CD8 binding agents

Abstract

To provide binding agents that bind CD8 and their use as therapeutic and diagnostic agents.SOLUTION: The present invention relates to, in part, agents that bind CD8 and their use as therapeutic and diagnostic agents. The present invention further relates to pharmaceutical compositions comprising the CD8 binding agents and their use in the treatment of various diseases, including, for example, cancers.SELECTED DRAWING: Figure 6B

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 542,920, filed Aug. 9, 2017. The content of this patent is hereby incorporated by reference in its entirety into this specification.

[0002] Field of the Invention The present invention relates, in part, to binding substances that bind CD8 (e.g., antibodies such as VHH, without limitation) and their use as therapeutic and diagnostic agents.

[0003] Description of the Text File Filed Electronically The content of the following text file filed electronically with this specification is hereby incorporated by reference in its entirety into this specification: computer - readable format copy of the sequence listing (file name: ORN - 032PC_ST25, creation date: Aug. 8, 2018, file size: 828 KB).

Background Art

[0004] Activated CD8 + T lymphocytes (also known as cytotoxic T cells or CTLs) are a major line of defense against a wide range of pathogenic microorganisms. Furthermore, CTLs also play an important role in anti - tumor immunity. Specifically, these effector cells can suppress carcinogenesis through mechanisms including the production of interferon (IFN) - γ and cytotoxins, the exocytosis of lytic proteins (e.g., perforin, granzyme), and the receptor - ligand binding of the FAS molecule.

[0005] Nevertheless, pathogenic microorganisms and tumor cells have developed various mechanisms to avoid immune destruction by CTLs. For example, tumors can evade immune surveillance by disabling the function of CTLs, for example, through the production of immunosuppressive cytokines by cancer cells themselves or non-cancer cells present in the tumor microenvironment and the involvement of immune checkpoint inhibition. Cancer cells have also been shown to eliminate CTLs by apoptosis.

[0006] Therefore, there remains a need for improved immunotherapeutic agents, including agents that can effectively deviate tumor avoidance and, for example, enhance anti-tumor immunity mediated by CTLs. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] In various aspects, the present invention relates to a CD8-binding substance having at least one targeting moiety that specifically binds to CD8. In various embodiments, these CD8-binding substances bind to CD8 but do not functionally regulate CD8 (including, but not limited to, not partially or completely neutralizing it). Thus, in various embodiments, the CD8-binding substances of the present invention are used, for example, to recruit CD8-expressing cells to a site of interest while signaling through CD8 to those CD8-expressing cells (i.e., the binding of the CD8-binding substance does not reduce or eliminate CD8 signaling at the site of interest). In one embodiment, the targeting moiety is a single-domain antibody (VHH). In various embodiments, the CD8-binding substance further comprises a signaling substance, such as, but not limited to, interferon, interleukin, and tumor necrosis factor, which can be modified to attenuate activity. In various embodiments, the CD8-binding substance comprises an additional targeting moiety that binds to another antigen of interest. In one embodiment, the other antigen of interest is present on tumor cells. In another embodiment, the other antigen of interest is present on immune cells. In these embodiments, the CD8-binding substances of the present invention can recruit immune cells, such as immune cells (e.g., cytotoxic T cells) that can kill and / or suppress tumors, directly or indirectly to the site of action (non-limiting examples include the tumor microenvironment, etc.).

[0008] In various embodiments, the CD8-binding substances of the present invention are used in the treatment of various diseases or disorders, such as cancer, infectious diseases, immune disorders, autoimmune diseases, and other diseases and disorders, and the present invention encompasses various treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0009]

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Mode for Carrying Out the Invention

[0010] The present invention is partly based on the discovery of substances (e.g., antibodies such as VHH, although non-limiting examples) that recognize and bind to CD8. In various embodiments, these CD8-binding substances bind to CD8 but do not functionally regulate CD8. In various embodiments, these CD8-binding substances bind to CD8 and recruit it directly or indirectly to sites where therapeutic action is required (e.g., tumors). The present invention further provides pharmaceutical compositions containing CD8-binding substances and their use in the treatment of various diseases.

[0011] CD8-binding substance In various embodiments, the CD8-binding substance of the present invention is a protein-based substance that can specifically bind to CD8. In various embodiments, the CD8-binding substance of the present invention is a protein-based substance that can specifically bind to CD8 without functionally regulating CD8 (e.g., partial or complete neutralization).

[0012] CD8 is a heterodimeric type I transmembrane glycoprotein, and its α and β chains are both composed of immunoglobulin (Ig)-like extracellular domains and short cytoplasmic tails linked to single-pass transmembrane domains by extended O-glycosylated stalks (Li et al., 2013). The cytoplasmic region of the CD8α chain contains two cysteine motifs that function as docking sites for the src tyrosine kinase p56lck (Lck). In contrast, this Lck-binding domain does not appear to be present in the β chain, suggesting that the CD8β chain is not involved in downstream signaling (Artyomov et al., 2010). CD8 functions as a coreceptor for the T cell receptor, and its basic role is to recruit Lck to the TCR-pMHC complex after coreceptor binding to MHC (Turner et al., 1990, Veillette et al., 1988). The increased local concentration of this kinase activates a signaling cascade that recruits and activates ζ chain-associated protein kinase 70 (ZAP-70), which then results in the amplification of T cell activation signals (Purbhoo et al., 2001, Laugel et al., 2007a).

[0013] In various embodiments, the CD8-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that recognizes an epitope present on the CD8α and / or β chain. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes on the CD8α and / or β chain. As used herein, a linear epitope means any continuous sequence of amino acids present on the CD8α and / or β chain. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on the CD8α and / or β chain. As used herein, a conformational epitope refers to a portion of one or more amino acids (which may be discontinuous) that forms a three-dimensional surface having features and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0014] In various embodiments, the CD8-binding substance of the present invention can bind to full-length and / or mature and / or isoform and / or splice variant and / or fragment and / or any other natural or synthetic analog, variant, or mutant of human CD8α and / or β chain. In various embodiments, the CD8-binding substance of the present invention can bind to any form of human CD8α and / or β chain, including monomer, dimer, heterodimer, multimer, and associated forms. In certain embodiments, the CD8-binding substance binds to a monomeric form of CD8α chain or CD8β chain. In another embodiment, the CD8-binding substance binds to a homodimeric form consisting of two CD8α chains or two CD8β chains. In a further embodiment, the CD8-binding substance binds to a heterodimeric form consisting of one CD8α chain or one CD8β chain.

[0015] In certain embodiments, the CD8-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on the human CD8α chain. In certain embodiments, the human CD8α chain comprises the amino acid sequence of isoform 1 (SEQ ID NO: 1).

[0016] In certain embodiments, the human CD8α chain comprises the amino acid sequence of isoform 2 (SEQ ID NO: 2).

[0017] In certain embodiments, the human CD8α chain comprises the amino acid sequence of isoform 3 (SEQ ID NO: 3).

[0018] In certain embodiments, the CD8-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on the human CD8β chain. In certain embodiments, the human CD8β chain comprises the amino acid sequence of isoform 1 (SEQ ID NO: 4).

[0019] In certain embodiments, the human CD8β chain comprises the amino acid sequence of isoform 2 (SEQ ID NO: 5).

[0020] In certain embodiments, the human CD8β chain comprises the amino acid sequence of isoform 3 (SEQ ID NO: 6).

[0021] In certain embodiments, the human CD8β chain comprises the amino acid sequence of isoform 4 (SEQ ID NO: 7).

[0022] In certain embodiments, the human CD8β chain comprises the amino acid sequence of isoform 5 (SEQ ID NO: 8).

[0023] In certain embodiments, the human CD8β chain comprises the amino acid sequence of isoform 6 (SEQ ID NO: 9).

[0024] In certain embodiments, the human CD8β chain comprises the amino acid sequence of isoform 7 (SEQ ID NO: 10).

[0025] In certain embodiments, the human CD8β chain comprises the amino acid sequence of isoform 8 (SEQ ID NO: 11).

[0026] In various embodiments, the CD8 binding substance of the present invention comprises a targeting moiety that can specifically bind. In various embodiments, the CD8 binding substance comprises a targeting moiety having an antigen recognition domain such as an antibody or a derivative thereof. In certain embodiments, the CD8 binding substance comprises a targeting moiety that is an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain comprises one variable region (V L ) and one constant region (C L ). The variable regions determine the specificity of the antibody. Each variable region comprises three highly variable regions, also known as complementarity-determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs are called CDR1, CDR2, and CDR3, and contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0027] In some embodiments, the CD8 binding substance comprises a targeting moiety that is a specific antibody derivative or antibody format. In some embodiments, the CD8 binding substance of the invention comprises the following targeting moieties: single domain antibody, recombinant heavy chain antibody (heavy chain antibody) (VHH), single chain antibody (scFv), shark heavy chain antibody (VNAR), microprotein (cysteine knot protein, knottin), DARPin; tetranectin; affibody; transbody; anticalin; adnectin; affilin; affimer; microbody; aptamer; alterase; plastic antibody; filomer; stradbody; maxibody; evibody; finomer; armadillo repeat protein; knotted domain, avimer, atrimer, probody, immunobody, triomab, tribody, pepbody, waxibody, unibody; duoibody, Fv, Fab, Fab’, F(ab’)2, peptidomimetic molecule, or synthetic molecule. These are described in U.S. Patent No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Patent No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Patent No. 7,250,297, U.S. Patent No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Patent No. 7,838,629, U.S. Patent No. 7,186,524, U.S. Patent No. 6,004,746, U.S. Patent No. 5,475,096, U.S. Patent Application Publication No. 2004 / 146938, U.S. Patent Application Publication No. 2004 / 157209, U.S. Patent No. 6,994,982, U.S. Patent No. 6,794,144, U.S. Patent Application Publication No. 2010 / 239633, U.S. Patent No. 7,803,907, U.S. Patent Application Publication No. 2010 / 119446, and / or U.S. Patent No. 7,166,697, the entire contents of which are incorporated herein by reference. See also Storz MAbs. 2011 May-Jun;3(3):310-317.

[0028] In some embodiments, the CD8-binding substance comprises a targeting moiety that is a single-domain antibody such as a VHH. The VHH can be derived, for example, from organisms that produce VHH antibodies such as camels, sharks, or the VHH can be a designed VHH. The VHH is a therapeutic protein derived from an antibody that includes the unique structural and functional properties of naturally occurring heavy-chain antibodies. The VHH technology is based on fully functional antibodies from camels that lack a light chain. These heavy-chain antibodies comprise a single variable domain (V H H) and two constant domains (CH2 and CH3).

[0029] In one embodiment, the CD8-binding substance comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

[0030] In some embodiments, the VHH comprises a fully human V H domain, such as a HUMABODY (Crescendo Biologics, Cambridge, UK). In some embodiments, the fully human V H domain, such as a HUMABODY, is monovalent, bivalent, or trivalent. In some embodiments, the fully human V H domain, such as a HUMABODY, is monospecific or multispecific, such as monospecific, bispecific, or trispecific. Exemplary fully human V H domains, such as HUMABODIES, are described, for example, in International Publication Nos. WO 2016 / 113555 and WO 2016 / 113557. The entire disclosures of these are incorporated herein by reference.

[0031] In some embodiments, the CD8 binding substance comprises a targeting moiety that is a VHH comprising a single amino acid chain having four "framework regions" or FRs and three "complementary determining regions" or CDRs. As used herein, "framework region" or "FR" means a region in the variable domain located between CDRs. As used herein, "complementary determining region" or "CDR" refers to a variable region in a VHH that comprises an amino acid sequence capable of specifically binding to an antigenic target.

[0032] In various embodiments, the CD8 binding substance comprises a VHH having a variable domain that comprises at least one of the CDR1, CDR2, and / or CDR3 sequences.

[0033] In some embodiments, the CDR1 sequence is selected from the following: GRSFSSYTLA (SEQ ID NO: 12); GRTFSSYTMG (SEQ ID NO: 13); GRTFSSYIMG (SEQ ID NO: 14); GRTFSSYTMG (SEQ ID NO: 15); GRTSGRTFSSYTMG (SEQ ID NO: 16); GRTFSSYAMG (SEQ ID NO: 17); GLTFSNYIMG (SEQ ID NO: 18); GRTFSSYTMG (SEQ ID NO: 19); GRTFSSDTMG (SEQ ID NO: 20); GLTFSNYIMG (SEQ ID NO: 21); GFTLDYYGIG (SEQ ID NO: 22); GHTFSSYTMG (SEQ ID NO: 23); GRTFSSYVIG (SEQ ID NO: 24); GFAFDGYAIG (SEQ ID NO: 25); GFAFGFFDMT (SEQ ID NO: 26); GRTFSNYVIG (SEQ ID NO: 27); GSIFSINVMG (SEQ ID NO: 28); GRTFSNYNVG (SEQ ID NO: 29); GHTFSSYTMG (SEQ ID NO: 30); GRTFSTYPVG (SEQ ID NO: 31); GRTFSNYAMG (SEQ ID NO: 32); GRTFSDYRMG (SEQ ID NO: 33); GLTFSNYIMA (SEQ ID NO: 34); GRTFSNSVMG (SEQ ID NO: 35); GRTFSSYIIG (SEQ ID NO: 36); GRTFSSYVMG (SEQ ID NO: 37); GGTFSNYVMG (SEQ ID NO: 38); GRTFSNYGIG (SEQ ID NO: 39); GFTFDDYAIA (SEQ ID NO: 40); GRTFSSYTVA (SEQ ID NO: 41); GFPFDDYAIA (SEQ ID NO: 42); GRTFSSYVMG (SEQ ID NO: 43); GRTLSSNPMA (SEQ ID NO: 44); GFTFDNYAIG (SEQ ID NO: 45); GRAFSSYFMG (SEQ ID NO: 46); TPTFSSYNMG (SEQ ID NO: 47); GFTFDDYAIA (SEQ ID NO: 48); GGTFSGYIMG (SEQ ID NO: 49); GRSFSSYTIA (SEQ ID NO: 50); GFSSDDYTIG (SEQ ID NO: 51); GFTFDDYTIG (SEQ ID NO: 52); GFSSDDYTIG (SEQ ID NO: 53); GFTFDQYTIA (SEQ ID NO: 54); GRTFSSYAMA (SEQ ID NO: 55); GFAFDGYAIG (SEQ ID NO: 56); GFSSDDYTIA (SEQ ID NO: 57); GFSSDDYTIG (SEQ ID NO: 58); GFTFDDYTIG (SEQ ID NO: 59); GFSSDDYTIG (SEQ ID NO: 60); GFSSDDYTIG (SEQ ID NO: 61); GFSFDDYAIA (SEQ ID NO: 62); GFSSDDYTIG (SEQ ID NO: 63);GFTGNDLAIG (SEQ ID NO: 64); GFSSDDYTIA (SEQ ID NO: 65); EGTLSSYGIG (SEQ ID NO: 66); GFSSDDYTIA (SEQ ID NO: 67); GFTFDDYAIA (SEQ ID NO: 68); GLSSDDYTIG (SEQ ID NO: 69); GLSSDDYTIG (SEQ ID NO: 70); GFSSDDYTIG (SEQ ID NO: 71); GFSFDDYTIG (SEQ ID NO: 72); GFTFDDYAIA (SEQ ID NO: 73); GFTFDDYAIG (SEQ ID NO: 74); GFTFGDYTIG (SEQ ID NO: 75); EGTFSSYGIG (SEQ ID NO: 76); GFSSDDYTIG (SEQ ID NO: 77); GVSIGDYNIG (SEQ ID NO: 78); GFTFDDYTIA (SEQ ID NO: 79); GFTFDDYTIA (SEQ ID NO: 80).;

[0034] In some embodiments, the CDR2 sequence is selected from the following: ASITWGGGNTY (SEQ ID NO: 81); AATVWTGAGTV (SEQ ID NO: 82); AAIGWSADITV (SEQ ID NO: 83); AFIDWSGGGTY (SEQ ID NO: 84); ATITWGGGSTY (SEQ ID NO: 85); AAISWSGGPTV (SEQ ID NO: 86); AAITWGGGSTV (SEQ ID NO: 87); AAITWSGVSTV (SEQ ID NO: 88); GAIMWSGAFTH (SEQ ID NO: 89); AAITWGGGSTV (SEQ ID NO: 90); SCISSSDRNTY (SEQ ID NO: 91); AFIDWSGGGTY (SEQ ID NO: 92); AVITWSGDSTY (SEQ ID NO: 93); ACISSKDGSTY (SEQ ID NO: 94); SGINSIGGSTT (SEQ ID NO: 95); AVVTWSGDSTY (SEQ ID NO: 96); AKITNFGITS (SEQ ID NO: 97); SFISWISDITY (SEQ ID NO: 98); AFIDWSGGGTY (SEQ ID NO: 99); AVILWSGVSTY (SEQ ID NO: 100); AAIVWSGGSTY (SEQ ID NO: 101); AAISSSGYHTY (SEQ ID NO: 102); SCISSPDGSTY (SEQ ID NO: 103); AAVLWSGVSTA (SEQ ID NO: 104); VAITWDGSATT (SEQ ID NO: 105); AAIGWNGGITY (SEQ ID NO: 106); GFITWSGASTY (SEQ ID NO: 107); AGINWSGESAD (SEQ ID NO: 108); SCIERSDGSTY (SEQ ID NO: 109); SCISNTDSSTY (SEQ ID NO: 110); SCISNTDSSTY (SEQ ID NO: 111); AQISWSAGSIY (SEQ ID NO: 112); AGMSWNPGPAV (SEQ ID NO: 113); SCISRSDGSTY (SEQ ID NO: 114); ANIGWTGDMTY (SEQ ID NO: 115); AAIIWSGSMTY (SEQ ID NO: 116); SCISNTDSSTY (SEQ ID NO: 117); AANTWSGGPTY (SEQ ID NO: 118); SCISSDGSTG (SEQ ID NO: 119); SCYSSSDGSTG (SEQ ID NO: 120); SCISSDGSTG (SEQ ID NO: 121); GCIKSSDGTTG (SEQ ID NO: 122); SCISNTDSSTY (SEQ ID NO: 123); AAIAWSAGSTY (SEQ ID NO: 124); SCISSKEGSTY (SEQ ID NO: 125); SCISSSDGSTG (SEQ ID NO: 126); SCYSSRDGTTG (SEQ ID NO: 127); SCISSDGSTG (SEQ ID NO: 128);SCYSSSDGSTG (SEQ ID NO: 129); SCFSSSDGSTG (SEQ ID NO: 130); SCISNTDSSTF (SEQ ID NO: 131); SCYSSSDGSTG (SEQ ID NO: 132); SCISNTDSSTY (SEQ ID NO: 133); SCISSSDGSTG (SEQ ID NO: 134); GGINWSGDSTD (SEQ ID NO: 135); SCFSSSDGSAG (SEQ ID NO: 136); SCISNTDSSTY (SEQ ID NO: 137); SCFSTRDGNAG (SEQ ID NO: 138); SCFSSRDGSTG (SEQ ID NO: 139); SCFSSRDGSTG (SEQ ID NO: 140); SCISSDGSTG (SEQ ID NO: 141); SCISNTDSSTY (SEQ ID NO: 142); SCISSPDGSTY (SEQ ID NO: 143); SCYSSSDGNTG (SEQ ID NO: 144); GGINWSGDSTD (SEQ ID NO: 145); SCFSSSDGSTG (SEQ ID NO: 146); SCISSGDGTTY (SEQ ID NO: 147); SCISSDGSTG (SEQ ID NO: 148); SCISSDGSTG (SEQ ID NO: 149); and SSISRSDGSTY (SEQ ID NO: 1221).;

[0035] In some embodiments, the CDR3 sequence is selected from the following: AKGLRNSDWDLRRGYEYDY (SEQ ID NO: 150); ADQASVPPPYGSERYDIASPSEYDY (SEQ ID NO: 151); ANSRAYYSSSYDLGRLASYDY (SEQ ID NO: 152); AAQRLGSVTDYTKYDY (SEQ ID NO: 153); ASVKVVAGSGIDISGSRNYDY (SEQ ID NO: 154); AKRLDYSATDKGVDLSDEYDY (SEQ ID NO: 155); AAGGSGRLRDLKVGQNYDY (SEQ ID NO: 156); ADSPPRTYSSGSVNLEDGSEYDY (SEQ ID NO: 157); VIPGRGSALPIDVGKSDEYEY (SEQ ID NO: 158); AAGASGRLRDLKVGQNYDY (SEQ ID NO: 159); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 160); AAQRLGSVTDYTKYDY (SEQ ID NO: 161); AIPPRAYSGGSYSLKDQSKYEY (SEQ ID NO: 162); ADGNVWSPPICSSAGPPPGGMDY (SEQ ID NO: 163); KSRSSYSNN (SEQ ID NO: 164); AMPPRAYTGRSVSLKDQSKYEY (SEQ ID NO: 165); LDTTGWGPPPYQY (SEQ ID NO: 166); AHPPDPSRGGEWRLQTPSEYDY (SEQ ID NO: 167); AAQRLGSVTDYTKYDY (SEQ ID NO: 168); VPRSHFTTAQDMGQDMGAPSWYEY (SEQ ID NO: 169); AVLIRYYSGGYQGLSDANEYDY (SEQ ID NO: 170); VVKYLSGSYSYAGQYNF (SEQ ID NO: 171); ADFNVWSPPICGSVGPPPGGMDY (SEQ ID NO: 172); AHESTYYSGTYYLTDPRRYVY (SEQ ID NO: 173); AVPARGLTMDLENSDIYDH (SEQ ID NO: 174); AATLQVTGSYYLDLSTVDIYDN (SEQ ID NO: 175); ATLFRSNGPKDLSSGYEYDY (SEQ ID NO: 176); AGESGVWVGGLDY (SEQ ID NO: 177); VGSANSGEFRFGWVLKPDLYNY (SEQ ID NO: 178); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 179); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 180); ERGYAYCSDDGCQRTQDYDY (SEQ ID NO: 181); GAARAWWSGSYDYTRMNNYDY (SEQ ID NO: 182);AETSADSGEFRFGWVLKPSLYDY (SEQ ID NO: 183); AAGSAYSGSYWNITMAANYDY (SEQ ID NO: 184); AQRIFGAQPMDLSGDYEY (SEQ ID NO: 185); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 186); ARDYRGIKDLDLKGDYDY (SEQ ID NO: 187); ADFNVWSPPICGSIWYGPPPRGMDY (SEQ ID NO: 188); ADSNVWSPPICGSRWYGPPPGGMAY (SEQ ID NO: 189); ADFNVWSPPICGSNWYGPPPGGMDY (SEQ ID NO: 190); ADFNVWSPPICGSIWYGPPPGGMDY (SEQ ID NO: 191); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 192); ARIITVATMRLDSDYDY (SEQ ID NO: 193); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 194); ADSNVWSPPICGRTWYGPPPGGMDY (SEQ ID NO: 195); ADFNVWSPPICGSIWYGPPPGGMAY (SEQ ID NO: 196); ADFNVWSPPICGSNWYGPPPGGMDY (SEQ ID NO: 197); ADFNVWSPPICGSSWYGPPPGGMDY (SEQ ID NO: 198); ADFNVWSPPICGSRWYGPPPGGMEY (SEQ ID NO: 199); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 200); ADFNVWSPPICGSRWYGPPPGGMAY (SEQ ID NO: 201); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 202); ADSNVWSPPICGKTWYGPPPGGMDY (SEQ ID NO: 203); AGESGVWVGGLDY (SEQ ID NO: 204); ADSNVWSPPICGSTWYGPPPGGMAY (SEQ ID NO: 205); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 206); ADFNVWSPPICGSRWYGPPPGGMDY (SEQ ID NO: 207); ADFNVWSPPICGSRWYGPPPGGMDY (SEQ ID NO: 208); ADFNVWSPPICGSRWYGPPPGGMDY (SEQ ID NO: 209); ADFNVWSPPICGSIWYGPPPGGMDY (SEQ ID NO: 210); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 211); ADFNVWSPPICGSVGPPPGGMDY (SEQ ID NO: 212);ADFNVWSPPICGSSWYGPPPGGMAY (SEQ ID NO: 213); AGESGVWVGGLDY (SEQ ID NO: 214); ADFNVWSPPICGSSWYGPPPGGMEY (SEQ ID NO: 215); ADGNVWSPPICGSAGPPPGGMDY (SEQ ID NO: 216); ADFNVWSPPICSSNWYGPPPRGMDY (SEQ ID NO: 217); ADFNVWSPPICGSIWYGPPPRGMDY (SEQ ID NO: 218).;

