 (SEQ ID NO: 1083); NO: 1083); SEQ ID No: 62 (SEQ ID NO: 1084) of WO2016 / 061142; SEQ ID No: 70 (SEQ ID NO: 1085) of WO2016 / 061142; SEQ ID No: 78 (SEQ ID NO: 1086) of WO2016 / 061142; and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 22, 26, 34, 42, 58, 66, 74, 82 and 86 of WO2016 / 061142: SEQ ID No: 22 (SEQ ID NO: 1087) of WO2016 / 061142; SEQ ID No: 26 (SEQ ID NO: 1088) of WO2016 / 061142; SEQ ID No: 34 (SEQ ID NO: 1089) of WO2016 / 061142. NO: 1089); SEQ ID No: 42 (SEQ ID NO: 1090) of WO2016 / 061142; SEQ ID No: 58 (SEQ ID NO: 1091) of WO2016 / 061142; SEQ ID No: 66 (SEQ ID NO: 1092) of WO2016 / 061142; SEQ ID No: 74 (SEQ ID NO: 1093) of WO2016 / 061142; SEQ ID No: 82 (SEQ ID NO: 1094) of WO2016 / 061142; or SEQ ID No: 86 (SEQ ID NO: 1095) of WO2016 / 061142.

[0624] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2016 / 022630, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID No: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WO2016 / 022630: SEQ ID No: 2 (SEQ ID NO: 1096) of WO2016 / 022630; SEQ ID No: 6 (SEQ ID NO: 1097) of WO2016 / 022630; SEQ ID No: 10 (SEQ ID NO: 1098) of WO2016 / 022630; SEQ ID No: 14 (SEQ ID NO: 1099) of WO2016 / 022630; SEQ ID No: 18 (SEQ ID NO: 1100) of WO2016 / 022630; SEQ ID No: 2 (SEQ ID NO: 1091) of WO2016 / 022630; No: 22 (SEQ ID NO: 1101); SEQ ID No: 26 (SEQ ID NO: 1102) of WO2016 / 022630; SEQ ID No: 30 (SEQ ID NO: 1103) of WO2016 / 022630; SEQ ID No: 34 (SEQ ID NO: 1104) of WO2016 / 022630; SEQ ID No: 38 (SEQ ID NO: 1105) of WO2016 / 022630; SEQ ID No: 42 (SEQ ID NO: 1106) of WO2016 / 022630; SEQ ID No: 46 (SEQ ID NO: 1107) of WO2016 / 022630; and / or a light chain comprising a SEQ ID NO selected from the group consisting of: Amino acid sequences of Nos. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48: SEQ ID No. 4 (SEQ ID NO: 1108) of WO2016 / 022630; SEQ ID No. 8 (SEQ ID NO: 1109) of WO2016 / 022630; SEQ ID No. 12 (SEQ ID NO: 1110) of WO2016 / 022630; SEQ ID No. 16 (SEQ ID NO: 1111) of WO2016 / 022630; SEQ ID No. 20 (SEQ ID NO: 1112) of WO2016 / 022630;SEQ ID No:24 (SEQ ID NO:1113) of WO2016 / 022630; SEQ ID No:28 (SEQ ID NO:1114) of WO2016 / 022630; SEQ ID No:32 (SEQ ID NO:1115) of WO2016 / 022630; SEQ ID No:36 (SEQ ID NO:1116) of WO2016 / 022630; SEQ ID No:40 (SEQ ID NO:1117) of WO2016 / 022630; SEQ ID No:44 (SEQ ID NO:1118) of WO2016 / 022630; or SEQ ID No:48 (SEQ ID NO:1119) of WO2016 / 022630.;

