NZ733157B2 - Agonistic tnf receptor binding agents - Google Patents

Abstract

The present invention relates to binding agents capable of binding to receptors of the TNF superfamily. In particular, binding agents capable of binding to CD40 and 4-1BB (CD137)receptors of the TNF superfamily, as well as to their use in medicine. The present invention further relates to nucleic acid molecules encoding such binding agents, to cells comprising such nucleic acid molecules and to pharmaceutical compositions and kits. id molecules encoding such binding agents, to cells comprising such nucleic acid molecules and to pharmaceutical compositions and kits.

Description

Agonistic TNF receptor binding agents cal Field of the Invention The present invention relates to binding agents g to receptors of the TNF superfamily, in ' particular binding agents binding to at least two different ors of the TNF superfamily, as well as to their use in medicine. The present invention further relates to nucleic acid molecules encoding such binding agents, to cells comprising such nucleic acid molecules and to pharmaceutical compositions and kits.

Background of the ion Receptors for tumor necrosis factor (TNF) superfamily ligands are oligomeric, mostly ic, type I or type ill transmembrane proteins and contain one to six cysteine-rich domains (CRDs) in their extracellular domain (Naismith J.H. and Sprang SR. (1998), TRENDS in Biochemical Sciences 23(2):74—79). Moreover, several receptors for TNF superfamily ligands contain death domains (DDs) that recruit caspase-interacting proteins following ligand binding to initiate the extrinsic pathway of caspase activation. us receptors of the TNF superfamily have been identified so far, including 4—1BB (CD137), BAFFR, BCMA, CD27, CD30, CD40, DCR3, DR3 (TRAMP), DR6, EDAR, Fas (CD95), FN14 (TWEAK-R), GITR, HVEM, LTBR, NGFR (CD271), OPG, 0X40 (CD134), RANK (TRANCE-R), RELT, TACI, TNFR], TNFRZ, R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, TROY and XEDAR (Bodmer, J.L. et a1. (2002), TRENDS in Biochemical Sciences, 27(1):19-26). h timely sion and intricate signaling, receptors of the TNF superfamily and their corresponding ligands act to regulate cell responses, including activation, proliferation, differentiation, and apoptosis. While critical to the regulation of beneficial processes, such as immune defense and hematopoiesis, TNF signaling is also implicated in genesis, transplant ion, virus replication, bone resorption, diabetes and autoimmune diseases.

W0 2016/110584 tic agents specifically binding to one or more receptors of the TNF amily are, therefore, considered as potential therapeutics in the prevention and/or ent of s diseases and disorders.

Accordingly, it was an object of the present invention to provide novel agonistic binding agents targeting ors of the TNF superfarnily, in particular agonistic binding agents targeting at least two different receptors of the TNF superfarnily.

Summary of the Invention In one aspect, the present invention relates to a binding agent comprising at least two binding s, wherein a first binding domain binds to a first or of the tumor necrosis factor (TNF) amily and a second binding domain binds to a second or of the TNF superfamily, wherein the first receptor and the second receptor are different.

In one embodiment, the binding agent is an agonistic binding agent.

In one embodiment, the binding agent is a bispecific molecule, preferably a bispecific antibody.

In one ment, the binding agent is in the format of a full-length antibody or an antibody fragment.

In one embodiment, the first receptor and the second receptor are selected from the group consisting of CD40, CD27, 0X40 (CD134) and 4-1BB (CD137).

In one embodiment, the first binding domain or the second binding domain binds to CD27, wherein the binding agent comprises (a) the heavy chain complementarity determining region(s) HCDRl, HCDRZ and/or HCDR3, and/or the light chain complementarity determining region(s) LCDRI, LCDRZ and/or LCDR3 of an antibody selected from the group consisting of ABZ7_1 to AB27_186 as shown in Table 7; and/or (b) the heavy chain variable domain (VH) or a variant or fragment thereof and/or the light chain variable domain (VL) or a variant or fragment thereof W0 20161110584 of an antibody selected from the group consisting of AB27_1 to AB27_186 as shown in Table 7.

In one embodiment, the first binding domain or the second binding domain binds to 0X40 (CD134), wherein the binding agent comprises (a) the heavy chain complementarity determining region(s) HCDRI, HCDR2 and/0r HCDR3, and/or the light chain complementarity determining region(s) LCDRl, LCDR2 and/or LCDR3 of an antibody selected from the group ting of AB134_1 to AB134_92 as shown in Table 8; and/or (b) the heavy chain variable domain (VH) or a variant or fragment thereof and/or the light chain variable domain (VL) or a variant or fragment thereof of an antibody selected from the group consisting of AB134__1 to AB} 34_92 as shown in Table 8.

In one embodiment, the first binding domain or the second binding domain binds to 4-1BB (CD137), n the binding agent comprises (a) the heavy chain complementarity determining region(s) HCDRl, HCDR2 and/or HCDR3, and/or the light chain complementarity determining region(s) LCDRl , LCDR2 and/or LCDR3 of an antibody selected from the group ting of AB137_1 to AB137_12 as shown in Table 9; and/or (b) the heavy chain le domain (VH) or a variant or fragment thereof and/or the light chain variable domain (VL) or a t or fragment thereof of an antibody selected from the group consisting of AB137_1 to ABl37_12 as showu in Table 9.

In one embodiment, the first g domain or the second binding domain binds to CD40, wherein the binding agent comprises (a) heavy chain complementarity determining region(s) HCDRI having the amino acid sequence of SEQ ID NO: 2323, HCDRZ having the amino acid sequence of SEQ ID NO: 2324 and/or HCDR3 having the amino acid sequence of SEQ ID NO: 2325, and/or light chain mentarity determining region(s) LCDRl having the amino acid “ sequence of SEQ ID NO: 2326, LCDR2 having the amino acid ce of SEQ ID NO: 2327 and/or LCDR3 having the amino acid sequence of SEQ ID NO: 2328; and/or W0 2016/110584 (b) heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 2321 or a variant or fragment thereof and/or light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 2322 or a variant or fragment f.

In one embodiment, (a) the first binding domain binds to CD40 and the second binding domain binds to CD27; (b) the first binding domain binds to CD40 and the second binding domain binds to 0X40 (CD134); (c) the first binding domain binds to CD40 and the second binding domain binds to 4-lBB (CD137); (d) the first binding dcrnm'n binds to 4-1 BB (C0137) and the second binding domain binds to CD27; (e) the first binding domain binds to 4-1BB (CD137) and the second binding domain binds to 0X40 ); or (t) the first binding domain binds to 0X40 (CD134) and the second binding domain binds to CD27.

In one ment, the binding agent comprises one or more heavy chain constant domains of an immunoglobulin and/or light chain constant domains ofan immunoglobulin.

In another , the present invention relates to a binding agent that binds to CD27, sing (a) the heavy chain complementarity determining (s) HCDRl, HCDR2 and/0r HCDR3, and/or the light chain mentarity determining region(s) LCDRI , LCDR2 and/or LCDR3 of an antibody selected from the group consisting of AB27_1 to AB27m186-as shown in Table 7; and/or (b) the heavy chain variable domain (VH) or a variant or fragment thereof and/or the light chain variable domain (VL) or a variant or fragment thereof of an antibody ed from the group consisting of AB27_1 to ABZ7_186 as shown in Table 7.

In another aspect, the present invention relates to a binding agent that binds to 0X40 (CD134), comprising (a) the heavy chain complementarity determining region(s) HCDRl, HCDR2 and/or HCDR3, and/or W0 20161110584 the light chain complementarity determining region(s) LCDRI, LCDRZ and/or LCDR3 of an antibody selected from the group consisting of AB134_1 to ABI34_92 as shown in Table 8; and/or (13) the heavy chain le domain (VH) or a variant or fragment thereof and/or the light chain variable domain (VL) or a variant or fragment thereof of an antibody selected from the group consisting of ABI34_1 to ABI34_92 as shown in Table 8.

In another aspect, the present invention relates to a binding agent that binds to 4-1BB ), comprising (a) the heavy chain complementarity ining region(s) HCDRI, HCDR2 and/0r HCDR3, and/or the light chain complementarity determining region(s) LCDRl, LCDR2 and/ or LCDR3 of an antibody selected from the group consisting of ABl37gl to AB137_l2 as shown in Table 9; and/or (b) the heavy chain variable domain (VH) or a t or fragment thereof and/or the light chain variable domain (VL) or a variant or fragment thereof of an antibody selected from the group consisting of ABI37_1 to AB137_12 as shown in Table 9.

In one embodiment, the binding agent is an agonistic binding agent.

In one embodiment, the binding agent is in the format of a flail-length antibody or an antibody fragment.

In one embodiment, the binding agent comprises one or more heavy chain constant s of an immunoglobulin and/or light chain nt domains of an immunoglobulin.

In another , the present invention relates to a binding agent g to the same epitope(s) as a binding agent as defined above.

In another aspect, the present invention relates to a c acid molecule encoding a binding agent as defined above, wherein, preferably, the nucleic acid molecule is contained in a vector.

W0 2016/110584 In another aspect, the t invention relates to a cell comprising a nucleic acid molecule as defined above.

Ln another , the present invention relates to a ceutical composition comprising, as an active agent, a binding agent as defined above, a nucleic acid le as defined above, or a cell as defined above.

In one ment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In another aspect, the present invention relates to a kit comprising a binding agent as defined above, a nucleic acid molecule as defined above, a cell as defined above, or a pharmaceutical composition as defined above.

In another aspect, the present invention relates to a binding agent as defined above, a nucleic acid molecule as defined above, a cell as defined above, or a pharmaceutical composition as defined above for use as a medicament.

In r aspect, the present invention relates to a binding agent as defined above, a nucleic acid molecule as defined above, a cell as defined above, or a pharmaceutical composition as defined above for use in the treatment of a disease selected from the group consisting of , infectious diseases, inflammatory diseases, metabolic diseases, autoimmune disorders, degenerative diseases, apoptosis—associated diseases and transplant rejections.

Brief Description of the Figures Figure 1 shows g curves of reference antibodies against CD27, CD134 and CD137 to the respective recombinant protein, as determined by enzyme-linked irnmunosorbent assay (ELISA).

Figure 2 shows the expression of CD27, CD134 and CD137 on the cell surface of HT1080 cell lines overexpressing CD27, CD134 and CD137, respectively, as determined by fluorescence- activated cell sorting (FACS).

W0 2016/110584 Figure 3 shows binding curves of reference antibodies against CD27, CD134 and CD137 to CD27, CD134 and CD137 expressed on the cell surface of corresponding HT1080 cell lines, as determined by a cellular binding assay.

Figure 4 shows the activation curve of an tic anti-CD27 nce antibody with or without cross-linking, as determined by ELISA measuring the IL-8 concentration in the supernatants of CD27-overexpressing HT1080 cells.

Figure 5 shows exemplary activation curves of agonistic anti-CD27, anti-CD134 and anti- CD137 dies under cross-linking conditions, as determined by ELISA measuring the IL-8 concentration in the supernatants of target-overexpressing HT1080 cells.

Figure 6 shows the expression of CD27, CD40, CD134 and CD137 on the cell surface of stable transfectants of HEK293_NFngfp_luc cells (A) and of stable transfectants of K562 cells (B) overexpressing CD 7, CD40, CD134 and CD137, respectively, as determined by FACS (white curves: control without antibody; grey curves: antibody ng).

Figure 7 shows the is of two examples (A and B) of a bispecific dy simultaneously targeting CD40 and CD137 (CD137xCD40). Activation of CD137 was measured by luciferase activity (relative light units, RLU) of HEK293mNFK_CD137_ _luc upon incubation with ted bispecific antibodies and D40 for trans-presentation or K562_wt for control.

Activation of CD40 was measured by luciferase activity (RLU) of HEK293_NFK_CD40_ fp_luc upon incubation with indicated bispecific antibodies and K562_CD137 for trans-presentation or t for control. The two parental monospecific, bivalent antibodies (CD137 IgGl or CD40 IgG1) and (ii) the two monovalent antibodies with one irrelevant arm (CD137 x control, CD40 x control) served as controls for the bispecific CD137xCD40 antibodies. Only bispecific CD137xCD4O antibodies resulted in activation of CD137 and CD40 upon trans-presentation by CD40 and CD137, respectively. Used sequences of variable domains of anti-CD137 and anti-CD40 antibodies are fied by the abbreviation AB137 and AB40, respectively.

Figure 8 shows the analysis of a bispecific antibody aneously targeting CD40 and CD27 (CD27xCD40). Activation of CD27 was measured by luciferase activity (RLU) of HEK293_NFK_CD27Mgfp_1uc upon incubation with the indicated bispecific antibody and K562_CD40 for presentation or K562_wt for control. Activation of CD40 was measured W0 10584 by rase activity (RLU) of HEK293_NFK_CD40_gfp_luc upon incubation with the indicated bispecific antibody and K562_CD27 for trans-presentation or t for control.

The two parental monospecific, bivalent antibodies (CD27 lgGl or CD40 IgG1) and (ii) the two monovalent dies with one irrelevant arrn (C027 x control, CD40 x control) served as controls for the bispecific CD27xCD4O antibody. The bispecific CD27xCD40 antibody resulted in superior activation of CD27 and CD40 upon trans-presentation by CD40 and CD27, respectively. Used sequences of variable domains of anti-CD27 and anti-CD40 antibodies are identified by the abbreviation AB27 and AB40, respectively.

Figure 9 shows the analysis of two examples (A and B) of a bispecific antibody aneously targeting CD40 and CD134 (CD134xCD40). Activation of CD134 was measured by luciferase activity (RLU) of _NFK_CD134_gfp_luc upon incubation with the indicated bispecific antibodies and D40 for trans-presentation or K562_wt for l. Activation of CD40 was measured by luciferase activity (RLU) of HEK293_NFK_CD40_gfp_luc upon incubation with antibodies and K562_CD134 for trans-presentation or K562_wt for control. The two parental monospecific, bivalent antibodies (CD134 IgGl or CD40 IgG1) and (ii) the two monovalent antibodies with one irrelevant arm (CD134 x control, CD40 x l) served as controls for the bispecific CD134xCD40 antibodies. Bispecific CD134xCD40 antibodies resulted in superior activation of CD134 and CD40 upon trans-presentation by CD40 and CD134, respectively. Used sequences of variable domains of anti-CD134 and anti-CD40 antibodies are identified by the abbreviation AB134 and A840, respectively.

Figure 10 shows the expression of CD27, CD134 and CD137 on the cell surface of double ectants of HEK293fiNFK_gfp_luc cells overexpressing CD27 + CD134 (HEK293wNFK_CD27_CD134_gfp__luc), CD134 + CD137 (HEK293_NFK_CD1 34_CD1 37_gfp_luc) and CD27 + CD137 (HEK293_NFK_CD27_CD137_gfp_luc), respectively, as determined by FACS (white curves: control without antibody; grey curves: antibody staining).

Figure 11 shows the analysis of a bispecific antibody simultaneously targeting CD27 and CD134 (CD134xCD27). Activation of CD27 and CD134 was ed by rase ty of HEK293*NFK_CD27_CD134_gfp_1uc upon incubation with the indicated bispecific antibody; maximum RLUs are shown. The two al monospecific, bivalent antibodies (CD134 IgGl or CD27 IgG1) and (ii) the two monovalent antibodies with one irrelevant arm (CD134 x control, CD27 x control) served as controls for the bispecific CD134XCD27 antibody. The bispecific W0 10584 CD134xCD27 antibody resulted in superior activation of HEK293_NFK_CD27_CD134_gfp_luc as compared to the four corresponding controls. Used sequences of variable domains of anti- CD134 and anti-CD27 antibodies are identified by the abbreviation AB134 and AB27, respectively.

Figure 12 shows the analysis of two es (A and B) of a ifie dy simultaneously targeting CD134 and CD137 xCDl37). Activation of CD134 and CD137 was measured by luciferase activity of HEK293_NFK__CD134_CD137_gfp_luc upon incubation with the indicated bispecific antibodies; maximum RLUs are shown. The two parental ecific, bivalent dies (CD134 IgGl or CD137 lgGl) and (ii) the two monovalent antibodies with one irrelevant arm (CD134 x control, CD137 x control) served as controls for the bispecific CD134xCDl37 antibodies. Bispecific CD134xCDl37 antibodies resulted in activation of HEK293_NFK_CD134‘CD137wgfp_luc, whereas the four corresponding controls did not result in measurable activation of the double transfectant. Used sequences of variable s of anti- CD134 and anti-CD137 antibodies are identified by the abbreviation A8134 and A3137, respectively.

Figure 13 shows the analysis oftwo examples (A and B) of a bispecific dy simultaneously targeting CD27 and CD137 (CD137xCD27). Activation of CD27 and CD137 was measured by luciferase activity of HEK293_NFK_CD27MCD137_gfp_luc upon incubation with the indicated ific antibodies; maximum RLUs are shown. The two parental ecific, bivalent antibodies (CD137 IgGl or CD27 IgG1) and (ii) the two monovalent antibodies with one irrelevant arm (CD137 x control, CD27 x control) served as controls for the bispecific CD137xCD27 antibodies. Bispecific CD134xCD27 antibodies ed in superior activation of HEK293_NFK_CD27_CD137_gfpflluc as compared to the two control antibodies with one irrelevant arm. Used sequences of variable domains of anti-CD137 and anti-CD27 antibodies identified by the abbreviation AB137 and AB27, respectively.

Detailed ntion of the Invention Although the t invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, the ts of the present invention will be described. These elements are listed with specific embodiments, however, it should be tood that they may be combined in any manner and in any number to create additional embodiments. The variously described es and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This ption should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered sed by the description of the present application unless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “A multilingual ry of biotechnological terms: (IUPAC Recommendations)”, H.G.W. erger, B. Nagel, and H.

Kblbl, Eds., Helvetica Chimica Acta, 0 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwise indicated, conventional methods of try, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., lar Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and ”comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group ofmembers, integers or steps although in some embodiments such other member, integer or step or group ofmembers, integers or steps may be ed, i.e. the subject—matter consists in the inclusion of a stated member, 3O r or step or group of members, integers or steps. The tenns “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be ued to cover both the singular and the plural, unless otherwise indicated herein or clearly dicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each dual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be med in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the invention and does not pose a tion on the scope of the invention otherwise d. No language in the specification should be construed as indicating any non-claimed element ial to the practice of the invention.

Several documents are cited throughout the text of this specification. Each of the nts cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such sure by virtue ofprior invention.

The term tor of the TNF amily” preferably refers to a receptor selected from the group consisting of 4-1BB (CD137), BAFFR, BCMA, CD27, CD30, CD40, DCR3, DR3 (TRAMP), DR6, EDAR, Pas (CD95), FNl4 (TWEAK-R), GITR, HVEM, LTBR, NGFR (CD271), OPG, 0X40 (CD134), RANK (TRANCE-R), RELT, TACI, TNFRI, TNFR2, TRAIL- R1, TRAIL-R2, TRAIL-R3, TRAIL—R4, TROY and XEDAR.

The term “receptor of the TNF superfamily”, as used herein, also includes variants of a given or of the TNF superfamily.

The term “variant” according to the invention refers, in patticular, to mutants, splice variants, conformation variants, ms, allelic variants, species variants and species gs, in particular those which are naturally present. An allelic variant relates to an alteration in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing ofien identifies numerous allelic ts for a given gene. A species homolog is a nucleic acid or amino acid sequence with a different species of origin from that of a given nucleic acid or amino acid sequence. The term “variant” shall encompass any posttranslationally modified variants and conformation variants.

According to the present invention, the first or and the second receptor of the TNF superfamily are preferably selected from the group consisting of CD40, CD27, 0X40 (CD134) and 4—1BB (CD137).

CD40, also referred to as tumor necrosis factor or superfamily member 5 FS), is the receptor for the ligand TNFSFS/CD40L. CD40 is known to transduce TRAF6- and MAP3K8-mediated signals that te ERK in macrophages and B-cells, leading to induction of immunoglobulin secretion. In one embodiment, CD40 is human CD40, having UniProt accession number P25942.

CD27, also referred to as TNFRSF7, is the receptor for the ligand CD70/CD27L. CD27 is believed to play a role in survival of activated T cells and a role in sis through association with SIVAl. In one embodiment, CD27 is human CD27, having UniProt accession number P26842. 0X40 ), also referred to as TNFRSF4, is the receptor for the ligand TNFSF4/OX40L/GP34. 0X40 (CD134) is a costimulatory molecule implicated in long-term T cell immunity. In one embodiment, 0X40 (CD134) is human 0X40 (CD134), having UniProt accession number P43489. 4-1BB (CD137), also referred to as TNFRSF9, is the receptor for the ligand /4-IBBL. 4- lBB (CD137) is believed to be involved in T cell activation. In one embodiment, 4-lBB (CD137) is human 4-lBB (CD137), having t accession number Q07011.

The term “binding domain” characterizes in connection with the present invention a structure, e. g., of an antibody, which binds to/interacts with a given target structure/antigen/epitope. Thus, the binding domain ing to the invention ates an “antigen-interaction site” or “antigen-binding site”.

The term “antigen” relates to an agent such as a protein or peptide comprising an epitope against which an immune response is directed and/or is to be directed.

The term “peptide” according to the invention comprises oligo- and polypeptides and refers to substances sing two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 more, ably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids joined covalently by peptide bonds. The term “protein” refers to large peptides, preferably to peptides with more than 100 amino acid residues, but in l the terms “peptides” and “proteins” are synonyms and are used interchangeably herein.

The term “epitope” refers to an antigenic determinant in a molecule, i.e., to the part in a molecule that is recognized by the immune system, for example, that is recognized by an antibody. For example, epitopes are the discrete, three-dimensional sites on an antigen, which are recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural teristics, as well as specific charge characteristics. Conformational and non- Conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the ce of denaturing solvents. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably n 5 and 100, preferably between 5 and 50, more ably between 8 and 30, most preferably between 10 and amino acids in , for example, the epitope may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

Mapping of epitopes recognized by binding agents can be med as described in detail in "Epitope Mapping Protocols” (Methods in Molecular Biology) by Glenn E. Morris ISBN- 0896039 and in "Epitope Mapping: A Practical Approach" Practical Approach Series, 248 by Olwyn M. R. Westwood, Frank C. Hay.

The term “agonistic binding agen ”, as used herein, refers to a binding agent, which activates the receptor(s) of the TNF superfarnily it is binding to. In one embodiment, the binding agent induces IL-8 release from and/or activates the NF-KB pathway in the ce11(s) expressing the receptor(s) of the TNF superfamily it is g to. In one embodiment, the binding agent is a activating binding agent, simultaneously activating receptors of the TNF amily, e.g., said first and second receptor, located on different cells, In another embodiment, the binding agent is a cis-activating binding agent, simultaneously activating receptors of the TNF superfamily, e.g., said first and second receptor, located on the same cell.

The term “binding” ing to the invention preferably relates to a c binding. A binding agent, such as an antibody or fragment thereof, is specific for a predetermined target if it is capable of binding to said predetermined target while it is not (substantially) capable of binding to other targets.