[0036] In various embodiments, the CD8 binding substance comprises an amino acid sequence selected from the following sequences: 1CDA7 (SEQ ID NO: 219) QVQLQESGGGLVQAGGSLRLSCAASGRSFSSYTLAWFRQAPGKEREFVASITWGGGNTYYPDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAAKGLRNSDWDLRRGYEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA12 (SEQ ID NO: 220) QVQLQESGGGLVQDGGSLRLSCAFSGRTFSSYTMGWFRQGPGKEREFVAATVWTGAGTVYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAADQASVPPPYGSERYDIASPSEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA14 (SEQ ID NO: 221) QVQLQESGGGLVQAGASLRLSCAASGRTFSSYIMGWFRQAPGKEREFVAAIGWSADITVYADSVKGRFTISRDNAENMVYLQMNSLNPEDTAVYYCAANSRAYYSSSYDLGRLASYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA15 (SEQ ID NO: 222) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYTMGWFRQAPGKEREFVAFIDWSGGGTYYDDSVKGRFTISRDNAENTVYLQMNNLEPEDTAVYYCAAAQRLGSVTDYTKYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA17 (Accession No. 223) QVQLQESGGGLVQAGGSLRLSCAASGRTSGRTFSSYTMGWFRQAPGKEREFVATITWGGGSTYYADSVKGRFTISRDNANNTVYLQMNSLKPEDTAVYYCAASVKVVAGSGIDISGSRNYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA18 (Accession No. 224) QVQLQESGGGLVQPGGSLRLSCLASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGPTVYADHVKGRFTISRDNAKNTVYLQVNSLKPEDTADYYCAAKRLDYSATDKGVDLSDEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA19 (Accession No. 225) QVQLQESGGGLVQAGDSLRLSCAASGLTFSNYIMGWFRQAPGKEREFVAAITWGGGSTVYADSVEGRFTISRDGTKNTVSLQMNSLLPEDTAVYYCAAAGGSGRLRDLKVGQNYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA24 (Accession No. 226) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYTMGWFRQAPGREREFVAAITWSGVSTVYTDSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAADSPPRTYSSGSVNLEDGSEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA26 (Accession No. 227) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSDTMGWFRQAPGKEREFVGAIMWSGAFTHYADSVKGRFTISRDNAKNTVYLQMNALKPEDTAVYYCAVIPGRGSALPIDVGKSDEYEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA28 (Accession No. 228) QVQLQESGGGLVQAGDSLRLSCAASGLTFSNYIMGWFRQAPGKEREFVAAITWGGGSTVYADSVEGRFTISRDGTKNTVSLQMNSLQPEDTAVYYCAAAGASGRLRDLKVGQNYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA37 (Accession No. 229) QVQLQESGGGLVQAGGSLRLSCAGSGFTLDYYGIGWFRQAPGKEREGVSCISSSDRNTYYADSVKGRFTISGDNAKNTVYLQMNNLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA43 (Accession No. 230) QVQLQESGGGLVQAGGSLRLSCVASGHTFSSYTMGWFRQAPGKEREFVAFIDWSGGGTYYANSVKGRFTISRDNAENTVYLQMNNLKPEDTAVYYCAAAQRLGSVTDYTKYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA45 (Accession No. 231) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYVIGWFRQAPGKEREFVAVITWSGDSTYSSDSLKGRFTISRDNAKNTVYLQMNALNPEDTAVYYCAAIPPRAYSGGSYSLKDQSKYEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA47 (Accession No. 232) QVQLQESGGGLVQAEGSLKLSCISGFAFDGYAIGWFRQAPGKEREGVACISSKDGSTYYADSVKGRFTMSVDKTKNTVYLQMSSLKPEDTAVYYCAADGNVWSPPICSSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA48 (Sequence number 233) QVQLQESGGGLVQPGGSLTLSCAASGFAFGFFDMTWVRQAPGKGLEWVSGINSIGGSTTYADSVKGRFTISRDNAKNELYLQMNSLKPDDTAVYYCAKSRSSYSNNWRPPGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA58 (Sequence number 234) QVQLQESGGGLVQARGSLTLSCAASGRTFSNYVIGWFRQAPGEEREFVAVVTWSGDSTYSSDSLKGRFTISRDNAKNTVYLQMNNLNPEDTAVYYCAAMPPRAYTGRSVSLKDQSKYEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA65 (Sequence number 235) QVQLQESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQTPGKERELVAKITNFGITSYADSAQGRFTISRGNAKNTVYLQMNSLKPEDTAVYYCNLDTTGWGPPPYQYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA68 (Sequence number 236) QVQLQESGGGLVQAGASLRLSCAASGRTFSNYNVGWFRQAPGKEREFVSFISWISDITYYSDSVKGRFIISRDNAKNMVYLQMNSLKPEDTAVYYCAAHPPDPSRGGEWRLQTPSEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA73 (Sequence number 237) QVQLQESGGGLVQAGGSLRLSCAASGHTFSSYTMGWFRQAPGKEREFVAFIDWSGGGTYYADSVKGRFTISRDNAENTVYLQMNNLKPEDTAVYYCAAAQRLGSVTDYTKYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA75 (Accession No. 238) QVQLQESGGGLVQAGGSLRLSCAASGRTFSTYPVGWFRQAPGKEREFVAVILWSGVSTYYADSVKGRFTISRDNAQNTVYLQMDSLKPEDTAVYYCAVPRSHFTTAQDMGQDMGAPSWYEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA86 (Accession No. 239) QVQLQESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVAAIVWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVLIRYYSGGYQGLSDANEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA87 (Accession No. 240) QVQLQESGGGLVQAGASLRLSCSASGRTFSDYRMGWFRQAPGKEREWVAAISSSGYHTYYADSVKGRFTISRDNAKNTGYLQMSSLKPEDTAVYYCAVVKYLSGSYSYAGQYNFWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA88 (Accession No. 241) QVQLQESGGGLVQAGDSLKLSCAASGLTFSNYIMAWFRQAPGKEREGVSCISSPDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSVGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA89 (Accession No. 242) QVQLQESGGGLVQAGGSLRLSCAASGRTFSNSVMGWFRQPPGKEREFVAAVLWSGVSTAYADSVKGRFTISRDNAKNTVYLQMNNLKPDDTAVYYCAAHESTYYSGTYYLTDPRRYVYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 1CDA92 (Sequence number 243) QVQLQESGGGLVQAGGSLRLSCVGDGRTFSSYIIGWFRQAPGNEREFVVAITWDGSATTYADSVKGRFTVSRDSAKNTAYLQMNSLKPEDTAVYYCAAVPARGLTMDLENSDIYDHWGRGTQVTVSSAAAYPYDVPDYGSHHHHH; 1CDA93 (Sequence number 244) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQALGKEREFVAAIGWNGGITYYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCAAATLQVTGSYYLDLSTVDIYDNWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA1 (Sequence number 245) QVQLQESGGGLVQAGGSLRLSCAASGGTFSNYVMGWFRQAPGKEREFVGFITWSGASTYYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAATLFRSNGPKDLSSGYEYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA5 (Sequence number 246) QVQLQESGGGLVQAGDSLRLTCTASGRTFSNYGIGWFRQAPGKEREFVAGINWSGESADYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGESGVWVGGLDYWXQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA22 (Sequence number 247) QVQLQESGGGLVQAGGSLRLSCAASGFTFDDYAIAWFRQAPGKEREGVSCIERSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAVGSANSGEFRFGWVLKPDLYNYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA28 (Accession No. 248) QVQLQESGGGLVQAGGSLRLSCTASGRTFSSYTVAWFRQSPGKEREGISCISNTDSSTYYADSVKGRFTISSDNAKSTVHLQMSSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA62 (Accession No. 249) QVQLQESGGGLVQPGGSLRLSCATFGFPFDDYAIAWFRQAPGKEREGVSCISNTDSSTYYADSVKGRFTISSDNAKNTVHLQMSSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA68 (Accession No. 250) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVAQISWSAGSIYYADSVKGRFTISNDNAKRTVYLQMNSLKPEDTAVYYCAERGYAYCSDDGCQRTQDYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA73 (Accession No. 251) QVQLQESGGGLVQAGGSLRLSCAASGRTLSSNPMAWFRQAAGKEREFVAGMSWNPGPAVYADSVKGRFTISRDSAENTVYLQMNSLKPEDTAVYYCAGAARAWWSGSYDYTRMNNYDYWGPGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA74 (Accession No. 252) QVQLQESGGGLVQAGGSLRLSCAVSGFTFDNYAIGWFRQAPGKEREGVSCISRSDGSTYYADSVRGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAETSADSGEFRFGWVLKPSLYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA74(C50S) (SEQ ID NO: 1216) QVQLQESGGGLVQAGGSLRLSCAVSGFTFDNYAIGWFRQAPGKEREGVSSISRSDGSTYYADSVRGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAETSADSGEFRFGWVLKPSLYDYWGQGTQVTVSS; 2CDA75(SEQ ID NO: 253) QVQLQESGGGLVQAGGSLRLSCAASGRAFSSYFMGWFRQTPGKEREFVANIGWTGDMTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAGSAYSGSYWNITMAANYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA77(SEQ ID NO: 254) QVQLQESGGGLVQAGGSLRLSCAASTPTFSSYNMGWFRQAPGKEREFVAAIIWSGSMTYYADSMKGRFTVSIDNAKNTVYLQMNSLKPEDTAVYYCAAQRIFGAQPMDLSGDYEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA81(SEQ ID NO: 255) QVQLQESGGGLVQAGGSLRLSCATFGFTFDDYAIAWFRQAPGKEREGISCISNTDSSTYYADSVKGRFTISSDSAKNTVHLQMSSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA87(SEQ ID NO: 256) QVQLQESGGGLVQAGGSLRLSCKASGGTFSGYIMGWFRQAPGKEREFVAANTWSGGPTYYSDSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYQCAARDYRGIKDLDLKGDYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA88 (Accession No. 257) QVQLQESGGGLVQAGDSLKLSCATSGRSFSSYTIAWFRQAPGKEREGISCISSDGSTGYADSVRGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSIWYGPPPRGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA89 (Accession No. 258) QVQLQESGGGLVQAGGYLRLSCAASGFSSDDYTIGWFRQAPGKEREGISCYSSSDGSTGFADSVKGRFTISSDNAKNTVYLQMNNLRPEDTAVYYCAADSNVWSPPICGSRWYGPPPGGMAYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA91 (Accession No. 259) QVQLQESGGGLAQVGGSLRLSCTASGFTFDDYTIGWFRQAPGKEREGISCISSDGSTGYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSNWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA92 (Accession No. 260) QVQLQESGGGLVQAGGSLRLSCAASGFSSDDYTIGWFRQAPGKEREGIGCIKSSDGTTGYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSIWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA93 (Accession No. 261) QVQLQESGGGLAQAGGSLRLSCAASGFTFDQYTIAWFRQAPGKEREGVSCISNTDSSTYYADSVKGRFTISSDNAKNTVYLQMSSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA94 (Sequence number 262) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVAAIAWSAGSTYYADSVKGRFAISRDNAENTVYLQMNSLKPEDTAVYYCAARIITVATMRLDSDYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 2CDA95 (Sequence number 263) QVQLQESGGGLVQAGGSLRLSCAASGFAFDGYAIGWFRQAPGKEREGVSCISSKEGSTYYADSVKGRFTISSDNAKNTVYLQMSSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA3 (Sequence number 264) QVQLQESGGGLVQAGGSLRLSCAASGFSSDDYTIAWFRRAPGKEREGISCISSSDGSTGYADSVKGRFTITSDSAKNTVYLQMNSLKPEDTAVYYCAADSNVWSPPICGRTWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA8 (Sequence number 265) QVQLQESGGGLVQPGGSLRLSCAASGFSSDDYTIGWFRQAPGKEREGISCYSSRDGTTGYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSIWYGPPPGGMAYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA11 (Sequence number 266) QVQLQESGGGLVQAGGSLRLSCAASGFTFDDYTIGWFRQAPGKEREGISCISSDGSTGYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSNWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA18 (Sequence number 267) QVQLQESGGGLVQAGGSLRLSCAASGFSSDDYTIGWFRQAPGKEREGISCYSSSDGSTGYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSSWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA19 (Sequence number 268) QVQLQESGGGLVQAGGSLRLSCAASGFSSDDYTIGWFRQAPGKEREGISCFSSSDGSTGFADSVKGRFTISSDNATNTVYLEMNSLKPEDTAVYYCAADFNVWSPPICGSRWYGPPPGGMEYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA21 (Sequence number 269) QVQLQESGGGLVQAGGSLRLSCATFGFSFDDYAIAWFRQAPGKEREGISCISNTDSSTFYADSVKGRFTISSDNAKNTVHLQMSSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA24 (Sequence number 270) QVQLQESGGGLVQAGGSLRLSCAASGFSSDDYTIGWFRQAPGKEREGISCYSSSDGSTGFADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAADFNVWSPPICGSRWYGPPPGGMAYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA28 (Sequence number 271) QVQLQESGGGLVQVGGSLRLSCTISGFTGNDLAIGWFRQAPGKDQREGISCISNTDSSTYYADSVKGRFTISSDNAKNTVHLQMSSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA29 (Accession No. 272) QVQLQESGGGLVQAGGSLRLSCAASGFSSDDYTIAWFRRAPGKEREGISCISSSDGSTGYADSVKGRFTISSDNAKNTVYLQMTSLKPEDTAVYYCAADSNVWSPPICGKTWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA31 (Accession No. 273) QVQLQESGGGLVQAGDSLRLSCAGSEGTLSSYGIGWFRQAPGKEREFVGGINWSGDSTDYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCAAGESGVWVGGLDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA32 (Accession No. 274) QVQLQESGGGLVQAGGSLRLSCAASGFSSDDYTIAWFRRAPGKEREGISCFSSSDGSAGYADSVKGRFTVSSDNAKNTVYLQMNSLKPEDTAVYYCAADSNVWSPPICGSTWYGPPPGGMAYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA33 (Accession No. 275) QVQLQESGGGLVQAGGSLRLSCATSGFTFDDYAIAWFRQAPGKEREGVSCISNTDSSTYYADSVKGRFTISSDNAKNTVYLQMSSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA37 (Accession No. 276) QVQLQESGGGLVQAGGSLRLSCEVSGLSSDDYTIGWFRQAPGKEREGFSCFSTRDGNAGYADSVKGRFTISSDNAKNTVYLQMNNLKPEDTAVYYCAADFNVWSPPICGSRWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA40 (Sequence number 277) QVQLQESGGGLVQAGGSLRLSCEVSGLSSDDYTIGWFRQAPGKKREGFSCFSSRDGSTGYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSRWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA41 (Sequence number 278) QVQLQESGGGLVQAGGSLRLSCAASGFSSDDYTIGWFRQAPGKEREGFSCFSSRDGSTGYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSRWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA48 (Sequence number 279) QVQLQESGGGLVQAGGSLRLSCAASGFSFDDYTIGWFRQVPGKEREGISCISSDGSTGYADSVKGRFTISSDNAKNTVYLQINSLKPEDTAVYYCAADFNVWSPPICGSIWYGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA57 (Sequence number 280) QVQLQESGGGLVQAGGSLRLSCATFGFTFDDYAIAWFRQAPGKEREGISCISNTDSSTYYADSVKGRFTISSDNAKNTVHLQMSSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA65 (Sequence number 281) QVQLQESGGGLVQAGGSLXLSCAASGFTFDDYAIGWFRQAPGKEREGVSCISSPDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSVGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA70 (Sequence number 282) QVQLQESGGGLVQAGASLRLSCKASGFTFGDYTIGWFRQAPGKEREGISCYSSSDGNTGYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAADFNVWSPPICGSSWYGPPPGGMAYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA73 (Sequence number 283) QVQLQESGGGLVQAGDSLRLSCAGSEGTFSSYGIGWFRQAPGKEREFVGGINWSGDSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGESGVWVGGLDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA83 (Sequence number 284) QVQLQESGGGLVQAGGSLRLSCAASGFSSDDYTIGWFRQAPGKEREGISCFSSSDGSTGFADSVKGRFTISSDNATNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSSWYGPPPGGMEYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA86 (Sequence number 285) QVQLQESGGGLVQAGDSLRLSCTASGVSIGDYNIGWFRQAPGKEREGVSCISSGDGTTYYTDSVKGRFTISTDNAKNTVYLQMNSLKPEDTAVYYCAADGNVWSPPICGSAGPPPGGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; 3CDA88 (Sequence number 286) QVQLQESGGGLVQAGGSLRLSCAASGFTFDDYTIAWFRQAPGGKEREGISCISSDGSTGYADSVKGRFTISSDNAKNMVYLQMNSLKPEDTALYYCAADFNVWSPPICSSNWYGPPPRGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH; or 3CDA90 (SEQ ID NO: 287) QVQLQESGGGLVQAGGSLRLSCAASGFTFDDYTIAWFRQAPGKEREGISCISSDGSTGYADSVRGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADFNVWSPPICGSIWYGPPPRGMDYWGKGTQVTVSSAAAYPYDVPDYGSHHHHHH.

[0037] In various exemplary embodiments, the CD8 binding substance comprises an amino acid sequence selected from any one of the above sequences that does not contain a terminal histidine tag sequence (i.e., HHHHHH: SEQ ID NO: 1213).

[0038] In some embodiments, the CD8 binding substance comprises an amino acid sequence selected from any one of the above sequences that does not contain a terminal HA tag (i.e., YPYDVPDYGS; SEQ ID NO: 1214).

[0039] In some embodiments, the CD8 binding substance comprises an amino acid sequence selected from any one of the above sequences that does not contain an AAA linker.

[0040] In some embodiments, the CD8 binding substance comprises an amino acid sequence selected from any one of the above sequences that does not contain an AAA linker, an HA tag, and a terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 1215).

[0041] In various embodiments, the invention contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the CD8 binding substances of the invention described herein. In various embodiments, the amino acid sequence of the CD8 binding substance further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0042] In various embodiments, the CD8 binding substance comprises a targeting moiety comprising a sequence that is at least 60% identical to any one of the sequences disclosed herein. For example, the CD8 binding substance is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99% or about 100% sequence identity) to any one of the sequences disclosed herein and may comprise a targeting moiety.

[0043] In various embodiments, the CD8 binding substance comprises a targeting moiety comprising an amino acid sequence having one or more amino acid mutations with respect to any one of the sequences disclosed herein. In various embodiments, the CD8 binding substance comprises a targeting moiety comprising an amino acid sequence having 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 15, or 20 amino acid mutations with respect to any one of the sequences disclosed herein. In some embodiments, one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0044] In some embodiments, the amino acid mutation is an amino acid substitution and can include conservative and / or non-conservative substitutions.

[0045] "Conservative substitutions" can be made, for example, based on similarities in the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the amino acid residues involved. The 20 natural amino acids can be classified into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0046] As used herein, "conservative substitution" is defined as the replacement of one amino acid by another amino acid described within the same group of the six standard amino acid groups above. For example, the replacement of Asp by Glu retains one negative charge in the polypeptide so modified. Further, glycine and proline can be substituted for each other based on their ability to disrupt α helices.

[0047] As used herein, "non-conservative substitution" is defined as the replacement of one amino acid by another amino acid described in a different group of the six standard amino acid groups (1)-(6) above.

[0048] In various embodiments, the substitution may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methylamino acids, C α-methylamino acids, N α-methylamino acids, and generally amino acid analogs).

[0049] In various embodiments, the amino acid mutation may be in the CDR of the targeting moiety (e.g., the CDR1, CDR2, or CDR3 region). In another embodiment, the amino acid change may be in the framework region (FR) of the targeting moiety (e.g., the FR1, FR2, FR3, or FR4 region).

[0050] Modification of the amino acid sequence can be achieved using techniques well known in any such art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.

[0051] In various embodiments, the mutation does not substantially reduce the ability of the CD8-binding substance of the invention that specifically binds to CD8. In various embodiments, the mutation does not substantially reduce the ability of the CD8-binding substance of the invention that specifically binds to CD8 without functionally regulating CD8.

[0052] In various embodiments, the binding affinity of the CD8-binding substance of the invention for the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other natural or synthetic analog, variant, or mutant (including monomers, dimers, heterodimers, multimers and / or associated forms) of human CD8α and / or β chain is characterized by the equilibrium dissociation constant (K D ). In various embodiments, the CD8-binding substance binds to the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other natural or synthetic analog, variant, or mutant (including monomers, dimers, heterodimers, multimers and / or associated forms) of human CD8α and / or β chain with a K D of about 1 μM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, or about 5 nM, or less than about 1 nM, and includes a targeting moiety.

[0053] In various embodiments, the CD8-binding substance includes a targeting moiety that binds to the antigen of interest, i.e., that binds to CD8 but does not functionally regulate it. For example, in various embodiments, the targeting moiety of the CD8-binding substance simply targets the antigen but does not substantially functionally regulate the antigen, e.g., it does not substantially inhibit, reduce, or neutralize the biological action of the antigen. In various embodiments, the targeting moiety of the CD8-binding substance binds to an epitope that is physically distant from the antigenic site important for biological activity (e.g., the active site of the antigen).

[0054] Such non-functional regulatory (e.g., non-neutralizing) binding is used in various embodiments of the invention including methods by which the CD8-binding substance of the invention directly or indirectly recruits effector immune cells to the required site via an effector antigen. For example, in various embodiments, in a method of reducing or removing a tumor, the CD8-binding substance of the invention can be used to directly or indirectly recruit cytotoxic T cells to tumor cells via CD8 (e.g., the CD8-binding substance can include a targeting moiety having an anti-CD8 antigen recognition domain and a targeting moiety having a recognition domain for a tumor antigen or receptor (e.g., an antigen recognition domain)). In such embodiments, it is desirable to directly or indirectly recruit CD8-expressing cytotoxic T cells without neutralizing CD8 activity. In these embodiments, CD8 signaling is an important part of the action of reducing or removing the tumor.

[0055] Therapeutic agent comprising the CD8-binding substance of the invention Chimeras and fusions with signaling substances In various embodiments, the CD8-binding substance of the invention is part of a chimera or fusion with one or more signaling substances. Accordingly, the invention provides, for example, chimeric or fusion proteins comprising a targeting moiety for CD8 and one or more signaling substances.

[0056] In various embodiments, the signaling molecule is modified to have a reduced affinity or activity for one or more of its receptors, thereby enabling attenuation of the activity of the chimeric or fusion protein (including agonism or antagonism), and / or preventing non-specific signaling or unwanted sequestration. In various embodiments, the signaling molecule is antagonistic in its wild-type form and has one or more mutations that weaken its antagonistic activity. In various embodiments, the signaling molecule is antagonistic due to one or more mutations, e.g., an agonist signaling molecule is converted to an antagonist signaling molecule, and such a converted signaling molecule may also have one or more mutations that weaken its antagonistic activity (e.g., as described in WO 2015 / 007520, the entire content of which is incorporated herein by reference).

[0057] Accordingly, in various embodiments, the signaling molecule is a modified (mutated) form of the signaling molecule having one or more modifications (e.g., mutations). In various embodiments, the mutation enables the modified signaling molecule to have one or more weakened activities such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity compared to the non-modified form, i.e., the wild-type form of the signaling molecule (e.g., by comparing the same signaling molecule in the wild-type form and the modified (e.g., mutated) form). In some embodiments, the mutation that weakens or reduces binding or affinity includes a mutation that substantially reduces or eliminates binding or activity. In some embodiments, the mutation that weakens or reduces binding or affinity is different from the mutation that substantially reduces or eliminates binding or activity. As a result, in various embodiments, the mutation enables the signaling molecule to have improved safety, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the non-modified form, i.e., the wild-type signaling molecule (e.g., by comparing the same signaling molecule in the wild-type form and the modified (e.g., mutated) form).

[0058] As described herein, a substance can have improved safety through one or more modifications, such as mutations. In various embodiments, improved safety means that the chimeric protein has lower toxicity (e.g., systemic toxicity and / or tissue / organ-related toxicity); and / or reduced or substantially eliminated side effects; and / or enhanced tolerance, reduced or substantially eliminated adverse events; and / or reduced or substantially eliminated; and / or an expanded therapeutic concentration range.

[0059] In various embodiments, the signaling molecule is modified to have one or more mutations that reduce the binding affinity or activity for one or more of its receptors. In some embodiments, the signaling molecule is modified to have one or more mutations that substantially reduce or eliminate the binding affinity or activity for the receptor. In some embodiments, the activity conferred by the wild-type signaling molecule is agonism for the receptor (e.g., activation of a cellular effect at the site of treatment). For example, the wild-type signaling molecule can activate its receptor. In such embodiments, the mutation results in a signaling molecule modified to reduce or eliminate the activating effect on the receptor. For example, the mutation can result in a signaling molecule modified to send a reduced activation signal to the target cell, or the activation signal can be eliminated. In some embodiments, the effect conferred by the wild-type signaling molecule is antagonism for the receptor (e.g., blocking or suppressing a cellular effect at the site of treatment). For example, the wild-type signaling molecule can antagonize or inhibit the receptor. In these embodiments, the mutation results in a signaling molecule modified to reduce or eliminate the antagonizing activity for the receptor. For example, the mutation can result in a signaling molecule modified to send a reduced inhibitory signal to the target cell, or the inhibitory signal can be eliminated. In various embodiments, the signaling molecule is antagonistic due to one or more mutations, e.g., an agonist signaling molecule is converted to an antagonist signaling molecule (e.g., as described in International Publication No. WO 2015 / 007520, the entire content of which is incorporated herein by reference), and such a converted signaling molecule optionally also has one or more mutations that reduce its binding affinity or activity for one or more of its receptors, or that substantially reduce or eliminate the binding affinity or activity for one or more of its receptors.

[0060] In some embodiments, the reduced affinity or activity for a receptor can be restored by the binding of one or more targeting moieties (e.g., a targeting moiety for CD8) described herein. In other embodiments, the reduced affinity or activity for a receptor is not substantially restorable by the activity of one or more targeting moieties.

[0061] In various embodiments, the chimeric proteins of the invention reduce off-target effects because their signaling moieties have mutations that weaken or eliminate the binding affinity or activity for a receptor. In various embodiments, for example, this reduction in side effects is observed compared to wild-type signaling moieties. In various embodiments, the signaling moiety is active against target cells. The reason is that the targeting moiety(ies) compensate for a defective / inadequate binding (e.g., but not limited to, and / or binding strength) required for substantial activation. In various embodiments, the modified signaling moiety is substantially inactive en route to the site of therapeutic action and has its effect substantially on specifically targeted cell types, thereby greatly reducing undesirable side effects.

[0062] In some embodiments, the signaling molecule can include one or more mutations that weaken or reduce binding or affinity for one receptor (i.e., the therapeutic receptor) and one or more mutations that substantially reduce or eliminate binding or activity for a second receptor. In such embodiments, these mutations can be at the same or different positions (i.e., the same mutation or multiple mutations). In some embodiments, the mutation(s) that reduce binding and / or activity for one receptor are different from the mutation(s) that substantially reduce or eliminate binding for another receptor. In some embodiments, the mutation(s) that reduce binding and / or activity for one receptor are the same as the mutation(s) that substantially reduce or eliminate binding for another receptor. In some embodiments, the chimeric protein has mutations that weaken binding and / or activity for the therapeutic receptor and thus enable a more controlled on-target therapeutic effect (e.g., as compared to the wild-type signaling molecule), and mutations that substantially reduce or eliminate binding and / or activity for another receptor and thus reduce side effects (e.g., as compared to the wild-type signaling molecule), of a modified signaling molecule.

[0063] In some embodiments, the substantial reduction or elimination of binding or activity is not substantially recoverable using a targeting moiety (a targeting moiety for CD8 or any other targeting moiety described herein). In some embodiments, the substantial reduction or elimination of binding or activity is recoverable using a targeting moiety. In various embodiments, the substantial reduction or elimination of binding or activity for the second receptor can also prevent adverse effects mediated by other receptors. Alternatively, or in addition, the substantial reduction or elimination of binding or activity for other receptors reduces or eliminates sequestration of the therapeutic chimeric protein away from the site of the therapeutic action, thus improving the therapeutic effect. For example, in some embodiments, this obviates the need for high doses of the chimeric protein of the invention to compensate for loss at other receptors. The ability to reduce such dosages further reduces the potential for side effects.