[0625] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2015 / 112900, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 38, 50, 82, and 86 of WO 2015 / 112900: SEQ ID No: 38 (SEQ ID NO: 1120) of WO 2015 / 112900; SEQ ID No: 50 (SEQ ID NO: 1121) of WO 2015 / 112900; SEQ ID No: 82 (SEQ ID NO: 1122) of WO 2015 / 112900; SEQ ID No: 86 (SEQ ID NO: 1123) of WO 2015 / 112900; and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 38, 50, 82, and 86 of WO 2015 / 112900: SEQ ID No: 38 (SEQ ID NO: 1120) of WO 2015 / 112900; SEQ ID No: 50 (SEQ ID NO: 1121) of WO 2015 / 112900; SEQ ID No: 82 (SEQ ID NO: 1122) of WO 2015 / 112900; SEQ ID No: 86 (SEQ ID NO: 1123) of WO 2015 / 112900. No: 42, 46, 54, 58, 62, 66, 70, 74 and 78 amino acid sequences: SEQ ID No: 42 (SEQ ID NO: 1124) of WO 2015 / 112900; SEQ ID No: 46 (SEQ ID NO: 1125) of WO 2015 / 112900; SEQ ID No: 54 (SEQ ID NO: 1126) of WO 2015 / 112900; SEQ ID No: 58 (SEQ ID NO: 1127) of WO 2015 / 112900; SEQ ID No: 62 (SEQ ID NO: 1128) of WO 2015 / 112900; SEQ ID No: 66 (SEQ ID NO: 1129) of WO 2015 / 112900; SEQ ID No: 70 (SEQ ID NO: 1121) of WO 2015 / 112900 NO: 1130); SEQ ID No: 74 (SEQ ID NO: 1131) of WO 2015 / 112900; or SEQ ID No: 78 (SEQ ID NO: 1132) of WO 2015 / 112900.

[0626] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2010 / 077634 and US 8,217,149, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain region comprising the amino acid sequence of SEQ ID No: 20 (SEQ ID NO: 1133) of WO 2010 / 077634 and / or a light chain variable region comprising the amino acid sequence of SEQ ID No: 21 (SEQ ID NO: 1134) of WO 2010 / 077634.

[0627] In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies obtainable from hybridomas obtainable under CNCM deposit numbers CNCM 1-4122, CNCM 1-4080, and CNCM 1-4081 as disclosed in US20120039906, the entire disclosure of which is hereby incorporated by reference.

[0628] In one embodiment, the targeting moiety comprises a VHH directed against PD-L1 as disclosed in, for example, US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the VHH against PD-L1 comprises SEQ ID Nos: 394-399 of US 8,907,065: SEQ ID No: 394 of US 8,907,065 (SEQ ID NO: 1135); SEQ ID No: 395 of US 8,907,065 (SEQ ID NO: 1136); SEQ ID No: 396 of US 8,907,065 (SEQ ID NO: 1137); SEQ ID No: 397 of US 8,907,065 (SEQ ID NO: 1138); SEQ ID No: 398 of US 8,907,065 (SEQ ID NO: 1139); or SEQ ID No: 399 of US 8,907,065 (SEQ ID NO: 1140).

[0629] In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 1135-1140 having one or more substitutions at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 (according to Kabat numbering). In some embodiments, the amino acid at position 11 is L, M, S, V, or W. In some embodiments, the amino acid at position 37 is F, Y, H, I, L, or V. In some embodiments, the amino acid at position 44 is G, E, A, D, Q, R, S, or L. In some embodiments, the amino acid at position 45 is L, R, C, I, L, P, Q, or V. In some embodiments, the amino acid at position 47 is W, L, F, A, G, I, M, R, S, V, or Y. In some embodiments, the amino acid at position 83 is R, K, N, E, G, I, M, Q, or T. In some embodiments, the amino acid at position 84 is P, A, L, R, S, T, D, or V. In some embodiments, amino acid position 103 is W, P, R, or S; 104 is G or D. In some embodiments, the amino acid at position 108 is Q, L, or R.

[0630] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the present invention have one or more targeting moieties that are directed against PD-L2. In some embodiments, the PD-1 or PD-L1 binding agents have one or more targeting moieties that selectively bind to a PD-L2 polypeptide. In some embodiments, the PD-1 or PD-L1 binding agents comprise one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L2 polypeptide.