According to the present invention, a binding agent, such as an antibody or fragment thereof, is capable of binding to a predetermined target if it has a significant y for said predetermined target and binds to said predetermined target in rd assays. “Affinity” or ng affinity” W0 20161110584 is ofien measured by equilibrium dissociation constant (Kn). Preferably, the term “significant affinity” refers to the binding to a predetermined target with a iation constant (Kn) of 10'5 M or lower, 106 M or lower, 10'7 M or lower, 10'3 M or lower, 10‘9M or lower, 10'10M or lower, 'll M or lower, or 10le or lower.

An agent is not (substantially) capable of binding to a target if it has no significant affinity for said target and does not bind significantly, in particular does not bind detectably, to said target in standard assays. Preferably, the agent does not detectably bind to said target if present in a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 1.1ng or higher. ably, an agent has no significant affinity for a target if it binds to said target with a Kn that is at least 10-fold, loo-fold, 103-fold, 104-fold, lOS-fold, or lOG-fold higher than the K0 for binding to the predetermined target to which the agent is capable of binding. For example, if the Kn for binding of an agent to the target to which the agent is capable of binding is 10'7 M, the Kn for binding to a target for which the agent has no significant affinity would be at least 1045 M, 10-5 M, 104 M, 10-3 M, 10-2 M, or 10-1 M.

Binding of an agent to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et a1., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, W. 13., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y (1992), and s described herein. ies may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the e 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled artisan. The affinity data may be analyzed, for example, by the method of Scatchard et al., Ann NY. Acad. ScL, 511660 (1949). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, e.g., salt tration, pH. Thus, measurements of affinity and other antigen-binding ters, e.g., K13, 1050, are preferably made with rdized solutions of antibody and antigen, and a rdized buffer. 3O The term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term “antibody” includes monoclonal antibodies, inant antibodies, human dies, humanized antibodies, chimeric dies and combinations of any of the foregoing. Each heavy chain is sed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be turther subdivided into regions of ariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy—terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The CDRs of a VB are termed HCDRl, HCDR2 and HCDR3, the CDRs of a VL are termed LCDRl, LCDRZ and LCDR3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

The nt regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further ided into constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from amino-terminus to carboxy- terminus in the ing order: CH1, CH2, CH3). The constant regions of the antibodies mediate the g of the immunoglObulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

Antibodies may be derived from different species, including but not limited to mouse, rat, , guinea pig and human.

Antibodies described herein include IgA such as IgAl or IgAZ, lgGl, IgGZ, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the dy is an IgGl antibody, more ularly an IgGl, kappa or IgGl, lambda isotype (i.e. IgGl, K, 1.), an IgG2a antibody (e.g.

IgGZa, K, it), an IgGZb antibody (e.g. IgGZb, K, 7t), an IgG3 antibody (e.g. IgG3, K, it) or an IgG4 antibody (e.g. lgG4, K, it).

As used herein, “isotype” refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes. “Isotype ing” refers to the phenomenon by which the class, or isotype, of an antibody changes from one 1g class to one of the other lg classes.

The term “monoclonal antibody”, as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal dy displays a single binding specificity and affinity. In one embodiment, the monoclonal antibodies are produced by a oma which includes a B-cell obtained from a non-human animal, e.g., a mouse, fused to an immortalized cell.

The term “recombinant antibody”, as used , includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal with respect to the immunoglobulin genes or a oma ed rom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (0) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.

The term “recombinant” in the context of the t invention means “made through genetic engineering”. Preferably, a binant object”, such as a inant antibody or nucleic acid“ is not lly occurring.

The term “naturally occurring”, as used herein, as d to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (cg, mutations introduced by random or site'specific mutagenesis in vitre or by somatic mutation in vivo).

The term “humanized antibody” refers to a molecule having an antigen binding site that is substantially d from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based upon the structure and/or sequence of a human immunoglobulin. The antigen binding site may either comprise complete variable regions (VH and VL) fused onto constant domains (e.g., the constant domains of IgGl/K), or only the mentarity determining regions (CDRs) grafted onto appropriate framework regions in the variable regions. n binding sites may be wild-type or modified by one or more amino acid substitutions, e.g. modified to resemble human globulins more y. Some forms of humanized antibodies preserve all CDR sequences (for example a humanized mouse antibody which contains all six CDRs from the mouse antibody). Other forms have one or more CDRs which are altered with respect to the original antibody.

The term “chimeric antibody” refers to those antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies d from a particular species or belonging to a particular class, while the remaining segment of the chain is gous to corresponding ces in another. Typically, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to sequences of antibodies derived from another. One clear advantage to such chimeric forms is that the variable region can conveniently be derived from tly known sources using readily available B-cells or hybridomas from non-human host organisms in combination with constant s derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation and the specificity is not affected by the source, the constant region being human, is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non human source. However, the definition is not limited to this particular example.

The antibodies described herein are preferably isolated. The term “isolated antibody”, as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds to an epitope, isoform or variant of a given receptor of the TNF superfamily may, r, have cross- reactivity to other related antigens, e.g., from other species (e.g., Species homologs of said receptor). er, an isolated dy may be substantially free of other cellular al andfor chemicals.

The term “antibody fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind to an n and may also be referred to as “antigen-binding fragmen ” of an antibody. It has been shown that the antigen-binding function of an dy can be perforated by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antibody fragment” include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH regions; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge ; (iii) Fd ' nts consisting of the VH and CH regions; (iv) Fv nts consisting of the VL and VH regions of a single arm of an dy, (v) dAb fragments (Ward et al., (1989) Nature 341: 544- 546), which consist of a VH region; (vi) isolated complementarity determining regions (CDR), and (vii) ations of two or more ed CDRs which may Optionally be joined by a synthetic linker. Furthermore, although the two regions/domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single n chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e. g., Bird et a1. (1988) Science 242: 423-426; and Huston et a1. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883).

Such single chain dies are also intended to be encompassed within the term “antibody fragment”. A further example is a binding-domain immunoglobulin fusion protein comprising (i) a g domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region.

The g-domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional ques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The terms “pa ” or “fragment” are used interchangeably herein and refer to a continuous element. For example, a part of a ure such as an amino acid sequence or protein refers to a continuous element of said structure. A portion, a part or a fragment of a structure preferably comprises one or more functional properties of said ure. For e, a portion, a part or a fragment of an epitope or peptide is preferably immunologically equivalent to the epitope or e it is derived fiom. A part or fragment of a n sequence preferably comprises a sequence of at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 utive amino acids of the protein sequence.

When used in connection with VH and VL domains, the term “fragment” preferably refers to one or more of the complementarity-determining regions (CDR3), preferably at least the CDR3 variable region, of the heavy chain variable region (VH) and/or of the light chain le region (VL). In one embodiment, said one or more of the complementarity-detennining regions (CDRs) are selected from a set of complementarity-determining regions CDRl, CDR2 and CDR3. In a particularly preferred embodiment, the term “fragment” refers to the complementarity- determining regions CDRl, CDR2 and CDR3 of the heavy chain variable region (VH), i.e.

HCDRI, HCDR2 and HCDR3, and/or of the light chain variable region (VL), i.e. LCDRl, LCDR2 and LCDR3.

WO 10584 The teaching given herein with respect to specific amino acid sequences, e.g., those shown in the sequence listing, is to be ued so as to also relate to variants of said specific sequences resulting in sequences which are functionally equivalent to said specific sequences, e.g., amino acid sequences exhibiting properties identical or similar to those of the specific amino acid sequences. One important preperty is to retain binding to a target.

For example, the sequences shown in the sequence listing can be modified so as to remove one or more, preferably all free cysteine residues, in particular by ing the cysteine residues by amino acids other than ne, preferably serine, alanine, threonine, glycine, tyrosine, leucine or methionine, most preferably e or serine.

It will be appreciated by those skilled in the art that, in particular, the ces of the CDRs and variable regions can be modified Without losing the ability to bind the first and/or the second receptor of the TNF amily. For example, CDR regions will be either identical or highly homologous to the regions of antibodies specified . By “highly homologous” it is plated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in the CDRs. In addition, the hypervariable and variable s may be modified so that they show substantial homology with the regions of antibodies Specifically disclosed herein. 2O For the es of the present invention, “variants” of an amino acid sequence, in particular variants of a heavy chain variable domain (VH) or of a light chain variable domain (VL), comprise amino acid insertion ts, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants. Amino acid deletion variants that comprise the deletion at the N—tenninal and/or C-terminal end of the protein are also called inal and/or C-terminal truncation variants. particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

Amino acid addition variants comprise amino- and/or y—terminal fiJSiOIlS of one or more amino acids, such as l, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by l of l, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein.

Amino acid tution variants are characterized by at least one residue in the sequence being removed and another e being inserted in its place. ence is given to modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to ing amino acids with other ones having similar properties.

Preferably, amino acid substitutions in protein variants are conservative amino acid substitutions.

A conservative amino acid substitution involves substitution of an amino acid with another one ofthe same family of amino acids, i.e., amino acids which are related in their side chains (e.g., in terms of the electrical charge and/or size). Naturally occurring amino acids are lly divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, , leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, ine, tyrosine) amino acids. alanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

Preferably the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the nce amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably continuous amino acids. In preferred embodiments, the degree of rity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 100, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acids that either are identical or that represent conservative amino acid tutions. “Sequence identity” n two amino acid sequences indicates the percentage of amino acids that are identical n the sequences.

The term “percentage identity” is intended to denote a percentage of amino acid residues which are identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two amino acid sequences are conventionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by "Window of comparison" in order to identify and compare local s of sequence similarity. The optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the rity search method of n and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin cs Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis).

The percentage identity is calculated by determining the number of identical positions between the two ces being compared, dividing this number by the number of positioris compared and multiplying the result obtained by 100 so as to obtain the percentage identity n these two sequences.

Binding agents in accordance with the present invention may be at least bispecific or multispecific, such as trispecific, tetraspecific and so on.

The term “bispecific molecule” refers to a le that has two different binding specificities and thus may bind to two ent types of antigen, such as a first and a second receptor of the TNF superfamily. Such bispecific molecule may be in the format of an antibody le or of an antibody-like le or of a protein scaffold with antibody-like properties or of a cyclic peptide with at least two binding specificities.

The term “bispecific antibody”, as used herein, refers to an antibody comprising two antigen- binding sites, a first binding site having affinity for a first antigen or epitope and a second 2016/050308 binding site having binding affinity for a second antigen or epitope distinct from the first. In particular, a bispecific antibody is an artificial protein that is composed of fragments of two different antibodies (said fi'agments of two different dies forming two binding domains) and consequently binds to two ent types of antigen.

A bispecific molecule/antibody according to the invention is engineered to simultaneously bind to a first receptor of the TNF superfamily and to a second receptor of the TNF superfamily, wherein the first receptor and the second receptor are different and may be located on the same cell (“cis”) or different cells (“trans”).

In one embodiment, the first or and the second receptor are located on the same cell and are selected from the group consisting of CD27, 0X40 (CD134) and 4-1BB (CD137). In one ment, said cell is a T cell.

In another embodiment, the first receptor and the second receptor are located on different cells, wherein the first or is CD40 and the second receptor is selected from the group consisting of CD27, 0X40 (CD134) and 4-1BB (CD137). In. one embodiment, said different cells are an antigen-presenting cell (APC), such as a dendritic cell, expressing CD40 and a T cell expressing the second receptor selected from the group consisting of CD27, 0X40 (CD134) and 4-1BB (CD137).

Bispecific (full-length) antibodies may be obtained by covalently/chemically linking two (monoclonal) antibodies or by conventional -hybridoma techniques. Covalent g of two monoclonal antibodies is described, for e, in Anderson (1992), Blood 80:2826-34. In the t of this invention, one of the antibodies is c for the first ” of the TNFlUVU}!IUI superfamily and the other one for the second receptor of the TNF superfamily.

Alternatively, bispecific (full-length) antibodies may be obtained by using the knobs-into-holes technology, which allows the hetero-dimen'zation of antibody heavy chains by introducing specific ons in the CH3 dimer interface, e.g., in the CH3 dimer interface of IgG1 or lgG4.

This technology is described, for example, in Spiess C. et a1. (2013), Journal of Biological Chemistry 288(37):26583-93; Labrijn A.F. et al. (2013), PNAS 110(13):5145—50; and WO 2008/119353 A1.

In one embodiment, the bispecific antibody is a bispecific single-chain antibody.

As used herein, a “bispecific single—chain antibody” denotes a single polypeptide chain comprising two binding domains. Each binding domain ses one VH region, wherein the VH region of the first binding domain specifically binds to the first receptor of the TNF superfamily (RECl), and the VH region of the second binding domain cally binds to the second receptor of the TNF superfamily (RECZ). The two binding domains are optionally linked to one another by a short peptide linker. Each binding domain may additionally comprise variable region from an antibody light chain (VL), the VH region and VL region within each of the first and second binding domains being linked to one another via along peptide linker, which is long enough to allow the VH region and VL region of the first g domain and the VH region and VL region of the second binding domain to pair with one another.

In one embodiment, the ponding variable heavy chain regions (VH) and the ponding variable light chain regions (VL) are ed, from N—terminus to C-terminus, in the order VH(REC1)—VL(REC1)-VH(REC2)-VL(REC2), VH(REC2)-VL(REC2)-V.H(REC l)-VL(REC1) or VH(REC2)—VL(REC2)-VL(REC1)—VH(REC1). It is, however, also envisaged that the ific single chain antibodies of the invention comprise other domain arrangements, such as 1)-VH(RECl)—VH(RECZ)-VL(REC2), VL(RECl)-VH(RECl)-VL(REC2)—VH(REC2), l)-VL(REC1)—VL(RECZ)-VH(REC2), VL(REC2)-VH(REC2)—VH(REC1)-VL(REC1), VL(REC2)-VH(REC2)—VL(RECl)—VH(REC1).

Peptide linkers are generally designed to provide flexibility and protease resistance. In one embodiment, the peptide linker is a e-serine-threonine-rich linker or glycine-serine-rich linker, wherein at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90% of the amino acids are a glycine or serine or threonine residue or a glycine or serine residue, respectively. In another embodiment, the amino acids are selected from e, serine and threonine, i.e., the peptide linker is exclusively composed of glycine, serine and ine residues (referred to as a glycine-serine- threonine linker). In yet another embodiment, the peptide linker is exclusively composed of glycine and serine residues (referred to as a glycine-serine linker). Short peptide linkers may 3O consist of 12 or less such as 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids, and preferably, 5 or 6 amino acids. Short peptide linkers preferably comprise the amino acid sequences SGGGGS or GGGGS. Long peptide linkers may consist of 12 or more, such as 15 to 25 or 15 to 20 or 15 to 18 amino acids, and may, for example, be ed from (GGGGS)3 ,(GGGGS)4 and (GGGGS)5.

In one embodiment, the binding agent of the invention is in the format of a bispecific antibody fragment. 2016/050308 The binding agent according to the invention may be one of various types of bivalent and trivalent single-chain variable nts (scFvs), filSlOtl proteins mimicking the le domains of two antibodies. A single~chain variable fragment (scFv) is a fusiOn protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a peptide linker of ten to about 25 amino acids. The peptide linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. Divalent (or nt) -chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. The invention also includes multispecific molecules comprising more than two scFvs binding domains. This makes it possible that the molecule comprises either multiple antigen specificities and is a trispecific, pecific, or multispecific molecule, or the molecule is a bispecific molecule comprising more than one scFv binding domain with specificity for the same antigen.

In ular, the molecule of the invention may be a pecific single chain Fv.

In one embodiment, the binding agent comprises two Fab regions, one being directed against the first receptor of the TNF amily and the other being directed against the second receptor of the TNF superfamily. In one embodiment, the binding agent is an antigen-binding fragment (Fab)2 complex. The (Fab)2 complex is composed of two Fab fragments, one Fab fragment comprising a Fv domain, i.e. VH and VL domains, binding to the first receptor of the TNF superfamily, and the other Fab fragment comprising a Fv domain binding to the second or of the TNF superfamily. Each of the Fab nts may be composed of two single chains, 3.

VL-CL module and a VH-CH module. Alternatively, each of the individual Fab fragments may be ed in a single chain, preferably, VL—CL—CH-VH, and the individual variable and constant domains may be connected with a e linker. In general, the individual single chains and Fab fragments may be connected via disulfide bonds, adhesive s, chemical linkage and/or peptide 1inker(s). The g agent may also se more than two Fab fragments, in particular, the molecule may be a Fab3, Fab4, or a multimeric Fab complex with specificity for 2, 3, 4, or more different antigens. The invention also includes ally linked Fabs.

Another possibility is the creation of scFvs with peptide linkers that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Still shorter peptide linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

A diabody (Kipriyanov, Int. J. Cancer 77 (1998), 763-772) is a small bivalent and bispecific antibody fragment. Diabodies comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain. This forces pairing with the complementary s of r chain and promotes the assembly of a dimeric molecule with two functional antigen-binding sites. To construct bispecific diabodies of the invention, the V-domains of an antibody binding to the first receptor of the TNF superfamily (RECl) and an antibody binding to the second receptor of the TNF superfami1y(RECZ) may be fused to create the two chains 1)—VL(REC2) and VH(REC2)-VL(REC1). Each chain by itself is not able to bind to the respective antigen, but recreates the functional antigen binding sites of an anti-RECI antibody and an anti-REC2 antibody on pairing with the other chain. To this end, a e linker that is too short to allow g between the two domains on the same chain is used. The two scFv molecules, with a linker between heavy chain variable domain and light chain variable domain that is too short for intramolecular dimerization, are co-expressed and self assemble to form bi-specific molecules with the two g sites at opposite ends.

In one embodiment, the bispecific antibody nt binding to the first receptor of the TNF superfamily (RECl) and the second receptor of the TNF superfamily (RECZ) is a minibody, preferably, a minibody sing two single VH-VL—C chains that are connected with each other via the constant domain (C) of each chain. According to this aspect, the corresponding variable heavy chain regions (VH), corresponding variable light chain regions (VL) and constant domains (C) are arranged, from N-terminus to C-terminus, in the order VH(REC1)-VL(RECI)- (C) and VH(REC2)-VL(REC2)—C, wherein C is preferably a CH3 . Pairing of the constant domains results in formation of the minibody. in one embodiment, the bispecific binding agent is in the format of an antibody—like molecule with a heavy chain containing two consecutive N-tenninal le domains with different specificities and a light chain with two consecutive variable domains with different specificities resulting in four binding s with two ent specificities (Wu et a1. , Nat.

Biotechnology 25(1 1)) g agents according to the invention may also comprises an amino acid sequence for tating secretion of the molecule, such as a N-terminal secretion signal, and/or one or more labels or tags facilitating binding, purification or detection of the molecule.

Preferably, the secretion signal is a signal sequence that allows a sufficient passage through the secretory pathway and/or secretion of the binding agent into the extracellular environment.

Preferably, the ion signal sequence is cleavable and is removed from the mature binding agent. The secretion sigial sequence preferably is chosen with respect to the cell or organism wherein the binding agent is produced in.

A “label or tag facilitating binding, purification or detection of the melee e” is meant to e any labels/tags known in the art for these purposes. Particularly preferred are affinity tags, such as chitin binding protein (CBP), e binding protein (MBP), glutatluone-S-transferase (GST) and poly(His) (e. g., Hiss); solubilization tags, such as thioredoxin (TRX) and polymANP); cinematography tags, such as a FLAG-tag; epitope tags, such as VS-tag, g and ; and fluorescent or luminescent labels or tags, such as fluorescent proteins (e.g., GFP, YFP, RFP etc), fluorescent dyes and luciferase.

The amino acid sequence of a peptide label or tag may be introduced at any position within the amino acid ce of the binding agent, and may, for example, take the shape of a loop within the encoded protein structure or it may be N-terminally or inally fused. The label or tag may further contain a cleavage site that allows a removal of the label or tag from the g agent. Similarly, non-peptidic labels or tags, e.g, fluorescent dyes, may be conjugated to the binding agent at any suitable site. g agents of the invention may contain, e.g., in addition to said first and second binding domain, a further binding domain which serves egg. to enhance selectivity for a specific cell type.

This can be achieved, e.g., by providing a binding domain that binds to another antigen sed on said specific cell type.

Binding agents of the invention may r comprise one or more modifications increasing the stability of the binding agent. The term “stability” of the binding agent relates to the “half-life” of the binding agent, e.g., in viva. “Half-life” relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules.

The binding agent may, for example, be conjugated to a half-life extension module. Such modules are known to a person skilled in the art and include, for example, albumin, an albumin- binding domain, an immunoglobulin-binding domain, an FeRn—binding motif, and, in particular, a polymer. Particularly preferred polymers include hylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid and metic e sequences.

The binding agents described herein may be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. A cytotoxin or cytotoxic agent includes any agent that is detrimental to and, in particular, kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, Vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, oxy anthracin dione, mitoxantrone, mithramycin, mycin D, l-dehydrotestosterone, glucocorticoids, procaine, aine, lidocaine, propranolol, and puromycin and analogs or gs thereof.

Suitable therapeutic agents for forming conjugates include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thi0guanine, bine, fludarabin, S-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, lan, carrnustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, ,..,. (A streptozotocin, mitomycin C, and cis-dichlorodiarnine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly ycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and mycin (AMC), and anti-mitotic agents (e.g., vincristine and vinblastine). In a red embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another ment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, cin, sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A.

Binding agents also can be conjugated to a radioisotope, e.g., iodine-131, yttrium-90 or indium- 111, to generate cytotoxic radiopharmaceuticals.

Techniques for conjugating such therapeutic moiety to binding agents, such as antibodies or fragments thereof, are well known, see, e.g., Amon et al., "Monoclonal dies For targeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds. ), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et 31., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal dies '84: Biological And Clinical Applications, Pineheraet al. (eds. ), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody—Toxin Conjugates", Immunol. Rev., 62: 119=58 (1982).

The present invention provides a binding agent comprising at least two binding domains, wherein a first binding domain binds to a first receptor of the tumor necrosis factor (TNF) superfamily and a second binding domain binds to a second receptor of the TNF superfarnily, wherein the first receptor and the second receptor are ent.

In one ment, the first binding domain binds to CD40 and the second g domain binds to CD27, wherein, preferably, the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1, and one or more CDRs or one or more le domains of an antibody AB27_X, wherein X is selected from the group consisting of 1 to 186, as shown in Table 7.

“AB40_1” refers to an antibody binding to CD40, which comprises (a) heavy chain complementarity determining regions HCDRl having the amino acid sequence of SEQ ID NO: 2323, HCDR2 having the amino acid sequence of SEQ ID NO: 2324 and HCDR3 having the amino acid ce of SEQ ID NO: 2325, and light chain complementarity determining s LCDRI having the amino acid sequence of SEQ ID NO: 2326, LCDR2 having the amino acid sequence of SEQ ID NO: 2327 and LCDR3 having the amino acid ce of SEQ ID NO: 2328; and (b) heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 2321 and light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 2322.