[0064] In various embodiments, the modified signaling molecule has reduced affinity, e.g., binding (e.g., K D ), and / or activation (e.g., when the modified signaling molecule is an agonist of its receptor, e.g., K A and / or EC 50 measurable as), and / or inhibition (e.g., when the modified signaling molecule is an antagonist of its receptor, e.g., K I and / or IC 50 measurable as) for one or more of its receptors, by one or more mutations that reduce, substantially reduce, or eliminate such affinity and / or activity. In various embodiments, the reduced affinity for the receptor of the immunomodulatory agent allows for attenuation of activity (including agonism or antagonism). In such embodiments, the modified signaling molecule has an affinity for the receptor that 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%, about 95%, or about 10% - 20%, about 20% - 40%, about 50%, about 40% - 60%, about 60% - 80%, about 80% - 100% that of the wild-type signaling molecule. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10 - 50-fold lower, about 50 - 100-fold lower, about 100 - 150-fold lower, about 150 - 200-fold lower, or 200-fold lower than the wild-type signaling molecule.

[0065] In some embodiments where the modified signaling substance has a mutation that reduces binding to one receptor and substantially reduces or eliminates binding to a second receptor, the attenuation or reduction of the binding affinity of the modified signaling substance for one receptor is less than the substantial reduction or elimination of the affinity for the other receptor. In some embodiments, the attenuation or reduction of the binding affinity of the modified signaling substance for one receptor is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% less than the substantial reduction or elimination of the affinity for the other receptor. In various embodiments, substantial reduction or elimination refers to a reduction in binding affinity and / or activity that is greater than the attenuation or reduction.

[0066] In various embodiments, the modified signaling substance comprises one or more mutations that reduce the intrinsic activity of the signaling substance to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1% compared to, for example, the wild-type signaling substance.

[0067] In some embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have a reduced affinity for its receptor that is lower than the binding affinity of the targeting moiety for that receptor. In some embodiments, this difference in binding affinity exists between the signaling agent / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity enables the signaling agent, e.g., the mutant signaling agent, to have a localized on-target effect and to minimize off-target effects that underlie side effects observed with the wild-type signaling agent. In some embodiments, this binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold lower, or at least about 25-fold, or at least about 50-fold lower, or at least about 100-fold, or at least about 150-fold lower.

[0068] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be evaluated by reflectometric interference spectroscopy under flow-through conditions, such as by Scatchard plot analysis and computer fitting of binding data (e.g., Scatchard, 1949) or as described by Brecht et al. (1993). The entire contents of these references are incorporated herein by reference.

[0069] In various embodiments, the signaling agent is one or more of an immunomodulatory agent, such as an interleukin, an interferon, and a tumor necrosis factor.

[0070] In some embodiments, the signaling molecule is an interleukin or a modified interleukin, for example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36 or a fragment, variant, analog, or family member thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulation of the proliferation of immune cells (e.g., helper T cells, B cells, eosinophils, and lymphocytes), chemotactic effects on neutrophils and T lymphocytes, and / or inhibition of interferons. Interleukin activity can be measured using assays known in the art (Matthews et al., in Lymphokines and Interferons: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20).

[0071] In some embodiments, the signaling molecule is an interferon or a modified interferon such as type I, II, and III interferons. Examples of interferons include, for example, interferon α-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21, interferon β, interferon γ, interferon κ, interferon ε, interferon τ, and interferon ω.

[0072] In some embodiments, the signaling agent is a tumor necrosis factor (TNF) or a modified form of tumor necrosis factor (TNF) or a protein of the TNF family, including but not limited to TNFα, TNFβ, LTβ, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TRAIL.

[0073] The amino acid sequences of the wild-type signaling substances described in this specification are well-known in the art. Thus, in various embodiments, the modified signaling substance has an amino acid sequence having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity) with the known wild-type amino acid sequences of the signaling substances described in this specification.

[0074] In various embodiments, the modified signaling molecule comprises an amino acid sequence having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity) to the amino acid sequence of any of the signaling molecules described herein.

[0075] 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 mutation is an amino acid substitution and can include conservative substitutions and / or non-conservative substitutions, as described elsewhere herein. In various embodiments, the substitution can also include non-classical amino acids, as described elsewhere herein.

[0076] As described herein, the modified signaling agent has mutations that affect the affinity and / or activity for one or more receptors. In various embodiments, there is a reduced affinity and / or activity for a therapeutic receptor, e.g., a receptor by which the desired therapeutic effect is mediated (e.g., agonism or antagonism). In various embodiments, the modified signaling agent has mutations that substantially reduce or eliminate the affinity and / or activity for a receptor, e.g., a receptor by which the desired therapeutic effect is not mediated (e.g., as a result of disrupted binding). Receptors for modified signaling agents, e.g., receptors for one of the cytokines, growth factors, and hormones described herein, are known in the art.

[0077] Examples of mutations that result in reduced affinity and / or activity (e.g., agonist activity) for a receptor are found in International Publication No. WO 2013 / 107791 and International Application No. PCT / EP2017 / 061544 (e.g., with respect to interferon), International Publication No. WO 2015 / 007542 (e.g., with respect to interleukin), and International Publication No. WO 2015 / 007903 (e.g., with respect to TNF), the entire contents of each of which are incorporated herein by reference. Examples of mutations that reduce the affinity and / or activity (e.g., antagonist activity) for a therapeutic receptor are found in International Publication No. WO 2015 / 007520, the entire contents of which are incorporated herein by reference.

[0078] In some embodiments, the modified signaling molecule comprises one or more mutations that reduce the affinity and / or activity for a type I cytokine receptor, type II cytokine receptor, chemokine receptor, receptor of the tumor necrosis factor receptor (TNFR) superfamily, TGF-beta receptor, receptor of the immunoglobulin (Ig) superfamily, and / or receptor of the tyrosine kinase superfamily of the signaling molecule.

[0079] In various embodiments, the receptor for the signaling molecule is a type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to, receptors for IL2 (beta subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and, similarly, receptors for thrombopoietin (TPO), prolactin, and growth hormone. Exemplary type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptor.

[0080] In various embodiments, the receptor for the signaling molecule is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits and are mainly receptors for interferons. This receptor family includes, but is not limited to, receptors for interferon alpha, interferon beta and interferon gamma, IL10, IL22, and tissue factor. Exemplary type II cytokine receptors include, but are not limited to, IFNα receptor (e.g., IFNAR1 and IFNAR2), IFNβ receptor, IFNγ receptor (e.g., IFNGR1 and IFNGR2), and type II IL receptor.

[0081] In various embodiments, the receptor for the signaling substance is a G protein-coupled receptor. The chemokine receptor has a seven-transmembrane structure and is a G protein-coupled receptor that binds to a G protein for signal transduction. Examples of chemokine receptors include, but are not limited to, CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors (XCR1). Representative chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.

[0082] In various embodiments, the receptor for the signaling substance is a TNFR family member. Tumor necrosis factor receptor (TNFR) family members share a cysteine-rich domain (CRD) formed from three disulfide bonds surrounding a CXXCXXC core motif that creates an elongated molecule. Representative tumor necrosis factor receptor families include the following: CD120a (TNFRSF1A), CD120b (TNFRSF1B), lymphotoxin beta receptor (LTBR, TNFRSF3), CD134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSF10A (TNFRSF10A), TNFRSF10B, (TNFRSF10B), TNFRSF10C (TNFRSF10C), TNFRSF10D (TNFRSF10D), RANK (TNFRSF11A), osteoprotegerin (TNFRSF11B), TNFRSF12A (TNFRSF12A), TNFRSF13B (TNFRSF13B), TNFRSF13C (TNFRSF13C), TNFRSF14 (TNFRSF14), nerve growth factor receptor (NGFR, TNFRSF16), TNFRSF17 (TNFRSF17), TNFRSF18 (TNFRSF18), TNFRSF19 (TNFRSF19), TNFRSF21 (TNFRSF21), and TNFRSF25 (TNFRSF25). In one embodiment, the TNFR family member is CD120a (TNFRSF1A) or TNF-R1. In another embodiment, the TNFR family member is CD120b (TNFRSF1B) or TNF-R2.

[0083] In various embodiments, the receptor for the signaling substance is a TGF-beta receptor. The TGF-beta receptor is a single-pass transmembrane serine / threonine kinase receptor. TGF-beta receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR3.

[0084] In various embodiments, the receptor for the signaling substance is an Ig superfamily receptor. Receptors of the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Examples of receptors of the Ig superfamily include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

[0085] In various embodiments, the receptor for the signaling substance is a tyrosine kinase superfamily receptor. Receptors of the tyrosine kinase superfamily are well known in the art. There are approximately 58 receptor tyrosine kinases (RTKs) classified into 20 subfamilies. Examples of receptors of the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms, e.g., FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0086] In one embodiment, the modified signaling substance is interferon alpha. In such embodiments, the modified IFNα substance has a reduced affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFNα substance has a substantially reduced or eliminated affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0087] Mutant forms of interferon alpha are known to those of skill in the art. In one exemplary embodiment, the modified signaling substance is an allelic form of IFNα2a having the amino acid sequence of IFNα2a (SEQ ID NO: 288).

[0088] In one exemplary embodiment, the modified signaling substance is an allelic form of IFNα2b having the amino acid sequence of IFNα2b (SEQ ID NO: 289), which differs from IFNα2a at amino acid position 23.

[0089] In some embodiments, the IFNα2 variant (IFNα2a or IFNα2b) has one or more amino acid mutations introduced at positions 144-154, for example, amino acid positions 148, 149 and / or 153. In some embodiments, the IFNα2 variant comprises one or more mutations selected from L153A, R149A, and M148A. Such variants are described, for example, in International Publication No. WO 2013 / 107791 and Piehler et al., (2000) J. Biol. Chem, 275:40425-33. The entire contents of these documents are incorporated herein by reference.

[0090] In some embodiments, the IFNα2 variant has a reduced affinity and / or activity for IFNAR1. In some embodiments, as described in International Publication No. WO 2010 / 030671, the IFNα2 variant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A. The entire contents of this patent are incorporated herein by reference.

[0091] In some embodiments, as described in International Publication No. WO 2008 / 124086, the IFNα2 variant comprises one or more mutations selected from K133A, R144A, R149A, and L153A. The entire contents of this patent are incorporated herein by reference.

[0092] In some embodiments, as described in International Publication No. WO 2015 / 007520 and International Publication No. WO 2010 / 030671, the IFNα2 variant comprises one or more mutations selected from R120E and R120E / K121E. The entire contents of these patents are incorporated herein by reference. In such embodiments, the IFNα2 variant antagonizes wild-type IFNα2 activity. In such embodiments, the mutant IFNα2 has a reduced affinity and / or activity for IFNAR1, but retains its activity for IFNAR2.

[0093] In some embodiments, the human IFNα2 variant comprises (1) one or more mutations selected from R120E and R120E / K121E (although not wishing to be bound by theory, these create an antagonist effect), and (2) one or more mutations selected from K133A, R144A, R149A, and L153A (although not wishing to be bound by theory, these enable, for example, a weakening effect on IFNAR2). In one embodiment, the human IFNα2 variant comprises R120E and L153A.

[0094] In some embodiments, the human IFNα2 variant comprises one or more mutations selected from L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, as disclosed in International Publication No. WO 2013 / 059885, the entire contents of which are incorporated herein by reference. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or L30A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or R33A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or M148A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations H57Y, E58N, Q61S, and / or L153A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations N65A, L80A, Y85A, and / or Y89A. In some embodiments, as disclosed in International Publication No. WO 2013 / 059885, the human IFNα2 variant comprises the mutations N65A, L80A, Y85A, Y89A and / or D114A.

[0095] In certain embodiments, the modified signaling agent is interferon beta. In such embodiments, the modified interferon beta agent has a reduced affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFNβ agent has a substantially reduced or eliminated affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0096] In certain embodiments, the modified signaling agent is IFNβ. In various embodiments, IFNβ includes functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of IFNβ. In various embodiments, IFNβ includes IFNβ from any species. In one embodiment, the chimeric protein includes a modified murine IFNβ. In one embodiment, the chimeric protein includes a modified human IFNβ. Human IFNβ is a polypeptide having a molecular weight of approximately 22 kDa and containing 166 amino acid residues. The amino acid sequence of human IFNβ is SEQ ID NO: 290.

[0097] In some embodiments, human IFNβ is IFNβ1a, which is a glycosylated form of human IFNβ. In some embodiments, IFNβ is IFNβ1b, which is a non-glycosylated form of human IFNβ having a Met-1 deletion and a mutation of Cys-17 to Ser.

[0098] In various embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity for the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFNβ has a reduced affinity and / or activity for IFNAR1. In various embodiments, the modified IFNβ is human IFNβ and has one or more mutations at positions F67, R71, L88, Y92, I95, N96, K123, and R124. In some embodiments, the one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G. In one embodiment, the modified IFNβ includes the F67G mutation. In one embodiment, the modified IFNβ includes the K123G mutation. In one embodiment, the modified IFNβ includes the F67G and R71A mutations. In one embodiment, the modified IFNβ includes the L88G and Y92G mutations. In one embodiment, the modified IFNβ includes the Y92G, I95A, and N96G mutations. In one embodiment, the modified IFNβ includes the K123G and R124G mutations. In one embodiment, the modified IFNβ includes the F67G, L88G, and Y92G mutations. In one embodiment, the modified IFNβ includes the F67S, L88S, and Y92S mutations.

[0099] In some embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity for the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFNβ has a reduced affinity and / or activity for IFNAR2. In various embodiments, the modified IFNβ is human IFNβ and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. In some embodiments, the one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In certain embodiments, the modified IFNβ comprises the W22G mutation. In certain embodiments, the modified IFNβ comprises the L32A mutation. In certain embodiments, the modified IFNβ comprises the L32G mutation. In certain embodiments, the modified IFNβ comprises the R35A mutation. In certain embodiments, the modified IFNβ comprises the R35G mutation. In certain embodiments, the modified IFNβ comprises the V148G mutation. In certain embodiments, the modified IFNβ comprises the R152A mutation. In certain embodiments, the modified IFNβ comprises the R152G mutation. In certain embodiments, the modified IFNβ comprises the Y155G mutation. In certain embodiments, the modified IFNβ comprises the W22G and R27G mutations. In certain embodiments, the modified IFNβ comprises the L32A and R35A mutations. In certain embodiments, the modified IFNβ comprises the L151G and R152A mutations. In certain embodiments, the modified IFNβ comprises the V148G and R152A mutations.

[0100] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with C17S or C17A.

[0101] In some embodiments, the modified IFNβ has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H, in combination with other IFNβ mutations described herein.

[0102] The crystal structure of human IFNβ is known and is described in Karpusas et al., (1998) PNAS, 94(22):11813-11818. In particular, the structure of human IFNβ has been shown to include five α helices (i.e., A, B, C, D, and E) and four loop regions (i.e., AB, BC, CD, and DE loops) that connect these helices. In various embodiments, the modified IFNβ has one or more mutations in the A, B, C, D, E helices and / or the AB, BC, CD, and DE loops that reduce its binding affinity or activity for a therapeutic receptor such as IFNAR. Representative mutations are described in International Publication No. 2000 / 023114 and U.S. Patent Application Publication No. 20150011732. The entire contents of these are incorporated herein by reference. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 28-30, 32, and 33. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 36, 37, 39, and 42. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 71-73. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 92, 96, 99, and 100. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 128, 130, 131, and 134. In a representative embodiment, the modified IFNβ is a human IFNβ that includes alanine substitutions at amino acid positions 149, 153, 156, and 159. In some embodiments, the mutant IFNβ includes mutations at SEQ ID NO: 290 and W22, and the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0103] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and R27, and the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0104] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and W22, the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R27, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0105] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and L32, and the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0106] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and R35, and the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0107] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and L32, the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R35, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0108] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and F67, and the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0109] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and R71, and the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0110] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and F67, and the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R71, and the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0111] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and L88, and the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0112] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and Y92, and the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0113] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and F67, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and comprises a mutation at L88, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and comprises a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0114] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and L88, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0115] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and I95, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and further comprises a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0116] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and N96, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at Y92, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0117] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and Y92, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and comprises a mutation at I95, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and comprises a mutation at N96, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0118] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and K123, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0119] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and R124, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0120] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and K123, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R124, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0121] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and L151, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0122] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and R152, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0123] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and L151, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0124] In some embodiments, the mutant IFNβ comprises mutations at SEQ ID NO: 290 and V148, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0125] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and V148, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further comprises a mutation at R152, where the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0126] In some embodiments, the mutant IFNβ comprises mutations at positions 290 and R155, where the mutations are aliphatic hydrophobic residues selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0127] In some embodiments, the present invention relates to a chimeric protein comprising (a) a modified IFNβ having the amino acid sequence of SEQ ID NO: 290 and a mutation at position W22, where the mutation is an aliphatic hydrophobic residue; and (b) one or more targeting moieties, where the targeting moiety comprises a recognition domain that specifically binds to an antigen or receptor of interest (e.g., CD8), and the modified IFNβ and the targeting moiety may optionally be linked by one or more linkers. In various embodiments, the mutation at position W22 is an aliphatic hydrophobic residue selected from G, A, L, I, M, and V. In various embodiments, the mutation at position W22 is G.

[0128] Additional representative IFNβ variants are provided in International Application No. PCT / EP2017 / 061544. The entire disclosure of this is incorporated herein by reference.

[0129] In certain embodiments, the modified signaling agent is interferon gamma. In such embodiments, the modified interferon gamma agent has a reduced affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains. In some embodiments, the modified interferon gamma agent has a substantially reduced or eliminated affinity and / or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains.

[0130] In some embodiments, the modified signaling substance is vascular endothelial growth factor (VEGF). VEGF plays an important role in both physiological and pathological angiogenesis, is a powerful growth factor that regulates vascular permeability and can act as a growth factor on cells expressing VEGF receptors, and further functions include, in particular, stimulation of cell migration of macrophage lineages and endothelial cells. In addition to at least three receptors (VEGFR1, VEGFR2, and VEGFR3), there are several members of the VEGF growth factor family. Members of the VEGF family can bind and activate two or more types of VEGFRs. For example, VEGF-A can bind VEGFR1 and VEGFR2, while VEGF-C can bind VEGFR2 and VEGFR3. Activation of VEGFR1 and VEGFR2 regulates angiogenesis, and activation of VEGFR3 is involved in lymphangiogenesis. Most angiogenesis-promoting signals are generated from activation of VEGFR2. It has been reported that VEGFR1 activation may be associated with a negative role in angiogenesis. It has also been reported that VEGFR1 signaling is important for in vivo progression through bone marrow-derived VEGFR1-positive cells in tumors (contributing to the formation of the pre-metastatic microenvironment in bone). Several VEGF-A-based therapies that target / neutralize therapeutic antibodies have been developed mainly for use in the treatment of various human tumors that depend on angiogenesis. However, these do not have no side effects. This is not surprising considering that they act as general non-cell / tissue-specific VEGF / VEGFR interaction inhibitors. Therefore, it would be desirable to limit VEGF (e.g., VEGF-A) / VEGFR2 inhibition to specific target cells (e.g., tumor vascular endothelial cells).

[0131] In some embodiments, the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E and VEGF 121 , VEGF 121 b, VEGF 145 , VEGF 165 , VEGF 165 b, VEGF 189, and VEGF 206 These isoforms include various isoforms of VEGF-A such as. In some embodiments, the modified signaling substance 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 substance has a reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In certain embodiments, the modified signaling substance has a reduced affinity and / or activity for VEGFR-2 (KDR / Flk-1) and / or a reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1). Such embodiments are used, for example, in wound healing methods or the treatment of ischemia-related diseases (not intended to be bound by theory, but mediated by the effect of VEGFR2 on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1 (Flt-1) associated with cancer and pro-inflammatory activity is avoided. In various embodiments, VEGFR-1 (Flt-1) functions as a decoy receptor, thus substantially reducing or eliminating the affinity for this receptor and avoiding sequestration of the therapeutic agent. In certain embodiments, the modified signaling substance has a reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or a reduced or eliminated affinity and / or activity for VEGFR-2 (KDR / Flk-1). In such embodiments, the modified signaling substance has a reduced affinity and / or activity for VEGFR3. Alternatively, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for VEGFR3.

[0132] Angiogenesis-promoting therapies are also important for various diseases (e.g., ischemic heart disease, bleeding, etc.) and include VEGF-based therapeutic agents. Activation of VEGFR2 is angiogenesis-promoting (acting on endothelial cells). VEGFR1 can cause stimulation of the migration of inflammatory cells (including, for example, macrophages) and lead to inflammation associated with vascular hyperpermeability. Activation of VEGFR1 can also activate bone marrow associated with tumor microenvironment formation. Therefore, VEGF-based therapeutic agents that are selective for VEGFR2 activation would be desirable in this case. Further, for example, cells that specifically target endothelial cells would be desirable.

[0133] In some embodiments, the modified signaling agent has a reduced affinity and / or activity (e.g., antagonistic) for VEGFR-2 and / or a substantially reduced or eliminated affinity and / or activity for VEGFR-1. When targeting tumor vasculature endothelial cells via a targeting moiety that binds to a tumor endothelial cell marker (e.g., PSMA, etc.), such constructs specifically inhibit VEGFR2 activation on such marker-positive cells, but do not activate VEGFR1 en route to and on the target cells (when the activity is eliminated), and thus, for example, eliminate the induction of an inflammatory response. This would provide a more selective and safer anti-angiogenic pharmacotherapy for many tumor types compared to VEGF-A neutralizing therapy.

[0134] In some embodiments, the modified signaling agent has a reduced affinity and / or activity (e.g., agonist activity) for VEGFR-2 and / or a substantially reduced or eliminated affinity and / or activity for VEGFR-1. By targeting vascular endothelial cells, in some embodiments, such constructs promote angiogenesis without causing the induction of an inflammatory response associated with VEGFR1. Therefore, such constructs would have a targeted angiogenesis-promoting effect with a substantially reduced risk of side effects resulting from systemic activation of VEGFR2 as well as VEGFR1.

[0135] In one exemplary embodiment, the modified signaling agent is VEGF having the amino acids of SEQ ID NO: 291 165 is.

[0136] In another exemplary embodiment, the modified signaling agent is VEGF having the amino acid sequence of SEQ ID NO: 292 165b is.

[0137] In these embodiments, the modified signaling agent has a mutation at amino acid I83 (e.g., a substitution mutation at I83, e.g., I83K, I83R, or I83H). Without intending to be bound by theory, such mutations are thought to result in reduced receptor binding affinity. See, for example, U.S. Patent No. 9,078,860. The entire content of this is incorporated herein by reference.

[0138] In one embodiment, the modified signaling agent is TNFα. TNF is a pleiotropic cytokine with many diverse functions, including regulation of cell proliferation, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and trafficking, inflammation, and septic shock. It binds to two separate membrane receptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits a very broad expression pattern, while TNFR2 is selectively expressed on specific populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. In response to receptor activation, entirely separate biological pathways are activated, although there is also some overlap. As a general principle, without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), and TNFR2 signaling is associated with the activation of cell survival signals (e.g., activation of the NFκB pathway). Administration of TNF is systemic toxicity, which is mainly due to the involvement of TNFR1. However, it should be noted that activation of TNFR2 is also associated with diverse effects, similar to TNFR1, and control of TNF targeting and activity is important in the development of TNF-based therapeutic agents.

[0139] In some embodiments, the modified signaling agent has a reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2. TNFR1 is expressed in most tissues and is involved in cell death signaling, whereas, in contrast, TNFR2 is involved in cell survival signaling. Thus, in embodiments relating to cancer therapies, the modified signaling agent has a reduced affinity and / or activity for TNFR1 and / or a substantially reduced or eliminated affinity and / or activity for TNFR2. In these embodiments, the chimeric protein can target cells in which apoptosis is desirable, such as tumor cells or tumor vascular endothelial cells. For example, in embodiments relating to methods for promoting cell survival in neurogenesis for the treatment of neurodegenerative disorders, the modified signaling agent has a reduced affinity and / or activity for TNFR2 and / or a substantially reduced or eliminated affinity and / or activity for TNFR1. Stated another way, the chimeric protein, in some embodiments, comprises a modified TNFα agent that can prioritize either the death or survival signal.

[0140] In some embodiments, the chimeric protein has a modified TNF having a reduced affinity and / or activity for TNFR1 and / or a substantially reduced or eliminated affinity and / or activity for TNFR2. Such chimeras are, in some embodiments, more potent inducers of apoptosis compared to chimeras having only mutations that result in a reduced affinity and / or activity for wild-type TNF and / or TNFR1. Such chimeras are, in some embodiments, used to induce tumor cell death or tumor vascular endothelial cell death (e.g., in the treatment of cancer). Also, in some embodiments, these chimeras, for example, via TNFR2, T regAvoid or reduce cell activation and thus further support TNFR1-mediated antitumor activity in vivo.

[0141] In some embodiments, the chimeric protein has a reduced affinity and / or activity for TNFR2 and / or has a modified TNF with substantially reduced or eliminated affinity and / or activity for TNFR1. Such chimeras are, in some embodiments, more potent activators of cell survival in some cell types, which can be specific therapeutic goals in various diseases, including, but not limited to, stimulation of neurogenesis. Further, such TNFR2-selective chimeras are also useful in the treatment of autoimmune diseases (e.g., Crohn's disease, diabetes, MS, colitis, etc., and many other diseases described herein). In some embodiments, the chimera targets autoreactive T cells. In some embodiments, the chimera is a T reg Promote cell activation and indirect suppression of cytotoxic T cells.

[0142] In some embodiments, the chimeric protein causes death of autoreactive T cells, for example, by activation of TNFR2 and / or avoidance of TNFR1 (e.g., by a modified TNF having a reduced affinity and / or activity for TNFR2 and / or a substantially reduced or eliminated affinity and / or activity for TNFR1). Without wishing to be bound by theory, these autoreactive T cells have altered apoptosis / survival signals due to changes in NFκB pathway activity / signal transduction. In some embodiments, the chimera causes death of autoreactive T cells having damage or alteration in the NFκB pathway underlying the imbalance in cell death (apoptosis) / survival signal properties and, optionally, altered sensitivity to specific death-inducing signals (e.g., TNFR2 activation).