[0631] In one embodiment, the targeting moiety comprises a VHH directed against PD-L2 as disclosed in, for example, US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In illustrative embodiments, the VHHs for PD-1 comprise SEQ ID Nos: 449-455 of US 8,907,065: SEQ ID No: 449 of US 8,907,065 (SEQ ID NO: 1141); SEQ ID No: 450 of US 8,907,065 (SEQ ID NO: 1142); SEQ ID No: 451 of US 8,907,065 (SEQ ID NO: 1143); SEQ ID No: 452 of US 8,907,065 (SEQ ID NO: 1144); SEQ ID No: 453 of US 8,907,065 (SEQ ID NO: 1145); SEQ ID No: 454 of US 8,907,065 (SEQ ID NO: 1146); or SEQ ID No: 455 of US 8,907,065 (SEQ ID NO: 1147). NO:1147).

[0632] In some embodiments, the PD-L2 targeting moiety comprises an amino acid sequence selected from SEQ ID NOs: 1141-1147 having one or more substitutions at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 (according to Kabat numbering). In some embodiments, the amino acid at position 11 is L, M, S, V, or W. In some embodiments, the amino acid at position 37 is F, Y, H, I, L, or V. In some embodiments, the amino acid at position 44 is G, E, A, D, Q, R, S, or L. In some embodiments, the amino acid at position 45 is L, R, C, I, L, P, Q, or V. In some embodiments, the amino acid at position 47 is W, L, F, A, G, I, M, R, S, V, or Y. In some embodiments, the amino acid at position 83 is R, K, N, E, G, I, M, Q, or T. In some embodiments, the amino acid at position 84 is P, A, L, R, S, T, D, or V. In some embodiments, amino acid position 103 is W, P, R, or S; 104 is G or D. In some embodiments, the amino acid at position 108 is Q, L, or R.

[0633] In various embodiments, the PD-L2 targeting moiety comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence. In various embodiments, the PD-L2 binding agent comprises a VHH having a variable region comprising at least one FR1, FR2, FR3, and FR4 sequence.

[0634] In some embodiments, the PD-L2 CDR1 sequence is selected from SEQ ID NO: 1148 to SEQ ID NO: 1154.

[0635] In some embodiments, the PD-L2 CDR2 sequence is selected from SEQ ID NO: 1155 to SEQ ID NO: 1161.

[0636] In some embodiments, the PD-L2 CDR3 sequence is selected from SEQ ID NO: 1162 to SEQ ID NO: 1168.

[0637] In one embodiment, the targeting moiety comprises any one of the anti-PD-L2 antibodies as disclosed in US 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the antibodies or antigen-binding fragments thereof used in the methods provided herein comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 43-47 of US2011 / 0271358: SEQ ID No: 43 (SEQ ID NO: 1169) of US2011 / 0271358; SEQ ID No: 44 (SEQ ID NO: 1170) of US2011 / 0271358; SEQ ID No: 45 (SEQ ID NO: 1171) of US2011 / 0271358; SEQ ID No: 46 (SEQ ID NO: 1172) of US2011 / 0271358; SEQ ID No: 47 (SEQ ID NO: 1173) of US2011 / 0271358; and / or a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 43-47 of US2011 / 0271358: SEQ ID No: 43 (SEQ ID NO: 1169) of US2011 / 0271358; SEQ ID No: 44 (SEQ ID NO: 1170) of US2011 / 0271358; Amino acid sequences of Nos. 48-51: SEQ ID No. 48 (SEQ ID NO: 1174) of US2011 / 0271358; SEQ ID No. 49 (SEQ ID NO: 1175) of US2011 / 0271358; SEQ ID No. 50 (SEQ ID NO: 1176) of US2011 / 0271358; or SEQ ID No. 51 (SEQ ID NO: 1177) of US2011 / 0271358.

[0638] In various embodiments, the targeting moiety of the present invention can comprise a sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, or at least about 77% identical to any of the sequences disclosed herein. , at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% , at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100% identical to any of the sequences disclosed herein or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99% or about 100% sequence identity) of a sequence targeting PD-1, PD-L1 and / or PD-L2.