In various embodiments, the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is l; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 2; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 3; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 4; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 5; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 6; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 7; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 8; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 9; the g agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 10; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40*1 and X is 11; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 12; the binding agent comprises or more CDRs or one or more variable domains of antibody AB40__I and X is 13; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 14; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 15; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 16; the binding agent ses one or more CDRs or one or more le domains of antibody AB40_1 and X is 17; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 18; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 19; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 20; the binding agent comprises one or more CDRs one or more variable domains of antibody AB40_1 and X is 21; the binding agent comprises or more CDRs or one or more variable domains of antibody AB40__1 and X is 22; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 23; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 24; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 25; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 26; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 27; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 28; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 29; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 30; the binding agent ses or more CDRs or one or more variable domains of antibody AB40_1 and X is 31; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40g1 and X is 32; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 33; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 34; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 35; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 36; W0 20161110584 the binding agent comprises one or more CDRs or one or more variable domains of dy AB40_1 and X is 37; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40'_1 and X is 38; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 39; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 40; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 41; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 42; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 43; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 44; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 45; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 46; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 47; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 48; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 49; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 50; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 51; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 52; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 53; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 54; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 55; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 56; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 57; the g agent comprises one or more CDRs or one or more le domains of antibody AB40fil and X is 58; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 59; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 60; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 61; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 62; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 63; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 64; the binding agent comprises one or more CDRs or one or more variable 2016/050308 domains of antibody AB40_1 and X is 65; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 66; the binding agent comprises or more CDRs or one or more variable domains of antibody AB4OAI and X is 67; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 68; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 69; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40”1 and X is ‘70-; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 71; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 72; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 73; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 74; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 75; the binding agent ses or more CDRs or one or more variable domains of antibody AB40_1 and X is 76; the binding agent comprises one or more CDRs or one or more le s of antibody AB40_,1 and X is 77; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_I and X is 78; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 79; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 80; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_l and X is 81; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 82; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 83; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 84; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 85; the g agent ses one or more CDRs or one or more variable domains of dy AB40_1 and X is 86; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 87; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 88; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 89; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 90; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 91; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 92; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40WI and X is 93; the binding agent comprises WO 10584 or more CDRs or one or more variable domains of antibody AB40_1 and X is 94; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 95; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 96; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 97; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 98; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 99; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 100; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 101; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40__1 and X is 102; the binding agent comprises o..e or more CDRs or one or more variable domains of antibody AB40_1 and X is 103; the binding agent comprises one or more CDRs or one or more le domains of dy AB40_1 and X is 104; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 105; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40m1 and X is 106; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 107; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 108; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 109; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40M1 and X is 110; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40~1 and X is 111; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 112; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40wl and X is 113; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 114; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 115; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40w1 and X is 116; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40m1 and X is 117; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 118; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 119; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 120; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 121; the binding agent comprises one or more CDRs or 2016/050308 one or more variable domains of dy AB40_1 and X is 122; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 123; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 124; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 125; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 126; the binding agent ses one or more CDRs or one or more variable s of antibody AB40_1 and X is 127; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40__1 and X is 128; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 129; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 130; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 131; the binding agent comprises one or more CDRs or one or more le s of antibody AB40_1 and X is 132; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 133; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 134; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 135; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40g1 and X is 136; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 137; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 138; the binding agent comprises one or more CDRs or one or more variable domains of dy AB40_1 and X is 139; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 140; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 141; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40WI and X is 142; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 143; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 144; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 145; the binding agent comprises one or more CDRs one or more variable domains of antibody AB40*1 and X is 146; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 147; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 148; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 149; the g agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 150; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 151; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 152; the binding agent comprises one or more CDRs or one or more variable domains of dy AB40ml and X is 153; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 154; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 155; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40ml and X is 156; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 157; the binding agent comprises one or more CDRs Or one or more variable domains of antibody AB40_1 and X is 158; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 159; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 160; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 161; the g agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 162; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 163; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40__1 and X is 164; the g agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 165; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 166; the binding agent campuses one or more CDRs or one or more variable domains of antibody AB40_”1 and X is 167; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 168; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 169; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 170; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 171; the binding agent ses one or more CDRs or one or more le domains of antibody AB40_1 and X is 172; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 173; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40__1 and X is 174; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 175; the g agent comprises one or more CDRs or one or more variable domains of dy AB40_1 and X is 176; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 177; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 178; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40__1 and X is 179; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 180; the binding agent comprises one or more CDRs or one or more variable domains of dy AB40_1 and X is 181; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_‘1 and X is 182; the binding agent comprises one or more CDRs or one or more le domains of antibody AB4G_1 and X is 183; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 184; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 185; or the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40__1 and X is 186.

In one embodiment, the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 5.

In one ment, the first binding domain binds to CD40 and the second binding domain binds to 0X40 ), wherein, preferably, the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1, as defined above, and one or more CDRs or one or more variable domains of an dy AB134_X, wherein X is selected from the group consisting of l to 92, as shown in Table 8.

In various embodiments, the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_I and X is 1; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 2; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 3; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 4; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 5; the g agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 6; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 7; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 8; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 9; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 10; the binding agent ses one or more CDRs or one or more variable s of antibody AB40_1 and X is 11; the binding agent comprises one or more CDRs or W0 20161110584 one or more variable domains of antibody AB40_1 and X is 12; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 13; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 14; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40#1 and X is 15; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40wl and X is 16; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40w1 and X is 17; the binding agent comprises one or more CDRs or one or more variable domains of dy AB40_1 and X is 18; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 19; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 20; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 21', the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 22; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 23; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 24; the binding agent ses one or more CDRs or one or more le domains of antibody AB40_1 and X is 25; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 26; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 27; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 28; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 29; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 30; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 31; the binding agent ses one or more CDRS or one or more variable domains of antibody AB40_1 and X is 32; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 33; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 34; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 35; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 36; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 37; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40fll and X is 38; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 39; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 40; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_I and X is 41; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 42; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 43; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 44; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 45; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_'1 and X is 46; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 47; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 48; the binding agent comprises or more CDRs or one or more variable domains of antibody AB40_1 and X is 49; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 50; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 51; the binding agent comprises one or more CDRs or one or more le domains of antibody AB40_1 and X is 52; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 53; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 54; the binding agent comprises one or more CDRs or one or more variable s of antibody AB40_1 and X is 55; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 56; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 57; the binding agent comprises or more CDRs or one or more le domains of antibody AB40_1 and X is 58; the g agent comprises one or more CDRs or one or more variable s of antibody 1 and X is 59; the binding agent ses one or more CDRs or one or more le domains of antibody AB40~1 and X is 60; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 61; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 62; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 63; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 64; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 65; the binding agent comprises one or more CDRs or one or more variable domains of dy AB40_1 and X is 66; the binding agent comprises or more CDRs or one or more le domains of antibody AB40_1 and X is 67; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 68; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 69; the binding agent comprises one or more CDRS or one or more variable domains of dy AB40_1 and X is 70; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40‘1 and X is 71; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40__] and X is 72; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 73; the g agent comprises one or more CDRS or one or more variable domains of antibody AB40A1 and X is 74; the binding agent ses one or more CDRS or one or more variable domains of dy AB40_1 and X is 75; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 76; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 77; the g agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 78; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 79; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 80; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 81; the binding agent comprises one or more CDRS or one or more variable domains of dy AB40_1 and X is 82; the binding agent comprises one or more CDRS or one or more variable s of antibody AB40_1 and X is 83; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 84; the binding agent comprises one 2O or more CDRS or one or more variable domains of antibody AB40_1 and X is 85; the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 86; the g agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 87; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40gl and X is 88; the binding agent comprises one or more CDRS or one or more variable domains of dy AB40_1 and X is 89; the binding agent ses one or more CDRS or one or more variable domains of antibody AB40_1 and X is 90; the g agent comprises one or more CDRs or one or more variable domains of dy AB40_1 and X is 91; or the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 92.

In one embodiment, the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 3. In another embodiment, the binding agent comprises one or more CDRS or one or more variable domains of antibody AB40_1 and X is 81.

WO 10584 In one embodiment, the first binding domain binds to CD40 and the second binding domain binds to 4-1BB (CD137), wherein, preferably, the binding agent comprises one or more CDRs or one or more variable s of dy AB40~_1 , as defined above, and one or more CDRs or one or more variable domains of an antibody AB137_X, wherein X is selected from the group consisting of 1 to 12, as shown in Table 9.

In various embodiments, the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 1; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 2; the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 3; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 4; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 5; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40__1 and X is 6; the binding agent comprises one or more CDRs or one or more variable s of dy AB40_1 and X is 7; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 8; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 9; the binding agent ses one or more CDRs or one or more variable domains of antibody AB40_1 and X is 10; the binding agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 11; or the binding agent comprises one or more CDRs or one or more variable domains of dy AB40_1 and X is 12.

In one embodiment, the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 2. In another embodiment, the g agent comprises one or more CDRs or one or more variable domains of antibody AB40_1 and X is 12.

In one embodiment, the first binding domain binds to 4-lBB (CD137) and the second binding domain binds to CD27, wherein, preferably, the binding agent comprises one or more CDRs or one or more variable domains of an antibody X, wherein X is seiected from the group consisting of 1 to 12, as shown in Table 9, and one or more CDRs or one or more variable domains of an antibody AB27_Y, wherein Y is selected from the group consisting of 1 to 186, as shown in Table 7.

In various embodiments, X is 1 and Y is selected from the group consisting of 1 to 186; X is 2 and Y is selected from the group consisting of 1 to 186; X is 3 and Y is selected from the group W0 10584 2016/050308 consisting of 1 to 186; X is 4 and Y is selected from the group consisting of 1 to 186; X is 5 and Y is selected from the group consisting of 1 to 186; X is 6 and Y is selected from the group consisting of 1 to 186; X is 7 and Y is selected from the group consisting of 1 to 186; X is 8 and Y is selected from the group consisting of 1 to 186; X is 9 and Y is selected from the group consisting of 1 to 186; X is 10 and Y is selected from the group consisting of 1 to 186; X is 11 and Y is selected from the group consisting of 1 to 186; or X is 12 and Y is selected from the group consisting of ‘1 to 186.