[0143] In some embodiments, TNFR2-based chimeras have additional therapeutic uses for treating various autoimmune diseases, particularly diseases including heart disease, demyelinating and neurodegenerative disorders, and infectious diseases.

[0144] In one embodiment, wild-type TNFα has the amino acid sequence of SEQ ID NO: 293.

[0145] In such embodiments, the modified TNFα substance has mutations at one or more amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, which results in a modified TNFα having reduced receptor-binding affinity. See, e.g., U.S. Patent No. 7,993,636. The entire content of this patent is incorporated herein by reference.

[0146] In some embodiments, as described in International Publication No. WO 2015 / 007903, the entire content of which is incorporated herein by reference, the modified human TNFα moiety has a mutation at one or more of amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, A109, P113, Y115, E127, N137, D143, A145, and E146 (numbering according to the human TNF sequence, GenBank accession number BAG70306, version BAG70306.1 GI:197692685). In some embodiments, the modified human TNFα moiety has a substitution mutation selected from L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145R, A145T, E146D, E146K, and S147D. In certain embodiments, the human TNFα moiety has a mutation selected from Y87Q, Y87L, Y87A, Y87F, and Y87H. In another embodiment, the human TNFα moiety has a mutation selected from I97A, I97Q, and I97S. In a further embodiment, the human TNFα moiety has a mutation selected from Y115A and Y115G. In certain embodiments, the human TNFα moiety has the E146K mutation. In certain embodiments, the human TNFα moiety has the Y87H and E146K mutations. In certain embodiments, the human TNFα moiety has the Y87H and A145R mutations. In certain embodiments, the human TNFα moiety has the R32W and S86T mutations. In certain embodiments, the human TNFα moiety has the R32W and E146K mutations. In certain embodiments, the human TNFα moiety has the L29S and R32W mutations. In certain embodiments, the human TNFα moiety has the D143N and A145R mutations. In certain embodiments, the human TNFα moiety has the D143N and A145R mutations. In certain embodiments, the human TNFα moiety has the A145T, E146D, and S147D mutations.In one embodiment, the human TNFα moiety has A145T and S147D mutations.

[0147] In some embodiments, as described in International Publication No. WO 2008 / 124086, the modified TNFα substance comprises one or more mutations selected from N39Y, S147Y, and Y87H. The entire content of this patent is incorporated herein by reference.

[0148] In some embodiments, the modified human TNFα moiety has mutations that confer receptor selectivity as described in International Application No. PCT / IB2016 / 001668. The entire content of this patent is incorporated herein by reference. In some embodiments, the mutation to TNF is TNFR1-selective. In some embodiments, the mutation to TNF that is TNFR1-selective is to one or more of positions R32, S86, and E146. In some embodiments, the mutation to TNF that is TNFR1-selective is one or more of R32W, S86T, and E146K. In some embodiments, the mutation to TNF that is TNFR1-selective is one or more of R32W, R32W / S86T, R32W / E146K, and E146K. In some embodiments, the mutation to TNF is TNFR2-selective. In some embodiments, the mutation to TNF that is TNFR2-selective is to one or more of positions A145, E146, and S147. In some embodiments, the mutation to TNF that is TNFR2-selective is one or more of A145T, A145R, E146D, and S147D. In some embodiments, the mutation to TNF that is TNFR2-selective is one or more of A145R, A145T / S147D, and A145T / E146D / S147D.

[0149] In certain embodiments, the modified signaling substance is TNFβ. TNFβ can form homotrimers or heterotrimers with LTβ (LTα1β2). In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2 and / or herpesvirus entry mediator (HEVM) and / or LTβR.

[0150] In certain embodiments, wild-type TNFβ has the amino acid sequence of TNF beta SEQ ID NO: 294.

[0151] In such embodiments, the modified soluble substance may contain mutations at one or more amino acid positions 106 - 113, which results in a modified TNFβ with reduced receptor binding affinity for TNFR2. In certain embodiments, the modified soluble substance has one or more substitution mutations at amino acid positions 106 - 113. In an exemplary embodiment, the substitution mutations are selected from Q107E, Q107D, S106E, S106D, Q107R, Q107N, Q107E / S106E, Q107E / S106D, Q107D / S106E, and Q107D / S106D. In another embodiment, the modified soluble substance has an insertion of about 1 to about 3 amino acids at positions 106 - 113.

[0152] In some embodiments, as described in WO 2015 / 007903, the modified substance is a TNF family member (e.g., TNFα, TNFβ), which can be of the single-chain trimer type. The entire content of this patent is incorporated herein by reference.

[0153] In some embodiments, the modifying agent is a TNF family member (e.g., TNFα, TNFβ), which has a reduced affinity and / or activity for TNFR1, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., International Publication No. WO 2015 / 007520, the entire content of which is incorporated herein by reference). In these embodiments, the modifying agent is a TNF family member (e.g., TNFα, TNFβ), which may also have a substantially reduced or eliminated affinity and / or activity for TNFR2. In some embodiments, the modifying agent is a TNF family member (e.g., TNFα, TNFβ), which has a reduced affinity and / or activity for TNFR2, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., International Publication No. WO 2015 / 007520, the entire content of this patent is incorporated herein by reference). In these embodiments, the modifying agent is a TNF family member (e.g., TNFα, TNFβ), which may similarly have a substantially reduced or eliminated affinity and / or activity for TNFR1. Constructs of such embodiments are used, for example, in methods of suppressing TNF responses in a cell-specific manner. In some embodiments, the antagonist TNF family member (e.g., TNFα, TNFβ) is of the single-chain trimer type as described in International Publication No. WO 2015 / 007903.

[0154] In certain embodiments, the modified signaling agent is TRAIL. In some embodiments, the modified TRAIL agent has a reduced affinity and / or activity for DR4 (TRAIL-RI) and / or DR5 (TRAIL-RII) and / or DcR1 and / or DcR2. In some embodiments, the modified TRAIL agent has a reduced affinity and / or activity for DR4 (TRAIL-RI) and / or DR5 (TRAIL-RII) and / or DcR1 and / or DcR2.

[0155] In certain embodiments, wild-type TRAIL has the amino acid sequence of SEQ ID NO: 295.

[0156] In such embodiments, the modified TRAIL agent may contain 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 based on the GenBank accession number NP_003801, version 10, NP_003801.1, GI: 4507593, human sequence, see above reference).

[0157] In some embodiments, the modified TRAIL agent may contain one or more mutations that substantially reduce its affinity and / or activity for TRAIL-R1. In such embodiments, the modified TRAIL agent may specifically bind to TRAIL-R2. Representative mutations include mutations at one or more of 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 certain embodiments, the modified TRAIL agent contains the mutations Y189Q, R191K, Q193R, H264R, I266L, and D267Q.

[0158] In some embodiments, the modified TRAIL substance may include one or more mutations that substantially reduce its affinity and / or activity for TRAIL-R2. In such embodiments, the modified TRAIL substance may specifically bind to TRIL-R1. Representative mutations include one or more mutations at amino acid positions G131, R149, S159, N199, K201, and S215. For example, the mutation(s) may be one or more of G131R, R149I, S159R, N199R, K201H, and S215D. In one embodiment, the modified TRAIL substance includes the mutations G131R, R149I, S159R, N199R, K201H, and S215D. Additional TRAIL mutations are described, for example, in Trebing et al., (2014) Cell Death and Disease, 5: e1035. The entire disclosures of these are incorporated herein by reference.

[0159] In one embodiment, the modified signaling substance is TGFα. In such embodiments, the modified TGFα substance has a reduced affinity and / or activity for the epidermal growth factor receptor (EGFR). In some embodiments, the modified TGFα substance has a substantially reduced or eliminated affinity and / or activity for the epidermal growth factor receptor (EGFR).

[0160] In certain embodiments, the modified signaling substance is TGFβ. In such embodiments, the modified signaling substance has a reduced affinity for TGFBR1 and / or TGFBR2. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for TGFBR1 and / or TGFBR2. In some embodiments, the modified signaling substance optionally has a substantially reduced or eliminated affinity and / or activity for TGFBR3, which, without intending to be bound by theory, may act as a reservoir of ligand for the TGF-beta receptor. In some embodiments, TGFβ selects TGFBR1 over TGFBR2 or TGFBR2 over TGFBR1. Similarly, without intending to be bound by theory, LAP may act as a reservoir of ligand for the TGF-beta receptor. In some embodiments, the modified signaling substance has a reduced affinity and / or activity for TGFBR1 and / or TGFBR2 and a substantially reduced or eliminated affinity and / or activity for latent associated peptide (LAP). In some embodiments, such chimeras are used in camurati-engelmann disease or other diseases associated with inappropriate TGFβ signaling.

[0161] In some embodiments, the modifying substance is a TGF family member (e.g., TGFα, TGFβ), which has a reduced affinity and / or activity, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are incorporated herein by reference), for one or more of TGFBR1, TGFBR2, TGFBR3. In these embodiments, the modifying substance is a TGF family member (e.g., TGFα, TGFβ), which also optionally has a substantially reduced or eliminated affinity and / or activity for one or more of TGFBR1, TGFBR2, TGFBR3.

[0162] In some embodiments, the modifying substance is a TGF family member (e.g., TGFα, TGFβ), which has a reduced affinity and / or activity for TGFBR1 and / or TGFBR2, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations; see, e.g., WO 2015 / 007520, the entire content of which is incorporated herein by reference). In these embodiments, the modifying substance is a TGF family member (e.g., TGFα, TGFβ), which also optionally has a substantially reduced or eliminated affinity and / or activity for TGFBR3.

[0163] In one embodiment, the modified signaling substance is an interleukin. In one embodiment, the modified signaling substance is IL1. In one embodiment, the modified signaling substance is IL1α or IL1β. In some embodiments, the modified signaling substance has a reduced affinity and / or activity for IL1R1 and / or IL1RAcP. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for IL1R1 and / or IL1RAcP. In some embodiments, the modified signaling substance has a reduced affinity and / or activity for IL1R2. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for IL1R2. For example, in some embodiments, the modified IL1 substance avoids interaction with IL1R2 and thus substantially reduces its function as a decoy and / or sink for therapeutic agents.

[0164] In one embodiment, wild-type IL1β has the amino acid sequence of SEQ ID NO: 296.

[0165] IL1 is a pro-inflammatory cytokine and an important immune system regulator. It is a potent activator of CD4 T cell responses, increasing the proportion of Th17 cells and enhancing the proliferation of IFNγ- and IL4-producing cells. IL1 is also a potent regulator of CD8 + T cells, enhancing antigen-specific CD8 + T cell proliferation, differentiation, migration to the periphery, and memory. The IL1 receptor includes IL1R1 and IL1R2. Binding to IL1R1 and signal transduction through IL1R1 constitute the mechanism by which IL1 mediates many of its biological (and pathological) effects. IL1R2 can function as a decoy receptor, thereby reducing the availability of IL1 for interaction and signal transduction through IL1R1.

[0166] In some embodiments, the modified IL1 has a reduced affinity and / or activity (e.g., agonist activity) for IL1R1. In some embodiments, the modified IL1 has a substantially reduced or eliminated affinity and / or activity for IL1R2. In such embodiments, recoverable IL1 / IL1R1 signal transduction and prevention of loss of the therapeutic chimera for ILR2 and resulting reduction in the required IL1 dosage (e.g., compared to chimeras having only wild-type or attenuated mutations for ILR1) are provided. Such constructs are used, for example, in methods of treating cancer, including stimulating the immune system to initiate an anti-cancer response.

[0167] In some embodiments, the modified IL1 has a reduced affinity and / or activity for IL1R1 (e.g., antagonist activity, e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520. The entire content of this patent is incorporated herein by reference). In some embodiments, the modified IL1 has a substantially reduced or eliminated affinity and / or activity for IL1R2. In such embodiments, IL1 / IL1R1 signaling is not recoverable, and there is prevention of loss of the therapeutic chimera for ILR2 and a resulting reduction in the IL1 dosage required (e.g., compared to a chimera having only a wild type or attenuating mutation for ILR1). Such constructs are used, for example, in methods of treating autoimmune diseases, including, for example, suppressing the immune system.

[0168] In such embodiments, the modified signaling substance has a deletion of amino acids 52-54, which results in the production of a modified human IL1β that has reduced binding affinity and reduced bioactivity for type I IL1R. See, for example, International Publication No. WO 1994 / 000491. The entire content of this patent is incorporated herein by reference. In some embodiments, the modified human IL1β has one or more substitution mutations selected from A117G / P118G, R120X, L122A, T125G / L126G, R127G, Q130X, Q131G, K132A, S137G / Q138Y, L145G, H146X, L145A / L147A, Q148X, Q148G / Q150G, Q150G / D151A, M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209X, K209A / K210A, K219X, E221X, E221S / N224A, N224S / K225S, E244K, N245Q (where X can be any change in an amino acid, e.g., a non-conservative change), which exhibit reduced binding to IL1R as described, for example, in International Publication No. WO 2015 / 007542 and International Publication No. WO 2015 / 007536, the entire contents of which are incorporated herein by reference (numbering based on GenBank accession number NP_000567, version NP-000567.1, GI:10835145, human IL1β sequence). In some embodiments, the modified human IL1β can have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S, and E221K. In one embodiment, the modified human IL1β includes the mutations Q131G and Q148G. In one embodiment, the modified human IL1β includes the mutations Q148G and K208E. In one embodiment, the modified human IL1β includes the mutations R120G and Q131G. In one embodiment, the modified human IL1β includes the mutations R120G and H146A. In one embodiment, the modified human IL1β includes the mutations R120G and H146N.In certain embodiments, the modified human IL1β comprises the mutations R120G and H146R. In certain embodiments, the modified human IL1β comprises the mutations R120G and H146E. In certain embodiments, the modified human IL1β comprises the mutations R120G and H146G. In certain embodiments, the modified human IL1β comprises the mutations R120G and K208E. In certain embodiments, the modified human IL1β comprises the mutations R120G, F162A, and Q164E.

[0169] In certain embodiments, the modified signaling molecule is IL2. In such embodiments, the modified signaling molecule has a reduced affinity and / or activity for IL2Rα and / or IL2Rβ and / or IL2Rγ. In some embodiments, the modified signaling molecule has a reduced affinity and / or activity for IL2Rβ and / or IL2Rγ. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for IL2Rα. Such embodiments may be suitable for the treatment of cancer, for example, when the modified IL2 is antagonistic to IL2Rβ and / or IL2Rγ. For example, the constructs of the present invention preferentially reduce the activation of CD8 + T cells (which can confer an anti-tumor effect), and do not preferentially reduce the activation of T cells having IL2 receptor α, β, and γ reg (which can confer an immunosuppressive effect and a tumor-promoting effect). Further, in some embodiments, the selectivity for IL2Rβ and / or IL2Rγ over IL2Rα avoids IL2 side effects such as pulmonary edema. Also, IL2-based chimeras are useful for the treatment of autoimmune diseases, for example, when the modified IL2 is an antagonist to IL2Rβ and / or IL2Rγ (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520. The entire contents of this patent are incorporated herein by reference). For example, the constructs of the present invention have CD8 with IL2 receptor β and γ +Prioritize the attenuation of T cell suppression (and thus the suppression of the immune response), and T having IL2 receptors α, β, and γ reg Do not prioritize. Alternatively, in some embodiments, the chimeric having IL2 prioritizes the activation of T reg and thus immunosuppression, and does not prioritize the activation of CD8 + T cells. For example, these constructs are used in the treatment of diseases that are thought to benefit from disease or immunosuppression, such as autoimmune disorders.

[0170] In some embodiments, the chimeric protein has the targeting moiety described herein that targets CD8 + T cells, and the modified IL2 substance has a reduced affinity and / or activity for IL2Rβ and / or IL2Rγ and / or a substantially reduced or eliminated affinity and / or activity for IL2Rα. In some embodiments, these constructs target CD8 + T cell activity and are typically inactive (or have substantially reduced activity) against T reg cells. In some embodiments, such constructs have an enhanced immune-stimulatory effect compared to wild-type IL2 (without wishing to be bound by theory, by not stimulating Tregs), while removing or reducing the systemic toxicity associated with IL2.

[0171] In one embodiment, wild-type IL2 has the amino acid sequence of SEQ ID NO: 297.

[0172] In such embodiments, the modified human IL2 substance has one or more mutations at the position of amino acid L72 (L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), the position of F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K) and the position of Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R or Y45K). Without wishing to be bound by theory, these modified IL2 substances are thought to have a reduced affinity for the high-affinity IL2 receptor and to maintain their affinity for the intermediate-affinity IL2 receptor as compared to wild-type IL2. See, for example, U.S. Patent Application Publication No. 2012 / 0244112. The entire contents of this patent are incorporated herein by reference.

[0173] In some embodiments, the modified IL2 substance has one or more mutations at amino acid positions R38, F42, Y45, and E62. For example, the modified IL2 substance may include one or more of R38A, F42A, Y45A, and E62A. In some embodiments, the modified IL2 substance may include a mutation at C125. For example, it may be C125S. In such embodiments, the modified IL2 substance may have a substantially reduced affinity and / or activity for IL2Rα, as described, for example, in Carmenate et al. (2013) The Journal of Immunology, 190:6230-6238. The entire disclosure is incorporated herein by reference. In some embodiments, the modified IL2 substance having mutations at R38, F42, Y45, and / or E62 can induce the proliferation of effector cells including CD8+ T cells and NK cells but not including Treg cells. In some embodiments, the modified IL2 substance having mutations at R38, F42, Y45, and / or E62 has less toxicity than the wild-type IL2 substance. Chimeric proteins comprising modified IL2 substances having a substantially reduced affinity and / or activity for IL2Rα may find use, for example, in oncology.

[0174] In other embodiments, the modified IL2 substance may have substantially reduced affinity and / or activity for IL2Rβ, for example, as described in International Publication No. WO 2016 / 025385. This entire disclosure is incorporated herein by reference. In such embodiments, the modified IL2 substance may induce the proliferation of Treg cells, but not the proliferation of effector cells such as CD8+ T cells and NK cells. Chimeric proteins containing a modified IL2 substance having substantially reduced affinity and / or activity for IL2Rβ may find use, for example, in the treatment of autoimmune diseases. In some embodiments, the modified IL2 substance may have one or more mutations at amino acid positions N88, D20, and / or A126. For example, the modified IL2 substance may include one or more of N88R, N88I, N88G, D20H, Q126L, and Q126F.

[0175] In various embodiments, the modified IL2 substance may contain a mutation at D109 or C125. For example, the mutation may be D109C or C125S. In some embodiments, the modified IL2 having a mutation at D109 or C125 may be utilized for attachment to a PEG moiety.

[0176] In one embodiment, the modified signaling substance is IL3. In some embodiments, the modified signaling substance has reduced affinity and / or activity for the IL3 receptor, and the IL3 receptor is a heterodimer having a specific alpha chain paired with a common beta (betac or CD131) subunit. In some embodiments, the modified signaling substance has substantially reduced or eliminated affinity and / or activity for the IL3 receptor, and the IL3 receptor is a heterodimer having a specific alpha chain paired with a common beta (betac or CD131) subunit.

[0177] In certain embodiments, the modified signaling agent is IL4. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for type 1 and / or type 2 IL4 receptors. In such embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for type 1 and / or type 2 IL4 receptors. The type 1 IL4 receptor is composed of an IL4Rα subunit having a common γ chain and specifically binds IL4. The type 2 IL4 receptor includes an IL4Rα subunit bound to a different subunit known as IL13Rα1. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for the type 2 IL4 receptor.

[0178] In certain embodiments, wild-type IL4 has the amino acid sequence of SEQ ID NO: 298.

[0179] In such embodiments, the modified IL4 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 IL4 agents maintain activity mediated by the type I receptor but significantly reduce the biological activity mediated by other receptors. See, for example, U.S. Patent No. 6,433,157. The entire content of this patent is incorporated herein by reference.

[0180] In certain embodiments, the modified signaling agent is IL6. IL6 signals through a cell surface type I cytokine receptor complex that includes a ligand-binding IL6R chain (CD126) and a signaling component gp130. IL6 can also bind to a soluble form of IL6R (sIL6R), which is the extracellular portion of IL6R. The sIL6R / IL6 complex is involved in neurite outgrowth and neuron survival and can thus be important for nerve regeneration by remyelination. Thus, in some embodiments, the modified signaling agent has a reduced affinity and / or activity for IL6R / gp130 and / or sIL6R. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for IL6R / gp130 and / or sIL6R.

[0181] In certain embodiments, wild-type IL6 has the amino acid sequence of IL6 (mature, wild-type) (SEQ ID NO: 299).

[0182] 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, these modified IL6 agents are thought to exhibit reduced binding affinity and reduced biological activity for IL6Rα. See, for example, WO 97 / 10338. The entire content of this patent is incorporated herein by reference.

[0183] In certain embodiments, the modified signaling agent is IL10. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for IL10 receptor 1 and IL10 receptor 2. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for IL10 receptor 1 and IL10 receptor 2.

[0184] In certain embodiments, the modified signaling agent is IL11. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for IL11Rα and / or IL11Rβ and / or gp130. In such embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for IL11Rα and / or IL11Rβ and / or gp130.

[0185] In certain embodiments, the modified signaling agent is IL12. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for IL12Rβ1 and / or IL12Rβ2. In such embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for IL12Rβ1 and / or IL12Rβ2.

[0186] In certain embodiments, the modified signaling agent is IL13. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for the IL4 receptor (IL4Rα) and IL13Rα1. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for the IL4 receptor (IL4Rα) or IL13Rα1.

[0187] In certain embodiments, wild-type IL13 has the amino acid sequence of IL13 (mature, wild-type) (SEQ ID NO: 300).

[0188] In such embodiments, the modified IL13 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, these modified IL13 agents are thought to exhibit reduced biological activity. See, e.g., International Publication No. WO 2002 / 018422. The entire contents of this patent are incorporated herein by reference.

[0189] In certain embodiments, the modified signaling substance is IL18. In some embodiments, the modified signaling substance has a reduced affinity and / or activity for IL18Rα and / or IL18Rβ. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for IL18Rα and / or IL18Rβ. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for IL18Rα type 2, an isoform of IL18Rα that lacks the TIR domain required for signaling.

[0190] In certain embodiments, wild-type IL18 has the amino acid sequence of IL18 (wild-type) (SEQ ID NO: 301).

[0191] In such embodiments, as described in International Publication No. 2015 / 007542, the entire contents of which are incorporated herein by reference, the modified IL18 substance may contain one or more mutations in the amino acids or amino acid regions selected from Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N127-K129, Q139-M149, K165-K171, R183 and Q190-N191 (numbering based on the GenBank accession number AAV38697, version AAV38697.1, GI: 54696650, human IL18 sequence).

[0192] In certain embodiments, the modified signaling substance is IL33. In such embodiments, the modified signaling substance has a reduced affinity and / or activity for ST2 receptor 1 and IL1RAcP. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for ST2 receptor and IL1RAcP.

[0193] In certain embodiments, wild-type IL33 has the amino acid sequence of SEQ ID NO: 302.

[0194] In such an embodiment, as described in International Publication No. 2015 / 007542, the entire content of which is incorporated herein by reference, the modified IL33 substance may contain one or more mutations in amino acids or amino acid regions selected from I113 - Y122, S127 - E139, E144 - D157, Y163 - M183, E200, Q215, L220 - C227, and T260 - E269 (numbering based on GenBank accession number NP_254274, version 254274.1, Gl:15559209, human sequence).

[0195] In one embodiment, the modified signaling substance is epidermal growth factor (EGF). EGF is a family of potent growth factors. Members include EGF, HB - EGF, and TGFα, amphiregulin, neuregulin, epiregulin, betacellulin, and others. EGF family receptors include EGFR (ErbB1), ErbB2, ErbB3, and ErbB4. These can function as homodimer and / or heterodimer receptor subtypes. Different EGF family members show different selectivities for various receptor subtypes. For example, EGF binds to ErbB1 / ErbB1, ErbB1 / ErbB2, ErbB4 / ErbB2, and several other heterodimer subtypes. HB - EGF has a similar pattern but binds to ErbB4 / 4. Regulation of EGF (EGF - like) growth factor signaling in the positive or negative direction is of interest from a major therapeutic perspective. For example, inhibition of EGFR signaling is of interest in the treatment of various cancers where EGFR signaling constitutes a major growth - promoting signal. Alternatively, stimulation of EGFR signaling is of interest from a therapeutic perspective, for example, in wound healing (acute and chronic), oral mucositis (a major side effect of various cancer therapies including radiotherapy).

[0196] In some embodiments, the modified signaling agent has a reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4. Such embodiments are used, for example, in methods of treating wounds. In some embodiments, the modified signaling agent binds to one or more of ErbB1, ErbB2, ErbB3, and ErbB4 and antagonizes the activity of the receptor. In such embodiments, the modified signaling agent has a reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4, whereby the activity of the receptor is antagonized in a manner that is attenuated. Such embodiments are used, for example, in the treatment of cancer. In certain embodiments, the modified signaling agent has a reduced affinity and / or activity for ErbB1. ErbB1 is a therapeutic target for kinase inhibitors - but in most cases, there are side effects because they are not very selective (e.g., gefitinib, erlotinib, afatinib, brigatinib, and icotinib). In some embodiments, the attenuated antagonistic ErbB1 signaling is more on-target and has fewer side effects than other substances that target the EGF receptor.

[0197] In some embodiments, the modified signaling agent has a reduced affinity and / or activity for ErbB1 (e.g., antagonist activity, e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are incorporated herein by reference) and / or substantially reduced or eliminated affinity and / or activity for ErbB4 or other subtypes with which it can interact. Specific targeting via the targeting moiety results in cell-selective inhibition of ErbB1 / ErbB1 receptor activation (antagonism, e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are incorporated herein by reference) without involving other receptor subtypes that may be associated with inhibition-related side effects. Thus, in contrast to EGFR kinase inhibitors that inhibit EGFR activity in all cell types in the body, such constructs will provide a cell-selective (e.g., tumor cells having activated EGFR signaling due to receptor amplification, overexpression, etc.) anti-EGFR (ErbB1) drug effect with reduced side effects.

[0198] In some embodiments, the modified signaling agent has a reduced affinity and / or activity (e.g., agonist activity) for ErbB4 and / or other subtypes with which it interacts. Targeting to specific target cells via the targeting moiety results in selective activation of ErbB1 signaling (e.g., epithelial cells). Such constructs are used in some embodiments for the treatment of wounds with reduced side effects (promotion of wound healing), particularly for the treatment of chronic conditions and applications other than local administration of therapeutic agents (e.g., systemic wound healing).