[0639] In various embodiments, the targeting moieties of the present invention may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences as disclosed herein that target PD-1, PD-L1, and / or PD-L2.

[0640] Other antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to or target PD-1, PD-L1, and / or PD-L2 are disclosed in WO 2011 / 066389, US 2008 / 0025980, US 2013 / 0034559, US 8,779,108, US 2014 / 0356353, US 8,609,089, US 2010 / 028330, US 2012 / 0114649, WO 2010 / 027827, WO 2011 / 066342, US 8,907,065, WO 2016 / 062722, WO 2009 / 101611, WO 2010 / 027827, WO 2011 / 066342, WO 2016061142, WO 2016,02263, WO 2010 / 077634, and WO 2015 / 112900, the entire disclosures of which are hereby incorporated by reference.

[0641] In various embodiments, the multispecific PD-1 or PD-L1 binders of the present technology comprise a targeting portion for signal regulatory protein alpha-1 (SIRP1α). SIRP1α (also known as SIRPα) belongs to a family of cellular immune receptors that include inhibitory (SIRPα) members, activating (SIRPβ) members, non-signaling (SIRPγ) members, and soluble (SIRPδ) members. SIRP1α is primarily expressed on myeloid cells, which include macrophages, granulocytes, myeloid dendritic cells (DCs), mast cells, and their precursors, including hematopoietic stem cells. SIRP1α acts as an inhibitory receptor that interacts with the widely expressed transmembrane glycoprotein CD47 to regulate phagocytosis. Specifically, CD47 expressed on target cells binds to SIRP1α on macrophages to produce an inhibitory signal that negatively regulates the phagocytosis of the target cells.

[0642] In various embodiments, the SIRP1α targeting moiety is a targeting moiety that specifically recognizes and binds to SIRP1α on macrophages.

[0643] In various embodiments, the SIRP1α targeting moiety is a targeting moiety that specifically recognizes and binds to SIRP1α on monocytes.

[0644] In various embodiments, the SIRP1α targeting moiety is a targeting moiety that specifically recognizes and binds to SIRP1α on TAMs (tumor-associated macrophages).

[0645] 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 cDC2 and pDC).

[0646] In various embodiments, the SIRP1α targeting portion comprises a targeting portion having a recognition domain that recognizes SIRP1α. In one embodiment, the recognition domain recognizes one or more linear epitopes present on SIRP1α. As used herein, a linear epitope refers to any continuous amino acid sequence present on SIRP1α. In another embodiment, the recognition domain recognizes one or more conformational epitopes present on SIRP1α. As used herein, a conformational epitope refers to one or more amino acid segments (which may be discontinuous) that form a three-dimensional surface having a feature and / or shape and / or tertiary structure that can be recognized by an antigen recognition domain.

[0647] In some embodiments, the SIRP1α targeting portion can bind to the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other naturally occurring or synthetic analog, variant or mutant of SIRP1α. In one embodiment, the SIRP1α is human SIRP1α. In various embodiments, the SIRP1α targeting portion can bind to any form of human SIRP1α, including monomeric form, dimeric form, heterodimeric form, multimeric form and related forms. In one embodiment, the SIRP1α targeting portion binds to the monomeric form of SIRP1α. In another embodiment, the SIRP1α targeting portion binds to the dimeric form of SIRP1α.

[0648] In one embodiment, the SIRP1α targeting moiety comprises a recognition domain that recognizes one or more epitopes present on human SIRP1α. In one embodiment, the SIRP1α targeting moiety comprises a recognition domain that recognizes human SIRP1α with a signal peptide sequence. An exemplary human SIRP1α polypeptide with a signal peptide sequence is SEQ ID NO: 1178.

[0649] In one embodiment, the SIRP1α targeting moiety comprises a recognition domain that recognizes human SIRP1α without a signal peptide sequence. An exemplary human SIRP1α polypeptide without a signal peptide sequence is SEQ ID NO: 1179.