In various embodiments, insl;Xis1aninsZ;Xislanins 3;Xislanins4;Xislanins S;Xisl anins6;Xislanins7;Xislanins8;Xis1anins9;Xislaninle;Xisland Yis 11;Xis 1 anins 12;Xislanins 13;Xislanins 14; Xislanins 15;Xisland Yis16;Xislanins 17;Xis1 aninsl8;Xis1anin319;Xis1anins20;Xis1 and Yile;Xis1anin822;Xislanin323;Xislanin524;XislaninsZS;Xisland Yi526;XislaninsZ7;Xislanins28;Xislanins29; in330;Xis1and Yis31;Xis1anins32;Xislanins33;Xis1anins34; Xislanins35;Xisland Yi336;Xislanins37;Xis1anins38;Xis1anins39; Xisl anins40;Xisland Yis41;Xis1anins42;Xisianins43;Xislanins44;Xis1anins45;Xisland Yis46;Xislanins47;Xislanins48;Xis1anins49; XislaninsSO;Xisland Yis51;Xis1anins 52;Xisl aninsS3;Xis1aninsS4;Xis1 anins55;Xisland Yis56;Xislanins 57;Xislanin358;Xislanins 59;Xislanins60;Xisl and Yi361;Xislanins62;Xislanins63;Xislanins64;Xisl anins65;Xisland Xis1anins67;Xis1anins68;Xislanins69;Xislanins70;Xisland Yis71;Xis 1anins72;XisI anins73;Xis1anins74;Xis1anins7S;Xisland Yis76;Xis1anins77;Xislanins78;Xislanins79;Xislanins80;Xisland Yis81;Xislanins 82;Xislanins 83;Xislanins84;Xislanins 85;Xis 1 and Yi586;Xislanin387;Xis1anin588;Xislanins89;Xis1anins9O;Xisland Yis91;Xis1anins92;Xislanins93;Xislanins94;Xisl anins95;Xis1and Yis96;Xis1anins97;Xislanin398;Xis 1anins99;Xisl anins100;Xisl anins 101;Xis 1 anin3102;Xislanins103;Xislanins104;Xis 1 anins 105; Xislanins 106;Xislanins 107;Xislanins 108;Xislanins slanins 110;Xislanins111;Xis1anins112;Xislanins113;Xisl anins 114;Xis1 anins 115;Xislanins slanins 117;Xis 1 aninsll8;Xislanins 119; Xislanins120;Xislanin3121;Xisl 22;Xislanin5123;Xislanins 124;Xislanin5125;Xislanins126;Xislanin8127;Xislanins128;Xisl 2016/050308 anins129;Xislanins 130;Xis 1 anins 131;Xis 1 anins 132;Xislanins 133; Xislanins 134;Xislanins 135;Xis1anins 136;Xisl anins 137;Xisl anins 138;Xis1anins139;Xislanins140;Xis1aninsl41;Xis1anins142;Xisl anins 143;Xislanins 144;Xislanins 145;Xis 1 anins 146;Xislanins 147; Xislanins 148;Xis 1 anins s 1 anins 150;Xisl anins 151;Xisl anins 152;Xis1anins153;Xislanins154;Xislanins155;Xislanin3156;Xisl anins157;Xis1anins 158;Xis 1 anins 159;Xisland‘z’is 160;Xis1anins 161; XislandY15162;Xis1anins 163;Xis1anins 164;Xisl 65;Xis1anins 166;Xis 1 anins 167;Xislaninsl68;Xis 1 deis 169; Xislanins 170;Xisl anins 171;Xis 1 anins 172;Xislanins 173;Xis 1 anins 174;Xis 1 anins 175; Xislaninsl76;Xislanins 177;Xislanins sl aninsl79;Xislanins 180;Xislanins 181;Xi31anins182;Xis 1 aninslS3gXis 1 anins 184;Xisl anin3185;Xis 1 anins 186; insl;XisZaninsZ;Xis2aninsS;XisZandYis4;Xis2and. - isS;XisZ aninsG;XisZanins7;XisZanin58;Xis2anin39;Xi52aninle;XisZand YisI];XisZanins12;Xis2anins13;Xis2a.nins14;Xis2anins15;XisZand Yisl6;Xis2anins17;XisZanins18;Xis2aninsl9;Xis2aninsZO;Xi52and Yis21;XisZanin322gXi32aninsZ3;XisZaninsZ4;Xi52anin325;Xi52 and Yi526;Xi52aninsZ7;XisZanins28;XisZaninsZ9;XisZanins30;XisZand Yis31;XisZanins32;Xi32aninsSS;Xis23nins34;XisZanins35;XisZ and XisZanins37;Xi32anins38;XisZanins39;Xi52anins40;Xis2and Yis41;Xis2anins42;Xi32anins43;XisZanins44;Xi52anins45;Xi52and Yis46;Xis2anins47;Xi32anins48;XisZanins49;Xis2anins 50;Xi52and YisSl;Xis2aninsSZ;XisZaninsS3;Xis2anins 54;XisZaninsSS;XisZand Yis56;X132aninsS7;XisZanin358;Xis2anins59;XisZanins60;XisZand Yis61;Xis2anins62;XisZanins63;XisZanins64;Xis2anins65;Xis2and Yis66;Xis2anins67;XisZanins68;XisZanin569;Xi52anins7O;Xi52and Yis71;Xi52anins72;XisZanins73;Xis2anins 74;Xis2anins75;Xi32and Yis76;XisZanins77;XisZanins78;Xi32anins79;Xi52anin580;Xis2 and Yis 2anins 82;Xi32anins 83;XisZanin384;Xi52anins85;Xis2and Yis 86;Xi32anins 87;XisZanins 88;XisZanins89;XisZanin590;Xi52and Yis9l;XisZanins92;XisZanins93;Xi52anins94;Xis2anins95;Xis2and Yis96;Xis2anins97;XisZandYis98;Xis2anins99;Xi52anins100;Xis2 anins 101;Xi32 anins 102;XisZanins 103;Xis221nins lO4;XisZanins 105; XisZanins106;Xi32anins107;Xi52anins108;XisZa.nins109;Xi32anins 110;XisZanins111;Xis2anins112;Xis2aninsll3;Xis2aninsll4;Xis2 anins115;Xis2anins116;XisZaninsll7;Xis2anins 118;Xi32anins119; Xis2anins120;Xi32anins121;Xi32anin5122;Xi52 anins 123;XisZanins 124;XisZanins125;XiSZanins126;XisZanin5127;Xis2 anins128;Xi32 anins129;Xis2anins130;Xi52anins131;Xi32anins132;Xi32aninsl33; XisZanins134;Xis2anins135;XisZaninsl36;Xi52 aninsl37;Xi32anins 138;Xi52anins 139;Xis23nins140;Xi32anins 141;XisZanins142;Xi52 anins143;XisZanins144;Xis2andYisl45;XisZanins 146;Xis2aninsI47; XiSZanins 148;Xi52anins149;Xis2anins 150;XisZa.ndYis151;Xis2anins 152;XisZanins153;XisZanins154;Xis2anins155;Xi32 anins 156;Xi52 anins 157;Xis2anins 158;XiSZanins 159;Xis2anins 160;XisZanins 161; XisZanins162;Xi82anin3163;XiSZanins l64;XisZanins165;XisZanins 166;Xi32anins167;XisZanins168;Xi32anins169;Xi32aninsl7O;Xis2 anins171;XisZanins172;XisZanins173;Xi52anins 174;Xi52aninsl75; XisZanins 176;XisZanins177;Xi82anins17S;XisZanins179;XisZanins 180;Xis2anins181;XisZanins182;Xi32anins183;XisZanin5184;Xis2 anin3185;Xi52anin3186; XisSanins1;Xis3aninsZ;Xis3anins3;Xis3anins4;Xis3anins5;Xis3 anins6;Xis3anins7;Xis3anin38;Xis3anins9;Xis3anins10;Xis3and Yis11;Xis3anins12;Xis3anins13;Xis3aninsl4;Xis3aninslS;XisSand Yis16;Xis3anins l7;XisBaninslS;Xis3anins l9;XisSa.ninsZO;Xis3and Yi321;Xis3anins22;Xis3anin323;Xis3aninsZ4;Xis3 anin325;Xi53and Yi526;Xi53anins27;Xis3anin328;Xis3anins29;Xis3anins 3and Xis3anins 32;Xis3anins33;Xis3anins34;Xis3 anins35;Xis3 and Yis36;Xis3anins37;Xi33anins38;Xis3anin339;Xis3anins40;Xis3&nd Yis41;Xis3anins42;Xi53anins43;Xis3anins44;Xis3anins45;Xis3and Yis46;Xis3anins47;XisSanins48;Xis3anins49;Xis3anins 50;Xis3and YisSl;Xis3anin352;Xis3anins53;Xis3aninsS4;Xis3aninsSS;Xis3and Xis3anins 57;Xis3anins 58;Xis3anins59;Xis3anins60;Xis3 and Yis61;Xis3anins 62;Xis3anins63;Xis3anins64;Xis3anins65;Xis3and Yis66;Xis3anins67;Xis3anins68;Xis3anins69;Xi53anins 70;Xis3 and Yis71;Xis3anins72;Xis3anins73;Xis3anins74;Xis3anins 75;Xis3and XisSanins 3anins78;Xis3anins79;Xis3anins 80;Xis3and Yis81;Xis3anin882;Xis3andY1883;Xis3anins84;Xis3anins85;Xis3and Yis 86;Xi53anins 87;XisSanins88;Xis3anins 89;Xis3anins 90;Xis3and Yis91;Xis3anins92;Xis3anin393;Xis3anins94;Xis3anins95;Xis3and Yis96;Xis3anin597;Xis3anins98;Xis3anins99;Xis3anin5100;Xi33 anins 101;Xis3anins 102.;Xis3anins 103;Xis3anins s3anins 105; Xis3anins106;Xis3aninle7;Xis3anins108;Xis3 anin5109;Xis3anins s3aninslll;Xis3anins112;Xis3aninsll3;Xis3 anins 114;Xi53 anins115;Xis3aninsll6;Xis3anins117;Xis3anins118;Xis3aninsll9; Xis3anins120;Xis3anins121;Xis3anins122;Xis3 aninle3;Xis3anins 124;Xis3anin8125;Xi53aninle6;Xis3anins127;Xis3anins128;Xis3 anins129;Xis3anin5130;Xis3anins131;Xis3anins132;Xi33aninsl33; Xis3anins134;Xis3anins135;Xis3anins136;Xis3 anins s3anins 138;Xi53aninsl39;Xis3anins l40;Xis3anins141;Xis3anins142;XisS anins 143;Xis3anins 144;Xi33anins 145;Xis3anins 146;Xis3anins 147; Xis3 aninsl48;Xis3 aninsl49;Xis3 anins 150;Xis3 anins 151;Xis3andY is 152;Xis3aninsIS3;Xis3anins154;Xis3aninS155;Xi53anins156;Xis3 aninslS7;Xis3anins 158;Xi53anins159;Xis3anins 160;Xis3anins 161; XisB anins162;Xis3anins163;Xis3anins164;Xis3 anins165;Xi53anins 166;Xi53anins167;Xi53anins168;Xis3anins169;Xis3aninsl70;Xis3 anins 171;Xis3 anins l72;Xis3anins 173;Xis3 anins 174;Xis3 anins 175; Xis3anins s3anins 177;Xis3anins 178;Xis3 anins l79;Xis3anins 180;Xis3anins181;Xis3anin5182;Xis3anins183;Xis3aninslS4;Xis3 anins s3anins 186; Xis4anins1;Xis4aninsZ;Xis4aninsS;Xis4andYis4;Xis4anins5;Xis4 anins6;Xis4anins7;Xis4anins8;Xis4anins9;Xis4anins10;Xis4and Yis11;Xis4anins12;Xis4anins l3;Xis4anins14;Xis4aninslS;Xis4and Yis16;Xis4anins17;Xis4anins18;Xis4anins19;Xis4aninsZO;Xis4and Yis21;Xis4aninsZZ;Xis4anins23;Xis4anins24;Xis4anin325;Xis4and Yis26;Xis4anins27;Xis4aninsZ8;Xis4anins29;Xis4anins30;Xis4and Yis31;Xis4anins 4anins33;Xis4anins34;Xis4anins35;Xis4and Yis36;Xis4anins37;Xis4anins38;Xis4anins39;Xis4anins40;Xis4and Yis41;Xis4anins42;Xis4anins43;Xis4anins44;Xis4anins45;Xis4and Yis46;Xis4anins47;Xis4anins48;Xis4anins49;Xis4aninsSO;Xis4and Yis51;Xis4anins 52;Xis4aninsS3;Xis4aninsS4;Xis4anins 55;Xis4and YisS6;Xis4anins57;Xis4anin558;Xis4aninsS9;Xis4anins60;Xis4and Yi361;Xis4anins 62;Xis4anin363;Xis4anins64;Xis4anins65;Xis4and Yis66;Xis4anins67;Xis4anins68;Xis4anins69;Xis4anins 70;Xis4and W0 2016/110584 Yis71;Xis4anins 72;Xis4anins73;Xis4anins74;Xis4anins75;Xis4and Xis4anins 77;Xis4andYis78;Xis4anins79;Xis4anins80;Xis4and Xis4anins 82;Xis4anins83;Xis4anin584;Xis4anins 85;Xis4and Yis86;Xis4anins87;Xis4andYis88;Xis4anin389;Xis4anins90;Xis4and Xis4andYis92;Xis4anins93;Xis4anins94;Xis4anins 95;Xis4and Yis96;Xis4anins 97;Xis4anins98;Xis4anins99;Xis4aninleO;Xis4 anins 101;Xis4anins 102;Xis4anins 1102%;Xis4a1r1ins 104;Xis4anins 105; Xis4anins106;Xis4anins107;Xis4anins108;Xis4anins109;Xis4anins 110;Xis4anins111;Xis4anins112;Xis4anins113;Xis4aninsll4;Xis4 15;Xis4anins116;Xis4anins 117;Xis4anins118;Xis4anins119; Xis4anins120;Xis4anins121;Xis4anins122;Xis4anins123;Xis4anins 124;Xis4anins 125;Xis4anins 126;Xis4anins 127;Xis4anins 128;Xis4 anins s4anins 130;Xis4anins 131;Xis4anins 132;Xis4anins 133; Xis4aninsl34;Xis4anins135;Xis4anins136;Xis4anin5137;Xis4anins 138;Xis4anins139;Xis4aninsl40;Xis4anins141;Xis4aninsl42;Xis4 anins 143;Xis4anins l44;Xis4anins 145;Xis4anins146;Xis4anins 147; Xis4anins148;Xis4anins149;Xis4anins150;Xis4anins151;Xis4anins 152;Xis4anins153;Xis4anins154;Xis4anins155;Xis4anins156;Xis4 anins 157;Xis4anins 158;Xis4anins 159;Xis4anins l60;Xis4anins 161; Xis4anins162;Xis4anins163;Xis4anins164;Xis4anins165;Xis4anins 166;Xis4anins 167;Xis4anins 168;Xis4anins 1693Xis4anins 170;Xis4 aninsl7l;Xis4anins172;Xis4anins173;Xis4anins174;Xis4anins 175; Xis4anins 176;Xis4anins177;Xis4anins178;Xis4anins179;Xis4anins 180;Xis4anins181;Xis4anins182;Xis4anins183;Xis4anin5184;Xis4 anins 185;Xis4andY is 186; XisSanins1;XisSaninsZ;XisSaninsS;XisSanins4;XisSaninsS;XisS aninsé;XisSanins7;Xis5anins8;Xis§aninsQ;XisSanins lO;XisSand Yisll;XisSanins12;XisSanins13;XisSanins14;XisSaninslS;XisSand Xis5anins17;XisSaninsl8;XisSanins19;XisSaninsZO;XisSand 3O Yile;XisSanin322;Xis5anins23;XiSSanin524;XisSaninsZS;XisSand Yi526;Xis5aninsZ7;XisSaninsZS;XisSanin329;XisSanins30;XisSand Yi331;XiSSanins32;XisSanins33;XisSanins34;XisSanins 35;XisSand Yis36;XisSanins37;XisSanins38;XisSanins39;XisSanins40;XisSand Yis41;Xis5anins42;XisSanins43;XisSanins44;XisSanins45;XiSSand Yis46;XisSanins47;XisSanins48;Xis5anins49;XisSanins 50;XisSand Yis51;XiSSanins 52;XisSaninsS3;XisSanins 54;Xis5 S;Xis5and YisS6;Xis5anins 57;XisSaninsSS;Xis5anins 59;Xi35 anins60;XisSand Yis6l;XisSanin362;Xi35anins63;XisSanins64;XisSanins65;XisSand Yis66;XisSanins67;XisSanins68;Xis5anins69;Xis5anins70;XisSand Yis7l;XisSanins72;XisSanins73;Xis5anins74;XisSanins75;XisSand Yis76;Xis5anins77;XisSanins78;Xis5anins79;XisS anins80;XisSand Yis81;XisSanins 82;XisSanins 83;Xis$anins84;XisSaninsSS;XisSand Yis86;Xis5anins 87;Xis$anins88;XisSaninsS9;XisSanins90;XisSand Yi391;Xis5anins92;Xi35anins93;XisSanins94;XisS anins95;XiSSand Yis96;Xis5anins97;Xis5anins98;XisSanins99;XisSanins100;Xi35 anins 101;Xis5anins102;XisSanins 103;XisSanins 104;XisSanins 105; Xis5anins106;Xis5aninslO7;XisSaninleS;XisSanins109;Xis5anins 110;Xis5anins111;XisSanins112;Xi35anin8113;Xi35anins114;Xis5 aninsllS;XisSanins lI6;Xis5aninsll7;Xis5anins118;Xis5aninsll9; XisSanins120;Xis$anins121;XiSSanin5122;Xi55anins123;XisSanins 124;Xis5anins125;XisSanins126;Xis5anins127;XisSanins128;Xi55 anins 129;Xi85anins 130;XisSanins l3l;XisSanins 132;Xi55anins 133; Xis5anins134;XisSanins13S;XisSanins136;XisSaninsl37;XisSanins 138;Xis5anins139;XisSanins140;Xis5anins141;XisSanins142;Xis5 anins s5anins l44;Xis5anins 145;Xi35aninsl46;XisSanins 147; XisSanins148;Xis5anins149;Xis5anins150;XiSSanins151;XisSanins 35anins153;XisSanins154;XisSanins155;XisSaninslS6;XisS anins 157;Xi55aninslS8;XisSanins 159;Xis5anins 160;XisSanins 161; XisSaninsl62;Xis5anins163;Xis53nins164;XisSanins165;XisSanins 166;XisSaninsl67;XisSanins168;Xi35aninsl69;XisSanins170;Xis5 anins 171;XisSanins172;Xis5anins l73;XisSanins l74;XisSanins 175; insl76;XisSanins177;XisSanins178;Xis$anins179;XisSanins s5anins181;XisSanins182;XisSanins183;XisSanins184;XisS anins185;Xi35anins 186; Xis6anins1;Xis6aninsZ;Xis6anins3;Xis6anins4;Xis6anins5;Xis6 anins6;Xis6anins7;Xis6aninsS;Xis6anins9;Xis6anins10;Xis6and Yisll;XisGanins12;Xis6aninsl3;Xis6aninsl4;Xi36anins15;Xi56and Yi516;Xi56anins17;Xis6anins18;Xis6aninsl9;Xis6anins20;Xi36and Yis21;Xis6anin522;Xis6anins23;Xis6anin824;Xis6anins25;Xis6and Xis6anin327;Xis6anins28;Xis6anin829;Xi36anins 30;Xis6and Yis31;Xis6anins32;Xi36anins33;Xis6anin534;Xiséanins35;Xi86and Yis36;Xis6anins37;Xis6anins38;Xiséanins39;Xis6anins40;Xis6and Yis41;Xi56anins42;Xi36anins43;Xis6anins44;Xis6anins45;Xis6and Yis46;Xis6anins47;Xi56anins48;Xis6anins49;Xis6anins 50;Xiséand Xis6anins 52;Xis6anins53;Xis6anins54;Xis6anins55;Xis6and YisS6;Xis6anins 57;Xis6aninsSS;XisGaninsS9;Xis6anins60;Xi56and Yis61;Xi36anins62;Xiséanin563;Xiséanins64;Xi36an Yi565;Xiséand Yis66;Xis6andYis67;Xis6andYis68;Xis6aninsfi9;Xis6anins70;Xis6and Yis71;Xis6anins 72;Xi56anins73;Xis6anins74;Xis6anins75;Xis6and Yis76;Xis6anins77;Xis6andYis78;Xis6anins'79;Xis6anins 80;Xis6and Yis81;Xis6anins82;Xis6aninsS3;Xis6anins84;Xis6anins85;Xis6and YisS6;Xis6anins 87;Xi56anins88;Xis6anins89;XisGanins90;Xis6and Yis91;Xis6anins92;Xis6anins93;Xis6anins94;Xis6anins95;Xis6and Yis96;Xis6andYis97;Xis6anins98;Xis6anins99;Xis6anins100;Xis6 anins 101;Xis6anins 102;Xis6anins s6anins 104;Xis6anins 105; 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Xis 10 anins s 10 anins 105;Xis 10 anins 106;Xis 10 anins s 10anins 108;Xis 10 anins 109;Xis 10anin3110;Xis 10 anins s 10 andY is 112;Xis 10 anins 113;Xis 10 anins 114;Xis 10 anins 115;Xis 10 anin5116;X W0 2016/1 10584 is 10aninsll7;Xis 10anins 118;Xis s 119;Xis 10 anins 120;Xis 10and Yis 121;Xis 10anins 122;Xis 10 anins 123;Xis 1021de is 124;Xileanins 125; Xis 10anins 126;Xis 10 anins 127;Xis 10 anins 128;Xis lOanins 129;Xis 10 anins 130;Xis 10 anins 131;Xis 10anins 132;Xis 10 anins 133;Xis 10anins 134;Xis 10 anins 135;Xis 10 anins 136;Xis 10 anins 137;XisIO anins 138;Xis anins s s 140;Xis 10 anins 141;Xis 10 anins 142;Xis 10 andY is 143;Xis 10 anins 144;Xis 10 anins 145;Xis 10 anins 146;Xis 10 anins 147;X is 10 anins 148;Xis 10 anins 149;Xis 10 anins 150;Xis 10 anins 151;Xis IOand Yis 152;Xis 10 anins 153;Xis 10 anins 154;Xis 10 aninslSS;Xis 10 anins 156; Xis 10 anins 157; Xis 10 anins 158;Xis 10 anins 159;Xis 10 anins s 10 anins s 10 anins 162;Xis 10 anins 163;Xis 10anins 164;Xis 10 anins s 10 anins 166;Xis 10 andY is 167;Xis 10 anins 168;Xis 10 anins 169;Xis IOaninsl70;X1310anins171;Xis10anins172;Xis10anins173;Xis10andY is 174;Xis 10 anins 175;Xis 10 anins s 10 anins 177;Xis 10 anins 178;X is IOanins s 10 anins 180;Xis 10 anins 181;Xis 10 anins 182;Xis 10 and Yis 183;Xis 10 anins s 10 anins 185;Xis 10 anins 186; Xisll aninsl;Xisllanin32;Xisll anins3;Xisll ;X1'sll aninsS; Xisllanins6;Xis 11 anins7;Xis 11 anins8;Xis 11 anin59;Xisll anins ;Xis 11 anins 11;Xi511 anins 12;Xis 11 anins 13;Xis 11 anins 14;Xisll anins 15;Xisll anins 16;Xisll anins 17;Xisll anins l8;Xisllanins 19;X isll aninsZO;Xis 11 anins21;Xisll 2;XisllaninsZ3;Xisll anins 24;Xisllanin525;Xisllanins26;Xis11anin327;XisllaninsZS;Xisll aninsZ9;Xisllanins30;Xis11anins31;Xis11anins32;Xisllanins33;X isllanins34gXis11anins35;Xisll anins36;Xis11anins37;Xisllanins 38;Xisll anins 39;Xisll anins40;Xis 11 1;Xis 11 anins42;Xis 11 anins43gXisllanins44;Xisllanins45;Xisl1anins46;Xisllanins47;X is 11 anins48;Xisll anins49;Xisll aninsSO;Xis 11 aninsSI;Xisll anins 52;Xisll anins 53;Xis 11 aninsS4;Xis 11 anins 55;Xisll anins56;Xis 11 anins57;Xisll anin358;XisllandY1359;Xis11 anins60;Xisll anins61;X 3O isllanins62;XisllaninsG3gXis11anins64;Xis11anins65;Xisllanins 66;Xis 11 anin567;Xis 11 anins68;Xis ll anins69;Xis 11 anins 70;Xis 11 anins71;Xis 11 anins72;Xis11 anins73;Xisll anins74;Xisllanins75;X isll anins76;Xisll anins77;Xis 11 anins78;Xisll anins79;Xis11 anins 80;Xis 11 anins 81;Xis 11 anins 82;Xis llanins83;Xisll anins84;Xis 11 aninsSS;Xis 11 anins86;Xisll anins87;Xis11 anins88;Xisllanins89;X is 11 anin590;Xis 11 anins91;Xis 11 anins92;Xis 11 anins 93;Xis 11 anins 94;Xis 11 anins95;Xis 11 anins96;Xis 11 7; Xisll anins98;Xisll anins 99;Xis 11 anins 100;Xis 11 anins 101;Xis 11 deis 102; Xis 11 anins 103;Xisllaninle4;Xi311anin3105;Xis11aninleé;Xis 11aninle7;Xis 11 08;Xisll aninle9;Xisll anins110;Xisll andY is 111;Xisll andY is 112;Xis 11 anins 113;Xisll l4;Xis 11 anins 115;Xis 11 anins 116;X i311 anins 117;Xis 11 anins 118;Xis 11 anins 119;Xis 11 anins 120;Xis 11 and Yisl21;Xis 11 anins122;Xisll anins123;Xis 11 andY is 124;Xis 11 anins 125; Xis 11 anins 126;Xis 11 anin5127;Xis 11 anins 128;Xis 11 anin5129;Xis11 anin5130;Xis 11 anins 131;Xi311 anins 132;Xis 11 anins 133;Xisll anins 134;Xis 11 anins 135;Xisll anin5136;Xis 11 anins 137;Xis 11 anins 138;Xis 11 anins 139;Xis 11 anins 140;Xis 11 anins 141;Xis11 anins 142;Xis 11 andY is 143;Xis11 anins 144;Xi311 anins 14S;Xisll anins 146;Xis 11 anins 147;X 1311 deis 148;Xis 11 anins 149;Xis 11 anins s 11 anins s 11 and Yis 152;Xis 11 anins 153;Xis 11 anins 154;Xis 11 anins 155;Xis 11 deis 156; Xis 11 anins s 11 anins 158;Xis 11 anins 159;Xis 11 anins 160;Xis 11 anins 161;Xis 11 anins 162;Xis 11 anins 163;Xis 11anins 164;Xisll anins 165;Xis11anins166;Xis11aninsl67;Xis11anin5168;Xisll aninsl69;Xis 11 anins 170;Xis 11 anins 171;Xisll anins 172;Xisll aninsl73;Xis 11 andY is 174;Xis 11 anins 175;Xis 11 anins 176;Xis 11 anins 177;Xisll anins 178;X is 11 anins 179;Xis 11 anin5180;Xisll anins 181;Xisll aninsl82;Xis 11 and Yis 183;Xisll anins 184;Xis 11 anins s 11 deis 186; Xis 12 anins 1;Xis 12anins2;Xis 12anins3;Xis 123nins4;Xis 12 aninsS; Xis 12 anins6;Xis 12 anins7;Xis 12 anins 8;Xis 12 anins 9;Xis 12 anins 10;Xis 12 anins 11;Xis IZanins 12;Xis 12 anins 13;Xi312and‘{is l4;Xis 12 anins 15;Xis 12 anins 16;Xis 12 anins l7;Xis 12anins 18;Xi312anins 19;X is 12anin520;Xis 12 aninle;Xis 12andY1322;Xis 12 anin523;Xis 12 anins 24;Xis 12anin525;Xis 12 anins26;Xis 12 aninsZ7;Xis 12 anins 28;Xi312 aninsZ9;Xis 12 anins 30;Xis 12 anins31;Xis 12 anins 32;Xis 12 anins 33;X is 534;Xis 12 anins35;Xis 12 anins36;Xis 12anins37;Xis 12 anins 38;Xis 12 anins 39;Xis 12 anins40;Xis 12anins41;XislZanins42;Xis 12 anins43;Xis 12 anins44;Xis s45;Xis 12 anins46;Xis 12 anins47;X is 12anins48;Xis 12 anins49;Xis s 50;Xis 12aninsSl;Xis 12 anins 52;Xis 12 anins 53;Xis 12 anins 54;Xis 12 aninsSS;Xis 12anins 56;Xis 12 anins 57;Xis 12 anin558;Xis 12anins 59;Xis 12 0;Xis 12anins61;X W0 2016/1 10584 is 12 anins 62;Xis 12 anins 63;Xis 12 anins 64;Xis 12 anins 65;Xis 12 anins 66;Xis 12 anins67;Xis 12 anins 68; Xis 123nins69; Xis 12 anins70; Xis 12 anins71;Xis 12 anins 72;Xis 12 anins73;Xis 12 anins 74;Xi512anins75;X is lZanins 76;Xis 12 anins 77;Xis 12 anins78;Xis 12 9;Xi312anins 80;Xis 12 anins 81; Xis 12 anins 82; Xis 12 anins 83; Xis 12 anins 84;Xis 12 anins 85;Xis 12 anins86;Xisl2anins 87;Xis12anins88;XislZanins89;X is 12 anins90;Xis 12 anins9l;Xis 12 anins 92;Xis 12 anins93;Xis 12 anins 94;Xis 12 andY is 95;Xis 12 anins 96;Xis 12 andY is 97; Xis 12 anins 98; Xis 12 anins 99; Xis 12 anins 100;Xis 12 anins 101; Xis 12 anins 102;Xis 12 anins 103; Xis 12 anins 104;Xis 12 anins 105; Xis 12 anins 106;Xis 12 anins 107; Xis 12 anins 108;Xis 12 anins 109;Xis 12 anins 110;Xis 12 anins s 12 andY is 112;Xis 12 anins 113; Xis 12 deis 114; Xis 12 anins 115;Xis 12 anins 116;X is 12 anins 117;Xis 12 anins 118;Xis 12 anins 119;Xis 12 anins 120;Xis 12 and Yis 121; Xis 12 anins 122; X is 12 anins 123; Xis 12 andY is 124; Xis 12 anins 125; X is 12 and Yis 126; Xis 12 andY is 127; Xis 12 andY is 128; X is 12 deis 129; Xis 12 ‘andY is 130; Xis 12 andY is 131; Xis 12 andY is 132; Xis 12 deis 133; Xis 12 andY is 134; Xis 12 anins 135; Xis 12 anins 136; Xis 12 deis 137; Xis 12 anins 138;Xis 12 andY is 139;Xis 12 anins 140;Xis 12 deis 141; Xis 12 anins 142; Xis 12 andY is 143;Xis 12 anins 144;Xis 12 deis 145; Xis 12 anins 146;Xis 12 anins 147; X is 12 anins 148;Xis 12 anins 149;Xis 12 anin8150;Xis 12 anins 151;Xis 12 and Yis 152;Xis 12 deis 153; Xis 12 deis 154; Xis 12 andY is 155;Xis 12 anins 156; Xis 12 anins 157;Xis 12 anins 158;Xis 12 anins 159;Xis 12 anins 160;Xis 12 anins 161; Xis 12 anins 162;Xis 12 anins I63; Xis 12 anins 164;Xis 12 anins s 12 andY is 166;Xis 12 andY is 167; Xis 12 anin5168;Xis 12 anins 169;Xis 12 anins s 12 anins 171;Xis 12 anins 172;Xis 12 anins 173;Xis IZandY is 174; Xis 12 anins 175; Xis 12 deis 176; Xis 12 anins 177;Xis 12 anins 178; X is 12 anins 179;Xis 12 anins s 12 anin3181;Xis 12andY15182;Xis 12 and Yis 183; Xis 12 anins 184; Xis 12 deis 185; orXis 12 and Yis 186.

In one ment, X is 2 and Y is 5. In another embodiment, X is 10 and Y is 164.

In one embodiment, the first binding domain binds to 4-1BB (CD137) and the second binding domain binds to 0X40 (CD134), wherein, preferably, the binding agent comprises one or more CDRS or one or more le domains of an antibody AB137_X, wherein X is selected from the group consisting of 1 to 12, as shown in Table 9, and one or more CDRs or one or more variable domains of an antibody AB 134_Y, wherein Y is selected from the group consisting of 1 to 92, as shown in Table 8.

In various ments, X is l and Y is ed from the group consisting of 1 to 92; X is 2 and Y is selected from the group consisting of 1 to 92; X is 3 and Y is selected from the group consisting of 1 to 92; X is 4 and Y is selected from the group consisting of 1 to 92; X is 5 and Y is selected from the group consisting of 1 to 92; X is 6 and Y is selected from the group consisting of 1 to 92; X is 7 and Y is selected from the group consisting of 1 to 92; X is 8 and Y is ed from the group consisting of 1 to 92; X is 9 and Y is selected from the group ting of 1 to 92; X is 10 and Y is selected from the group consisting of 1 to 92; X is 11 and Y is selected from the group consisting of 1 to 92; or X is 12 and Y is ed from the group consisting of 1 to 92.