[0199] In certain embodiments, the modified signaling substance is insulin or an insulin analog. In some embodiments, the modified insulin or insulin analog has a reduced affinity and / or activity for the insulin receptor and / or the IGF1 or IGF2 receptor. In some embodiments, the modified insulin or insulin analog has a reduced or eliminated affinity and / or activity for the insulin receptor and / or the IGF1 or IGF2 receptor. The attenuated response to the insulin receptor enables control of diabetes, obesity, metabolic disorders, etc., while avoiding cancer-promoting effects by dissociating from the IGF1 or IGF2 receptor.

[0200] In certain embodiments, the modified signaling substance is insulin-like growth factor-I or insulin-like growth factor-II (IGF1 or IGF2). In certain embodiments, the modified signaling substance is IGF1. In such embodiments, the modified signaling substance has a reduced affinity and / or activity for the insulin receptor and / or the IGF1 receptor. In certain embodiments, the modified signaling substance binds to the IGF1 receptor and antagonizes the activity of the receptor. In such embodiments, the modified signaling substance has a reduced affinity and / or activity for the IGF1 receptor, thereby enabling the activity of the receptor to be antagonized in a weakened form. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for the IGF1 receptor. In some embodiments, the modified signaling substance has a reduced affinity and / or activity for the IGF2 receptor, thereby enabling the activity of the receptor to be antagonized in a weakened form. In certain embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for the insulin receptor and thus does not interfere with insulin signaling. In various embodiments, this is applied to cancer treatment. In various embodiments, the substance may prevent the IR isoform A from developing resistance to cancer treatment.

[0201] In one embodiment, the chimeric protein has (i) a CD8 binding substance and (ii) a targeting moiety that targets tumor cells, together with any modified (e.g., variant) form of a signaling substance described herein. In certain embodiments, the chimeric protein has a targeting moiety that targets CD8 on T cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0202] In various embodiments, the signaling substance is a toxin or a toxic enzyme. In some embodiments, the toxin or toxic enzyme is derived from plants and bacteria. Examples of toxins or toxic enzymes include, but are not limited to, ribosome-inactivating proteins (RIPs) such as diphtheria toxin, Pseudomonas toxin, anthrax toxin, ricin and saporin, modeccin, abrin, gelonin, and pokeweed antiviral protein. Additional toxins include those disclosed in Mathew et al., (2009) Cancer Sci 100(8):1359-65, the entire disclosure of which is incorporated herein by reference. In such embodiments, the chimeric protein of the invention can be utilized to induce cell death in a cell type-specific manner. In such embodiments, the toxin can be modified, e.g., mutagenized, to reduce the affinity and / or activity of the toxin to attenuate its effect, as described herein with respect to other signaling substances.

[0203] Multispecific chimeras and fusions with signaling substances In various embodiments, the CD8 binding substance of the invention is part of a chimera or fusion of one or more signaling substances and / or one or more additional targeting moieties described herein. Thus, the invention provides, for example, a chimeric or fusion protein comprising one or more signaling substances and a targeting moiety for CD8 and / or one or more additional targeting moieties.

[0204] In various embodiments, the CD8-binding substance of the present invention is multispecific, i.e., the CD8-binding substance has two or more targeting moieties having recognition domains that recognize and bind two or more targets, such as antigens, or receptors, or epitopes. In such embodiments, the CD8-binding substance of the present invention may comprise two or more targeting moieties having recognition domains that recognize and bind two or more epitopes on the same antigen or different antigens. In various embodiments, such multispecific CD8-binding substances exhibit advantageous properties such as improved binding affinity and / or improved selectivity. In certain embodiments, the CD8-binding substance of the present invention comprises two targeting moieties and is bispecific, i.e., binds to and recognizes two epitopes on the same antigen or different antigens.

[0205] In various embodiments, the multispecific CD8-binding substance of the present invention comprises two or more targeting moieties, each targeting moiety being an antibody or antibody derivative described herein. In certain embodiments, the multispecific CD8-binding substance of the present invention comprises at least one type of VHH comprising an antigen recognition domain for CD8 and one type of antibody or antibody derivative comprising an antigen recognition domain for a tumor antigen.

[0206] In various embodiments, the multispecific CD8-binding substance has two or more targeting moieties that target different antigens or receptors, and one targeting moiety may be attenuated for its antigen or receptor, e.g., the targeting moiety binds to its antigen or receptor with low affinity or binding strength (e.g., including binding with lower affinity or binding strength than the affinity or binding strength that other targeting moieties have for the antigen or receptor, e.g., the difference in binding affinity may be about 10-fold, or 25-fold, or 50-fold, or 100-fold, or 300-fold, or 500-fold, or 1000-fold, or 5000-fold; e.g., the targeting moiety with lower affinity or binding strength may bind to its antigen or receptor in the range of mid-high nM or low-mid μM K D and bind, while the targeting moiety with higher affinity or binding strength has a K in the range of mid-high pM or low-mid nM Dcapable of binding). For example, in some embodiments, the present multispecific CD8 binding substance comprises a deattenuated targeting moiety that targets an indiscriminate antigen or receptor, which improves targeting to the cell of interest (e.g., via other targeting moieties) and can prevent effects that span multiple cell types, including those that are not the therapeutic target (e.g., by binding to an indiscriminate antigen or receptor with a higher affinity than that provided in these embodiments). In various embodiments, the chimeric proteins of the present invention have one or more targeting moieties that target different antigens or receptors with low affinity or low binding avidity, and the other binding can be enhanced, for example, cooperatively.

[0207] The multispecific CD8-binding substances of the present invention can be constructed using methods known in the art. See, for example, U.S. Patent No. 9,067,991, U.S. Patent Application Publication No. 20110262348, and International Publication No. 2004 / 041862. The entire contents of these patents are incorporated herein by reference. In an exemplary embodiment, the multispecific CD8-binding substance of the present invention comprising two or more targeting moieties can be constructed by chemical cross-linking, for example, by reacting an amino acid residue with an organic derivatization reagent as described in Blattler et al., Biochemistry 24, 1517-1524 and European Patent No. 294703, the entire contents of which are incorporated herein by reference. In another exemplary embodiment, the multispecific CD8-binding substance comprising two or more targeting moieties is constructed by gene fusion, i.e., by constructing a single polypeptide comprising the polypeptides of the individual targeting moieties. For example, a single polypeptide construct can be formed encoding a first VHH having an antigen recognition domain for CD8 and a second antibody or antibody derivative having an antigen recognition domain for a tumor antigen. Methods for producing bivalent or multivalent VHH polypeptide constructs are disclosed in International Publication No. 96 / 34103, the entire contents of which are incorporated herein by reference. In a further exemplary embodiment, the multispecific CD8-binding substance of the present invention can be constructed using a linker. For example, the carboxy terminus of a first VHH having an antigen recognition domain for CD8 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). Representative linkers that can be used are described herein. In some embodiments, the components of the multispecific CD8-binding substance of the present invention are directly linked to each other without using a linker.

[0208] In various embodiments, the multispecific CD8-binding substance of the present invention recognizes and binds to CD8 and one or more antigens found on one or more immune cells. Immune cells can include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer cells, T lymphocytes (e.g., cytotoxic T lymphocytes, helper T cells), B lymphocytes, plasma cells, dendritic cells, or subsets thereof. In some embodiments, the CD8-binding substance specifically binds to an antigen of interest and effectively mobilizes one or more immune cells either directly or indirectly.

[0209] In various embodiments, the multispecific CD8-binding substance of the present invention recognizes and binds to CD8 and one or more antigens found on tumor cells. In these embodiments, the CD8-binding substance can mobilize immune cells either directly or indirectly to tumor cells or the tumor microenvironment. In some embodiments, the CD8-binding substance can mobilize immune cells, such as immune cells (e.g., CTLs) that can kill and / or suppress tumors, either directly or indirectly to the site of action (such as, but not limited to, the tumor microenvironment).

[0210] In some embodiments, the CD8-binding substance can alter the balance of immune cells in a manner favorable for an immune attack on a tumor or is used in a method that includes altering the balance of immune cells. For example, the CD8-binding substance can advantageously alter the ratio of immune cells at clinically important sites to cells that can kill and / or suppress tumors (e.g., T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), neutrophils, B cells, dendritic cells, or subsets thereof), and cells that protect tumors (e.g., myeloid-derived suppressor cells (MDSC), regulatory T cells (T reg); can be changed to be disadvantageous to tumor-associated neutrophils (TAN), M2 macrophages, tumor-associated macrophages (TAM), or subsets thereof. In some embodiments, the CD8-binding substance of the present invention can increase the ratio of effector T cells to regulatory T cells.

[0211] In some embodiments, the multispecific CD8-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a tumor cell-associated antigen. In some embodiments, the targeting moiety recruits tumor cells directly or indirectly. For example, in some embodiments, the recruitment of tumor cells is to one or more effector cells (e.g., immune cells described herein) that can kill and / or suppress the tumor cells. In some embodiments, the targeting moiety recruits T cells directly or indirectly to tumor cells by two targeting moieties that interact with their respective antigens on the tumor and CD8-positive immune cells (e.g., T cells).

[0212] A tumor cell, or cancer cell, refers to an uncontrolled proliferation of cells or tissues and / or an abnormal increase in cell survival and / or an abnormal increase in the suppression of apoptosis that interferes with the normal functioning of the body's organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Examples of tumor cells include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancers, endometrial cancer, esophageal cancer, eye cancer, head and neck cancers, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), melanoma, multiple myeloma, neuroblastoma, oral cancer (lip, glossal, tongue, intraoral, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancers, salivary gland tumors, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancers, vulvar cancer, Hodgkin lymphoma and non-Hodgkin lymphoma, and lymphomas including B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL)), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large tumor lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, as well as other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and cells of abnormal blood vessel growth associated with nevus syndrome, edema (e.g., those associated with brain tumors), and the Meigs syndrome, but are not limited thereto.

[0213] Examples of tumor cells or cancer cells include, but are not limited to, carcinomas such as various subtypes (including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myeloid, acute lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell), lymphomas and myelomas (including, for example, Hodgkin and non-Hodgkin lymphomas, light chain type, non-secretory MGUS, and plasmacytoma), and central nervous system cancers (including, for example, brain tumors (such as gliomas (such as astrocytomas, oligodendrogliomas, and ependymomas), meningiomas, pituitary adenomas, and neuromas), and spinal cord tumors (such as meningiomas and neurofibromas)).

[0214] Examples of tumor antigens include MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, tumor antigens of the MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), tumor antigens of the GAGE family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin and gamma-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, tumor antigens of the Smad family, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, and BCMA (TNFRSF17) are included, but not limited to these. In various embodiments, the CD8-binding substance comprises a targeting moiety that binds one or more of these tumor antigens.

[0215] In some embodiments, the present multi-specific CD8-binding substance recognizes and binds CD8 as well as antigens on tumor cells. In some embodiments, the multi-specific CD8-binding substance directly or indirectly recruits CTLs to tumor cells or the tumor microenvironment.

[0216] In various embodiments, the present multi-specific CD8-binding substance has a targeting moiety that targets two different cells (e.g., to form a synapse) or the same cell (e.g., to obtain a higher concentration of signaling effector effect).

[0217] In some embodiments, the multi-specific CD8-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a T cell-related target (e.g., an antigen, a receptor). In some embodiments, the targeting moiety 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 to, for example, in some embodiments, a treatment site (e.g., a site having one or more diseased cells or cells to be regulated to obtain a therapeutic effect). Examples of effector T cells include cytotoxic T cells (e.g., αβTCR, CD3 + , CD8 + , CD45RO + ); CD4 + effector T cells (e.g., αβTCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL7R / CD127 +); CD8 + Effector T cells (e.g., αβ TCR, CD3 + , CD8 + , CCR7 + , CD62Lhi, IL7R / CD127 + ); Effector memory T cells (e.g., CD62Llow, CD44 + , TCR, CD3 + , IL7R / CD127 + , IL15R + , CCR7low); Central memory T cells (e.g., CCR7 + , CD62L + , CD27 + ; or CCR7hi, CD44 + , CD62Lhi, TCR, CD3 + , IL7R / CD127 + , IL15R + ); CD62L + Effector T cells; Early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L - )(TemE and TemL, respectively) of CD8 + Effector memory T cells (TEM); CD127( + )CD25(low / - ) Effector T cells; CD127( - )CD25( - ) Effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca( + )); TH1 effector T cells (e.g., CXCR3 + , CXCR6 + and CCR5 + ; or αβ TCR, CD3 + , CD4 + , IL12R + , IFNγR + , CXCR3 + ), TH2 effector T cells (e.g., CCR3+ and CCR4 + and CCR8 + ; or αβ TCR, CD3 + , CD4 + , IL4R + , IL33R + , CCR4 + , IL17RB + , CRTH2 + ); TH9 effector T cells (e.g., αβ TCR, CD3 + , CD4 + ); TH17 effector T cells (e.g., αβ TCR, CD3 + , CD4 + , IL23R + , CCR6 + , IL1R + ); CD4 + CD45RO + CCR7 + effector T cells, ICOS + effector T cells; CD4 + CD45RO + CCR7( - ) effector T cells; and effector T cells that secrete IL2, IL4 and / or IFN-γ are included.

[0218] Examples of target T cell antigens include, for example, the following (when applicable, including the extracellular domain): CD8, CD3, SLAMF4, IL2Rα, 4-1BB / TNFRSF9, IL2Rβ, ALCAM, B7-1, IL4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL6R, CCR3, IL7Rα, CCR4, CXCRl / ILSRA, CCR5, CCR6, IL10Rα, CCR7, IL10Rβ, CCRS, IL12Rβ1, CCR9, IL12Rβ2, CD2, IL13Rα1, IL13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrinα4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common γ chain / IL2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, thrombopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAILRl / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL1R1 and TSLPR. In various embodiments, the CD8 binding substance comprises a targeting moiety that binds to one or more of these exemplary T cell antigens.

[0219] In some embodiments, the multi-specific CD8-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a B cell-related target (e.g., an antigen, a receptor). In some embodiments, the targeting moiety, for example, in some embodiments, recruits B cells directly or indirectly to a treatment site (e.g., a site having one or more diseased cells or cells to be regulated to obtain a therapeutic effect). Examples of B cell antigens of interest include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, CDw150, and B cell maturation antigen (BCMA). In various embodiments, the CD8-binding substance comprises a targeting moiety that binds to one or more of these exemplary B cell antigens.

[0220] In some embodiments, the multi-specific CD8 binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a natural killer cell-related target (e.g., an antigen, a receptor). In some embodiments, the targeting moiety, for example, in some embodiments, mobilizes natural killer cells directly or indirectly to a site of treatment (e.g., a site having one or more diseased cells or cells to be modulated to obtain a therapeutic effect). Examples of natural killer cell antigens of interest include, for example, TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, Kir1alpha, DNAM-1, LMIR5 / CD300LB, Fc-epsilonRII, LMIR6 / CD300LE, Fc-gammaRl / CD64, MICA, Fc-gammaRIIB / CD32b, MICB, Fc-gammaRIIC / CD32c, MULT-1, Fc-gammaRIIA / CD32a, Nectin-2 / CD112, Fc-gammaRIII / CD16, NKG2A, FcRH1 / IRTA5, NKG2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-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 CD8 binding substance comprises a targeting moiety that binds to one or more of these exemplary NK cell antigens.

[0221] In some embodiments, the multispecific CD8 binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a macrophage / monocyte-related target (e.g., an antigen, a receptor). In some embodiments, the targeting moiety, for example, in some embodiments, recruits macrophages / monocytes directly or indirectly to a site of treatment (e.g., a site having one or more diseased cells or cells to be modulated to obtain a therapeutic effect). Examples of macrophage / monocyte antigens of interest include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin alpha4 / CD49d, BLAME / SLAMF8, integrin alphaX / CDllc, CCL6 / C10, integrin beta2 / CD18, CD155 / PVR, integrin beta3 / CD61, CD31 / PECAM-1, Latexin, CD36 / SR-B3, leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, endoglin / CD105, osteoactivin / GPNMB, Fc-gammaRI / CD64, osteopontin, Fc-gammaRIIB / CD32b, PD-L2, Fc-gammaRIIC / CD32c, Siglec-3 / CD33, Fc-gammaRIIA / CD32a, SIGNR1 / CD209, Fc-gammaRIII / CD16, SLAM, GM-CSFRalpha, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-gammaRl, TLR4, IFN-gannnaR2, TREM-1, IL-1RII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4. IL10Rα, ALCAM, IL10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, ClqR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / 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-CSFR, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD105, NCAM-L1, Fc-γRI / CD64, PSGL-1, Fc-γRIIICD16, RP105, G-CSFR, L-selectin, GM-CSFRα, siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL6R, TREM-2, CXCR1 / IL8RA, TREM-3 and TREML1 / TLT-1. In various embodiments, the CD8 binding substance comprises a targeting moiety that binds to one or more of these exemplary macrophage / monocyte antigens.

[0222] In some embodiments, the multispecific CD8 binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to dendritic cell-related targets (e.g., antigens, receptors). In some embodiments, the targeting moiety, for example, in some embodiments, recruits dendritic cells directly or indirectly to a treatment site (e.g., a site having one or more diseased cells or cells to be modulated to obtain a therapeutic effect). Examples of dendritic cell antigens of interest include, for example, CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-Pl / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL12 / IL23p40, 4-Amino-1,8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4 Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, ClqR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBCCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLl, CD2F-10 / SLAMF9, OsteoactivinGPNMB, Chern23, PD-L2, CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, DEC205, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, DEP-1 / CD148, SIGNR4, DLEC, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, DEC205, and vanilloid R1. In various embodiments, the CD8-binding substance comprises a targeting moiety that binds to one or more of these exemplary DC antigens.

[0223] In some embodiments, the multispecific CD8-binding substance of the invention comprises a targeting moiety for Clec9A that is a VHH comprising a single amino acid chain having four "framework regions" or FRs and three "complementary determining regions" or CDRs. As used herein, "framework region" or "FR" means the regions in the variable domain located between CDRs. As used herein, "complementary determining region" or "CDR" refers to the variable region in a VHH that comprises an amino acid sequence capable of specifically binding to an antigenic target.

[0224] In various embodiments, the multispecific CD8-binding substance of the invention comprises a VHH for Clec9A having a variable domain that comprises at least one of the CDR1, CDR2, and / or CDR3 sequences.

[0225] In an exemplary embodiment, the CDR1 sequence is selected from SEQ ID NOs: 303 to 322.

[0226] In an exemplary embodiment, the CDR2 sequence is selected from SEQ ID NO: 323 to SEQ ID NO: 344.

[0227] In an exemplary embodiment, the CDR3 sequence is selected from SEQ ID NO: 345 to SEQ ID NO: 359; or LGR; or VIK.

[0228] In various embodiments, the Clec9A targeting moiety comprises an amino acid sequence selected from the following sequences: R2CHCL8 (SEQ ID NO: 360); R1CHCL50 (SEQ ID NO: 361); R1CHCL21 (SEQ ID NO: 362); R2CHCL87 (SEQ ID NO: 363); R2CHCL24 (SEQ ID NO: 364); R2CHCL38 (SEQ ID NO: 365); R1CHCL16 (SEQ ID NO: 366); R2CHCL10 (SEQ ID NO: 367); R1CHCL34 (SEQ ID NO: 368); R1CHCL82 (SEQ ID NO: 369); R2CHCL3 (SEQ ID NO: 370); R2CHCL69 (SEQ ID NO: 371); R1CHCL56 (SEQ ID NO: 372); R2CHCL32 (SEQ ID NO: 373); R2CHCL49 (SEQ ID NO: 374); R2CHCL53 (SEQ ID NO: 375); R2CHCL22 (SEQ ID NO: 376); R2CHCL25 (SEQ ID NO: 377); R2CHCL18 (SEQ ID NO: 378); R1CHCL23 (SEQ ID NO: 379); R1CHCL27 (SEQ ID NO: 380); R2CHCL13 (SEQ ID NO: 381); R2CHCL14 (SEQ ID NO: 382); R2CHCL42 (SEQ ID NO: 383); R2CHCL41 (SEQ ID NO: 384); R2CHCL94 (SEQ ID NO: 385); or R2CHCL27 (SEQ ID NO: 386).

[0229] 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.

[0230] In some embodiments, the CDR1 sequence is selected from SEQ ID NO: 387 to SEQ ID NO: 452.

[0231] In some embodiments, the CDR2 sequence is selected from SEQ ID NO: 453 to SEQ ID NO: 518.

[0232] In some embodiments, the CDR3 sequence is selected from SEQ ID NO: 519 to SEQ ID NO: 584.

[0233]

[0234] In some embodiments, the Clec9A-binding substance comprises an amino acid sequence selected from any one of the above sequences that does not contain a terminal HA tag (i.e., YPYDVPDYGS; SEQ ID NO: 1214).

[0235] In some embodiments, the Clec9A-binding substance comprises an amino acid sequence selected from any one of the above sequences that does not contain an AAA linker.

[0236] In some embodiments, the Clec9A-binding substance comprises an amino acid sequence selected from any one of the above sequences that does not contain an AAA linker, an HA tag, and a terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 1215).

[0237] In one embodiment, the targeting moiety comprises an anti-Clec9A antibody disclosed in Tullett et al., JCI Insight. 2016;1(7):e87102. The entire disclosure of this document is incorporated herein by reference.

[0238] In various embodiments, the present invention contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the targeting moieties directed to Clec9A of the present invention described herein. In various embodiments, the amino acid sequence of the targeting moiety directed to Clec9A further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0239] In various embodiments, the chimeric protein of the present invention comprises a targeting moiety comprising an amino acid sequence that is at least 60% identical to any one of the sequences disclosed herein. For example, the chimeric protein may be at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99% or about 100% sequence identity) to any one of the sequences disclosed herein and may comprise a targeting moiety.

[0240] In some embodiments, the multi-specific CD8-binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a target (e.g., an antigen, a receptor) on immune cells selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, and eosinophils. In some embodiments, the antigen recognition domain mobilizes megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, and eosinophils, for example, in some embodiments, to a site of treatment (e.g., a site having one or more diseased cells or cells to be modulated to obtain a therapeutic effect), either directly or indirectly.

[0241] In some embodiments, the multi-specific CD8-binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a megakaryocyte and / or platelet-related target (e.g., an antigen, a receptor). Examples of megakaryocyte and / or platelet antigens include, for example, GPIIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the CD8-binding substance comprises a targeting moiety that binds to one or more of these exemplary megakaryocyte and / or platelet antigens.

[0242] In some embodiments, the multi-specific CD8-binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to an erythrocyte-related target (e.g., an antigen, a receptor). Examples of erythrocyte antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb / IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2 (MHC-II), and Rh antigens. In various embodiments, the CD8-binding substance comprises a targeting moiety that binds to one or more of these exemplary erythrocyte antigens.

[0243] In some embodiments, the multi-specific CD8-binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a mast cell-related target (e.g., an antigen, a receptor). Examples of mast cell antigens of interest include, for example, SCFR / CD117, Fcε Examples include RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMA1, FCER1A, FCER2, and TPSAB1. In various embodiments, the CD8-binding substance comprises a targeting moiety that binds to one or more of these exemplary mast cell antigens.

[0244] In some embodiments, the multispecific CD8-binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a basophil-related target (e.g., an antigen, a receptor). Examples of target basophil antigens include, for example, FcεRI, CD203c, CD123, CD13, CD107a, CD107b, and CD164. In various embodiments, the CD8-binding substance comprises a targeting moiety that binds to one or more of these basophil antigens.

[0245] In some embodiments, the multispecific CD8-binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to a neutrophil-related target (e.g., an antigen, a receptor). Examples of 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 CD8-binding substance comprises a targeting moiety that binds to one or more of these neutrophil antigens.

[0246] In some embodiments, the multispecific CD8-binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to an eosinophil-related target (e.g., an antigen, a receptor). Examples of target eosinophil antigens include, for example, CD35, CD44, and CD69. In various embodiments, the CD8-binding substance comprises a targeting moiety that binds to one or more of these eosinophil antigens.

[0247] In various embodiments, the multispecific CD8-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that specifically binds to any suitable antigen or receptor or cell surface marker known to those of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Examples of tissue-specific markers include endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, 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, K6IRS2, KRT10, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUC1, TACSTD1; neovascular markers such as CD13, TFNA, alpha-v beta-3 (α V β3), E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN, but are not limited thereto. In various embodiments, the CD8-binding substance comprises a targeting moiety that binds one or more of these antigens.

[0248] In various embodiments, the multispecific CD8-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that specifically binds to one or more of the checkpoint markers expressed on T cells, such as PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

[0249] In various embodiments, the multispecific CD8-binding substance of the present invention comprises a targeting moiety having an antigen recognition domain that specifically binds to one or more of checkpoint markers, such as 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.

[0250] By way of non-limiting example, in various embodiments, the multispecific CD8-binding substance comprises: (i) CD8; (ii) a targeting moiety that targets one or more of checkpoint markers expressed on T cells, such as PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, Cd27, CD40L, TIM3, and A2aR; and / or (iii) the targeting moiety targets tumor cells in addition to any of the modified (e.g., mutant) signaling substances described herein.

[0251] In various embodiments, the multispecific CD8-binding substance of the present invention has one or more targeting moieties that target PD-1. In some embodiments, the CD8-binding substance has one or more targeting moieties that selectively bind to the PD-1 polypeptide. In some embodiments, the CD8-binding substance comprises one or more of an antibody, antibody derivative or format, peptide or polypeptide, or fusion protein that selectively binds to the PD-1 polypeptide.

[0252] In various embodiments, the multispecific CD8-binding substance of the present invention comprises a VHH for PD1 having a variable domain comprising at least one of the CDR1, CDR2, and / or CDR3 sequences.

[0253] In some embodiments, the CDR1 sequence is selected from SEQ ID NOs: 651 to 664.

[0254] In some embodiments, the CDR2 sequence is selected from SEQ ID NO: 665 to SEQ ID NO: 678.

[0255] In some embodiments, the CDR3 sequence is selected from SEQ ID NO: 679 to SEQ ID NO: 692.