[0650] In one embodiment, the SIRP1α targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP1α isoform 2 (SEQ ID NO: 1180).

[0651] In one embodiment, the SIRP1α targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP1α isoform 4 (SEQ ID NO: 1181).

[0652] In various embodiments, the SIRP1α targeting moiety can be any protein-based agent capable of specific binding, such as an antibody or a derivative thereof. In one embodiment, 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 a variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain includes a variable region (V L ) and a constant region (C L ). The variable region determines the specificity of the antibody. Each variable region includes three hypervariable regions flanked by four relatively conserved framework regions (FR), also known as complementarity determining regions (CDRs). The three CDRs (referred to as CDR1, CDR2, and CDR3) contribute to the antibody's binding specificity. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0653] In some embodiments, the SIRP1α targeting moiety comprises an antibody derivative or antibody form. In some embodiments, the SIRP1α targeting moiety is a single domain antibody, a heavy chain only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain only shark antibody (VNAR), a microprotein (cysteine ​​knot protein, knottin), a DARPin; a tetranectin; an affibody; a transbody; an anticalin; an adnectin; an affilin; a microbody; a peptide aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer; an armadillo repeat protein; a Kunitz domain; a phylobody; a stradobody; a maxibody; an evibody; a fynomer; a Kunitz domain; a phylobody; a stradobody; a maxibody; a evibody; a fynomer ... domain), avimer, atrimer, probody, immunobody, triomab, troybody; pepbody; vaccibody, unibody; affimer, duobody, Fv, Fab, Fab′, F(ab′)2, peptide mimetic molecule or synthetic molecule as described in the following U.S. Patent Nos. or Patent Publication Nos.: US 7,417,130, US2004 / 132094, US 5,831,012, US2004 / 023334, US7,250,297, US 6,818,418, US2004 / 209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US2004 / 146938, US2004 / 157209, US 6,994,982, US 6,794,144, US 2010 / 239633, US 7,803,907, US2010 / 119446 and / or US 7,166,697, the contents of which are hereby incorporated by reference in their entirety. See also Storz MAbs. 2011 May-June; 3(3):310-317.

[0654] In one embodiment, the SIRP1α targeting moiety comprises a single domain antibody, such as a VHH from an organism that produces VHH antibodies (such as camels, sharks), or a designed VHH. VHHs are therapeutic proteins derived from antibodies that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional antibodies lacking light chains from camels. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3).

[0655] In one embodiment, the SIRP1α targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

[0656] In some embodiments, the VHH comprises a fully human V H domain, such as HUMABODY (Crescendo Biologics, Cambridge, UK). In some embodiments, the fully human V H The domain (e.g., HUMABODY) is monovalent, bivalent, or trivalent. In some embodiments, the fully human V H The domain (eg, HUMABODY) is monospecific or multispecific, such as monospecific, bispecific, or trispecific. H Domains such as HUMABODY are described, for example, in WO2016 / 113555 and WO2016 / 113557, the entire contents of which are incorporated by reference.

[0657] For example, in some embodiments, the SIRP1α targeting moiety comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, VHHs or fusion proteins that selectively bind to SIRP1α. In some embodiments, the SIRP1α targeting moiety comprises an antibody or derivative thereof that specifically binds to SIRP1α. In some embodiments, the SIRP1α targeting moiety is a camelid heavy chain antibody (VHH) that specifically binds to SIRP1α.

[0658] In various embodiments, the SIRP1α targeting moiety 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 known to recognize and bind to SIRP1α.

[0659] In various embodiments, the present technology contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs of the SIRP1α targeting moieties described herein (collectively referred to herein as "analogs"). 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.

[0660] 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 one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, twenty, thirty, forty, or fifty 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.

[0661] In some embodiments, the amino acid mutation is an amino acid substitution, and can include conservative and / or non-conservative substitutions.