In various embodiments, Xis1aninsl;XisianinsZ;Xis1anins3;Xislanins4;XislaninsS;Xisl anins6;Xis1anins7;Xislanins8;Xislanins9;Xislanins 10;Xisland Yisll;Xislanins 12;Xislaninsl3;Xislanins 14; Xislanins 15;Xisland Yisl6;Xislanins l7;Xisl anins18;Xislanins19;Xislanins20;Xisland Yis21;Xislanins22;Xisl anin523;Xislanins24; Xislanins 25;Xis 1 and Yi326;Xislanin527;Xis1anin328;Xis 1 dei529; Xisl anins30;Xisland Yis31;Xislanins 32;Xislanins33;Xislanins34;Xislanins35;Xisland Yis36;Xislanin537;Xis l anins38;Xislanins39;Xisl anins40;Xis1and Xislanins42;Xislanins43;Xislanins44;Xislanins45;Xisland Yis46;Xislanins47;Xislanins48;Xisl anins49;Xisl anins50;Xisland YisSl;Xislanins 52;Xis i 3;Xis1aninsS4;Xislanins 55;Xisiand Yi556;Xislanins 57;Xis1anin358;XislaninsS9;Xislanins60;Xisland Yis61;Xislanins 62;Xis1anins63;Xis1anins64;Xislanin565;Xis l and Yi366;Xislanins67;Xislanins68;Xislanins69;Xislanins70;Xisland Yis71;Xis1anins72;Xislanins73;Xis1anins74;Xislanins75;Xisland Yis76;Xis1anins77;Xislanins78;Xislanins79;Xislanins80;Xisland Yi581;Xislanins 82;XislaninsS3;Xis1anins84;Xislanins 85;Xisl and Yis86;Xis1 anin387;Xis1anins88;Xis1anins89;Xislanins90;Xisland Yis91;Xislanin392; XisZanins1;XisZanins2;Xis2anins3;Xi52anins4;Xi52anins5;Xi32 anins6;XisZanins7;XisZandYis8;Xis2anins9;Xi52anins10;Xi52and W0 2016/1 10584 Yis Zanins 12;Xi32aninsl3;XisZanins14;Xis2anin515;Xi52and Yis 16;XisZanins l7;XisZanins18;XisZanins 19;XisZaninsZO;XisZand Yis21;Xis2anin322;XiSZanins23;XisZaninsZ4gXi52anin325;Xi32and YisZ6;XisZanin327;Xis2aninsZS;XisZaninsZ9;XisZanins30;XisZand Yis3l;Xi52anins32;Xi52anins33;XisZanins 34;XisZanins35;XisZand Yis36;XisZaninsS7;XisZanins38;Xi82anins39;Xis2anins40;Xi32and Yis41;XisZanins42;XisZanins43;XisZanins44;Xi32and‘x’is45;Xis2and Yis46;Xi52andYi547;XisZandYis48;XisZanins49;XisZanins50;Xis2and Yis51;XisZaninsSZ;XisZandYisS3;Xis2aninsS4;Xi52aninsSS;Xis23nd YisS6;XisZanin557;Xis2anins58;Xi32anins 59;XisZanins60;Xi32and Yis61;Xis2anins62;Xis2anins63;Xi52anin364;Xi32anins65;XisZand Yis66;Xis2anins67;XisZandYis68;XisZanins69;Xi52anins70;Xi32and XisZanins72;Xiszanins73;Xis2anins'/4;Xis2anins75;XisZand Yis76;XisZanins77;Xiszanins78;Xis2anins79;Xis2anins80;Xi52 and Yis 81;XisZanins 82;Xi32anins83;Xis23nins84;XisZanins85;XisZand Yis86;Xi82anins87;Xis2anins88;Xis2aninsS9;Xis2anins90;XisZand Yis91;XisZanins92; Xis3anins1;Xis3aninsZ;Xis3anins3;Xis3anins4;Xis3aninsS;Xis3 anins6;Xis3anins7;Xis3 ;Xis3anins9;Xis3anins 10;Xis3and Yis11;Xis3anins 12;Xis3anins13;Xis3aninsl4;Xis3 anins15;Xis3and Yis16;Xis3anins 3anins18;XisBanins19;Xis3anins20;Xis3and Yile;Xi53anin522;Xis3aninsZ3;Xis3aninsZ4;Xis3anin525;Xis3and Yi526;Xis3anin527;Xis3anin328;Xi33anin329;Xis3 anins 30;Xis3and Yis3l;Xis3anins32;Xis3anins33;Xis3anins34;Xis3anins35;Xis3and Yi536;Xis3anins37;Xis3anins38;Xis3anins39;Xis3anins40;Xis3and Yis4l;Xis3anins42;Xis3anins43;XisSanins44;Xis3anins45;Xis3and Yis46;Xis3anins47;Xi53anins48;Xis3anins49;Xis3anin550;Xis3and YisSl;Xis3a.nins 52;XisSaninsS3;Xis3anins54;Xis3 anins 55;Xis3and Yis56;Xis3aninSS7;Xis3anin358;Xis3aninsS9;Xis3anins60;Xis3 and Yis6l;Xis3anin562;Xis3anins63;Xis3anins64;Xis3anins65gXis3 and Yi366;Xis3a.nins 67;Xis3anin568;Xis3anins69;Xis3anins70;Xis3and Xis3anins 72;Xis3anins73;Xis3anins74;Xis3 anins75;Xis3and Yis76;Xis3anins77;Xis3anins78;Xis3anins79;Xis3 anins 80;Xis3and YisSl;Xis3anins 82;Xis3anins83;Xis3aninsS4;Xis3 aninsSS;Xis3 and Yis86;Xi53deis 87;Xis3anins 88;Xis3anins89;Xis3anins90;Xis3and Xis3anins92; Xis4aninzs1;Xis4andYisZ;Xis4aninsS;Xis4andY is4;Xis4anins 5;Xis4 anins6;Xis4anins7;Xis42deis8;Xis4anins9;Xis4anins10;Xis4and Yisll;Xis4anins 12;Xis4aninsl3;Xis4anins 14; Xis4anins 15;Xis4and Yis16;Xis4anins17;Xis4anins18;Xis4anins19;Xis4anin520;Xis4and Yis21;Xis4anins22;Xis4aninsZ3;Xis4anins24; Xis4anin525;Xis4and Yis26;Xis4aninsZ7;Xis4anins28;Xis4anins29; Xis4anins30;Xis4and Xis4anins32;Xis4anins33;Xis4anins34;Xis4anins35;Xis4and Yis36;Xis4anins37;Xis4anins38;Xis4anins39; Xis4anins40;Xis4and Yis41;Xis4anins42;Xis4anins43;Xis4a.nins44; X154 anins45;Xis4and Yis46;Xis4anins47;Xis4anins48;Xis4anins49; Xis4aninsSO;Xis4and Yis 51;Xis4anins 4aninsS3gXis4anin354; Xis4anin555;Xis4and YisS6;Xis4anins 5.7;Xis4anins 4aninsS9; Xis4anins60;Xis4and Yi561;Xis4anins62;Xis4anins63;Xis4anins64-;Xis4anins65;Xis4and Yis66;Xis4anins67;Xis4anins68;Xis4anins69; Xis4anins 70;Xis4and Yis71;Xis4anins72;Xis4anins73;Xis4anins74;Xis4anins75;Xis4and Yis76;Xis4anins 77;Xis4anins78;Xis4a.nins79;Xis4anins 80;Xis4and YisSl;Xis4anins 82;Xis4anins 83;Xis4aninsS4;Xis4anin585;Xis4and Xis4anins 87;Xis4anins 88;Xis4aninsS9;Xis4anins90;Xis4and Yi591;Xis4anins92; XisSanins1;XisSanin32;Xis5adeis3;XisSanins4;XisSaninsS;Xis5 ;XisSanins7;Xi55aninsS;Xis5anin59;XisSaninle;Xis5and Yis]1;XisSanins12;XisSaninsl3;XisSaninsl4;Xis5anins15;XisSand Yis 16;XisSanins 17;XisSaninslS;XisSanins l9;Xis5aninsZO;XisSand Yile;XisSanins22;XisSanins23;Xis5anin524;XisSaninsZS;Xis5and YisZ6;XisSanins 27;XisSanin328;Xi35anins29;XisSanins30;Xi55and Yis31;XisSanins 5anins33;XisSanins34;XisSanins 35;XisSand Yis36;XisSanins 37;Xis$anins38;XisSanins39;XisSanins40;XisSand Yis41;XisSanins42;XisSanins43;XisSanins44;XisSanins45;XisSand Yis46;XisSanins 47;Xi55anins48;XisSanins49;Xis5anin550;XisSand YisSl;XisSanins 52;Xis$anins 53;XisSaninsS4;XisSanin555gXiSSand YisS6;XisSanins 57;Xis5anin558;Xis5aninsS9;XisSanins60;XisSand Yis61;XisSanin362;Xi35anins 63;XisSanin564;Xis5anins 653Xi85and Yis 66;XisSanins67;XisSanins68;XisSandYis69;XisSanins70;XisSand W0 2016/1 10584 Yis7l;Xis5anins72;XisSanins73;XisSanins74;XisSanins75;XisSand Yis76;XisSanins77;XisSanins78;XisSanins79;XisSanin580;Xi35and Yis81;XisSanins82;Xis§aninsS3;XisSanin884;Xis5aninsSS;XisSand YisS6;XisSanins 87;Xis$anins88;XisSanins 89;XisSanins90;XisSand Yis91;XisSanins92; Xis6aninsl;Xis6aninsZ;Xis6anins3;Xis6anins4;Xis6anins5;Xis6 anins6;Xis6anins7;Xis6anins anin59;Xis6anins10;Xis6and Yisll;Xi56anins12;Xis6anins13;Xiséaninsl4;Xis6anins15;Xi36and Yisl6;Xis6anins17;Xis6anins18;Xi56anins19;Xis6anin320;Xis6and Yis21;Xis6anins22;Xi56anin523;Xis6aninsZ4;Xis6anins25;Xis6and Xis6anins27;XiSGanin528;Xis6anin529;Xis6anin330;Xis6and Yis31;Xis6anins32;Xis6anins33;Xis6anins34;Xi36anins35;Xis6and Yis36;Xis6anins37;Xis6anin338;Xis6anin539;Xi36anins40;Xis6and Yis4l;Xi36anins42;Xis6anins43;Xis6anins44;Xis6anins45;Xis6and ,_.. (J! Yis46;Xis6anins47;Xiséanins48;XisGanins49;Xis6anins50;Xiséand YisSl;Xi36anins 52;Xis6anins53;Xis6aninsS4;Xis6anins 55;Xis6and Yis56;Xis6anins57;Xis6anin558;Xis6aninSS9;Xis6aninsfiO;Xis6and Yis61;Xis6anins62;Xis6anins63;Xis6anins64;Xis6anins65;Xis6 and Yi566;Xis6anins67;Xis6anin368;Xis6anins69;Xis6anins 70;Xis6and Yis71;Xis6anins72;Xis6andYis73;Xis6anins74;Xis6anins 75;Xi56and Yis76;Xis6anins77;Xis6anins78;Xis6anins79;XisGanins 80;Xis6and Yis 81;Xis6anins 6andYi383;Xis6anins84;Xis6anins85;Xis6and Yis 86;Xiséanins 87;Xis6anin388;Xis6anin589;Xi36anins90;Xis6and Yis91;Xi36anins92; Xis7anins1;Xis7aninsZ;Xis7anin53;Xis7anins4;Xis7aninsS;Xis7 anin56;Xis7anins7;Xis7aninsS;Xis7anins9;Xis7anins 10;Xis7and Yis11;Xis7anins12;Xis7anins l3;Xis7anins14;Xis7anins15;Xis7and Yis 16;Xis7anins 17;Xis7anins18;Xis7anins l9;Xis7anins20;Xis7and Yis21;Xis7anin322;Xis7anin323;Xis7anin324;Xis7anin525;Xis7and YisZ6;Xis7aninsZ7;Xis7anins28;Xis7anin829;Xis7anins30;Xis7and Xis7anins 32;Xis7anins33;Xis7anins34;Xis7anins35;Xis7and Yis36;Xis7anins 37;Xis7anin538;Xis7anins39;Xis7anins40;Xis7and Yis41;Xis7anins42;Xis7anins43;Xis7anins44;Xis7anins4S;Xis7and Yis46;Xis7anins47;Xis7anins48;Xis?anins49;Xis7aninsSO;Xis7and YisSl;Xis7anins 7aninsS3;Xis7anins54;Xis7anins 55;Xis7and Yis 56;Xis7anins 57;Xis7anin358;Xis7anins59;Xis7anin360;Xis7and Yis61;Xis7anins62;Xis7andYis63;Xis7anins64;Xis7anins65;Xis7and Yi566;Xis7zdeis67;Xis7andYis68;Xis7anins69;Xis7anins70;Xis7and Yis71;Xis7andYis72;Xis7anins73;Xis7andYis74;Xis7anins75;Xis7and Yis76;Xis7anins 77;Xis7anins78;Xis7anins79;Xis7anins 80;Xis7and Yis 81;Xis7anins 82;Xis’?aninsS3;Xis7aninsS4;Xis7anins 85;Xis7and Yis86;Xis7anins 7anins 88;Xis7aninsS9;Xis7anin390;Xis7and Yis91;Xis7anins92; Xis8anins1;Xi38anins2;Xi58anins3;Xis8anins4;Xi38anins5;Xis8 ;Xi38anins7;XisSanins8;XisSanins9;Xis8anins 10;Xis8and XisSanins12;Xis8aninsl3;Xis8anins14;Xis8anins15;Xi38and Yis16;Xis83nins17;Xis$aninsi8;XisSaninle;XisSanin520;Xis8and Yis21;Xis8aninsZZ;XisSaninsZ3;XisSaninsZ4;XisSanins25;Xis8and YisZ6;XisSanins27;XisSanin328;Xis8anin829;Xis8anins30;Xi58and Yis31;Xis8anins32;Xis8anins33;XisSanins34;XisSanins35;Xi58and Yis36;XisSanins37;Xis8anins38;Xis8anins39;Xis8anins40;XisSand Yis41;Xis8anins42;Xis8anins43;Xis8anins44;XisSanins45;Xi38and Yis46;Xis8anins47;XisSanins48;Xi38anins49;Xi38aninsSO;Xis8and Yis51;Xis8anin352;Xi38anins53;Xis8aninsS4;Xis8anins 55;XisSa.nd YisS6;Xis8aninsS7;XisSanin358;Xi58anins 59;XisSanins60;XisSand Yis61;Xis8anins62;Xis8anins63;XisSanins64;Xi58anins65;Xis8and Yis66;Xi58anin567;XiSSanins68;XisSanin569;Xi88anins 70;Xi38and Xis8anins 72;XisSanins73;Xis8anins74;Xis8anins75;Xi38and Yis76;XisSanins77;Xis8anins78;Xis8anins79;Xis8anins 80;Xis8and Yis81;XisSanins 82;Xi58anins 83;Xis8aninsS4;XisSanins85;Xis8and Xis8anins 87;Xi58anins 88;XisSanins89;Xis8anins90;Xi58and Yis9l;Xi38anins92; XisQanins1;Xis9aninsZ;Xi59anins3;Xis9anins4;Xis9aninsS;Xis9 anins6;Xis9anins7;Xis9anins8;Xis9anins9;Xis9anins10;Xis9and Yis11;Xis9anins12;Xis9anins13;Xis9anins14;Xis9aninslS;Xi39and Yis16;Xis9anins17;Xis9anin518;Xis9anins l9;Xis9aninsZO;Xis9and Yi521;Xis9anins22;Xis9anins23;Xis9aninsZ4;Xis9andY13253Xis9and Yis26;Xis9anin327;Xis9anin528;Xis9aninsZ9;Xis9anins 30;Xis9and Yis31;Xis9anins32;Xi59anins 33;Xis9anin534;Xis9anins35;Xis9and Yis36;Xis9anins 37;Xis9anins38;Xis9anins39;Xis9anins40;XiS9and W0 2016/1 10584 2016/050308 Yis41;Xis9anins42;Xis9anins43;Xis9anins44;Xis9anins4S;Xis9and Yis46;Xis9anins47;Xis9anins48;Xis9anins49;Xis9anin550;Xis9and Yis51;Xi59anins52;Xis9anins53;Xis9aninsS4;Xis9aninsSS;Xis9and YisS6;Xis9anins 57;Xi59anins 58;Xis9anins 59;Xis9anins60;Xis9and Yis61;Xis9anins 9anins63;Xis9anins64;Xis9anins65;Xis9and Yis66gXis9anins 67;XisQanins68;Xis9anins69;Xis9anins 70;Xis9and Yis71;XisQanins72;Xi39anins73;Xis9anins74;Xis9anins75;Xi59and Yis76;Xis9anins77;Xis9anins78;Xi59anins79;Xis9anins 80;Xis9and YisSl;Xis9anins 82;Xis9anins 83;Xis9aninsS4;Xis9anins 85;Xi39and Yis 86;Xi39anins 87;Xi39anin588;Xis9anins 9anins90;Xis9and Yis91;Xis9anins92; Xileaninsl;Xis10anins2;XisIOanin33;Xis‘10anins4;Xis10aninss; Xis10anins6;Xis10anins7;Xis10anins8;Xis10anins9;Xis10anins ;Xis 10anins 11;Xis 10 anin512;Xis 10anins 13; Xis 10anins 14;Xis 10 anins 15;Xis 10 anins 16;Xis lOanins 17;Xis 10aninsl8;Xis lOanins 19;X is 10anin520;Xis IOanin321;Xis IOanin322;Xis 10 anin523;Xis IOanins 24;Xis 10anins25;Xis10anin326;Xi510anin327; Xis 10aninsZS;Xile aninsZ9;Xis 10anins30;Xis 10anins31;Xileanins32;Xis lOanins33;X is 10anins34;Xis10anins35;Xis 10anins36;Xis 10 anins37;Xis 10 anins 38;Xis 10 anins 39;Xis 10 anins40;Xis 10 anins41; Xis 10 anins42;Xis 10 anins43;Xis 10 anins44;Xis 10anins45;Xis 10 anins46;Xis 10 7;X is 10anins48;Xis 10anins49;Xis 10aninSSO;Xis 10 anin551;Xis IOanins 52;Xis loanins 53;Xis 10 anins 54;Xis 10 aninsSS;Xis 356;Xis 10 aninsS7;Xis 10 anins 58;Xis 10 anins 59;Xis 10 anin360;Xis 10 anins6l;X is 10anins62;Xis s63;Xis IOanins64;Xis 10 anins65;Xis 10 anins 66;Xis 10anins67;Xis10anins68;Xis lOanins69;Xis 10anins70;Xis 10 anins7l;Xis 10anins72;Xis 10anins73;Xis 10anins74;Xis 10 €deiS75;X is 10anins76;Xis IOanins77;Xis 10 anins78;Xis 10 anins79;Xileanins 80;Xis10anin381;Xis102deis82;Xis10aninsS3;Xis10adeisS4;Xile anins 85;Xis lOaninsS6;Xis 10anins87;Xis 10andY1588;Xis 10aninsS9;X is 10anins90;Xis10anins91;Xi310anins92; Xis11anins1;Xisllanin52;Xisllanins3;Xisllanins4;XisllaninsS; Xis 11 anins6;Xisllanins 7;Xis 11 anins 8;Xis 11 ;Xisll anins ;Xisll anins 11;Xisl] anins 12;Xisll anins 13;Xis llanins 14;Xisll anins15;Xisllanins16;Xisllanins17;Xisllanins18;Xisll andY1519gX isllanins2O;Xis11anin521;Xisll anins22;XisllaninsZ3;Xisll anins 24;Xis 11 anins25;Xis 11 anins26;Xis 11 anins27;Xis 11 aninsZ8;Xis 11 anin529;Xisll anins30;Xisll anins31;Xisll anins32;Xisll anins33;X is 11 anins34;Xis 11 anins35;Xisll anins36;Xis 11 anins 11 anins 38;Xis 11 anins39;Xis 11 anins40;Xisll anins41; Xis 11 anins42;Xisll anins43;Xisllanins44;Xisllanins45;Xisllanins46;Xis11anins47;X is 11 and‘x'is48;Xis 11 anins49;Xisl‘i and‘x’isSO;Xis‘:1 anins 51;Xisli anins 52;Xis 11 andY1553;Xis 11 4;Xis 11 S;Xisll anins56gXis 11 aninsS7;Xisll anin558;Xisll anins 59;Xisll anin560;Xisll anins61;X is11 anins62;Xisll anins63;Xisll anins64;Xisll anin565;Xisll anins 66;Xis 11 anins67;Xisll anin568;Xisll aninsG9; Xisll anins70;Xis 11 anins71;Xisllanins72;Xis!1anins73;Xi311anins74;Xisllanins75;X isllanins76;Xisllanins77;Xi311anins78;Xisll anins79;Xisll anins 11 anins81;Xisll andY1582;Xisll aninsS3;Xisll aninsS4;XisIl aninsSS;Xis11anin386;Xisll anins87gXisllandY1388;Xisllanins89;X isllanins90;Xisll anins9l;Xisllanins92; Xis12anins1;Xis123nin32;Xis12anins3;Xis12anins4;Xis12aninsS; Xis 12 aninsé;Xis 12 anins7;Xis 12 anins 8;Xis 12 ;Xis 12 anins ;Xis 12 anins 11;Xis 12 anins 12;Xis 12anins 13;Xis 12anins 14;Xis 12 anins 15;Xis s 16;Xis 12 anins 17;Xis 1in3 18;Xis lZanins 19;X is 12 anin320;Xis 12 anins21;Xis l2 anin322;Xis 12 anins23;Xis12anins 24;Xis 525;Xis 12 anins26;Xis 12anins27;Xis lZanin528;Xi312 aninsZ9;Xis 12anins30;Xis12anins31;Xis12anins32;Xis12anins33;X is 12 aninsS4;Xis 12 anins35;Xis 12 anins36;Xis 12 anins37;Xis 12anins 38;Xis 12 anins 39;Xis 12 anins40;Xis 12 anins41;Xis 12anins42;Xis 12 anins43;Xis12anins44;Xis12anins45;Xis12anins46;Xis12anins47;X is 12 anins48;Xis 12anins49;Xis 12 anins 50;Xis 12 anins 51;Xis 12 anins 52;Xis 12 anins53;Xis 12aninsS4;Xis 12anins 55;Xi312 anins 56;Xis12 aninsS7;Xis 12 anins58;Xis 12anins 59;Xi512anins60;Xis 12 anins61;X is 12 anins62;Xis 12anins63;Xis 12 anins64;Xis 12 5;Xis 12 anins 66;Xis 12 anin567;Xis 12 anins68;Xis 12anins69;Xis 12anins 70;Xis 12 anins7l;Xis 12 anins72;Xis 12 anins73;Xis 12anins74;Xis 12 anins75;X is 12 6;Xis 12 7;Xis 12 anins78;Xis 12 anins79;Xis 12 anins 80;Xis 12 anins 81;Xi312andY1382;Xis 12anin883;Xis12anins 84;Xis 12 W0 2016/1 10584 anins85;Xis 12 anins 86;Xis 12 anins 87;Xis 12 andY is 88;Xis 12 anins 89;X is 12 anin590;Xis 12 anins 91;orXis 12 anins92.

In one embodiment, X is 2 and Y is 3. In another embodiment, X is 12 and Y is 9.

In one embodiment, the first binding domain binds to 0X40 (CD134) and the second binding domain binds to CD27, wherein, preferably, the binding agent comprises one or more CDRs or one or more variable domains of an dy X, wherein X is selected from the group consisting of 1 to 92, as shown in Table 8, and one or more CDRs or one or more variable domains of an antibody AB27_Y, wherein Y is selected from the group consisting of l to 186, as shown in Table 7.

In various embodiments, X is l and Y is ed from the group consisting of 1 to 186; X is 2 and Y is selected from the group ting of 1 to 1.86; X is 3 and Y is selected item the group consisting of 1 to 186; X is 4 and Y is selected from the group consisting of l to 186; X is 5 and Y is selected frOm the group consisting of l to 186; X is 6 and Y is selected from the group consisting of 1 to 186; X is 7 and Y is selected from the group consisting of l to 186; X is 8 and Y is selected from the group consisting of l to 186; X is 9 and Y is ed from the group consisting of l to 186; X is 10 and Y is selected from the group ting of l to 186; X is 11 and Y is selected from the group consisting of 1 to 186; X is 12 and Y is selected from the group consisting of 1 to 186; X is 13 and Y is selected from the group consisting of 1 to 186; X is 14 and Y is selected from the group consisting of l to 186; X is 15 and Y is ed from the group consisting of 1 to 186; X is 16 and Y is selected from the group consisting of l to 186; X is 17 and Y is selected fi'om the group consisting of 1 to 186; X is 18 and Y is selected fi'om the group consisting of 1 to 186; X is 19 and Y is selected from the group consisting of 1 to 186; X is 20 and Y is selected fi’om the group consisting of l to 186; X is 21 and Y is selected from the group consisting of 1 to 186; X is 22 and Y is selected from the group consisting of 1 to 186; X is 23 and Y is selected from the goup consisting of 1 to 186; X is 24 and Y is selected from the group consisting of 1 to 186; X is 25 and Y is selected from the group consisting of l to 186; X is 26 and Y is selected from the group consisting of l to 186; X is 27 and Y is selected from the group consisting of l to 186; X is 28 and Y is selected from the group ting of 1 to 186; X is 29 and Y is ed from the group consisting of l to 186; X is 30 and Y is ed from the group consisting of 1 to 186; X is 31 and Y is selected from the group consisting of 1 to 186; X is 32 and Y is selected from the group consisting of 1 to 186; X is 33 and Y is selected from the group consisting of 1 to 186; X is 34 and Y is selected fiom the group consisting of 1 to 186; X is 35 and Y is ed from the group consisting of 1 to 186; X is 36 and Y is selected from the group consisting of 1 to 186; X is 37 and Y is selected from the group consisting of 1 to 186; X is 38 and Y is selected from the group consisting of 1 to 186; X is 39 and Y is selected from the group consisting of 1 to 186; X is 40 and Y is ed from the group consisting of 1 to 186; X is 41 and Y is selected from the group consisting of 1 to 186; X is 42 and Y is selected from the group consisting of 1 to 186; X is 43 and Y is selected from the group consisting of 1 to 186; X is 44 and Y is selected from the group consisting of l to 186; X is 45 and Y is selected from the group consisting of 1 to 186; X is 46 and Y is selected fi‘om the group consisting of 1 to 186; X is 47 and Y is selected from the group ting of 1 to 186; X is 48 and Y is selected from the group consisting of 1 to 186; X is 49 and Y is selected from the group consisting of 1 to 186; X is 50 and Y is selected from the group consisting of 1 to 186; X is 51 and Y is selected from the group consisting of l to 186; X is 52 and Y is selected from the group ting of 1 to 186; X is 53 and Y is selected from the group consisting of 1 to 186; X is 54 and Y is selected from the group consisting of l to 186; X is 55 and Y is selected from the group consisting of 1 to 186; X is 56 and Y is selected from the group consisting of l to 186; X is 57 and Y is selected fi'om the group consisting of 1 to 186; X is 58 and Y is selected from the group consisting of 1 to 186; X is 59 and Y is selected from the group consisting of 1 to 186; X is 60 and Y is selected from the group consisting of 1 to 186; X is 61 and Y is selected from the group consisting of 1 to 186; X is 62 and Y is selected from the group consisting of 1 to 186; X is 63 and Y is selected from the group consisting of 1 to 186; X is 64 and Y is selected from the group ting of 1 to 186; X is 65 and Y is selected from the group consisting of 1 to 186; X is 66 and Y is selected from the group consisting of 1 to 186; X is 67 and Y is selected from the group consisting of 1 to 186; X is 68 and Y is ed from the group consisting of l to 186; X is 69 and Y is selected from the group consisting of l to 186; X is 70 and Y is selected from the group consisting of 1 to 186; X is 71 and Y is ed from the group consisting of 1 to 186; X is 72 and Y is ed fiom the group consisting of 1 to 186; X is 73 and Y is selected from the group consisting of 1 to 186; X is 74 and Y is selected from the group consisting of 1 to 186; X is 75 and Y is selected from the group consisting of 1 to 186; X is 76 and Y is selected from the group consisting of 1 to 186; X is 77 and Y is selected from the group consisting of 1 to 186; X is 78 and Y is selected from the group consisting of l to 186; X is 79 and Y is selected from the group consisting of 1 to 186; X is 80 and Y is selected from the group consisting of l to 186; X is 81 and Y is selected from the group consisting of 1 to 186; X is 82 and Y is selected from the group consisting of 1 to 186; X is 83 and Y is selected from the group consisting of l to 186; X is 84 and Y is selected from the group consisting of l to 186; X is 85 and Y is selected from the group consisting of 1 to 186; X is 86 and Y is selected from the group ting of 1 to 186 X is 87 and Y is selected from the group consisting of 1 to 186; X is 88 and Y is selected from the group consisting of l to 186; X is 89 and Y is selected fiom the group consisting of 1 to 186; X is 90 and Y is selected from the group consisting of 1 to 186; X is 91 and Y is selected from the group consisting of 1 to 186; or X is 92 and Y is selected from the group consisting of 1 to 186.