[0256] In various exemplary embodiments, the PD1 targeting moiety comprises an amino acid sequence selected from the following sequences: 2PD23 (SEQ ID NO: 693); or 2PD26 (SEQ ID NO: 694); or 2PD90 (SEQ ID NO: 695); or 2PD106 (SEQ ID NO: 696); or 2PD16 (SEQ ID NO: 697); or 2PD71 (SEQ ID NO: 698); or 2PD152 (SEQ ID NO: 699); or 2PD12 (SEQ ID NO: 700); or 3PD55 (SEQ ID NO: 701); or 3PD82 (SEQ ID NO: 702); or 2PD8 (SEQ ID NO: 703); or 2PD27 (SEQ ID NO: 704); or 2PD82 (SEQ ID NO: 705); or 3PD36 (SEQ ID NO: 706).

[0257] In various exemplary embodiments, the PD1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include a terminal histidine tag sequence (i.e., HHHHHH: SEQ ID NO: 1213).

[0258] In some embodiments, the PD1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include a terminal HA tag (i.e., YPYDVPDYGS; SEQ ID NO: 1214).

[0259] In some embodiments, the PD1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include an AAA linker.

[0260] In some embodiments, the PD1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include an AAA linker, an HA tag, and a terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 1215).

[0261] In certain embodiments, the targeting moiety comprises the anti-PD-1 antibody pembrolizumab (also known as MK-3475, Keytruda), or a fragment thereof. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, et al. (2013) New England Journal of Medicine 369(2):134-44, US 8,354,509, and International Publication No. WO 2009 / 114335. The entire disclosures of these are incorporated herein by reference. In an exemplary embodiment, the pembrolizumab or an antigen-binding fragment thereof provided herein for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 707 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 708.

[0262] In certain embodiments, the targeting moiety comprises the anti-PD-1 antibody nivolumab (also known as BMS-936558, MDX-1106, ONO-4538, Opdivo), or a fragment thereof. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Patent No. 8,008,449 and International Publication No. WO 2006 / 121168. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, nivolumab or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 709 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 710.

[0263] In certain embodiments, 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-I monoclonal antibodies are disclosed in U.S. Patent Application Publication No. 2008 / 0025980 and International Publication No. 2009 / 101611. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 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 SEQ ID NOs: 15-18 of U.S. Patent Application Publication No. 2008 / 0025980; SEQ ID NO: 15 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 711); SEQ ID NO: 16 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 712); SEQ ID NO: 17 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 713); SEQ ID NO: 18 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 714), and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 20-24 of U.S. Patent Application Publication No. 2008 / 0025980; SEQ ID NO: 20 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 715); SEQ ID NO: 21 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 716); SEQ ID NO: 22 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 717); SEQ ID NO: 23 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 718); or SEQ ID NO: 24 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO: 719).

[0264] In certain embodiments, the targeting moiety comprises a light chain comprising SEQ ID NO: 18 of U.S. Patent Application Publication No. 2008 / 0025980 and a heavy chain comprising SEQ ID NO: 22 of U.S. Patent Application Publication No. 2008 / 0025980.

[0265] In certain embodiments, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0266] In certain embodiments, the targeting moiety comprises the pd-l2-Fc fusion protein AMP-224, which is disclosed in International Publication Nos. WO 2010 / 027827 and WO 2011 / 066342, the entire disclosures of which are incorporated herein by reference. In such embodiments, the targeting moiety may comprise a targeting domain comprising SEQ ID NO: 4 (SEQ ID NO: 720) of International Publication No. WO 2010 / 027827 and / or a B7-DC fusion protein comprising SEQ ID NO: 83 (SEQ ID NO: 721) of International Publication No. WO 2010 / 027827.

[0267] In certain embodiments, the targeting moiety comprises peptide AUNP12 or any other peptide disclosed in US Patent Application Publication No. US 2011 / 0318373 or US Patent No. 8,907,053. For example, the targeting moiety may comprise AUNP12 (i.e., Compound 8 or SEQ ID NO: 49 of US Patent Application Publication No. US 2011 / 0318373), which has the following sequence of SEQ ID NO: 722: SNTSESFK(SNTSESF)FRVTQLAPKAQIKE-NH2

Chemical formula

[0268] In certain embodiments, as disclosed in US Patent Application Publication No. US 2014 / 0044738, the targeting moiety comprises the anti-PD-1 antibody 1E3 or a fragment thereof. The entire disclosure of this patent is incorporated herein by reference. In an exemplary embodiment, 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 723; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 724.

[0269] In certain embodiments, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the targeting moiety comprises the anti-PD-1 antibody 1E8 or a fragment thereof. The entire disclosure of this patent is incorporated herein by reference. In exemplary 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: 725; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 726.

[0270] In certain embodiments, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the targeting moiety comprises the anti-PD-1 antibody 1H3 or a fragment thereof. The entire disclosure of this patent is incorporated herein by reference. In exemplary 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: 727; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 728.

[0271] In certain embodiments, the targeting moiety comprises a VHH that targets PD-1, as disclosed, for example, in U.S. Patent No. 8,907,065 and International Publication No. 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In exemplary embodiments, the VHH against PD-1 comprises SEQ ID NOs: 347-351 of U.S. Patent No. 8,907,065; SEQ ID NO: 347 of U.S. Patent No. 8,907,065 (SEQ ID NO: 729); SEQ ID NO: 348 of U.S. Patent No. 8,907,065 (SEQ ID NO: 730); SEQ ID NO: 349 of U.S. Patent No. 8,907,065 (SEQ ID NO: 731); SEQ ID NO: 350 of U.S. Patent No. 8,907,065 (SEQ ID NO: 732); or SEQ ID NO: 351 of U.S. Patent No. 8,907,065 (SEQ ID NO: 733).

[0272] In certain embodiments, the targeting moiety comprises any one of the anti-PD-1 antibodies or fragments thereof as disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and International Publication No. 2010 / 036959, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 25-29 of U.S. Patent Application Publication No. 2011 / 0271358: SEQ ID NO: 25 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 734); SEQ ID NO: 26 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 735); SEQ ID NO: 27 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 736); SEQ ID NO: 28 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 737); SEQ ID NO: 29 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 738); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 30-33 of U.S. Patent Application Publication No. 2011 / 0271358: SEQ ID NO: 30 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 739); SEQ ID NO: 31 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 740); SEQ ID NO: 32 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 741); SEQ ID NO: 33 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 742).

[0273] In various embodiments, the multi-specific CD8-binding moiety comprises one or more antibodies or antibody fragments directed to PD-1 selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene Ltd.).

[0274] In various embodiments, the present multispecific CD8-binding substance has one or more targeting moieties that target PD-L1. In some embodiments, the CD8-binding substance has one or more targeting moieties that selectively bind to the PD-L1 polypeptide. In some embodiments, the CD8-binding substance comprises one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds to the PD-L1 polypeptide.

[0275] In various embodiments, the multispecific CD8-binding substance of the present invention comprises a VHH against PD-L1 having a variable domain comprising at least one of the CDR1, CDR2, and / or CDR3 sequences.

[0276] In some embodiments, the CDR1 sequence is selected from SEQ ID NOs: 743 to 773.

[0277] In some embodiments, the CDR2 sequence is selected from SEQ ID NOs: 774 to 804.

[0278] In some embodiments, the CDR3 sequence is selected from SEQ ID NOs: 805 to 835.

[0279] In various exemplary embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from the following sequences: 2LIG2 (SEQ ID NO: 836); or 2LIG3 (SEQ ID NO: 837); or 2LIG16 (SEQ ID NO: 838) or 2LIG22 (SEQ ID NO: 839) or 2LIG27 (SEQ ID NO: 840) or 2LIG29 (SEQ ID NO: 841) or 2LIG30 (SEQ ID NO: 842) or 2LIG34 (SEQ ID NO: 843) or 2LIG35 (SEQ ID NO: 844) or 2LIG48 (SEQ ID NO: 845) or 2LIG65 (SEQ ID NO: 846) or 2LIG85 (SEQ ID NO: 847) or 2LIG86 (SEQ ID NO: 848) or 2LIG89 (SEQ ID NO: 849) or 2LIG97 (SEQ ID NO: 850) or 2LIG99 (SEQ ID NO: 851) or 2LIG109 (SEQ ID NO: 852) or 2LIG127 (SEQ ID NO: 853) or 2LIG139 (SEQ ID NO: 854) or 2LIG176 (SEQ ID NO: 855) or 2LIG189 (SEQ ID NO: 856) or 3LIG3 (SEQ ID NO: 857) or 3LIG7 (SEQ ID NO: 858) or 3LIG8 (SEQ ID NO: 859) or 3LIG9 (SEQ ID NO: 860) or 3LIG18 (SEQ ID NO: 861) or 3LIG20 (SEQ ID NO: 862) or 3LIG28 (SEQ ID NO: 863) or 3LIG29 (SEQ ID NO: 864) or 3LIG30 (SEQ ID NO: 865) or 3LIG33 (SEQ ID NO: 866).

[0280] In various exemplary embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include a terminal histidine tag sequence (i.e., HHHHHH: SEQ ID NO: 1213).

[0281] In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include a terminal HA tag (i.e., YPYDVPDYGS; SEQ ID NO: 1214).

[0282] In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include an AAA linker.

[0283] In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from any one of the above sequences that does not include an AAA linker, an HA tag, and a terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 1215).

[0284] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab), or a fragment thereof. MEDI4736 is selective for PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80 receptors. MEDI4736 and antigen-binding fragments thereof for use in the methods provided herein comprise a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. The sequence of MEDI4736 is disclosed in International Publication No. WO 2016 / 06272, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 867 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 868.

[0285] In an exemplary embodiment, 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: 869) of International Publication No. WO 2016 / 06272; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 (SEQ ID NO: 870) of International Publication No. WO 2016 / 06272.

[0286] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (also known as MPDL3280A, RG7446), or a fragment thereof. In an exemplary embodiment, atezolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 871; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 872.

[0287] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody avelumab (also known as MSB0010718C), or a fragment thereof. In an exemplary embodiment, the atezolizumab or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 873; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 874.

[0288] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105), or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the BMS-936559 or 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: 875; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 876.

[0289] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3G10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the 3G10 or 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: 877; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 878.

[0290] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10A5, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the 10A5 or 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: 879; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 880.

[0291] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 5F8, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 5F8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 881; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 882.

[0292] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 10H10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 10H10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 883; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 884.

[0293] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 1B12, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 1B12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 885; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 886.

[0294] In certain embodiments, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, the targeting moiety comprises the anti-PD-L1 antibody 7H1, or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 7H1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 887; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 888.

[0295] In certain embodiments, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, the targeting moiety comprises the anti-PD-L1 antibody 11E6, or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 11E6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 889; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 890.

[0296] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 12B7, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 12B7 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 891; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 892.

[0297] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 13G4, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the 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: 893; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 894.

[0298] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 1E12, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738. The entire disclosure of this patent is incorporated herein by reference. In an exemplary embodiment, the 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: 895; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 896.

[0299] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 1F4, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738. The entire disclosure of this patent is incorporated herein by reference. In an exemplary embodiment, the 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: 897; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 898.

[0300] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2G11, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738. The entire disclosure of this patent is incorporated herein by reference. In an exemplary embodiment, the 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: 899; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 900.

[0301] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3B6, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738. The entire disclosure of this patent is incorporated herein by reference. In an exemplary embodiment, 3B6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 901; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 902.

[0302] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3D10, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738 and International Publication No. 2012 / 145493. The entire disclosure of these patents is incorporated herein by reference. In an exemplary embodiment, 3D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 903; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 904.

[0303] In certain embodiments, the targeting moiety comprises an anti-PD-L1 antibody of either of the types disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and International Publication No. 2010 / 036959. The entire contents of these patents are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 34-38 of U.S. Patent Application Publication No. 2011 / 0271358: SEQ ID NO: 34 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 905); SEQ ID NO: 35 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 906); SEQ ID NO: 36 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 907); SEQ ID NO: 37 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 908); SEQ ID NO: 38 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 909); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 39-42 of U.S. Patent Application Publication No. 2011 / 0271358: SEQ ID NO: 39 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 910); SEQ ID NO: 40 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 911); SEQ ID NO: 41 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 912); SEQ ID NO: 42 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 913).

[0304] In certain embodiments, as disclosed in International Publication No. 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the targeting moiety comprises anti-PD-L1 antibody 2.7A4 or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.7A4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 of International Publication No. 2011 / 066389 (SEQ ID NO: 914); and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 of International Publication No. 2011 / 066389 (SEQ ID NO: 915).

[0305] In certain embodiments, as disclosed in International Publication No. WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the targeting moiety comprises an anti-PD-L1 antibody 2.9D10 or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.9D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (SEQ ID NO: 916) of International Publication No. WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (SEQ ID NO: 917) of International Publication No. WO 2011 / 066389.

[0306] In certain embodiments, as disclosed in International Publication No. WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the targeting moiety comprises an anti-PD-L1 antibody 2.14H9 or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.14H9 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 (SEQ ID NO: 918) of International Publication No. WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27 (SEQ ID NO: 919) of International Publication No. WO 2011 / 066389.

[0307] In certain embodiments, as disclosed in International Publication No. WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the targeting moiety comprises an anti-PD-L1 antibody 2.20A8 or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.20A8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32 (SEQ ID NO: 920) of International Publication No. WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 (SEQ ID NO: 921) of International Publication No. WO 2011 / 066389.

[0308] In certain embodiments, as disclosed in International Publication No. WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the targeting moiety comprises the anti-PD-L1 antibody 3.15G8 or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the 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: 922) of International Publication No. WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 923.

[0309] In certain embodiments, as disclosed in International Publication No. WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the targeting moiety comprises the anti-PD-L1 antibody 3.18G1 or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the 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: 924) of International Publication No. WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 (SEQ ID NO: 925) of International Publication No. WO 2011 / 066389.

[0310] In certain embodiments, as disclosed in International Publication No. WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4OPT or a fragment thereof. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the 2.7A4OPT or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62 (SEQ ID NO: 926) of International Publication No. WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 67 (SEQ ID NO: 927) of International Publication No. WO 2011 / 066389.

[0311] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9OPT or a fragment thereof, as disclosed in International Publication No. WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, 2.14H9OPT or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 (SEQ ID NO: 928) of International Publication No. WO 2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 77 (SEQ ID NO: 929) of International Publication No. WO 2011 / 066389.

[0312] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 061142. The entire content of this patent is incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods provided herein has the amino acid sequence of SEQ ID NO: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of WO 2016 / 061142; SEQ ID NO: 18 (SEQ ID NO: 930) of WO 2016 / 061142; SEQ ID NO: 30 (SEQ ID NO: 931) of WO 2016 / 061142; SEQ ID NO: 38 (SEQ ID NO: 932) of WO 2016 / 061142; SEQ ID NO: 46 (SEQ ID NO: 933) of WO 2016 / 061142; SEQ ID NO: 50 (SEQ ID NO: 934) of WO 2016 / 061142; SEQ ID NO: 54 (SEQ ID NO: 935) of WO 2016 / 061142; SEQ ID NO: 62 (SEQ ID NO: 936) of WO 2016 / 061142; SEQ ID NO: 70 (SEQ ID NO: 937) of WO 2016 / 061142; SEQ ID NO: 78 (SEQ ID NO: 938) of WO 2016 / 061142, and comprises a heavy chain; and / or the amino acid sequence of SEQ ID NO: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO 2016 / 061142; SEQ ID NO: 22 (SEQ ID NO: 939) of WO 2016 / 061142; SEQ ID NO: 26 (SEQ ID NO: 940) of WO 2016 / 061142; SEQ ID NO: 34 (SEQ ID NO: 941) of WO 2016 / 061142; SEQ ID NO: 42 (SEQ ID NO: 942) of WO 2016 / 061142; SEQ ID NO: 58 (SEQ ID NO: 943) of WO 2016 / 061142; SEQ ID NO: 66 (SEQ ID NO: 944) of WO 2016 / 061142; SEQ ID NO: 74 (SEQ ID NO: 945) of WO 2016 / 061142; SEQ ID NO: 82 (SEQ ID NO: 946) of WO 2016 / 061142; SEQ ID NO: 86 (SEQ ID NO: 947) of WO 2016 / 061142, and comprises a light chain.

[0313] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 022630. The entire content of this patent is incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods provided herein has the amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WO 2016 / 022630; SEQ ID NO: 2 of WO 2016 / 022630 (SEQ ID NO: 948); SEQ ID NO: 6 of WO 2016 / 022630 (SEQ ID NO: 949); SEQ ID NO: 10 of WO 2016 / 022630 (SEQ ID NO: 950); SEQ ID NO: 14 of WO 2016 / 022630 (SEQ ID NO: 951); SEQ ID NO: 18 of WO 2016 / 022630 (SEQ ID NO: 952); SEQ ID NO: 22 of WO 2016 / 022630 (SEQ ID NO: 953); SEQ ID NO: 26 of WO 2016 / 022630 (SEQ ID NO: 954); SEQ ID NO: 30 of WO 2016 / 022630 (SEQ ID NO: 955); SEQ ID NO: 34 of WO 2016 / 022630 (SEQ ID NO: 956); SEQ ID NO: 38 of WO 2016 / 022630 (SEQ ID NO: 957); SEQ ID NO: 42 of WO 2016 / 022630 (SEQ ID NO: 958); SEQ ID NO: 46 of WO 2016 / 022630 (SEQ ID NO: 959) for the heavy chain; and / or SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 of WO 2016 / 022630; SEQ ID NO: 4 of WO 2016 / 022630 (SEQ ID NO: 960); SEQ ID NO: 8 of WO 2016 / 022630 (SEQ ID NO: 961); SEQ ID NO: 12 of WO 2016 / 022630 (SEQ ID NO: 962); SEQ ID NO: 16 of WO 2016 / 022630 (SEQ ID NO: 963); SEQ ID NO: 20 of WO 2016 / 022630 (SEQ ID NO: 964); SEQ ID NO: 24 of WO 2016 / 022630 (SEQ ID NO: 965); SEQ ID NO: 28 of WO 2016 / 022630 (SEQ ID NO: 966); SEQ ID NO: 32 of WO 2016 / 022630 (SEQ ID NO: 967); SEQ ID NO: 36 of WO 2016 / 022630 (SEQ ID NO: 968);It includes a light chain comprising an amino acid sequence selected from SEQ ID NO: 40 (SEQ ID NO: 969) of International Publication No. WO2016 / 022630; SEQ ID NO: 44 (SEQ ID NO: 970) of International Publication No. WO2016 / 022630; SEQ ID NO: 48 (SEQ ID NO: 971) of International Publication No. WO2016 / 022630;

[0314] In certain embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in International Publication No. WO2015 / 112900. The entire content of this patent is incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 38, 50, 82, and 86 of International Publication No. WO2015 / 112900; SEQ ID NO: 38 (SEQ ID NO: 972) of International Publication No. WO2015 / 112900; SEQ ID NO: 50 (SEQ ID NO: 973) of International Publication No. WO2015 / 112900; SEQ ID NO: 82 (SEQ ID NO: 974) of International Publication No. WO2015 / 112900; SEQ ID NO: 86 (SEQ ID NO: 975) of International Publication No. WO2015 / 112900; and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of International Publication No. WO2015 / 112900; SEQ ID NO: 42 (SEQ ID NO: 976) of International Publication No. WO2015 / 112900; SEQ ID NO: 46 (SEQ ID NO: 977) of International Publication No. WO2015 / 112900; SEQ ID NO: 54 (SEQ ID NO: 978) of International Publication No. WO2015 / 112900; SEQ ID NO: 58 (SEQ ID NO: 979) of International Publication No. WO2015 / 112900; SEQ ID NO: 62 (SEQ ID NO: 980) of International Publication No. WO2015 / 112900; SEQ ID NO: 66 (SEQ ID NO: 981) of International Publication No. WO2015 / 112900; SEQ ID NO: 70 (SEQ ID NO: 982) of International Publication No. WO2015 / 112900; SEQ ID NO: 74 (SEQ ID NO: 983) of International Publication No. WO2015 / 112900; SEQ ID NO: 78 (SEQ ID NO: 984) of International Publication No. WO2015 / 112900.

[0315] In certain embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in International Publication No. WO 2010 / 077634 and U.S. Patent No. 8,217,149. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the anti-PD-L1 or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 20 (SEQ ID NO: 985) of International Publication No. WO 2010 / 077634; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21 (SEQ ID NO: 986) of International Publication No. WO 2010 / 077634.

[0316] In certain embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies obtainable from hybridomas available under CNCM Accession Nos. CNCM I-4122, CNCM I-4080, and CNCM I-4081, as disclosed in U.S. Patent Application Publication No. 20120039906. The entire disclosures of these patents are incorporated herein by reference.

[0317] In certain embodiments, the targeting moiety comprises a VHH directed against a PD-L1 antibody, for example, as disclosed in U.S. Patent No. 8,907,065 and International Publication No. WO 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the VHH against PD-L1 comprises SEQ ID NOs: 394-399 of U.S. Patent No. 8,907,065; SEQ ID NO: 394 (SEQ ID NO: 987) of U.S. Patent No. 8,907,065; SEQ ID NO: 395 (SEQ ID NO: 988) of U.S. Patent No. 8,907,065; SEQ ID NO: 396 (SEQ ID NO: 989) of U.S. Patent No. 8,907,065; SEQ ID NO: 397 (SEQ ID NO: 990) of U.S. Patent No. 8,907,065; SEQ ID NO: 398 (SEQ ID NO: 991) of U.S. Patent No. 8,907,065; SEQ ID NO: 399 (SEQ ID NO: 992) of U.S. Patent No. 8,907,065.

[0318] In various embodiments, the present multi-specific CD8 binding substance has one or more targeting moieties that target PD-L2. In some embodiments, the CD8 binding substance has one or more targeting moieties that selectively bind to the PD-L2 polypeptide. In some embodiments, the CD8 binding substance comprises one or more of an antibody, an antibody derivative or format, a peptide or polypeptide, or a fusion protein that selectively binds the PD-L2 polypeptide.

[0319] In certain embodiments, the targeting moiety comprises, for example, a VHH that targets PD-L2, as disclosed in U.S. Patent No. 8,907,065 and International Publication No. 2008 / 071447. The entire disclosures of these patents are incorporated herein by reference. In an exemplary embodiment, the VHHs against PD-1 include SEQ ID NOs: 449 - 455 of U.S. Patent No. 8,907,065: SEQ ID NO: 449 of U.S. Patent No. 8,907,065 (SEQ ID NO: 993); SEQ ID NO: 450 of U.S. Patent No. 8,907,065 (SEQ ID NO: 994); SEQ ID NO: 451 of U.S. Patent No. 8,907,065 (SEQ ID NO: 995); SEQ ID NO: 452 of U.S. Patent No. 8,907,065 (SEQ ID NO: 996); SEQ ID NO: 453 of U.S. Patent No. 8,907,065 (SEQ ID NO: 997); SEQ ID NO: 454 of U.S. Patent No. 8,907,065 (SEQ ID NO: 998); SEQ ID NO: 455 of U.S. Patent No. 8,907,065 (SEQ ID NO: 999).

[0320] In one embodiment, the targeting moiety comprises an anti-PD-L2 antibody of either of the types disclosed in US Patent Application Publication No. 2011 / 0271358 and International Publication No. 2010 / 036959. The entire contents of these patents are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 43-47 of US Patent Application Publication No. 2011 / 0271358: SEQ ID NO: 43 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1000); SEQ ID NO: 44 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1001); SEQ ID NO: 45 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1002); SEQ ID NO: 46 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1003); SEQ ID NO: 47 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1004); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 48-51 of US Patent Application Publication No. 2011 / 0271358: SEQ ID NO: 48 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1005); SEQ ID NO: 49 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1006); SEQ ID NO: 50 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1007); SEQ ID NO: 51 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 1008).

[0321] In various embodiments, the targeting moiety of the invention can comprise a sequence that targets PD-1, PD-L1, and / or PD-L2 and is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99% or about 100% sequence identity) to any of the sequences disclosed herein.

[0322] In various embodiments, the targeting moiety of the invention can include any combination of heavy chains, light chains, heavy chain variable regions, light chain variable regions, complementarity determining regions (CDRs), and framework region sequences that target PD-1, PD-L1, and / or PD-L2 disclosed herein.

[0323] Additional antibodies, antibody derivatives or formats, 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 2007 / 005874, WO 2001 / 014556, US 2011 / 0271358, WO 2010 / 036959, WO 2010 / 077634, US 8,217,149, US 2012 / 0039906, WO 2012 / 145493, US 2011 / 0318373, US 8,779,108, US 2014 / 0044738, WO 2009 / 089149, WO 2007 / 00587, WO 2016 / 061142, WO 2016 / 02263, WO 2010 / 077634, and WO 2015 / 112900. The entire disclosures of these patents are incorporated herein by reference.

[0324] In various embodiments, the multispecific CD8-binding substance of the invention comprises a targeting moiety having an antigen recognition domain that specifically binds to XCR1, e.g., on DCs. In various embodiments, the multispecific CD8-binding substance of the invention comprises a targeting moiety having an antigen recognition domain that comprises all or part of XCL1.

[0325] In various embodiments, the multispecific CD8-binding substance has a targeting moiety having a recognition domain that specifically binds to a target that is part of a non-cellular structure (e.g., an antigen, a receptor). In some embodiments, the antigen or receptor is not an essential component of an intact cell or cellular structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-proteinaceous non-cellular marker, which includes, but is not limited to, nucleic acids such as DNA or RNA, including, for example, DNA released from necrotic tumor cells, or extracellular deposits such as cholesterol.

[0326] In some embodiments, the target of interest (e.g., an antigen, a receptor) is part of a non-cellular component of the stroma or extracellular matrix (ECM) or a marker associated therewith. As used herein, stroma refers to the connective and supportive framework of a tissue or organ. Stroma can include a collection of cells such as fibroblasts / myofibroblasts / glial cells, epithelium, adipose, immune, vascular, smooth muscle, and immune cells, along with the extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target of interest (e.g., an antigen, a receptor) is part of a non-cellular component of the stroma such as the extracellular matrix and extracellular molecules. As used herein, ECM refers to the non-cellular components present in all tissues and organs. The ECM consists of a multitude of biochemically distinct components including, but not limited to, proteins, glycoproteins, proteoglycans, and polysaccharides. These ECM components are typically produced by adjacent cells and secreted into the ECM by exocytosis. Once secreted, the ECM components often aggregate to form a complex network of macromolecules. In various embodiments, the chimeric proteins of the invention include a targeting moiety that recognizes a target (e.g., an antigen or receptor or non-protein molecule) located on any component of the ECM. Examples of ECM components 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).