[0662] "Conservative substitutions" can be made, for example, based on similarities in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0663] As used herein, a "conservative substitution" is defined as the exchange of one amino acid listed in the six standard amino acid groups shown above for another amino acid within the same group. For example, exchanging Asp for Glu results in a negative charge being retained in the polypeptide thus modified. In addition, glycine and proline can be substituted for each other based on their ability to disrupt alpha helices.

[0664] As used herein, a "non-conservative substitution" is defined as the exchange of one amino acid listed in different of the six standard amino acid groups (1) to (6) shown above for another amino acid.

[0665] In various embodiments, the substitutions may also include non-classical amino acids. Exemplary non-classical amino acids generally include, but are not limited to, selenocysteine, pyrrolysine, N-formylmethionine, beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoroamino acids, designer amino acids such as beta-methyl amino acids, C alpha-methyl amino acids, N alpha-methyl amino acids, and amino acid analogs.

[0666] In various embodiments, the amino acid mutation can be in a CDR (e.g., CDR1, CDR2, or CDR3) of the targeting moiety. In another embodiment, the amino acid change can be in a framework region (FR) (e.g., FR1, FR2, FR3, or FR4) of the targeting moiety.

[0667] Modification of the amino acid sequence can be achieved using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0668] In various embodiments, the mutation does not substantially reduce the ability of the SIRP1α targeting moiety to specifically recognize and bind to SIRP1α. In various embodiments, the mutation does not substantially reduce the ability of the SIRP1α targeting moiety to specifically bind to SIRP1α and does not functionally modulate (e.g., partially or fully neutralize) SIRP1α.

[0669] In various embodiments, the SIRP1α targeting moiety binds to the target antigen but does not functionally modulate the target antigen, i.e., SIRP1α. For example, in various embodiments, the SIRP1α targeting moiety merely targets the antigen but does not substantially functionally modulate (e.g., substantially inhibit, reduce, or neutralize) the biological effect of the antigen. In various embodiments, the SIRP1α targeting moiety binds to an epitope that is physically separated from an antigenic site that is important for the biological activity of the antigen (e.g., the active site of the antigen).

[0670] In other embodiments, the SIRP1α targeting portion binds to the target antigen and functionally regulates the target antigen, i.e., SIRP1α. For example, in various embodiments, the SIRP1α targeting portion targets the antigen (i.e., SIRP1α) but does not functionally regulate (e.g., inhibit, reduce, or neutralize) the biological effect of the antigen. Such binding accompanied by functional regulation can be used in various embodiments of the present invention, including methods of using the chimeric protein of the present invention to directly or indirectly recruit active immune cells to a desired site via an effector antigen.

[0671] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention comprise a targeting moiety having an antigen recognition domain that specifically binds to, for example, XCR1 on DCs. In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the invention comprise a targeting moiety having an antigen recognition domain that comprises all or part of XCL1.

[0672] In various embodiments, the multispecific PD-1 or PD-L1 binding agents of the present invention are present with a targeting portion having 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 a component of an intact cell or a cellular structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-protein non-cellular marker, including but not limited to nucleic acids, including DNA or RNA, such as, for example, DNA released from necrotic tumor cells or extracellular deposits such as cholesterol.

[0673] In some embodiments, the target of interest (e.g., antigen, receptor) is a part of the non-cellular component of the matrix or extracellular matrix (ECM) or a marker associated with the non-cellular component. As used herein, matrix refers to the connection and support structure of a tissue or organ. Matrix can include cells such as fibroblasts / myofibroblasts, glial cells, epithelial cells, fat, immune cells, blood vessels, smooth muscle and immune cells, as well as the collection of extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target of interest (e.g., antigen, receptor) is a part of the non-cellular component of the matrix such as the extracellular matrix and extracellular molecules. As used herein, ECM refers to the non-cellular components present in all tissues and organs. ECM is composed of biochemically unique components, including but not limited to a large collection of proteins, glycoproteins, proteoglycans and polysaccharides. These components of ECM are usually produced by adjacent cells and secreted into the ECM via exocytosis. Once secreted, the ECM components tend to aggregate and form a complex macromolecular network. In various embodiments, the chimeric protein of the present invention comprises a targeting moiety that recognizes a target (e.g., antigen or receptor or non-protein molecule) on any component of the ECM. Illustrative components of the ECM include, but are not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers and other ECM proteins or ECM non-proteins such as polysaccharides and / or lipids, or ECM-associated molecules (e.g., proteins or non-proteins such as polysaccharides, nucleic acids and / or lipids).