In various embodiments, Xislanins l;Xislanins2;Xislanins3;Xis 1. anins4;Xislanins5;Xisl ;Xislanins7;Xisl aninsS;Xisl anins9;Xis1anins 10;Xisland Yis11;Xislanins 12;Xislanins 13;Xislanins l4;Xislanins 15;Xisland Yis l6;Xislanins l7;Xis 1 aninsl8;Xis 1 anins l9;Xis1anin320;Xisland Yis21;Xislanin322;XislaninsZ3;Xislanin524gXislanins25;Xisland Xislanins27;Xislanin328;XislaninsZ9;Xislanins30;Xisland Yis31;Xislanins 32;Xis1anins33;Xislanins34;Xisl anins35;Xis1 and Yis36;XislaninsS7;Xislanins38;Xislanins39;Xisl anins40;Xisl and Yis41;Xis1anins42;Xislanins43;Xislanins44;Xislanins45;Xisland Yis46;Xis1anins47;Xislanins48;Xislanins49;XislaninsSO;Xisland YisSl;Xis l anin552;XislaninsS3;XislaninsS4;XislaninsSS;Xisland Xislanins 57;Xis 1 anins 58;Xislanin359;Xislanins60;Xis1and Yis61;Xislanins62;Xislanins63;Xislanins64;Xislanins65;Xisland Yis66;Xislanins67;XislaninsG8;Xislanins69;Xis1anins70;Xisland Yis71;Xislanins72;Xislanins73;Xislanins74;Xisl anins75;Xisl and Yis76;Xislanins 77;Xis1 anins78;Xislanins79;Xis 1 anins80;Xisl and Yi581;Xislanins 82;Xis1anins 83;Xislanins84;Xis1anins 85;Xisland YisS6;Xis l anins87;Xis1anins88;Xislanins89;Xislanins90;Xisland Yis91;Xis1anins92;Xislanins93;Xislanin894;Xis 1 anins 95;Xisland Xis 1 andY1597;Xis l anins98;Xis 1 aninsQ9;Xis 1 anins100;Xisl anins 101;Xis 1 anins 102;Xis 1 anins 103;Xis 1 anins 104;Xislanins 105; Xislaninle6;Xislanins 107;Xis1anins 108;Xis1anins slanins slaninslll;Xis1anins112;Xislanins113;Xis1aninsll4;Xisl anins 115;Xis 1 anins ll6;Xisla.ninsll7;Xis 1 anins 118;Xis l anins119; Xislanins120;Xislanins121;Xis 1anins122;Xisl anin5123;Xis 1 anins 124;Xis1anins 125;Xis 1 anins slanin5127;Xis 1 anin3128;Xisl andY15129;Xis 1 anins 130;Xislanins s1anins slanins 133; Xislaninsl34;Xis1anins135;Xislanins 136;Xis1 anins 137;Xis 1 anins 138;Xis 1 anins 139;Xis 1 anins 140;Xis 1 anins 141;Xislanins 142;Xisl anins slanins 144;Xis 1 anins 145;Xislanins 146;Xis 1 deis 147; insl48;Xislanins 149;XislaninslSO;Xisl andY is 151;Xislanins 152;Xislanins153;Xislanin3154;Xis1aninslSS;Xis 1 anin5156;Xisl aninslS7;Xis1 anin5158;Xisl aninslS9;Xislanins160;Xis 1aninsl61; ins 162;Xis 1 anins 163;Xis 1 anins 164;Xisl andY isl65;Xis1 anins 166;Xislanins167;Xislanins168;Xislaninsl69;Xis1aninsl70;Xisl anins 171;Xis1anins 172;Xis 1 aninsl73;Xislanins 174;Xis 1 anins 175; Xis 1 anins 176;Xis 1 anins slanins 178;Xisl andY is 179;Xislanins 180;Xisl anins181;Xis1anins182;Xis1anins183;Xis 1 anin5184;Xisl anin5185gXislanin3186; 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Xis 17 anins156; Xis 17 deis 157; Xis 17 anin3158;Xis 17 anins 159; Xis 17 anins 160; Xis 17 anins 161;Xis 17 anins 162; Xis 17 anins 163;Xis 17 anins 164;Xis 17 anins 165; Xis 17 anins 166;Xis 17 anins167;Xis 17 anins 168; Xis 17 anins 169;Xis 17 anins 170;Xis 17 anins 171;Xis 17 anins 172;Xis 17anins 173;Xis 17 andY is 174; Xis 17 anins 175; Xis 17 anins 176; Xis 17 deis 177; Xis 17 andY is 178; X is 17 anins 179;Xis 17 anins 180;Xis 17 anins 181;Xis 17 anins 182;Xis l7 and Yis s 17 anins 184;Xis 17 anins s 17 anins 186; Xis 18 anins 1;Xis 18 anins 8 anin53;Xis '18 anins4;Xis 18 aninsS; Xis 18 anins 6;Xis 18 anins7;Xis 18 anins 8;Xis 18 anins 9;Xis 18 anins 10;Xis 18 anins 11;Xis 18 anins 12;Xis 18 anins 13;Xis 18 anins 14;Xis 18 anins 15;Xis 18 anins 16;Xis 18 anins 17;Xis 18 anins 18;Xis 18 anins 19;X is 18 anin320;Xis 18 anin521;Xis 18 anins 22;Xis 18 anins23;Xis 18 anins 24;Xis 18 anins25;Xis 18 6;Xis 18 anins27;Xis 18 anins 28;Xis 18 aninsZ9;Xis 18 anins 30;Xis 18 anins 31;Xis 18 anins32;Xis 18 anins33;X is 18 aninsS4;Xis 18 anins 35;Xis 18 anins36;Xis 18 anins 37;Xis 18 anins 38;Xis 18 anins 39;Xis 18 anins 40;Xis 18 anins 41;Xis 18 anins 42;Xis 18 anins43;Xis 18 anins44;Xis 18 anins45;Xis 18 anins46;Xis 18 anins47;X is 18 anins48;Xis 18 anins49;Xi318 anins 50;Xis 18anins 51;Xis 18 anins 52;Xis 18 anins 53;Xis 18 deis 54; Xis 18 anins 55;Xis 18 aninS 56;Xis 18 aninsS7;Xis 18 anins58;Xis 18 anins 59;Xis 18 anins60;Xis 18 anins61;X 2016/050308 is 18 andY1562;Xis 18 anins63;Xis l8anins64;Xis 18 anins65;Xis 18 anins 66;Xis 18 anins 67;Xis 18 anins68;Xis l8 anins69;Xis 18 anins 70;Xis 18 anins71;Xis 18 anins 72.;Xis 18 anins 73;Xis 18 andY is 74;Xis 18 anins 75;X is 18 6;Xis 183nins77;Xis 18anins78;Xis 18 anins 79;Xis 18 anins 80;Xis 18anins 81;Xis 18 anins 82;Xis 18anins 83; Xis 18 4;Xis 18 anins 85;Xis 18 aninsS6;XislSaninsS7;Xis 18 andY is 18aninsS9;X is 18 anins90;Xis 18 anins91;Xis 18anins92;Xis 18 aninsQ3;Xisl8 anins 94;Xis 18 anin595;Xi518anins96;Xis 18anins97;Xis 18 anins98;Xis 18 anin599;Xi318anins100;Xis18anins101;Xisl8anins102;Xi3182deis 103;Xis 18 anins s 18 anins 105;Xis 18 anins 106;Xis l8anins 107;Xis 18 anins 108;Xis 18 anins 109;Xis 18 anins 110;Xis 18 anins 111;Xis 18 andY is 112;Xis 18 anins 113;Xis 18 anins 114;Xis 18 anins 115;Xis 18 anins 116;X is 18 anins 117;Xis 18 anins s 18 anins 119;Xis 18 anins 120;Xis 18 and Yis 121;Xis 18 anins 122;Xis 18 anins 123;Xis 18 anins 124;Xis 18 deis 125; Xis 18 anins 126;Xis 18 anins 127;Xis 18 anins 128;Xis 18 anins 129;Xis 18 anins 130;Xis 18 anins s 18 anins 132;Xis 18 deis 133; Xis 18 anins 134;Xis 18 anins 135;Xis 18 anins 136;Xis 18 aninS 137;Xis 18 anins138;Xis 18:111in5 139;Xis 18 anins 140;Xis 18 anins 141;Xis 18 anins s 18 andY is 143;Xis 18 anins 144;Xis 18 anins 145;Xis 18 anins 146;Xis 18 anins I47;X is 18 anins 148;Xis 18 anins 149;Xis 18 anins 150;Xis 18 anins 151;Xis 18 and Yi5152;X1318 anins 153;Xis 18 anins154;Xis 18 anins 155;Xis 18 anins 156; Xis 18 anins 1.57;Xis 18 anins 158;Xis 18 anins 159;Xis 18 anins s 18 deis 161; Xis 18 anins 162;Xis 18 anins 163;Xis 18 anins 164;Xis 18 anins 165;Xis 18 anins 166; Xis 18 anins 167;Xis 18 anins s18 anins 169;Xis 18 anins 170;Xis 18 aninsl71;Xis 18 anins 172;Xis 18 aninsl73;Xis18andY is 174;Xis 18 anins 175;Xis 18 anins 176;Xis 18 anins 177;Xis 18 anins 178;X is 18 anins 179;Xis 18 anins 180;Xis 18 anins 181;Xis 18 anins s 18 and Yis183;Xis18anins184;Xis18anins185;Xis18aninsl86; Xis19aninsl;Xis19aninsZ;Xis19anins3;Xis19anins4;Xisl9anin55; 3O Xis 19 anins6;Xis 19 anins7;Xis 19anin38;Xis 19anins 9;Xis 19 anins ;Xis 19 anins 11;Xis 19anins 12;Xis 19 anins 13;Xis 19anins 14;Xis l9 aninslS;Xisl9anins16;Xisl9anins17;Xis19anin518;Xisl9aninsl9;X is l9anin520;Xis19andY1'321;Xis19anin522;Xis 19anins23;Xi519anins 24;Xis l9anin525;Xis l9anins26;Xis 19andY1327;Xis19aninSZ8;Xis 19 anins29;Xis l9 anins30;Xis 19 anins31;Xis 19anins 32;Xis 19 anins33;X is 19 anins 34;Xis 19 anins 35;Xis 19anins 36;Xis 19 andY is 37;Xis 19 anins 38;Xis 19 anins 39;Xis 19 anins40;Xis 19 anins 41; Xis 19 anins42;Xis 19 anins 43;Xis 19 anins44;Xis 19 anins45;Xis 19 anins 46; Xis 19 anins47;X is 19 anins48;Xis 19 anins49;Xis 19 aninsSO;Xis 19 andY is 51;Xisl9 anins 52;Xis 19 anins 53;Xis 19 anins 54;Xis 19 anins 55; Xis 19 aninsS6;Xis 19 aninsS7;Xis l9 8;Xis 19 anins 59;Xis 19 anins60; Xis 19 anins61;X is 19 anins 62;Xis 19and‘1’is 63;Xis 19 anins64;Xis 19 andY is 65;Xis 19 anins 66;Xis 19 andY is 67;Xis 19 anins 68;Xis 19 andY is 69; Xis 19 anins 70;Xis 19 anins71;Xis 19 2;Xis 19anins73;Xis 19anins74; Xis 19 anins75;X is 19 anins76;Xis 19 anins77;Xis 19anins78;Xis 19 anins 79;Xisl9 anins 80;Xis 19 anins 81;Xis 19 anins 82;Xis 19 anins83; Xis 19 aninsS4;Xis 19 anins85;Xis 19 anins 86;Xis 19 anins 87;Xis 19anins 88; Xis 19 aninsS9;X is 19 anins90;Xis 19 1;Xis 19 anins92;Xis 19 andY is93;Xis 19 anins 94; Xis 19 anins 95; Xis 19 anins 96;Xis 19 anins 97;Xis 19 anins98;Xis 19 anins 99;Xis 19 anins 100;Xis 19 anins 101;Xis 19 anins 102;Xis 19 anins 103;Xis 19 anins 104; Xis 19anins 105; Xis 19 anins 106; Xis 19 anins 107;Xis 19anins 108;Xis 19 anins 109;Xis 19 anins 110;Xi519anins 111;Xis 19andY is 112;Xis 19 anins 113;Xis 19 anins 114;Xis 19 anins 115;Xis 19 anins 116;X is 19 anins 117;Xis 19 anins 118;Xis 19 anins 119;Xis 19 anins 120;Xis 19and Yis 121;Xis 19 anins 122;Xis 19 anins 123;Xis 19 anins 124;Xis 19 anins 125; Xis 19 deis 126; Xis 19 deis 127; Xis 19 and Yis 128; Xis 19 deis 129; Xis 19 anins 130;Xis 19 anins 131;Xis 19 anins 132;Xis 19 anins 133;Xis 19 anins 134;Xis 19 anins 135;Xis 19 anins 136;Xis 19 anins 137;Xis 19 anins s 19 anins 139;Xis 19 anins 140;Xis 19 anins 141; Xis 19 anins 142;Xis 19 andY is 143; Xis 19 and Y is s 19 anins 145; Xis 19 anins 146;Xis 19 anins 147;X is 19 anins 148;Xis 19 anins 149;Xis 19 anins 150;Xis 19 anins s 19 and Yis 152;Xis 19 anins s 19 anins s 19 anins 155;Xis 19 deis 156; Xis 19 anins 157;Xis 19 anins 158; Xis 19 anins 159;Xis 19 anins 160;Xis 19 anins 161;Xis 19anins 162;Xis 19 anins 163;Xis 19 anins 164;Xis 19 anins 165; Xis 19 deis 166; Xis 19 deis 167; Xis 19 anins 168; Xis 19 andY is 169; Xis 19 anins 170;Xis 19 anins 171;Xis 19 anins 172;Xis 19 anins 173;Xis l9 andY is s 19 deis 175; Xis 19 anins 176;Xis 19 andY is s 19 anins 178;X is 19 anins 179;Xis 19 anins 180;Xis 19 anins 181;Xis 19 anins 182; Xis 19 and Yis 183;Xis 19 anins 184;Xis 19 anins 185;Xis 19 anins 186; Xiszoanins1;XisZOanins2;XisZOanins3;XisZOanins4;Xis20anins5; XisZOanins6;Xi520anins7;XisZOanins8;Xis20 anin39;Xis20anins IO;XisZOanins 11;Xis20anins12;Xi320anins 13; Xis 20anins 14;Xi520 anins15;Xi320anins16;XisZOaninsl7;Xi520anins18;Xi520anins 19;X is20aninsZO;Xis20anin521;Xi320anins22;Xis20 aninsZ3;XisZOanins 24;Xi520anin525;Xis20anin526;Xis20anins27;XisZOanins28;Xis20 anin529;Xis20anins30;XisZOanins31;Xi320anins32;Xis20anins33;X 1320andYis34;Xis20anins3S;XisZOanins36;Xis20 anins37;Xi520anins 38;Xi520 anins 39;XisZOanins40;Xis20anins41;Xis20anins 42;Xis20 anins43;Xi520anins44;Xi520anins45;Xis20anins46;Xis20anins47;X isZOanins48;XisZOanins49;Xi520andYisSO;Xi320 aninsSl;XisZOanins 52;Xi320anins53;Xis20aninsS4;Xi520anins55;Xis20anins56;Xis20 anins57;Xis20aninsSS;Xi320andYisS9;Xi520andYiséO;Xis20anin561;X isZOanins62;XisZOanins63;Xis20anins64;Xis20 5;XisZOanins 20 anins 67;XisZOanins68;Xis20anins 69;Xi320anins 70;XisZO anins71;Xi320anins72;Xi820anins73;XisZOanins74;Xis20anins75;X isZOanins76;XisZOanins77;Xis20anins78;XisZOanins79;XisZO anins 80;Xi320 anins Sl;XisZOanins 82;Xi520anins83;XisZOanins 20 anin385;Xi520anins86;Xi520anin387;Xis20anins88;Xis20anins89;X isZOanins90;Xis20anins91;Xi320anins92;Xis20 anins93;XisZOanins 94;Xis20 anins 95;Xis20anins96;Xis20anins97;XisZOanins 98;Xi520 anins99;Xi520anins100;Xis20anins101;Xi520anins102;XisZOanins 103;Xis 20 anins 104;Xis 20 anins 105;Xis 20 anins 106;Xis 20 anins 107;Xis 20anin5108;Xi820anins109;Xis20aninsllO;Xis20anins111;Xi320andY is112;Xis20anins113;Xis20anins114;Xi520anins 115;XisZOaninsll6;X isZOanins117;Xi820anins118;XisZOanins119;Xi820anins120;Xis20and Yis 121;Xis 20 anins 122; Xis 20 anins s 20 anins 124;Xis 20 deis 125; Xis 20 anins 126;Xis 20 anins 127;Xis 20 anins s 20 andY is 129; Xis 20 anins 130;Xi320 anins 131;Xis 20 anins 132;Xis20anins s 20 anins 134;Xis 20 anins 135;Xis 20 anins 136;Xis 20 anins 137;Xis 20 anins I38;Xis 20anin5139;Xi520anins140;Xi320anins141;XisZOanins142;Xis20andY is 143;Xis 20 anins 144;Xis20 anins 145;Xis 20 anins 146;Xis 20 anins 147;X i320 anins I48;Xis 20 anins 149;Xis20anin3150;Xis 20 anins 151;Xis20 and Yis s 20 anins 153;Xis 20 anins 154;Xis 20 anins s 20 anins 156; XisZOanins157;Xis20anins158;Xis20anins159;Xi320anins160;Xis20 WO 10584 anins 161;Xis 20 anins 162;Xis 20 anins 163;Xis 20 anins 164;Xis20 anins 165;X is 20 deis 166; Xis 20 andY is 167; Xis 20 andY is 168; Xis 20 anins 169; Xis 20anins 170;Xi320aninsl71;XisZOanins l72;XisZO anins 173;X1520andY is 174;Xis 20 anins 175; Xis 20 anins 176; Xis 20 anins 177; X is 20 anins 178;X isZOanins 179;XisZOanins180;Xi320anins 181;Xis 20 anin3182;Xi320and Yis183;Xi320anins184;Xi320anins185;Xi320anins186; Xis21aninsl;Xis21anins2;Xi321anins3;Xis21anins4;XileaninsS; X1321anins6;Xis21anins7;Xis21aninsS;Xis21 anins9;Xile anins ;Xi521aninsll;Xis2lanins12;Xis21aninsl3;Xi321aninsl4;Xis21 aninslS;Xi521aninsl6;Xi521aninsl7;XileandYi518;Xi521aninsl9;X insZO;Xileanin521;Xi521anins22;Xi521anin323;Xi521 anins 24;Xi521 anin325;Xis 21 anins26;Xile 7; Xile anin328;Xi521 anins29;Xis21anins30;Xis21anins31;Xi521anin532;Xis21anin333;X i521anin534;Xis21anins35;Xi521anins36;Xis21anins37;Xileanins 38;Xileanins39;XisZI anins40;Xi521anins41;Xi521anins42;Xi521 anins43;Xi321anins44;Xileanins45;Xi521anins46;Xis21anins47;X is21anins48;Xis2lanins49;Xi521aninsSO;Xi321anins51;Xi521anins 52;Xi321aninsS3;XileaninsS4;Xi521aninsSS;Xis21anin556;Xi521 aninsS7;Xis21aninsS8;Xis21aninsS9;Xileanins60;Xis21anin361;X i321anins62;Xi521anins63;Xileanins64;Xi821 anins65;Xi521anins 66;Xileanins67;Xi521anins68;Xis21anin569;Xi321anins70;Xile an Yis71;Xi521anins72;Xi321anins73;Xis21anins74;Xis21anins75;X i521anins76;Xileanins77;Xi521anins78;Xis21 anins79;Xi521anins 80;XileaninsSl;Xileanin382;Xi521anin383;Xileanins84;Xis21 andY is 85;Xis21 mlins 86;Xis 21 andY is 87; Xis 21 anins 88;Xis 21 anins 89;X i521anins90;Xis21anin591;Xi321anins92;Xis21anins93;Xi521anins 94;Xileanins95;Xi321anins96;Xis21anins97;Xileanins98;Xis21 anins 99; Xis 21 deis 100; Xis 21 anins 101; Xis 21 anins 102; Xis 21 anins 103; Xis 21 anins 104; Xis 21 anins 105; Xis 21 andY is 106; Xis 21 andY is 107; Xis 21anins108;Xi521anin3109;Xis21aninsllO;Xi821anins111;Xis2landY is112;Xis2lanins113;Xi321aninsll4;Xileanins115;Xis21 16;X i521 aninsll7;Xis21 anin5118;Xi521anins119;Xi821anin5120;Xi821 and Y is 121;Xis 21 anins 122;Xis 21 deis 123; Xis 21 anins 124;Xis 21 anins 125; Xis 21 deis 126; Xis 21 anins 127; Xis 21 andY is 128; X is 21 andY is 129; X is 21 anins 130;Xis 21 deis 131; Xis 21 anins 132;Xis 21 anins 133;Xis 21 anins W0 2016/1 10584 134;Xis 21 anins 135;Xis 21 anins 136; Xis 21 anins 137;Xis 21 anins 138;Xis 21 anins 139;Xi521 anins 140;Xis 21 anins 141;Xi321 anins 142;Xi521 andY is 143;Xis 21 anins 144;Xis 21 anins 145;Xis 21 anins 146;Xis 21 anins 147;X is 21 anins 148; 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Xis 22 anins 105;Xis 22 anins 106;Xis 22 anins 107;Xis 2016/050308 22anin5108;Xis223nins109;Xi522anins110;Xi822 andY is 111;Xi322 andY is 112;Xi522anins113;Xis22anins 114;Xis22anins 115;X is22 anins 116;X is 22 anins 117;Xi322 anins 118;Xis 22 anins 119;Xis 22 and Yis 120;Xis 22 and Yis 121;Xis 22 anins 122;Xis 22 anins 123;Xis 22 andY is 124; Xis 22 anins 125; Xis 22 deis 126; Xis 22 andY is 127; Xis 22 anins 128;Xis 22 anins 129;Xis 22 ;Xis22anins131;Xi522anins132;Xi522anins 133;Xis22anins 134;Xis 22 anins 135;Xis 22 anins 136;Xis 22 anins 137;Xis 22 anins 138;Xis 22anins139;X1522anins140;Xis22anins141;Xi522 andY is 142;Xis 22 andY is s 22 anins 144;Xis 22 anins 145;Xis 22 anins 146;X is 22 anins 147;X i522 anins 148;X1522 anins 149;Xis 22 anins 150;Xis 22 aninslSl;Xi522 and Yis 152;Xis 22 anins 153;Xis 22 anins 154;Xis 22 andY is 15S;Xis 22 anins 156; Xis 22 anins 157;Xis 22 and Y is 158; Xis 22 andY is 159;Xis 22 anins 522 anins 161;Xis 22 anins 162;Xis 22 anins 163;Xis22 anins 164;Xis22 anins s 22 anins 166;Xis 22 anins 167;Xis 22 anins 168;Xis 22 anins 169;Xis 22 aninsl70;Xi522 anins 171;Xis22 anins 172;Xis 22 anins 173;Xis 22 andY is 174;Xi322 anins 175;Xis 22 anins 176;Xis 22 anins 177;Xis 22 anins 178;X is22 anins 179;Xis 22 anins 180;Xis 22 anins 18];Xis 22 anins 182;Xis22 and Yis 183;Xis 22 anins 184;Xis 22 anins 185;Xis22 anins 186; 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Xis 37 anins 163;Xis37 anins 164;Xis 37 anins 165;Xis 37 anins 166; Xis 37 anins 167;Xis 37 anins I68;Xis 37 anins 169;Xis W0 2016/110584 37 anins 170;Xis 37 anins 171;Xis 37 anins 172;Xis 37 andY is 173;Xis37 andY is 174;Xis 37 anins 175;Xis 37 anins 176;Xis 37 anins 177;X is 37 anins 178;X is 37 anins 179;Xis 37 anin3180;Xis 37 anins 181;Xis 37 anins 182;Xis 37 and Yis 183;Xis 37 anins 184;Xis 37 anins 185;Xis 37 anins 186; Xis38anins1;Xis38aninsZ;Xis38anin33;Xis38anins4;Xi538aninSS; Xis38’anins 6;Xis38 anins 7;Xis38 ;Xis38 anins 8 anins ;Xis38 anins 11;Xis 38 anins 12;Xis 38 anins 13; X1338 anins 14;Xis38 anins 15;Xis 38 anins 16;Xis38 anins 17;Xis38 anins 18;Xis 38 anins 19;X is38anin520;Xis38 aninle;Xis38 aninsZ2;Xis38 anin523;Xis38 anins 24;Xis38anins 38 anin526;Xis 38 anins27; Xis38 anin528;Xis38 anin529;Xis38 anins 38 anins31;Xis38 anins32;Xis 38 anins33;X is38anins34;Xis38 anins35;Xis38 anins36;Xis38 anins37;Xis38 anins 38;Xis38 anins39;Xis38 anins40;Xis38anins41;Xis38 anins42;Xis38 anins43;Xis 38 anins44;Xis38 anins45;Xis38 anins46;Xis38 anins47;X is38anins48;Xis38anins49;Xis38 aninsSO;Xis38 aninsSl;Xi538 anins 38 aninsS3gXis 38 anins S4;Xi538 anins 55; Xis383nin556;Xis38 anins57;Xis38 8;XisSSaninsS9;Xis38 anin360;Xis38 anins61;X is38 anins 62;Xis 38 anins63;Xis38 anins64;Xis38 anins 65;Xis38 anins 66;Xis s67;Xis38 anins68;Xis 38 anins69;Xis 38 anins 70;Xis38 anins71;Xis38 anins 72;Xis38 anins73;Xis 38 anins74;Xis38 anins75;X i338 anins76;Xis38 anins77;Xis38 anins78;Xis38 anins79;Xis38 anins 38 anins 81;Xis38 anins 82;Xis 38 anins 83;Xis38anins 84;Xis38 anins 85;Xis38 anin386;Xis 38 anins87;Xis38 anins 88;Xis 38 anins 89;X is 38 anins90;Xis 38 anins91;Xis 38 anins92;Xis 38 anins 93;Xis38 anins 94;Xis38 anins 95;Xi338 anins96;Xis 38 anin397;Xis38 anin598;Xis38 9;Xis 38 anins 100;Xis38anins 101;Xis38anin5102;Xis 38anins s 38 anins 104;Xis 38 anins 105;Xis38 anin8106;Xis38 anins 107;Xis 38 anins 108;Xis 38 anins 109;Xis38 anins 110;Xis38 aninslll;Xis 38 andY is 112;Xis38anins113;Xis38anins 114;Xis38aninsllS;Xis38anins116;X is38anins117;Xis38aninsll8;Xis3Sanins119;Xis38anins120;Xis38 and Yis 121;Xis 38 anins 122;Xis 38 anins 123;Xis 38 anins 124;Xis 38 deis 125; Xis 38 anins 126; Xis 38 deis 127; Xis 38 anins 128;Xis 38 anins 129;Xis 38 anins s 38 anins 131;Xis 38 anins 132;Xis 38 anins 133;Xis 38 anins 134;Xis 38 anins 135;Xis 38 anins s 38 anins 137;Xis38 anins s 38 anins 139;Xis 38 anins 140;Xis 38 anins 141;Xis 38 anins 142;Xis 38 andY W0 2016/110584 is 143;Xis 38 anins 144;Xis 38 anins 145;Xis 38 anins 146;Xis 38 anins 147;X is 38 anins 148;Xis 38 anins 149;Xis 38 anin3150;Xis 38 anins 151;Xis 38 and Yis 152;Xis 38 anins 153;Xis 38 anins 154;Xis 38 anins 155;Xis 38 anins 156; Xis 38 andY is 157;Xis 38 anins 158;Xis 38 anins 159;Xis 38 anins I60;Xis 38 anins 161;Xis 38 anins 162;Xis 38 anins 163;Xis 38 anins 164;Xis 38 anins 165;Xis 38 anins 166;Xis 38 anins 167;Xis 38 anins 168;Xis 38 anin5169;Xis 38 anins 170;Xis 38 anins s 38 anins s38 anins 173;Xis38 andY is 174;Xis 38 anins 17S;Xis 38 anins 176;Xis 38 anins s 38 anins 178;X is 38 anins s 38 anins 180;Xis 38 anins 181;Xis 38 anins 182;Xis 38 and Yis 183;Xis38 anins 184;Xis38 anins 185;Xis 38 anins 186; Xis39 aninsl;Xis 39 anin32;Xis39 anins3;Xis39anins4;Xis39anin55; Xis39anins6;Xis39 anins7;Xis39anins8;Xis39 anins9;Xis39anins ;Xis39 anins11;Xis39anins12;Xis39aninsl3;Xis39aninsl4;Xis39 anins15;Xi539anins16;Xis39anins17;Xis39anins18;Xis39aninsI9;X is 39 and Yis 20;Xis 39 anins 21;Xis 39 anins 22;Xis 39 anins 23;Xis 39 anins 39 aninsZS;Xis39aninsZ6;Xis39aninsZ7;Xis39anins 28;Xi339 anins29;Xis39 anins30;Xis39 anins31;Xis39anins32;Xis39anins33;X is39anin534;Xis39anins3S;Xis 39anins36;Xis39anins37;Xis39 anins 38;Xis 39 anins39;Xis 39andYis40;Xis39anins4l;Xis39anins42;Xis39 anins43;Xis39anins44;Xis39anins45;Xis39andYis46;Xi339anins47;X is39anins48;Xis39anins49;Xis39aninsSO;Xis39anins51;Xis39anins 52;Xis 39 anins 53;Xis39anins 54;Xis39anin855;Xis 39 anins 56;Xis39 7;Xis39anins58;Xis39andYis59;Xis39andYis60;Xis39anin561;X is39 2;Xis39anin363;Xis 39anins64;Xis39anins65;Xis39anins 66;Xis 39anins 67;Xis39anins68;Xis39anin369;Xis39 anins70;Xis39 anins71;Xis39anins72;Xis39andYis73;Xis39 anins74;Xis39anins75;X is39anins76;Xis39anins77;Xis39anins78;Xis39anins79;Xis39anins 80;Xis 39anins 81;Xis39 anins82;Xis39anin883;Xis 393nins 84;Xis39 anins85;Xis39anin386;Xis39anins87;Xis39anin588;Xis39anins89;X is39anins90;Xis39anins91;Xi839anins92;Xis 39anins93;Xis39 anins 94;Xis39anins 95;Xis39 anins96;Xis39 7;Xis39anins 98;Xis39 anins99;Xis 39 anins 100;Xis39anins s39anins102;Xis39anins 103;Xis 39 anins 104;Xis 39 anins 105;Xis 39 anins 106;Xis 39 anins 107;Xis 39anins108;Xis39anins109;Xis39anins110;Xis39anins111;Xis39andY is 112;Xis39anins113;Xis39anins114;Xis39aninsllS;Xis39anins 116;X W0 2016/110584 