[0327] In some embodiments, the targeting moiety recognizes a target (e.g., an antigen, a receptor) on an ECM proteoglycan. A proteoglycan is a glycosylated protein. A basic proteoglycan unit comprises a core protein having one or more covalently attached glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+), which attracts water via osmosis and keeps the ECM and resident cells hydrated. Proteoglycans can also capture growth factors and store them within the ECM. Examples of proteoglycans that can be targeted by the chimeric proteins of the present invention include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In certain embodiments, the targeting moiety recognizes a target (e.g., an antigen, a receptor) on a non-proteoglycan polysaccharide such as hyaluronic acid.

[0328] In some embodiments, the targeting moiety recognizes a target (e.g., an antigen, a receptor) on an ECM fiber. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety recognizes one or more epitopes on collagen or a collagen fiber. Collagen is the most abundant protein in the ECM. Collagen exists as a fibrous protein in the ECM and provides structural support to resident cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagen present within the ECM, including, but not limited to, fibrillar collagen (types I, II, III, V, XI), FACIT collagen (types IX, XII, XIV), short-chain collagen (types VIII, X), basement membrane collagen (type IV), and / or type VI, VII, or XIII collagen. Elastin fibers give tissues elasticity, allowing the tissue to stretch and contract as needed and then return to its original state. In some embodiments, the targeting moiety recognizes one or more epitopes on elastin or an elastin fiber.

[0329] In some embodiments, the targeting moiety recognizes one or more ECM proteins including, but not limited to, tenascin, fibronectin, fibrin, laminin, or nidogen / entactin.

[0330] In one embodiment, the targeting moiety recognizes and binds to tenascin. The tenascin (TN) family of glycoproteins includes at least four members: tenascin-C, tenascin-R, tenascin-X, and tenascin-W. The primary structure of tenascin proteins includes several common motifs ordered in the same continuous sequence: a seven-amino acid terminal repeat, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain. Each protein member is associated with typical variations in the number and nature of the EGF-like and fibronectin type III repeats. Isoform variants also exist, particularly for tenascin-C. Over 27 splice variants and / or isoforms of tenascin-C are known. In certain embodiments, the targeting moiety recognizes and binds to tenascin-CA1. Similarly, tenascin-R also has various splice variants and isoforms. Tenascin-R typically exists as a dimer or trimer. Tenascin-X is the largest member of the tenascin family and is known to exist as a trimer. Tenascin-W exists as a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on the tenascin protein. In some embodiments, the targeting moiety recognizes monomeric and / or dimeric and / or trimeric and / or hexameric forms of the tenascin protein.

[0331] In certain embodiments, the targeting moiety recognizes and binds to fibronectin. Fibronectin is a glycoprotein that links cells to collagen fibers in the ECM and enables cells to move through the ECM. When binding to integrins, fibronectin unfolds to form a functional dimer. In some embodiments, the targeting moiety recognizes monomeric and / or dimeric forms of fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. In an exemplary embodiment, the targeting moiety recognizes fibronectin extracellular domain A (EDA) or fibronectin extracellular domain B (EDB). Elevated levels of EDA are associated with various diseases and disorders including psoriasis, rheumatoid arthritis, diabetes, and cancer. In some embodiments, the targeting moiety recognizes fibronectin comprising the EDA isoform and can be used to target the chimeric protein to diseased cells including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin comprising the EDB isoform. In various embodiments, such targeting moieties can be used to target the chimeric protein to tumor cells including tumor neovessels.

[0332] In certain embodiments, the targeting moiety recognizes and binds to fibrin. Fibrin is another proteinaceous substance often found in the matrix network of the ECM. Fibrin is formed by the action of the protease thrombin on fibrinogen, thereby polymerizing fibrin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes monomeric as well as polymeric forms of fibrin.

[0333] In some embodiments, the targeting moiety recognizes and binds to laminin. Laminin is a major component of the basement membrane and serves as a protein network infrastructure for cells and organs. Laminin is a heterotrimeric protein that includes an α-chain, a β-chain, and a γ-chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes monomeric, dimeric, and trimeric forms of laminin.

[0334] In some embodiments, the targeting moiety recognizes and binds to nidogen or entactin. Nidogen / entactin is a highly conserved family of sulfated glycoproteins. They form major structural components of the basement membrane and function to link laminin to the collagen IV network in the basement membrane. Members of this family include nidogen-1 and nidogen-2. In various embodiments, the targeting moiety recognizes epitopes on nidogen-1 and / or nidogen-2.

[0335] In various embodiments, the targeting moiety includes an antigen recognition domain that recognizes an epitope present on any of the targets (e.g., ECM proteins) described herein. In some embodiments, the antigen recognition domain recognizes one or more linear epitopes present on a protein. As used herein, a linear epitope refers to any continuous sequence of amino acids present on a protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on a protein. As used herein, a conformational epitope refers to a portion (which may be discontinuous) of one or more amino acids that forms a three-dimensional surface with features and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0336] In various embodiments, the targeting moiety can bind to the full-length and / or mature and / or isoform and / or splice variant and / or fragment and / or any other natural or synthetic analog, variant, or mutant of any of the targets described herein (e.g., an ECM protein). In various embodiments, the targeting moiety can bind to any type of protein described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers, and associated forms. In various embodiments, the targeting moiety can bind to any post-translationally modified form of a protein described herein, such as a glycosylated and / or phosphorylated form.

[0337] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an extracellular molecule such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In one embodiment, the DNA is shed into the extracellular space from necrotic cells or apoptotic tumor cells or other diseased cells.

[0338] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaques are known. Fibro-lipid (fibro-fatty) plaques are characterized by the accumulation of lipid-laden cells beneath the intima of the artery. Beneath the endothelium, there is a fibrous cap covering the atherosclerotic plaque core. The core contains lipid-laden cells (macrophages and smooth muscle cells), fibrin, proteoglycans, collagen, elastin, and necrotic cell debris with increased tissue cholesterol and cholesterol ester content. In advanced plaques, the central core of the plaque usually contains extracellular cholesterol deposits (released from dead cells), which form regions of cholesterol crystals with empty needle-like spaces. At the periphery of the plaque, there are younger foam cells and capillaries. Fibrous plaques are also located beneath the intima within the arterial wall, causing focal narrowing of the lumen that is sometimes associated with wall thickening and proliferation and sometimes with some atrophy of the muscular layer. Fibrous plaques contain collagen fibers (eosinophilic), calcium deposits (hematoxylinophilic), and lipid-laden cells. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular components of these plaques, such as fibrin, proteoglycans, collagen, elastin, necrotic cell debris, and calcium or other mineral deposits or precipitates. In some embodiments, the necrotic cell debris is nucleic acid, such as DNA or RNA released from dead cells.

[0339] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures found in brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures localized in amyloid plaques found in the brains of patients with Alzheimer's disease. For example, the targeting moiety recognizes and binds to peptide amyloid beta, which is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in brain plaques found in patients with Huntington's disease. In various embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in plaques associated with other neurodegenerative diseases or musculoskeletal diseases such as Lewy body dementia and inclusion body myositis.

[0340] Linker and functional group In various embodiments, the CD8 binding substance may comprise one or more functional groups, residues, or moieties. In various embodiments, the one or more functional groups, residues, or moieties are attached or genetically fused to any of the signaling substances or targeting moieties described herein. In some embodiments, such functional groups, residues or moieties impart one or more desirable properties or functional groups to the CD8 binding substance of the invention. Examples of such functional groups and techniques for introducing them into CD8 binding substances are known in the art; see, for example, Remington’s Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

[0341] In various embodiments, the CD8 binding substance can be conjugated and / or fused with another substance to extend its half-life or otherwise improve its pharmacodynamic and pharmacokinetic properties. In some embodiments, the CD8 binding substance can be fused or conjugated with one or more of PEG, XTEN (e.g., as rPEG), polyxen, albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, etc. In some embodiments, the CD8 binding substance can be fused or conjugated with an antibody or an antibody fragment such as an Fc fragment. For example, the chimeric protein can be fused to the N-terminus or C-terminus of the Fc domain of human immunoglobulin (Ig) G. In various embodiments, each individual chimeric protein is fused to one or more substances described in BioDrugs (2015) 29:215-239, the entire contents of which are incorporated herein by reference.

[0342] In some embodiments, the functional group, residue, or moiety comprises a suitable pharmaceutically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly(ethylene glycol) or mPEG). In some embodiments, the attachment of the PEG moiety extends the half-life and / or reduces the immunogenicity of the CD8-binding protein. For example, any suitable form of pegylation, such as that used in the art for antibodies and antibody fragments (including, but not limited to, single domain antibodies such as VHH), is commonly used; see, e.g., Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and WO 04 / 060965. The entire contents of these references are incorporated herein by reference. Various reagents for the pegylation of proteins are also commercially available, e.g., from Nektar Therapeutics, USA. In some embodiments, site-specific pegylation via cysteine residues is used in particular (see, e.g., Yang et al., Protein Engineering, 16, 10, 761-770 (2003), the entire contents of which are incorporated herein by reference). For example, for this purpose, it can be attached to the native cysteine residues in the CD8-binding substances of the present invention. In some embodiments, the CD8-binding substances of the present invention are modified to appropriately introduce one or more cysteine residues for the attachment of PEG, or an amino acid sequence containing one or more cysteine residues for the attachment of PEG can be fused to the amino terminus and / or carboxy terminus of the CD8-binding substance using techniques known in the art. In some embodiments, the functional group, residue, or moiety comprises N-linked or O-linked glycosylation. In some embodiments, the N-linked or O-linked glycosylation is introduced as part of a co-translational and / or post-translational modification.

[0343] In some embodiments, a functional group, residue, or moiety includes one or more detectable labels or other signal generating groups or moieties. Suitable labels and techniques for their attachment, use, and detection are known in the art and include, but are not limited to, fluorescent labels (e.g., fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine and fluorescent metals such as Eu or other metals of the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (e.g., luminol, isoluminol, aromatic acridinium esters, imidazole, acridinium salts, oxalate esters, dioxetanes or GFP and its analogs), radioisotopes, metals, metal chelates or metal cations or other metals or metal cations particularly suitable for use in in vivo, in vitro or in situ diagnosis and imaging, as well as chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase). Other suitable labels include moieties detectable using NMR or ESR spectroscopy. The VHHs and polypeptides of the invention so labeled can be used for in vitro, in vivo or in situ assays (immunological assays known per se as ELISA, RIA and EIA and other "sandwich methods" etc.) and for in vivo diagnosis and imaging purposes, depending on the choice of the particular label.

[0344] In some embodiments, the functional group, residue, or moiety includes a tag that is bound or genetically fused to the CD8 binding substance. In some embodiments, the CD8 binding substance can include a single tag or multiple tags. For example, the tag is a peptide, sugar, or DNA molecule that does not inhibit or interfere with the binding of the CD8 binding substance to a target antigen such as CD8 or any other tumor antigen. In various embodiments, the tag is at least about: 3-5 amino acids in length, 5-8 amino acids in length, 8-12 amino acids in length, 12-15 amino acids in length, or 15-20 amino acids in length. Representative tags are described, for example, in U.S. Patent Application Publication No. 2013 / 0058962. In some embodiments, the tag is an affinity tag such as glutathione-S-transferase (GST) and histidine (His) tag. In one embodiment, the CD8 binding substance includes a His tag.

[0345] In some embodiments, the functional group, residue, or moiety includes a chelating group for chelating, for example, one of a metal or a metal cation. Suitable chelating groups include, for example, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0346] In some embodiments, the functional group, residue, or moiety comprises a functional group that is part of one member of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such functional groups can be used to link the CD8-binding substance of the invention to another protein, polypeptide, or chemical compound that is conjugated to the other half of the binding pair, i.e., via the binding of the binding pair. For example, the CD8-binding substance of the invention can be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin. For example, in a diagnostic system in which a detectable signal-producing substance is conjugated to avidin or streptavidin, such a conjugated CD8-binding substance can be used, for example, as a reporter. For example, such a binding pair can be used to bind the CD8-binding substance to a carrier such as a carrier suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described in Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Also, such a binding pair can be used to link a therapeutically active agent to the CD8-binding substance of the invention.

[0347] In some embodiments, the present CD8-binding substance optionally comprises one or more linkers. In some embodiments, the present CD8-binding substance comprises a linker that links a targeting moiety and a signaling substance. In some embodiments, the chimeric protein of the invention comprises a linker within the signaling substance (for example, in the case of single-chain TNF, it can comprise two linkers that give rise to a trimer).

[0348] In some embodiments, the CD8-binding substance comprises a linker that links the binding region and / or targeting moiety, respectively. In some embodiments, linkers can be utilized to link the various functional groups, residues, or moieties described herein to the CD8-binding substance. In some embodiments, the linker is a single amino acid or multiple amino acids that do not affect or decrease the stability, orientation, binding, neutralization, and / or excretion characteristics of the binding region and the binding protein. In various embodiments, the linker is selected from a peptide, a protein, a sugar, or a nucleic acid.

[0349] The present invention contemplates the use of various linker sequences. In various embodiments, the linker can be derived from natural multi-domain proteins or can be, for example, empirical linkers as described in Chichili et al., (2013), Protein Sci. 22(2):153-167; Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369. The entire contents of these references are incorporated herein by reference. In some embodiments, the linker can be designed using a linker design database and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369 and Crasto et al., (2000), Protein Eng. 13(5):309-312. In various embodiments, the linker can be functional. For example, but not limited to, the linker can function to improve folding and / or stability, to improve expression, to improve pharmacokinetics, and / or to improve the biological activity of the present CD8-binding substance.

[0350] In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids in length. For example, the linker can be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is flexible. In another embodiment, the linker is rigid.

[0351] In some embodiments, the linker is a polypeptide. In some embodiments, the linker is greater than about 100 amino acids in length. For example, the linker can be greater than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is flexible. In another embodiment, the linker is rigid.

[0352] In various embodiments, the linker is substantially composed of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine). For example, in some embodiments, the linker is (Gly4Ser) n wherein n is from about 1 to about 8, for example, 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NOs: 1009 - 1016). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 1017). Examples of additional linkers include, but are not limited to, linkers having the following sequences: LE, GGGGS (SEQ ID NO: 1009), (GGGGS) n(n = 1 - 4) (SEQ ID NO: 1009 - 1012), (Gly)8 (SEQ ID NO: 1018), (Gly)6 (SEQ ID NO: 1019), (EAAAK) n (n = 1 - 3) (SEQ ID NO: 1020 - 1022), A(EAAAK) n A(n = 2 - 5) (SEQ ID NO: 1023 - 1026), AEAAAKEAAAKA (SEQ ID NO: 1027), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 1028), PAPAP (SEQ ID NO: 1029), KESGSVSSEQLAQFRSLD (SEQ ID NO: 1030), EGKSSGSGSESKST (SEQ ID NO: 1031), GSAGSAAGSGEF (SEQ ID NO: 1032), and (XP) n , where X represents any amino acid, for example, Ala, Lys, or Glu. In various embodiments, the linker is (GGS) n (n = 1 - 20) (SEQ ID NO: 1176 - SEQ ID NO: 1195). In some embodiments, the linker is G. In some embodiments, the linker is AAA. In some embodiments, the linker is (GGGGS) n (n = 9 - 20) (SEQ ID NO: 1196 - SEQ ID NO: 1207).

[0353] In some embodiments, the linker is one or more of GGGSE (SEQ ID NO: 1208), GSESG (SEQ ID NO: 1209), GSEGS (SEQ ID NO: 1210), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 1211), and linkers of G, S, and E randomly arranged at every 4 - amino - acid interval.

[0354] In some embodiments, the linker is the hinge region of an antibody (including, for example, IgG, IgA, IgD, and IgE, subclasses (such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linker is the hinge region of an antibody (including, for example, IgG, IgA, IgD, and IgE, subclasses (such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge regions found in IgG, IgA, IgD, and IgE class antibodies function as flexible spacers, allowing the Fab portions to move freely in space. In contrast to the constant regions, the hinge domain is structurally diverse and varies in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region vary within the IgG subclasses. The hinge region of IgG1 encompasses amino acids 216 - 231, and because it is freely flexible, the Fab fragments can rotate around their axis of symmetry and move within a sphere centered on the first position of the inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1 and has 12 amino acid residues and 4 disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix stabilized by additional inter-heavy chain disulfide bridges. These properties limit the flexibility of the IgG2 molecule. IgG3, unlike other subclasses, forms a polyproline double helix lacking flexibility due to its unique extended hinge region (about 4 times the length of the IgG1 hinge) that contains 62 amino acids (including 21 prolines and 11 cysteines). In IgG3, the Fab fragments are relatively far from the Fc fragment, giving the molecule greater flexibility. The extended hinge of IgG3 is also responsible for its higher molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between IgG1 and IgG2. The flexibility of the hinge region has been reported to decrease in the following order: IgG3 > IgG1 > IgG4 > IgG2.

[0355] According to crystallographic studies, the immunoglobulin hinge region can be functionally further subdivided into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region includes the carboxyl terminus of C H1 to the first residue in the hinge that restricts motion, usually the amino acids of the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the flexibility of the antibody segment. The core hinge region contains the interheavy chain disulfide bridges, and the lower hinge region connects to the amino terminus of the C H2 domain and contains residues in C H2 (ibid.). The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 1212), which is thought to confer flexibility by generating a cyclic octapeptide upon dimerization by disulfide bond formation and functioning as a pivot axis. In various embodiments, the linker of the present invention comprises one, or two, or three upper hinge regions, core regions, and lower hinge regions of any antibody (including, for example, IgG, IgA, IgD, and IgE, subclasses (such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region may also include one or more glycosylation sites, which include many structurally different types of carbohydrate attachment sites. For example, IgA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, which is considered an advantageous property for secretory immunoglobulins. In various embodiments, the linker of the present invention includes one or more glycosylation sites. In various embodiments, the linker is the hinge-CH2-CH3 domain of a human IgG4 antibody.

[0356] Optionally, the CD8 binding substance may be linked to the antibody Fc region, C H 2 and C HSuch polypeptides can be prepared using a vector encoding such a CD8 binding substance linked to the Fc region as a single nucleotide sequence, including one or both of the 3 domains and optionally a hinge region.

[0357] In some embodiments, the linker is a synthetic linker such as PEG.

[0358] In various embodiments, the linker can be functional. For example, without limitation, the linker can function to improve folding and / or stability, to improve expression, to improve pharmacokinetics, and / or to improve the biological activity of the CD8 binding substance. In another example, the linker can function to target the CD8 binding substance to a specific cell type or site.

[0359] Modification and Production of CD8 Binding Substances In various embodiments, the CD8 binding substance comprises a targeting moiety that is a VHH. In various embodiments, the VHH is not limited to a particular biological source or a particular preparation method. For example, the VHH can generally be obtained by: (1) isolating the V H H domain of a natural heavy chain antibody; (2) expressing a nucleotide sequence encoding the natural V H H domain; (3) "humanizing" the natural V H H domain, or such a humanized V Hby expression of a nucleic acid encoding an H domain; (4) "camelization" of a natural VH domain from any animal species, such as from mammalian species including human, or by expression of a nucleic acid encoding such a camelized VH domain; (5) "camelization" of a "domain antibody" or "Dab" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for known proteins, polypeptides or other amino acid sequences in the art; (7) preparing a nucleic acid encoding VHH using nucleic acid synthesis techniques known in the art and subsequently expressing the thus obtained diffusion; and / or (8) by any one or more of the foregoing combinations.

[0360] In certain embodiments, the CD8 binding substance comprises a VHH corresponding to the VH domain of a natural heavy chain antibody that targets human CD8. H In some embodiments, such VH sequences can typically be generated or obtained by appropriately immunizing a camelid animal species with a CD8 molecule (i.e., immunizing to generate an immune response to CD8 and / or to produce a heavy chain antibody to CD8), obtaining a suitable biological sample (e.g., a blood sample, or any sample of B cells) from the camelid animal, and starting from the sample, generating a VH sequence to CD8 using any suitable known technique. H In some embodiments, the natural VH domain to CD8 is a camelid animal V H H domain. H H domain. HFrom an untreated library of H sequences, such a library can be obtained, for example, by screening using one or more screening techniques known in the art, using CD8, or at least one portion, fragment, antigenic determinant, or epitope thereof. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. The entire contents of these patents are incorporated herein by reference. In some embodiments, for example, techniques such as random mutagenesis and / or CDR shuffling described in WO 00 / 43507 are used to obtain an untreated V H H library-derived V H H library, such as an untreated V H H library-derived improved synthetic or semi-synthetic libraries can be used. The entire contents of this patent are incorporated herein by reference. In some embodiments, for V against CD8 H Another technique for obtaining an H sequence is to appropriately immunize a transgenic mammal capable of expressing a heavy chain antibody (i.e., immunize to generate an immune response against CD8 and / or produce a heavy chain antibody against CD8), obtain a suitable biological sample (e.g., a blood sample or any sample of B cells) from the transgenic mammal, and then, starting from the sample, use any suitable known technique to generate a V H H sequence against CD8. For example, for this purpose, the heavy chain antibody-expressing mice and further methods and techniques described in WO 02 / 085945 and WO 04 / 049794 (the entire contents of these patents are incorporated herein by reference) can be used.

[0361] In certain embodiments, the CD8-binding substance is "humanized", i.e., a natural V HIt includes a VHH in which one or more amino acid residues in the amino acid sequence of the H array (and in particular, in the framework array) are substituted with one or more amino acid residues at the corresponding position(s) in the VH domain from a conventional human four-chain antibody. This can be carried out using humanization techniques known in the art. In some embodiments, possible humanizing substitutions or combinations of humanizing substitutions can be determined by methods known in the art, for example, by comparison between the sequence of the VHH and the sequence of the native human VH domain. In some embodiments, the humanizing substitutions are selected such that the resulting humanized VHH still retains advantageous functional properties. Usually, as a result of humanization, the VHH of the present invention can become more "human-like", but still retains favorable properties such as reduced immunogenicity compared to the corresponding native V H H domain. In various embodiments, the humanized VHH of the present invention can be obtained by any suitable method known in the art, and thus is not strictly limited to polypeptides obtained using a polypeptide containing the native V H H domain as a starting material.

[0362] In one embodiment, the CD8-binding substance is "camelized", i.e., one or more amino acid residues in the amino acid sequence of the native VH domain from a conventional four-chain antibody are replaced with the V of the heavy-chain antibody of an animal of the family Camelidae HIt includes a VHH substituted with one or more amino acid residues at the corresponding position(s) in the H domain. In some embodiments, such "camelization" substitutions are made at amino acid positions that form the VH-VL interface and / or are present therein and / or at positions of prominent characteristic residues of so-called camelids (see, for example, WO 94 / 04678, the entire content of which is incorporated herein by reference). In some embodiments, the VH sequence used as a starting material or starting point for generating or designing a camelized VHH is a VH sequence derived from a mammal, such as a human VH sequence, for example, a VH3 sequence. In various embodiments, the camelized VHH can be obtained by any suitable method known in the art (i.e., as shown in (1)-(8) above), and thus is not strictly limited to a polypeptide obtained using a polypeptide containing a natural VH domain as a starting material.

[0363] In various embodiments, both "humanization" and "camelization" involve preparing a nucleotide sequence encoding a natural V H H domain or VH domain, respectively, and then modifying one or more codons in the nucleotide sequence in a manner such that the new nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively, by methods known in the art. This nucleic acid can then be expressed by methods known in the art to obtain the VHH of the present invention for the purpose. Alternatively, based on the amino acid sequence of each of the natural V H H domain or VH domain, the amino acid sequence of each of the humanized or camelized VHH of the present invention of interest can be designed and then newly synthesized using peptide synthesis techniques known in the art. Also, natural V HBased on the amino acid sequence or nucleotide sequence of each of the H domain or VH domain, a nucleotide sequence encoding each of the humanized or camelized VHHs of interest is designed, and then it can be newly synthesized using nucleic acid synthesis techniques known in the art. Thereafter, the nucleic acid thus obtained can be expressed by methods known in the art so that the VHH of the present invention can be obtained. Starting from the natural VH sequence or V H H sequence, other suitable methods and techniques for obtaining the VHH of the present invention and / or the nucleic acid encoding it are known in the art. For example, one or more parts in one or more natural VH sequences (such as one or more FR sequences and / or CDR sequences), one or more natural V H H sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences may be combined in a suitable manner to obtain the VHH of the present invention or the nucleotide sequence or nucleic acid encoding it.

[0364] The method for producing the CD8 binding substance of the present invention is described herein. For example, the DNA sequence encoding the CD8 binding substance of the present invention can be chemically synthesized using methods known in the art. The synthetic DNA sequence can be ligated to other appropriate nucleotide sequences including expression control sequences, for example, to produce a gene expression construct encoding the CD8 binding substance of interest. Thus, in various embodiments, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the CD8 binding substance of the present invention.

[0365] The nucleic acid encoding the CD8-binding substance of the present invention may be incorporated (linked) into an expression vector, and this vector can be introduced into a host cell by gene transfer, transformation, or transduction techniques. For example, the nucleic acid encoding the CD8-binding substance of the present invention can be introduced into a host cell by retroviral transduction. Examples of host cells are Escherichia coli cells, Chinese hamster ovary (CHO) cells, human fetal kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cultured cells (COS), or human hepatocarcinoma cells (e.g., Hep G2), and myeloma cells. The transformed host cells can be grown under conditions that allow the host cells to express the gene encoding the CD8-binding substance of the present invention. Thus, in various embodiments, the present invention provides an expression vector comprising the nucleic acid encoding the CD8-binding substance of the present invention. In various embodiments, the present invention further provides a host cell comprising such an expression vector.

[0366] Specific expression and purification conditions vary depending on the expression system used. For example, when the gene is expressed in E. coli, the gene is first inserted into an expression vector by placing the engineered gene downstream of a bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. In another example, when the engineered gene is expressed in a eukaryotic host cell, such as a CHO cell, the gene is first inserted into an expression vector containing, for example, a suitable eukaryotic promoter, a secretion signal, a transcription enhancer, and various introns. The gene construct can be introduced into a host cell using gene transfer, transformation, or transduction techniques.