[0674] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on an ECM proteoglycan. Proteoglycans are glycosylated proteins. The basic proteoglycan unit includes a core protein with one or more covalently attached glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge, thereby attracting positively charged sodium ions (Na+), which attract water molecules via osmotic action, thereby keeping the ECM and resident cells hydrated. Proteoglycans may also help capture and store growth factors within the ECM. Illustrative proteoglycans that the chimeric protein of the present invention can target include but are not limited to heparan sulfate, chondroitin sulfate, and keratan sulfate. In one embodiment, the targeting moiety recognizes a target (e.g., antigen, receptor) on a non-proteoglycan polysaccharide such as hyaluronic acid.

[0675] In some embodiments, the targeting moiety identifies a target (e.g., antigen, receptor) on an ECM fiber. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety identifies one or more epitopes on collagen or collagen fibers. Collagen is the most abundant protein in ECM. Collagen is present in ECM in the form of fibrous proteins and provides structural support to resident cells. In one or more embodiments, the targeting moiety identifies and is bound to various types of collagen present in the ECM, including but not limited to fibrous collagen (type I, type II, type III, type V, type XI), interspersed with triple-helical fibril-related collagen (facit collagen) (type IX, type XII, type XIV), short-chain collagen (type VIII, type X), basement membrane collagen (type IV) and / or type VI, type VII or type XIII collagen. Elastin fibers provide elasticity to tissues, allowing them to stretch and then return to their original state when needed. In some embodiments, the targeting moiety identifies one or more epitopes on elastin or elastin fibers.

[0676] In some embodiments, the targeting moiety recognizes one or more ECM proteins, including but not limited to tenascin, fibronectin, fibrin, laminin, or nidogen / entactin.

[0677] In one embodiment, the targeting moiety recognizes and binds to tenascin. The glycoproteins of the tenascin (TN) family include at least four members, namely tenascin C, tenascin R, tenascin X and tenascin W. The primary structure of tenascin inc...

Claims

1. A PD-L1 binding agent comprising an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 301, 287, 290, 300, 305, 306, and 309.

2. A PD-L1 binding agent, comprising at least one targeting moiety, wherein the at least one targeting moiety comprises three complementarity determining regions (CDR1, CDR2 and CDR3), wherein: (a) CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 199, 181, 178, 183, 189, 196, 201 and 206; (b) CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 259, 260, 241, 242, 245, 246, 263 and 264; and (c) CDR3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 268, 272 and 277.

3. The PD-L1 binding agent of claim 1 or 2, wherein the targeting moiety is a single domain antibody.

4. The PD-L1 binding agent of claim 3, wherein the targeting moiety comprises VHH, humanized VHH or camelized VHH.

5. The PD-L1 binding agent of any one of the preceding claims, wherein the PD-L1 binding agent comprises one or more signaling agents.

6. The PD-L1 binding agent of claim 5, wherein the signaling agent is selected from one or more of interferon, interleukin and tumor necrosis factor, any of which is optionally modified.

7. The PD-L1 binding agent of any one of the preceding claims, wherein the PD-L1 binding agent comprises one or more additional targeting moieties.

8. The PD-L1 binding agent of claim 7, wherein the one or more additional targeting moieties recognize and optionally functionally modulate a tumor antigen.

9. The PD-L1 binding agent of claim 8, wherein the one or more additional targeting moieties recognize and optionally functionally modulate an antigen on an immune cell.

10. The PD-L1 binding agent of claim 9, wherein the immune cell is selected from the group consisting of T cells, B cells, dendritic cells, macrophages, neutrophils and NK cells.

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