is 39 anins 117;Xis 39 anins s39 aninsll9;Xis 39 anins 120;Xis 39 and Yis 121;Xis 39 anins 122;Xis 39 anins s 39 anins 124; Xis 39 anins 125; Xis 39 anin8126;Xis 39 andY is 127; Xis 39 anins 128;Xis 39 anins 129;Xis 39 anins 130;Xis 39 anins s 39 anins 132;Xis 39 anins 133;Xis 39 anins 134;Xis 39 anins 13S;Xis 39 anins 136;Xis 39 anins137;Xis 39 anins 138;Xis 39 anins 139;Xis 39 anins s 39 anins s 39 andY is 142;Xis 39 andY is 143;Xis 39 anins 144;Xis 39 anins 145;Xis 39 anins 146;Xis 39 anins 147;X is 39 anins 148;Xis 39 anins 149;Xis 39 anins150;Xis 39 anins 151;Xis 39 and Yis152;Xis 39 anins 153;Xis 39 anins 154;Xis39 andY is 155;Xis 39 aninslSG; Xis 39 anins s 39 deis 158; Xis 39 anins 159;Xis 39 anins 160;Xis 39 anins 161; Xis 39 anins s 39 anins 163;Xis 39 and Y is s 39 anins I65;Xis 39 anins 166;Xis 39 anins 167; Xis 39 anins 168;Xis 39 anins 169;Xis 39 anins 170;Xis 39 anins s 39 anins 172;Xis 39 andY is is39 andY is 174; Xis 39 anins 175;Xis 39 anins 176;Xis 39 anins 177;Xis 39 anins 178;X is 39 anins 179;Xis 39 anins 180;Xis 39 anins 181;Xis 39 anins 182;Xis 39 and Yis 183;Xis 39 anins 184;Xis 39 anins 185;Xis 39 andY is 186; Xis40anins1;Xis40anins2;Xis40anins3;Xis40andYis4;Xis40anins5; Xis40anin56;Xis40 anins 7;Xis40 anins 8;Xis40 anins9;Xis40anins ;Xis40anins 11;Xis40anins 12;Xis40anins 13; Xis40anins 14;Xis40 anins15;Xis40anins16;Xis40anins 17;Xis40anins18;Xis40anins19;X i340 anins 20;Xis 4O aninle;Xis40 anin522;Xis4O anins23;Xis40 anins 24;Xis40andYi325;Xis40aninsZ6;Xis40anins27;Xis40anin528;Xis40 anins29;Xis40 anins30;Xis40 anins31;Xis40 anins 32;Xis40 3;X is40anins34;Xis40anins35;Xis40anin836;Xis40 anins37;Xis40anins 38;Xis40 anins39;Xis40anins40;Xis40anins41;Xis40anins42;Xis40 anins43;Xis40 anins44;Xis40andYis45;Xis40anins46;Xis40 anins47;X is40anins48;Xis40anins49;Xis40anins SO;Xis4O aninsSl;Xis40anins 52;Xis40anins 53;Xis40anins 54;Xis40aninSSS;Xis40anin556;Xis40 anins 57;Xis40 aninsS8;Xis40 anins 59;Xis40anins60;Xis40 anins61;X is40anins62;Xis40anins63;Xis40 anins64;Xis40anins65;Xis40anins 66;Xis40 anins67gXis40anins68;Xis40anins69;Xis40anins70;Xis40 anins71;Xis40 anins72;Xis40anins73;Xis40anins74;Xis40 anins75;X is40anins76;Xis40anins77;Xis40anins78;Xis40anins79;Xis40anins 80;Xis 40anins 81;Xis40anins 82;Xis40 anins 83;Xis40anins 84;Xis40 anins85;Xis40aninsS6;Xis40anins 87;Xis40anins88;Xis40aninsS9;X W0 2016/110584 is40anins90;Xis40anins91;Xis40 anins92;Xis40 anins93;Xis40anins 94;Xis40anins95;Xis 40anins96;Xis40anins 97; Xis40anins98;Xis40 anins 99;Xis 40 anins 100;Xis 40anins 101;Xis 40 anins 102;Xis 40anins 103;Xis 40 anins 104;Xis 40 anins 105;Xis 40 anins 106;Xis 40 anins s 40aninSIO8;Xis40anins109;Xis40anins110;Xis40anins111;Xis40andY is112;Xis40anins113;Xis40édeis114;Xis40anins115;Xis40aninsll6;X is40anins 117;Xis40anins 118;Xis40anins s 40anins 120;Xis40 and Yi5121;Xis40anins122;Xis403nins123;Xis40anins 124;Xis40anins 125; Xis 40 anins 126; Xis 40 deis 127; Xis 40anin5128;Xis 40 anins 129;Xis 40 anins s40 anins 131;Xis40 anins 132;Xis 40 anins 133;Xis40 anins 134;Xis40 anins 135;Xis 40 anins s 40 anins 137;Xis 40 anins 138;Xis 40anins 139;Xis 40 anins 140;Xis40 anins 141;Xis40 anins 142;Xis40 andY is 143;Xis40 anins 144;Xis 40 anins 145;Xis 40 anins 146;Xis 40 anins 147;X is 40 anins 148;Xis 40 anins 149;Xis 40 anins 150; Xis 40 anins 151;Xis 40 and Yi3152;Xis 40 anins 153;Xis40 anins s40 anins 155;Xis40 anins 156; Xis 40 anins 157;Xis 40 anins 158;Xis 40 anins 159;Xis40 deis 160; Xis 40 anins161;Xis40anins162;Xis40anins 163;Xis40anins164;Xis40anins 165;Xis 40 anins 166;Xis 40 anins 167;Xis 40 anins l68;Xis40anins 169;Xis 40anins 170;Xis 40 anins 171;Xis40 anins 172;Xis40 anins 173;Xis 40 andY is 174;Xis 40 anins 175;Xis 40 anins 176;Xis 40 anins 177;Xis40 anins 178;X is40anins179;Xis40anins180;Xis40anins181;Xis40anins182;Xis40and Yis s 40 anins s 40 anins 185;Xis40 anins 186; Xis4laninsl;Xis41aninsZ;Xis4lanins3;Xis41anins4;Xis41aninsS; Xis4lanins6;Xis41anins7;Xis4lanins8;Xis41anins9;Xis41anins 10;Xis41anins11;Xis41anin312;Xis4laninsl3;Xis41aninsl4;Xis41 anins 15;Xis41 anins 16;Xis4l anins 17;Xis41 anins 41 anins 19;X is41aninsZO;Xis41aninle;Xis41anins22;Xis41aninsZ3;Xis41anins 24;Xis 41 anins 2S;Xis 41 aninsZ6;Xis4l anin327;Xis41 anins28;Xis4l anin529;Xis41anins30;Xis41anins31;Xis41anins32;Xis41anins33;X is4lanins34;Xis41anins35;Xis41anins36;Xis41anins37;Xis41anins 38;Xis41anins39;Xis41anins40;Xis41anins41;Xis41anins42;Xis41 anins43;Xis41anins44;Xis41anins45;Xis41anins46;Xis41anins47;X is41anins48;Xis4lanins49;Xis41aninsSO;Xis41aninsSl;Xis41anins 52;Xis41aninsS3;Xis41aninsS4;Xis41anins55gXis4laninsS6;Xis4l aninsS7;Xis41 anins58;Xis41aninsS9;Xis4l anins60;Xis4l anins61;X W0 2016/110584 1 10 is41anins62;Xis41anins63;Xis41anins64;Xis41anins65;Xis4lanins 66;Xis 41 anins 67;Xis41 anins68;Xis4l anins69; Xis41 anins70;Xis41 1;Xis41anins72;Xis41anins73;Xis41anins74;Xis41anins75;X i541anins76;Xis41anins77;Xis41anins78;Xis41anins79;Xis41anins 80; Xis 41 anins 81;Xis 41 anins 82;Xis41 anins 83; Xis41 anins84;Xis41 anins85;Xis41anins86;Xis41aninsS7;Xis41anins88;Xis41anins89;X is 41 and Y is 90; X is 41 2111in5 91; Xis 41 anins 92; Xis 41 and Y is 93; Xis 41 anins 94;Xis41anins95;Xis41anins96;Xis41anins97;Xis41anins98;Xis41 andY is 99; Xis 41 andY is 100; X is 41 andY is 101; X is 41 andY is 102;-X is 41 anins 103;Xis 41 anins 104; Xis 41 deis 105; Xis 41 anins 106; Xis 41 anins 107; Xis 41 anins 108;Xis41 anins 109;Xis41 anins 110;Xis41 anins 111;Xis41 andY is!12;Xis4l l3;Xis41aninsll4;Xis4l anins 115;Xis4l anin5116;X i541 anins117;Xis41aninsll8;Xis41 aninsll9;Xis 41 anins 120;Xis41 and Yis s 41 anins 122; Xis 41 deis 123; Xis 41 andY is 124; Xis 41 deis 125; Xis 41 anins 126;Xis 41 anins 127; Xis 41 anins 128;Xis 41 anins 129;Xis 41 aninsl30;Xis4l anins131;Xis41 anins 132;Xis41 anins 133;Xis41 anins 134;Xis 41 anins 135;Xis 41 anins s 41 deis 137; Xis 41 anins 138;Xis 41aninsl39;Xis41anins140;Xis41anins141;Xis41anins142;Xis41andY is 143; Xis 41 deis 144; Xis 41 andY is 145; Xis 41 deis 146; X is 41 andY is 147; X is 41 anins 148;Xis41 anins 149;Xis 41 anins 150;Xis 41 anins s 41 and Yis 152;Xis41 anins 153;Xis 41 anins 154;Xis 41 anins 155;Xis 41 deis 156; Xis41 anins 157;Xis 41 deis 158; Xis41 anins 159; Xis4l anins 160; Xis41 anins 161;Xis 41 deis 162; Xis41 anins 163;Xis 41 anins 164; Xis 41 anins 165;Xis 41 anins 166;Xis 41 anins 167;Xis 41 deis 168; Xis 41 anins 169;Xis 41 anins 170;Xis 41 anins 171;Xis 41 anins 172;Xis41 anins 173;Xis 41 andY is 174;Xis 41 anins 175; Xis 41 andY is 176;Xis 41 anins 177;Xis 41 anins 178;X is 41 anins 179;Xis 41 anins 180;Xis 41 anins 181;Xis41 deis 182; Xis 41 and Y is 183;Xis 41 anins 184;Xis 41 andY is 185; Xis 41 andY is 186; nins1;Xis42aninsZ;Xis42anins3;Xis42anins4;Xis42aninsS; Xis42anins6;Xis42anins7;Xis423ninsS;Xis42anins9;Xis42anins ;Xis42 anins 42 anins12;Xis42anins13;Xis42anins14;Xis42 anins15;Xis42anins16;Xis42anins l7;Xis42anins18;Xis42aninsl9;X is42anin520;Xis42anin321;Xis42anins22;Xis423nins23;Xis42anins 24;Xis42 anins25;Xis42aninsZ6;Xis42aninsZ7;Xis42anins28;Xis42 anin529;Xis42anins30;Xis42anin531;Xis42anins32;Xis42anin533;X W0 2016/110584 1 1 l is42 anins34gXis42anins35;Xis42anins36;Xis42 anins37;Xis42 anins 38;Xis42 anins 39;Xis 42 anins40;Xis42 anins41; 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Xis48 anins77;Xis48 anins78; Xis48 andY is 79;Xis48 anins 80;Xis48 anins 81;Xis48 anins 82;Xis48 anins 83; Xis48 anin584;Xis48 anins 85;Xis48 anins 86;Xis48anins 87;Xis48 anins 88;Xis48 anins 89;X i548 anins90;Xis48 anins91;Xis48 anins92;Xis 48 andY is 93;Xis48 deis 94; Xis 48 anins 95;Xis 48 deis 96; Xis 48 anins 97;Xis 48 deis 98; Xis 48 andY is 99; Xis 48 deis 100; Xis 48 andY is 101; Xis 48 anins 102; Xis 48 anins s 48 anins 104;Xis 48 anins 105;Xis 48 deis 106; Xis 48 anins 107;Xis 48 anins 108;Xis 48 anins 109;Xis48 anins 110;Xis48 anins 111;Xis48 andY is 112;Xis48 anins 113;Xis48 anins 114;Xis48 anins 115;Xis48 anins 116;X i348 anins 117;Xis48 anins 118;Xis48 anins 119;Xis48anins 120;Xis48 and Yis 121; Xis 48 andY is 122;Xis 48 anins 123;Xis 48 anins 124;Xis 48 anins 125; Xis 48 deis 126; Xis 48 andY is 127; Xis 48 deis 128; Xis 48 and Y is 129; X is 48 anins 130;Xis 48 deis 131; Xis48 anins s48 anin3133;Xis48 anins 134;Xis 48 andY is 135;Xis 48 andY is 136; Xis48 andY is 137;Xis 48 andY is 138;Xis 48 anins 139;Xis 48 anins 140;Xis 48 anins 141;Xis 48 anins 142;Xis 48 andY is 143; Xis 48 anins 144;Xis 48 anins 145; Xis 48 anins 146;Xis 48 anins 147;X 1548 deis 148; Xis48 anins 149;Xis48 anins 150;Xis48 anins 151;Xis48 and Yis s 48 anins 153;Xis 48 anins 154; Xis 48 anins 155;Xis 48 anins 156; Xis 48 anins 157;Xis 48 andY is 158; Xis 48 anins 159;Xis48 deis 160; Xis48 anins 161;Xis 48 anins 162;Xis 48 anins 163; Xis 48 deis 164; Xis 48 deis 165; Xis 48 anins 166;Xis 48 anins 167; Xis 48 anins 168;Xis 48 anin8169;Xis 48 andY is 170; Xis 48 deis 171; Xis 48 anins 172; Xis 48 anins 173;Xis 48 andY is 174;Xis 48 anins 175;Xis 48 deis 176; Xis 48 andY is 177;Xis 48 anins 178;X W0 2016/110584 1 19 i548 anins 179;Xis 48 anins 180;Xis48 anins 181;Xis 48 anins I82;Xis 48 and Yis 183;Xis48anins184;Xis483nins 185;Xis48anins 186; Xis49anins1;Xis49aninsZ;Xis49anins3;Xis49anins4;Xis49aninsS; Xis49anins6;Xis49anins 7;Xis49anins 8;Xis49 anins9;Xis49anins ;Xis49anins11;Xis49anins12;Xis49anins13;Xis49aninsl4;Xis49 anins 15;Xis49anins16;Xis49 anins 17;Xis49anins18;Xis49anins l9;X i349anins20;Xis49anins21;Xis49anins22;Xis49aninsZ3;Xis49anins 49 anins 25;Xis49 aninsZ6;Xis49 anin827;Xis49 anins28;Xis49 anins29;Xis49anins30;Xis49 anins31;Xis49anins32;Xis49anins33;X is49anins34;Xis49anins35;Xis49anins36;Xis49anins37;Xis49anins 38;Xis49&nins 39;Xis49anins40;Xis49anins4l;Xis49anins42;Xis49 anins43;Xis49anins44;Xis49 anins45;Xis49anins46;Xis49anins47;X i349 anins48;Xis49anins49;Xis49 anins50;Xis49aninsSl;Xis49 anins 52;Xis 49 anins 53; Xis 49 anins 54;Xis 49 anins 55;Xis 49 anins 56; Xis49 7;Xis49anins58;Xis49 anins59;Xis49andYis60;Xis49anins61;X is49anins62;Xis49anins63;Xis49anins64;Xis49anins65;Xis49 anins 66;Xis49anins67;Xis49 anins68;Xis49anins69;Xis49 anins 70;Xis49 anins71;Xis49 anins72;Xis49anins73;Xis49 anins 74;Xis49 anins75;X i349anins76;Xis49anins77;Xis49anins78;Xis49anins79;Xis49anins 80;Xis49anins 81;Xis49anins82;Xis49anins83;Xis49anins 84;Xis49 anins85;Xis492deis86;Xis49anin587;Xis49anin588;Xis49anins89;X ins 90;Xis49anins91;Xis49anins92;Xis49anins93;Xis49anins 49anins95;Xis49andYis96;Xis49anins97;Xis49anins98;Xis49 9;Xis49anins100;Xis49anins101;Xis49anins102;Xis49anins 103;Xis 49 anins s 49 anins 105;Xis49 anins 106;Xis 49 anins 107;Xis 49anins108;Xis49anins109;Xis49anins110;Xis49anins111;Xis49andY is112;Xis49anins113;Xis49anins114;Xis49anins115;Xis49anins116;X is49anins 117;Xis49 anins118;Xis49anins119;Xis493nins 120;Xis49 and Yis 121;Xis 49 anins 122;Xis 49 anins 123;Xis 49 anins 124;Xis 49 anins 125; 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XisSOanins1;XisSOanin32;Xis50anins3;XisSOanins 4;Xis 50aninSS; Xis50anins6;XisSOanins7;Xis50anins8;XisSOandY i39;Xis50anins ;Xis50anins11;Xi550anins12;XisSOanins13;XisSOaninsl4;XisSO anin315;Xis 50 anins 16;Xis 50 anins 17;Xis 50 anins 18; Xis SOanins 19;): is 50anins20;Xis SOanin321;Xis SOanin322;Xis 50 anins23;XisSO anins 24;Xis 50 aninsZS;Xis sZ6;XisSO anins27;Xis 50anin528;Xis 50 aninsZ9;Xis 50anins30;Xis SOanins31;Xi350anins 32;XisSOanins33;X is 50anins34;XisSOanins35;Xis 50anins36;XisSOanins37;XisSO anins SOanins39;Xis 50anins40;Xis 50anins41;Xis 50anins42;Xis 50 anins43;Xis SOanins44;Xis 50 anins45;Xis 50 anins 46;Xis 50 anins47;X isSOanins48;Xis50anins49;Xis SOaninsSO;Xis50aninsSl;XisSOanins 52;Xis 50 aninsSS;XisSOanins 54;Xis 50anins 55;Xis 50 anins 56;Xi350 anins 57;Xis 50 aninsS8;XisSO anins 59;Xis 50anins 60;Xis SOanins61;X isSOanins62;XisSOanins63;Xis 50anins64;XisSOanins65;XisSOanins 66;XisSOandYis67;XisSOanins68;Xis 50anins69;Xis 50anins70;XisSO anins 71;Xis 50anins72;Xis SOanins 73;XisSOanins74;Xis 50 anins75;X is 50 anins76;Xis 50 anins77;Xis SOanins 78;Xis 50anins79;Xis 50 anins 80;Xis 50 anins 81;Xi350 anins 82;Xis SOanin583;Xis 50 anins 84;XisSO anin585;Xis 50anins86;XisSOanins 87;XisSOanins88;Xis$0anin389;X isSOanins90;XisSOanins91;Xi350 anins92gXiSSOanin593;Xis 50anins 94;Xis 50 anins95;XisSOanins96;Xis 50anins97;XisSOanins 98;Xi550 deis 99; 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XisSl aninsl;Xis 51 aninsZ;XisSl anins 3;XisSl anins4;XisSl aninsS; XisSlanins6;XisSl anins7;XisSl anins8;XisSl anins9;Xis51anins ;Xi351aninsll;XisSlanins12;Xi351anin313;Xis51aninsl4;XiSSI anins15;Xi851 anins 16;XisSl anins 17;Xi351 anins 18;XisSl anins 19;X i551aninsZO;XisSlaninle;XisSlanin522;Xis51anins23;Xi551anins 24;Xi551 anins25;Xis 51 anins26;Xi351 anin827;Xis 51 anins 28;XisSl aninsZ9;XisSlanin530;Xi551anins31;XisSlanins32;XisSlanins33;X i551 anins34;XisSl 5;XisSl anins36;Xis 51 anins37;XisSl anins 38;X1551 anins39;Xis 51 anins40;XisSl anins41;XisSl 2;Xi351 anins43;Xis51anins44;XisSlanins45;XisSlanins46;XisSlanins47;X i351anins48;XisSlanins49;XisSlanin550gXi851aninsSl;XisSIanins 52;Xis51 anins 53;XisSl anins 54;Xis 51 aninsSS;Xis 51 anins 56;Xis51 anins57;Xi351anin358;Xis51aninsS9;XisSlanins60;XisSl aninsfil;X i351 2;XisSl anins63;XisSl anins64;Xis 51 anins65;XisSl anins 66;Xi351 7;Xis 51 anins68;XisSl anins69;Xis 51 0;XisSl anins71;XisSl anins72;XisSl anins73;Xis51anins74;Xi551anins75;X i551 anins76;XisSl anins77;Xi351 anins78;Xis 51 anins79;Xis 51 anins 80;XisSl anin581;Xi551anins82;Xis51aninsS3;XisSlaninsS4;XisSl anins85;XisSlanins86;Xis51anins87;XisSlanins88;XisSl anins89;X is 51 aninsQO;XisSl anins9l;Xi351 anins92;Xis 51 anin593;X1351 anins 94;Xi351 5;Xis 51 anins96;Xis 51 anins97;Xis 51 anins 98;Xis51 W0 2016/110584 anins 99; 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Xis 52 and Y is 103;Xis 52 anins 104;Xis 52 anins 105;Xis 52 anins 106;Xis 52 anins 107;Xis 52 anins 108;Xis 52 anins 109;Xis 52 anins 110;Xi352 anins 111;Xis 52 andY is 112;Xi352anins 113;Xis 52 anins 114;Xi552 anins 115;Xis 52 anins 116;X is 52 anins 117;Xis 52 anins 118;Xis 52 anins 119;Xis 52 anins s 52 and Yi5121;Xis 52 anins 122;Xis 52 anins 123;Xis 52 anins 124;Xis 52 andY is 125; Xis 52 anins 126;Xis 52 anins 127;Xis 52 anins 128;Xis 52 anins 129;Xis 52 anins s 52 anins s 52 anins 132;Xis 52 anins 133;Xis 52 anins 134;Xis 52 anins 135;Xis 52 anins 136;Xis 52 anins 137;Xis 52 anins 138;Xis 52 anins 139;Xis 52 anins 140;Xis 52 anins 141;Xis 52 anins 142;Xis 52 andY is 143;Xis 52 anins 144;Xis 52 andY is I45;Xis 52 anins 146;Xis 52 anins 147;X is 52 anins 148;Xis 52 anins 149;Xis 52 anins 150;Xis 52 anins151;Xis 52 and Yis152;Xis 52 anins 153;Xis 52 anins s 52 anins 155;Xis 52 anins 156; Xis 52 anins 157;Xis 52 anins 158;Xis 52 anins 159;Xis 52 anins 160;Xis 52 anins 161;Xis 52 anins 162;Xis 52 anins 163;Xis 52 anins s 52 anins 165;Xis 52 anins 166;Xis 52 anins s 52 anins 168;Xis 52 anins s 52 anins 170;Xis 52 anins s 52 anins 172;Xis 52 anins 173;Xis 52 andY is 174;Xis 52 deis 175; 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Xis 89 anins47;X isS9anins48;Xi589anins49;Xis 89 aninsSO;Xis89 andY is 51;Xi589anins 52;Xis89 anins 53;Xis 89 anins 54;Xis 89 aninsSS; Xis89 aninsS6;XisS9 anins 57;Xis 89 anins 89 anins 59;Xi589 0; Xi389 anins6l;X is 89anins 62;Xis 89anins63;Xi589anins64;Xis 89 anins65gXi589 anins 66;Xis 89anins 67;Xi389anins68;Xis89anins69; Xis 89 anins70;Xi389 anins71;Xis 89anins72;Xis 89 anins 73;Xis 89anins74;Xis89anins75;X is 89 anins76;Xis89anins77;Xis89 anins78;Xis 89 9;XisS9anins 80;Xis 89anins 81;Xis 89 anins 82;Xis 89 deis 83; Xis 89 anins 84;Xis 89 aninsSS;Xis 89 anins 86;Xis89 anin587;Xis89anins 88;Xis 89 anins 89;X i589 anins90;Xis 89adeis91;Xis 89 anins 92;Xis 89 anins 93;Xis 89 anins 94;Xis 89 anins95;Xis 89anins96;Xis89anins 97;Xis89anins98;Xi389 anins99;Xi389anins100;Xi389a.nins101;XisS9anins102;Xi589anins 103;Xis 89 anins 104;Xis 89 anins 105;Xis 89 anins 106;Xis 89 anins 107;Xis 89 anins 108;Xis 89 anins 109;Xis 89 anins 110;Xis89 anins 111;Xis 89andY Xis89aninsii3;Xis89anins114;Xis89anins 115;Xis89anins 116;X is 89 anins 117;Xis 89anins 118;Xis 89 anins 119;Xis 89 anins 120;Xis89 and Yis 121;Xis 89 anins 122;Xis 89 anins 123;Xis 89 anins 124;Xis 89 anins 125; Xis 89 anins 126;Xis 89 andY is 127; Xis 89 deis 128; Xis 89 anins 129;Xis 89 anin5130;Xis 89 anins s 89 anins 132;Xis 89 anins 133;Xis 89 anins 134;Xis 89 anins 135;Xis 89 anins 136;Xis 89 anins137;Xis 89 anins 138;Xis 89 anins 139;Xis 89 anins 140;Xis 89 anins 141;Xis 89 anins 142;Xis 89 andY is 143;Xis 89 anins 144;Xis 89 anins 145;Xis 89 anins s 89 anins 147;X is 89 anins 148;Xis 89 anins 149;Xis 89 anins 150;Xis 89 deis 151; Xis 89 and Yis 152;Xis 89 anins s 89 anins 154;Xis 89 anins 155;Xis 89 anins 156; Xis 89 anins 157;Xis 89 anins 158;Xis 89 anins 159; Xis 89 anins 160;Xis 89 W0 2016/110584 anins 16I;Xis 89 anins 162;Xis 89 anins 163;Xis 89anins 164;Xis 89 anins 165;Xis 89 anins 166;Xis 89 anins 167;Xis 89 68;Xis 89 anins 169;Xis 89aninsl70;Xi389anins171;XisS9anins172;X1889aninsl73;Xis89andY is 174; Xis 89 anins 175;Xis 89 anins 176;Xis 89 anins 177;Xis 89 anins 178;X is 89 anins 179;Xis 89 anin5180;Xis 89 anins s 89 anins s 89 and Yis 183;Xis 89 aninsl84;Xis 89 85;Xis 89 andY is 186; XisQOanins1;Xis90aninsZ;Xis90anins3;Xis90anins4;Xis90aninsS; Xis90anins6;Xis90anins7;Xis90anins8;Xis90 anins9;Xis90anins ;Xis90anins11;Xis90anins12;Xis90anins l3; Xis90anins 14;Xis90 anins15;Xis90anins16;Xis90anins l7;Xi390anins18;Xis90aninsl9;X is 90anins20;Xis90anin321;Xis90anin322;Xis90 aninsZ3;Xis90anins 24;Xis90andY1325;Xis 9Oanin526;Xis 527; Xis 90anins28;Xis90 aninsZQ;Xis90anins30;Xis90anins31;Xis9OandYi332;Xis90anin533;X i390anins34;Xis9Oanins35;Xis90anins36;Xis90anins37;Xis90anins 38;Xis90anin539;Xis90anins40;Xis90anins41;Xis90anins42;Xis9O anins43;Xis90anins44;Xis90anins45;Xis90anins46;Xi390anins47;X is90anins48;Xis90anins49;Xis90aninsSO;Xis 90 aninsSl;Xis90anins 52;Xis 90 aninsS3;Xis 90aninSS4;Xis90anins 55; Xis 90 anins 56;Xi890 aninsS7;Xis90aninsS8;Xis90anins59;Xis90andYis60;Xis90anins61;X i390anins62;Xis90anins63;Xis90anins64;Xis90andYi365;Xis90anins 66;Xis90anins67;Xis90anins68;Xis90anins69; Xis903nins70;Xis90 anins71;Xis90anins72;Xis90anins73;Xis90anins74;Xis90anins75;X is90anins76;Xis90anins77;Xis90anins78;Xis90anins79;Xis90anins 80;Xis90anins 81;Xis90anin582;Xis90anin883;Xis90anins 84;Xis90 anin385;Xis90anins86;Xis9Oanin587;Xis9Oanins 88;Xi590anins89;X i390 anins 90anins91;Xis90anins92;Xis 90anins93;Xis90 anins 94;Xis9Oanins95;Xis90anin396;Xis90anins97;Xis 90anins98;Xis90 anins99;XisQOanins100;Xis90anins101;Xis90anins102;Xis90anins s 90 anins 104;Xis 90 anins 105;Xis 90 anins 106;Xis 90 anins 107;Xis 3O 90anin5108;Xis90anins109;Xis90anins110;Xis90anins111;Xis90andY is 112;Xis90anins113;Xis90anins114;Xis90anins115;Xis90aninsll6;X is90anins117;Xis90anins118;Xi390anins119;Xis9Oanins120;Xis90and Yis 121; Xis 90 anins 122;Xis 90 anins 123;Xis 90 anins 124;Xis 90 anins 125; Xis 90 anins 126;Xis 90 anins 127; Xis 90 anins s 90 anins 129;Xis 90 anins 130;Xis 90:111in3 131;Xis 90anins 132;Xis90aninsl33;Xis90anins W0 2016/110584 134;Xis 90 anins 135;Xis 90 anins 136;Xis 90 anins 137;Xis 90 anins 138;Xis 90 anins 139;Xis 90 anins s 90 anins 141;Xis 90 andY is s 90 andY is 143;Xis 90 anins 144;Xis 90 anins 145;Xis 90 anins 146;Xis 90 anins 147;X is 90 anins 148;Xis 90 anins 149;Xis90 anins 150;Xis 90 anins 151;Xis 90 and Yis 152;Xis 90 anins 153;Xis 90 anins s 90 anins 155; Xis 90 anins 156; Xis 90 anins 157;Xis 90 anins 158;Xis 90 anins 159;Xis 90 anins 160; Xis 90 anins 161; Xis 90 anins 162;Xis 90anins s 90 anins 164;Xis 90 anins 165;Xis 90 anins 166;Xis 90 anins 167;Xis 90 anins 168;Xis 90 anins s 90 anins 170;Xis 90 anins 171;Xis 90anins 172;Xis90 andY is 173;Xis 90 andY is 174;Xis 90 anins 175;Xis 90 anins 176;Xis 90 anins 177;Xis 90 anins 178;X is 90 anins s 90 anins 180;Xis 90 anin8181;Xis 90 andY is 182;Xis 90 and Yis 183;Xis 9O anins‘184;Xis 90 andY is 185;Xis90 anins 186; 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In one embodiment, X is 3 and Y is 5.