[0367] The CD8-binding substance of the present invention can be produced by growing a host cell transfected with an expression vector encoding the CD8-binding substance under conditions that permit the expression of the protein. After expression, the protein can be collected and purified using techniques well known in the art, such as affinity tags like glutathione-S-transferase (GST) and histidine (His), or chromatography. In certain embodiments, the CD8-binding substance comprises a His tag (which can be cleaved by an engineered protein cleavage site, if desired).

[0368] Accordingly, in various embodiments, the present invention provides a nucleic acid encoding the CD8-binding substance of the present invention. In various embodiments, the present invention provides a host cell comprising a nucleic acid encoding the CD8-binding substance of the present invention.

[0369] In various embodiments, the CD8 binding substance of the present invention or the chimeric protein comprising the CD8 binding substance can be expressed in vivo, for example, in a patient. For example, in various embodiments, the CD8 binding substance of the present invention or the chimeric protein comprising the CD8 binding substance can be administered in the form of a nucleic acid encoding the CD8 binding substance of the present invention or the chimeric protein comprising the CD8 binding substance. In various embodiments, the nucleic acid is DNA or RNA. In some embodiments, the CD8 binding substance of the present invention or the chimeric protein comprising the CD8 binding substance is encoded by a modified mRNA, i.e., an mRNA comprising one or more modified nucleotides. In some embodiments, the modified mRNA comprises one or more modifications found in U.S. Patent No. 8,278,036. The entire content of this patent is incorporated herein by reference. In some embodiments, the modified mRNA comprises one or more of m5C, m5U, m6A, s2U, Ψ, and 2'-O-methyl-U. In some embodiments, the present invention relates to the administration of a modified mRNA encoding one or more chimeric proteins of the present invention. In some embodiments, the present invention relates to a gene therapy vector comprising a modified mRNA. In some embodiments, the present invention relates to a gene therapy vector comprising a modified mRNA. In various embodiments, the nucleic acid is in the form of an oncolytic virus, such as an adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus or vaccinia.

[0370] Pharmaceutically acceptable salts and excipients The CD8-binding substances described in this specification have sufficiently basic functional groups that can react with inorganic or organic acids, or carboxyl groups that can react with inorganic or organic bases, and can form pharmaceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids, as is well known in the art. Such salts include, for example, those pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P.H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002. These documents are hereby incorporated by reference in their entirety into this specification.

[0371] Pharmaceutically acceptable salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharinate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, terephthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate.

[0372] The term "pharmaceutically acceptable salts" also refers to salts of the compositions of the invention having acidic functional groups such as carboxylic acid functional groups, and bases. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, and organic amines such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine such as mono-, bis-, or tris-(2-OH-lower alkylamine), N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amine such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine and lysine, etc.

[0373] In some embodiments, the compositions described herein are in the form of pharmaceutically acceptable salts.

[0374] Pharmaceutical Compositions and Formulations In various embodiments, the invention relates to pharmaceutical compositions comprising a CD8 binding substance and a pharmaceutically acceptable carrier or excipient described herein. Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally contain an appropriate amount of a pharmaceutically acceptable excipient to provide a suitable form for administration, as needed.

[0375] In various embodiments, the pharmaceutical excipient can be a liquid such as water and oil, including those of petroleum, animal, plant, or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil. The pharmaceutical excipient can be, for example, saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. Further, adjuvants, stabilizers, thickeners, lubricants, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. When any of the agents described herein is administered intravenously, water is a useful excipient. Saline and aqueous dextrose and glycerin solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, silica gel, sodium stearate, glycerin monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene glycol, water, ethanol, and the like. Any of the agents described herein may optionally contain a small amount of a wetting or emulsifying agent, or a pH buffering agent. Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995). This reference is incorporated herein by reference.

[0376] The present invention includes the pharmaceutical composition (and / or additional therapeutic agent) described in various formulations. Any of the pharmaceutical compositions (and / or additional therapeutic agents) of the present invention described herein can take the form of solutions, suspensions, emulsions, drip solutions, tablets, pills, pellets, capsules, liquid-containing capsules, gelatin capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, frozen suspensions, dry powders, or any other suitable form for use. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In a further embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a solution.

[0377] Optionally, the pharmaceutical composition (and / or additional agent) of the present invention may also include solubilizing agents. Also, the agent can be delivered using any suitable vehicle or delivery device known in the art. The combination therapy agents outlined herein can be co-delivered in a single delivery vehicle or delivery carrier.

[0378] Formulations containing the pharmaceutical composition (and / or additional agent) of the present invention can be conveniently provided in unit dosage form and can be prepared by any method well known in the pharmaceutical arts. Such methods generally include the step of mixing the therapeutic agent with a carrier, which comprises one or more accessory ingredients. Usually, the formulation is prepared by uniformly and completely mixing the therapeutic agent with a liquid carrier, a micronized solid carrier, or both, and then, if necessary, shaping the product into the dosage form of the desired formulation (e.g., wet or dry granulation, powder blend, etc., followed by tableting using conventional methods known in the art).

[0379] In various embodiments, any of the pharmaceutical compositions (and / or additional agents) described herein are formulated according to routine procedures as compositions adapted to the methods of administration described herein.

[0380] Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical. Administration can be local or systemic. In some embodiments, administration is by oral route. In another embodiment, administration is by parenteral injection. The method of administration can be left to the discretion of the attending physician, in part depending on the site of the medical condition. In most cases, administration results in the release of any of the agents described herein into the bloodstream.

[0381] In one embodiment, the CD8 binding substance described herein is formulated according to conventional methods as a composition adapted for oral administration. Compositions for oral delivery may be, for example, in the form of tablets, troches, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Compositions for oral administration may contain one or more agents, such as sweeteners like lactose, aspartame or saccharin, flavoring agents like peppermint, wintergreen or cherry oil, coloring agents and preservatives, in order to provide a pharmaceutically palatable formulation. Further, in tablet or pill form, the composition can be coated to enable sustained action over a long period by delaying disintegration and absorption in the gastrointestinal tract. Selectively permeable membranes surrounding osmotically active substances carrying any of the CD8 binding substances described herein are also suitable as oral administration compositions. In these latter platforms, liquid from the environment around the capsule is absorbed by the transport compound, which swells and expels the drug or drug composition through an opening. These delivery platforms can provide essentially a zero-order delivery profile, in contrast to the rapid rise profile of immediate release formulations. Time-delay substances such as glycerol monostearate or glycerol stearate can also be used. Oral compositions may contain standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are of pharmaceutical grade. In addition to the active compound, the suspension may contain, for example, precipitation inhibitors such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc., and mixtures thereof.

[0382] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injections and infusions) include, for example, solutions, suspensions, dispersions, emulsions, etc. They may be manufactured in the form of a sterile solid composition (e.g., a lyophilized composition), which can be dissolved or suspended in a sterile injectable medium immediately before use. They may contain, for example, known suspending or dispersing agents in the art. Suitable formulation ingredients for parenteral administration include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as EDTA, buffering agents such as acetate, citrate, or phosphate, and osmotic pressure regulators such as sodium chloride or dextrose.

[0383] In the case of intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier must be stable under the conditions of manufacture and storage and must be protected against microorganisms. The carrier may be, for example, a solvent or dispersion medium including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0384] The compositions provided herein can be used alone or in combination with other suitable ingredients to prepare aerosol formulations (i.e., "nebulized" formulations) for administration by inhalation. The aerosol formulations can be placed in acceptable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, etc.

[0385] Any pharmaceutical composition (and / or additional agent) of the present invention described herein can be administered by controlled release known to those skilled in the art or by sustained release means or delivery devices. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,556. Each of these patents is hereby incorporated by reference in its entirety. Such dosage forms are useful, for example, for enabling controlled or sustained release of one or more active ingredients using hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, other polymer matrices, gels, osmotic membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, and can provide the desired release profiles at various rates. Suitable controlled or sustained release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein. The present invention thus provides, without limitation, unit dosage forms suitable for oral administration such as tablets, capsules, gelcaps, and caplets adapted for controlled or sustained release.

[0386] Controlled or sustained release of the active ingredient can be stimulated by various conditions including, but not limited to, pH changes, temperature changes, stimulation by light of an appropriate wavelength, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0387] In another embodiment, the sustained release system can be placed in the vicinity of the target area to be treated and thus requires only a portion of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other release control systems discussed in the review in Langer, 1990, Science 249:1527-1533 can be used.

[0388] The pharmaceutical preparation is preferably sterile. Sterilization is achieved, for example, by filtration through a sterile filtration membrane. If the composition is lyophilized, filter sterilization can be carried out before or after lyophilization and reconstitution.

[0389] Administration and Dosage It will be understood that the actual dosage of the CD8 binding substance administered according to the present invention will vary depending on the particular dosage form and method of administration. One of ordinary skill in the art can take into account many factors that can alter the action of the CD8 binding substance (e.g., body weight, gender, diet, time of administration, route of administration, rate of excretion, condition of the subject, combination of drugs, genetic factors, and sensitivity to the response). Administration can be carried out continuously or in one or more separate dosages within the range of the maximum tolerated dose. The optimal rate of administration for a given set of conditions can be determined by one of ordinary skill in the art using conventional dose administration tests.

[0390] In some embodiments, suitable dosages of the CD8 binding substance are in the range of about 0.01 mg / (kg body weight of the subject) to about 10 g / (kg body weight of the subject), about 0.01 mg / (kg body weight of the subject) to about 1 g / (kg body weight of the subject), about 0.01 mg / (kg body weight of the subject) to about 100 mg / (kg body weight of the subject), about 0.01 mg / (kg body weight of the subject) to about 10 mg / (kg body weight of the subject), for example, about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, 1.9 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg body weight, about 100 mg / kg body weight, about 1 g / kg body weight, about 10 g / kg body weight (including all values and ranges therebetween).

[0391] The individual dosages of the CD8-binding substance can be administered in unit dosage forms containing, for example, from about 0.01 mg to about 100 g, from about 0.01 mg to about 75 g, from about 0.01 mg to about 50 g, from about 0.01 mg to about 25 g, from about 0.01 mg to about 10 g, from about 0.01 mg to about 7.5 g, from about 0.01 mg to about 5 g, from about 0.01 mg to about 2.5 g, from about 0.01 mg to about 1 g, from about 0.01 mg to about 100 mg, from about 0.1 mg to about 100 mg, from about 0.1 mg to about 90 mg, from about 0.1 mg to about 80 mg, from about 0.1 mg to about 70 mg, from about 0.1 mg to about 60 mg, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 30 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 0.1 mg to about 3 mg, from about 0.1 mg to about 1 mg, or from about 5 mg to about 80 mg per unit dosage form. For example, the unit dosage form can be about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 200 mg, about 500 mg, about 1 g, about 2.5 g, about 5 g, about 10 g, about 25 g, about 50 g, about 75 g, about 100 g (including all values and ranges therebetween).

[0392] In one embodiment, the CD8 binding substance is administered in an amount of about 0.01 mg to about 100 g per day, about 0.01 mg to about 75 g per day, about 0.01 mg to about 50 g per day, about 0.01 mg to about 25 g per day, about 0.01 mg to about 10 g per day, about 0.01 mg to about 7.5 g per day, about 0.01 mg to about 5 g per day, about 0.01 mg to about 2.5 g per day, about 0.01 mg to about 1 g per day, about 0.01 mg to about 100 mg per day, about 0.1 mg to about 100 mg per day, about 0.1 mg to about 95 mg per day, about 0.1 mg to about 90 mg per day, about 0.1 mg to about 85 mg per day, about 0.1 mg to about 80 mg per day, about 0.1 mg to about 75 mg per day, about 0.1 mg to about 70 mg per day, about 0.1 mg to about 65 mg per day, about 0.1 mg to about 60 mg per day, about 0.1 mg to about 55 mg per day, about 0.1 mg to about 50 mg per day, about 0.1 mg to about 45 mg per day, about 0.1 mg to about 40 mg per day, about 0.1 mg to about 35 mg per day, about 0.1 mg to about 30 mg per day, about 0.1 mg to about 25 mg per day, about 0.1 mg to about 20 mg per day, about 0.1 mg to about 15 mg per day, about 0.1 mg to about 10 mg per day, about 0.1 mg to about 5 mg per day, about 0.1 mg to about 3 mg per day, about 0.1 mg to about 1 mg per day, or about 5 mg to about 80 mg per day. In various embodiments, the CD8 binding substance is administered in a daily amount of about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 200 mg, about 500 mg, about 1 g, about 2.5 g, about 5 g, about 7.5 g, about 10 g, about 25 g, about 50 g, about 75 g, about 100 g (including all values and ranges therebetween).

[0393] In certain embodiments of the present invention, a pharmaceutical composition comprising a CD8 binding substance may be administered, for example, more than twice a day (e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times a day), about once a day, about every other day, about every three days, about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every six months, or about once a year.

[0394] Combination therapies and additional therapeutic agents In various embodiments, the pharmaceutical compositions of the present invention are co-administered with additional therapeutic agents. The co-administration may be simultaneous or sequential.

[0395] In one embodiment, the additional therapeutic agent and the CD8 binding substance of the present invention are co-administered to a subject. As used herein, the term "simultaneously" means that the additional therapeutic agent and the CD8 binding substance are administered at time intervals of about 60 minutes, e.g., about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute. The administration of the additional therapeutic agent and the CD8 binding substance can be accomplished by co-administration of a single formulation (e.g., a formulation comprising the additional therapeutic agent and the CD8 binding substance) or separate formulations (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising the CD8 binding substance).

[0396] Co-administration does not require that the timing of their administrations overlap over time such that the pharmacological activities of the additional therapeutic agent and the CD8-binding substance overlap, and thereby the combined therapeutic effect is exerted. For example, the additional therapeutic agent and the CD8-binding substance can be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and the CD8-binding substance are administered at time intervals greater than about 60 minutes. For example, the time interval between sequential administrations of the additional therapeutic agent and the CD8-binding substance can be greater than about 60 minutes, greater than about 2 hours, greater than about 5 hours, greater than about 10 hours, greater than about 1 day, greater than about 2 days, greater than about 3 days, greater than about 1 week, greater than about 2 weeks, or greater than about 1 month. The optimal administration time will depend on the metabolism, excretion rate, and / or pharmacodynamic activity of the additional therapeutic agent and the CD8-binding substance being administered. Either the additional therapeutic agent or the CD8-binding substance can be administered first.

[0397] Co-administration also does not require that the therapeutic agents be administered to the subject by the same route of administration. Rather, each therapeutic agent can be administered by any suitable route, e.g., non-parenterally or orally.

[0398] In some embodiments, the CD8-binding substances described herein act synergistically when co-administered with another therapeutic agent. In such embodiments, the CD8-binding substance and the additional therapeutic agent can be administered at lower doses than the doses employed when the therapeutic agent is used in monotherapy.

[0399] In some embodiments, the invention relates to chemotherapeutic agents as additional therapeutic agents. For example, without limitation, such combinations of the present CD8 binding substance and chemotherapeutic agents are used for the treatment of cancer as described elsewhere herein. Examples of chemotherapeutic agents include alkylating agents such as thiotepa, CYTOXAN (cyclophosphamide), alkyl sulfonates such as busulfan, improsulfan and piposulfan, aziridines such as benzodopa, carbocon, meturedopa, and uredopa, ethyleneimine, methylmelamine such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine, acetogenins (e.g., bratasin, brattacinone), camptothecins (including the synthetic analog topotecan), bryostatin, callystatin, CC-1065 (including adozelesin, carzelesin and bizelesin synthetic analogs), cryptophycins (e.g., cryptophycin 1, cryptophycin 8, etc.), dolastatin, duocarmycins (including the synthetic analog KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobenbitin, phenesterine, prednimustine, trofosfamide, uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine, antibiotics such as enediyne antibiotics (calicheamicin, particularly calicheamicin gamma II and calicheamicin omega II (see Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)), dynemicin, dynemicin A, bisphosphonates such as clodronate, esperamicin, and neocarzinostatin chromophore and related pigment protein enediyne antibiotic chromophore), aclacinomycin, actinomycin, aurodox, azaserine, bleomycin,Cactinomycin, carabicin, caminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin (doxorubicin) (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, queramycin, rhodrubicin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin, antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU), folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethoprim, purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, androgens, e.g., calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone, antiadrenal substances, e.g., aminoglutethimide, mitotane, trilostane, folic acid supplements, e.g., frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, demeclocycline, diaziquone, elformithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, e.g., maytansine and ansamitocin, mitoguazone, mitoxantrone, mopidamol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid,2-ethylhydrazide, procarbazine, PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.), razoxane, rizoxin, schizophyllan, spirigermanium, tenuazonic acid, 2,2’,2”-trichlorotriethylamine, trichothecene (e.g., T2 toxin, verracurin A, loridine A, and anguidine), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (ara-C), cyclophosphamide, thiotepa, taxoid, e.g., taxol paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), Abraxane Cremophor-free, albumin-processed nanoparticle-forming paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), Taxotere docetaxel (Rhone-Poulenc Rorer, Antony, France), chlorambucil, Gemzar (gemcitabine), 6-thioguanine, mercaptopurine, methotrexate, platinum analogs, e.g., cisplatin, oxaliplatin and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, navelbine (vinorelbine), novantrone, teniposide, edatrexate, daunomycin, aminopterin, Xeloda, ibandronate, irinotecan (Camptosar, CPT-11) (including the treatment of irinotecan with 5-FU and leucovorin), topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoid, e.g., retinoic acid, capecitabine, combretastatin, leucovorin (LV), oxaliplatin, including the oxaliplatin treatment (FOLFOX), lapatinib (Tykerb), PKC-α, Raf, H-Ras, inhibitors of EGFR (e.g., erlotinib (Tarceva) and VEGF-A that suppress cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above-mentioned agents, but are not limited thereto. Further, the treatment method may further include the use of radiation. Further, the treatment method isIt may further include the use of photomechanical therapy.

[0400] In some embodiments, including but not limited to infectious disease applications, the present invention relates to anti-infective agents as additional therapeutic agents. In some embodiments, the anti-infective agent is an antiviral agent, which includes but is not limited to abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, and foscarnet. In some embodiments, the anti-infective agent is an antibacterial agent, which includes but is not limited to cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, levaquin, floxin, tequin, avalox and norfloxacin); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem / cilastatin, and meropenem). In some embodiments, the anti-infective agent includes anti-malarial drugs (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether / lumefantrine, atovaquone / proguanil and sulfadoxine / pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.

[0401] Although not limited, in some embodiments including autoimmune disease applications, the additional therapeutic agent is an immunosuppressant. In some embodiments, the immunosuppressant is an anti-inflammatory agent such as a steroidal anti-in...

Claims

1. A CD8-binding substance comprising an amino acid sequence having at least 90% identity with one of SEQ ID NOs: 235, 246, 250, 252, 268, and 1216.

2. A CD8-binding substance comprising at least one targeting moiety comprising three complementarity-determining regions (CDR1, CDR2, and CDR3), wherein (a) CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 29, 37, 39, 45, and 51; (b) CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 97, 108, 112, 114, 130, and 1221; and (c) CDR3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 166, 177, 181, 183, and 199, the CD8-binding substance.

3. The targeting moiety is a full-length antibody, single domain antibody, recombinant heavy chain antibody (VHH), single-chain antibody (scFv), shark heavy chain antibody (VNAR), microprotein, darpin, anticalin, adnectin, aptamer, Fv, Fab, Fab’, F(ab’) ２ , peptidomimetic molecule, natural ligand for a receptor, or synthetic molecule, the CD8-binding substance according to claim 1 or 2.

4. The CD8-binding substance according to any one of Claims 1 to 3, wherein the targeting moiety is a single-domain antibody.

5. wherein the targeting moiety is V Ｈ H, humanized V Ｈ H, or camelized V Ｈ The CD8-binding substance according to claim 3, comprising H.

6. The CD8-binding substance according to any one of Claims 1 to 5, comprising one or more types of signal transduction substances.

7. The CD8-binding substance according to Claim 6, wherein the signal transduction substance is selected from one or more of interferon, interleukin, and tumor necrosis factor, and any of these may be optionally modified.

8. The CD8-binding substance according to any one of Claims 1 to 7, comprising one or more additional targeting moieties.

9. The CD8-binding substance according to Claim 8, wherein the one or more additional targeting moieties recognize a tumor antigen and may functionally regulate the tumor antigen.

10. The CD8-binding substance according to Claim 9, wherein the one or more additional targeting moieties recognize an antigen on an immune cell and may optionally functionally regulate the antigen on the immune cell.

11. The CD8-binding substance according to Claim 10, wherein the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, and NK cells.

12. The CD8-binding substance according to any one of Claims 1 to 11, which recruits cytotoxic T cells to tumor cells or the tumor microenvironment.

13. The CD8-binding substance according to any one of Claims 1 to 12, which binds to CD8 without substantially functionally regulating its activity.

14. A recombinant nucleic acid composition encoding a CD8-binding substance according to any one of claims 1 to 13.

15. A host cell comprising the nucleic acid according to claim 14.

16. A CD8-binding substance comprising at least one targeting moiety comprising three complementarity-determining regions (CDR1, CDR2, and CDR3), wherein (a) CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 12 to 80; (b) CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 81 to 149 or 1221; and (c) CDR3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 150 to 218, a CD8-binding substance.

17. The targeting moiety is a full-length antibody, single-domain antibody, recombinant heavy-chain antibody (VHH), single-chain antibody (scFv), shark heavy-chain antibody (VNAR), microprotein, darpin, anticalin, adnectin, aptamer, Fv, Fab, Fab', F(ab') ２ , peptidomimetic molecule, natural ligand for a receptor, or synthetic molecule, the CD8-binding substance according to claim 16.

18. The CD8-binding substance according to claim 16 or claim 17, wherein the targeting moiety is a single-domain antibody.

19. wherein the targeting moiety is V Ｈ H, humanized V Ｈ H, or camelized V Ｈ The CD8-binding substance according to claim 18, comprising H.

20. The CD8-binding substance according to claim 19, comprising an amino acid sequence having at least 90% identity with one of SEQ ID NOs: 219 to 287 or SEQ ID NO: 1216.

21. The CD8-binding substance according to any one of claims 16 to 20, comprising one or more signal transduction substances.

22. The CD8-binding substance according to claim 21, wherein the signal transduction substance is selected from one or more of interferon, interleukin, and tumor necrosis factor, and any of these may be optionally modified.

23. The CD8-binding substance according to any one of claims 16 to 22, comprising one or more additional targeting moieties.

24. The CD8-binding substance according to claim 23, wherein the one or more additional targeting moieties recognize a tumor antigen and may functionally regulate the tumor antigen.

25. The CD8-binding substance according to claim 24, wherein the one or more additional targeting moieties recognize an antigen on an immune cell and may optionally functionally regulate the antigen on the immune cell.

26. The CD8-binding substance according to claim 25, wherein the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, and NK cells.

27. The CD8-binding substance according to any one of claims 16 to 26, which recruits cytotoxic T cells to tumor cells or the tumor environment.

28. The CD8-binding substance according to any one of claims 16 to 27, which recognizes CD8 and binds to CD8 without substantially and functionally regulating its activity.

29. A recombinant nucleic acid composition encoding the CD8-binding substance according to any one of claims 16 to 28.

30. A host cell containing the nucleic acid according to claim 29.

31. The CD8-binding substance according to any one of claims 1 to 13 or claims 16 to 28, which is suitable for use in a patient having one or more of cancer, infectious diseases, immune disorders, and / or autoimmune diseases.

32. A method for treating or preventing cancer, comprising administering to a patient in need of administration an effective amount of a chimeric comprising a targeting moiety comprising an antigen or receptor recognition domain targeting CD8 and a signaling substance selected from one or more of interferon, interleukin, and tumor necrosis factor.

33. The method according to claim 32, wherein the signaling substance is modified.

34. The method according to claim 32 or 33, wherein the CD8-binding substance is the CD8-binding substance according to any one of claims 1 to 13 or claims 16 to 28.

35. The CD8-binding substance comprises at least one targeting moiety comprising three complementarity-determining regions (CDR1, CDR2, and CDR3), wherein (a) CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 12 to 80; wherein (b) CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 81 to 149 or 1216; and wherein (c) CDR3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 150 to 218, The method according to any one of claims 32 to 34.

36. The method according to any one of claims 32 to 35, wherein the cancer is selected from one or more of basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), melanoma, myeloma, neuroblastoma, oral cancer (lip, glossal, tongue, intraoral, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland adenocarcinoma, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, Hodgkin lymphoma and non-Hodgkin lymphoma, and lymphoma including B cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL)), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large tumor lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and one or more of nevus syndrome, edema (e.g., related to brain tumors), and abnormal angiogenesis related to the Meigs syndrome.

37. The CD8 binding substance according to any one of claims 1 to 36 for use in the treatment of one or more of cancer, infectious diseases, immune disorders, and / or autoimmune diseases.

38. Use of the CD8 binding substance according to any one of claims 1 to 37 for the manufacture of a drug for treating one or more of cancer, infectious diseases, immune disorders, and / or autoimmune diseases described herein.

39. (a)Any one of the CD8 binding substances according to claims 1 to 13 or claims 16 to 28; and (b)Modified human IFNα2 having one or more mutations that confer improved safety compared to wild-type IFNα2 A chimeric protein comprising, A chimeric protein in which the targeting moiety and the modified signaling substance may optionally be linked by one or more linkers. **Claim 40** The chimeric protein according to claim 39, wherein the modified human IFNα2 comprises one or more mutations at positions R120, M148, R149, and L153. **Claim 41** The chimeric protein according to claim 40, wherein the modified human IFNα2 comprises one or more mutations selected from R120E, R149A, and L153A. **Claim 42** The chimeric protein according to claim 40, wherein the modified human IFNα2 comprises the R120E mutation and either the R149A or L153A mutation. **Claim 43** (a) Any one of the CD8-binding substances according to claims 1 to 13 or claims 16 to 28; and (b) A modified human IFNβ having one or more mutations that confer improved safety compared to wild-type IFNβ, A chimeric protein comprising, A chimeric protein in which the targeting moiety and the modified signaling substance may optionally be linked by one or more linkers. **Claim 44** The chimeric protein according to claim 43, wherein the modified human IFNβ comprises one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. **Claim 45** The chimeric protein according to claim 44, wherein the modified human IFNβ comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G.

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