The term “one or more CDRs”, as used in connection with the above embodiments, refers to one of the CDRS HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and LCDR3 (e.g., HCDR3), or several (i.e., 2, 3, 4, 5 or 6) of the CDRs, such as a set of HCDRI, HCDRZ and HCDR3 or a set of LCDRl, LCDR2 and LCDR3. In a preferred embodiment, the term “one or more CDRs” refers to all six CDRs, i.e., HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and LCDR3.

W0 10584 The term “one or more le domains”, as used in connection with the above embodiments, ably refers to one of the VH domain and the VL domain or, more preferably, to both the VH domain and the VL domain.

The term “nucleic acid molecule”, as used herein, is ed to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.

A nucleic acid molecule may be -stranded or double-stranded. RNA includes in vitro ribed RNA (IVT RNA) or synthetic RNA.

In the context of the present invention, the term “DNA” relates to a molecule which comprises deoxyribonucleotide residues and preferably is entirely or substantially composed of deoxyribonucleotide residues. “Deoxyrihonucleotide” relates to a nucleotide which lacks a hydroxyl group at the 2’-position of a bofuranosyl group. The term "DNA" comprises isolated DNA such as partially or completely purified DNA, ially pure DNA, synthetic DNA, and inantly generated DNA and includes modified DNA which s from naturally occurring DNA by addition, deletion, substitution and/or alteration of one or more tides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a DNA or internally, for example at one or more nucleotides of the DNA. Nucleotides in DNA molecules can also comprise non-stande tides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides. These altered DNAs can be referred to as analogs or analogs of naturally-occurring DNA.

In the context of the present invention, the term “RNA” relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide es. “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2'—position of a B- D-ribofuranosyl group. The term includes double ed RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations 3O include on of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA. According to the present invention, the term “RNA” includes and preferably relates to “mRNA” which means “messenger RNA” and relates to a W0 2016/110584 transcript which may be produced using DNA as template and encodes a peptide or protein. mRNA typically comprises a 5‘ non translated region (5'-UTR), a protein or peptide coding region and a 3' non translated region {3‘-UTR). ...R A has a limited halftime in cells and in vitro, Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription or al synthesis. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available.

The nucleic acid molecule according to the t invention may be contained/comprised in a vector. The term “vector”, as used herein, includes any s known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral or baculoviral vectors, or ial some vectors such as bacterial artificial chromosomes (BAC), yeast artificial somes (YAC), or P1 artificial chromosomes (PAC). Said s include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the ly linked coding ce in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and y a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

The term “cell” or “host cell” ably relates to an intact cell, i.e. a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material. An intact cell ably is a viable cell, ie a living cell capable of carrying out its normal metabolic functions. Preferably said term relates according to the ion to any cell which can be transfected with an exogenous nucleic acid. Preferably, the cell when transfected with an exogenous c acid and transferred to a recipient can express the nucleic acid in the recipient. The term "cell" includes bacterial cells; other useful cells are yeast cells, fimgal cells or mammalian cells. Suitable ial cells include cells from gram-negative bacterial strains such as strains of Escherichia coli, Proteus, and Pseudomonas, and gram- positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylocowus, and Lactococcus. Suitable fimgal cell include cells from s of Trichoderma, Neurospora, and Aspergillus. le yeast cells include cells fi'om species of Saccharomyces (Tor example Saccharomyces cerevisiae), saccharomyces (for example Schizosaccharomyces pombe), Pichia (for example Pichia pastoris and Pichia methanolica), and Hansenula. Suitable W0 2016/110584 mammalian cells e for example CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK and the like. However, ian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can be used as well. Mammalian cells are particularly preferred for adoptive transfer, such as cells fi‘om humans, mice, hamsters, pigs, goats, and primates. The cells may be derived from a large number of tissue types and include primary cells and cell lines such as cells of the immune system, in ular antigen-presenting cells (APCs), such as dendritic cells, and T cells, stem cells, such as hematopoietic stem cells and mesenchymal stem cells, and other cell types. An antigen-presenting cell is a cell that displays antigen in the context of major histocompatibility complex on its surface. T cells may recognize this complex using their T cell receptor (TCR).

The binding agents of the invention can be produced either intracellularly (e. g. in the cytosol, in the periplasma or in inclusion ) and then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. Methods and reagents used for the recombinant tion of ptides, such as specific le expression vectors, transformation or transfection methods, selection markers, methods of induction of protein expression, culture conditions, and the like, are known in the art. Similarly, n isolation and purification techniques are well known to the skilled person.

The binding agents, c acid molecules or cells described herein may be administered in the form of any suitable pharmaceutical composition.

The ceutical compositions of the invention are preferably sterile and contain an effective amount of the binding agents, nucleic acid molecules or cells described herein to generate the desired reaction or the desired effect.

Pharmaceutical compositions are usually provided in a uniform dosage form and may be prepared in a manner known per so. A ceutical composition may, e.g., be in the form of a solution or suspension.

A pharmaceutical composition may further se one or more carriers and/or excipients, all ofwhich are preferably phannaceutically acceptable. The term "pharmaceutically acceptable", used herein, refers to the non-toxicity of a material which, preferably, does not interact with the action of the active agent of the ceutical composition.

W0 2016/110584 The term “carrier" refers to an organic or inorganic component, of a l or synthetic nature, in which the active ent is combined in order to facilitate, enhance or enable application.

According to the invention, the term “carrier” also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a subject.

Possible r substances for parenteral administration are, e.g., sterile water, Ringer, Ringer e, sterile sodium chloride on, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glyoolide oopolymers or p0lyoxyethylene/polyoxy—propylene copolymers.

The term “excipien ”, as used herein, is intended to include all sub s which may be present in a pharmaceutical ition and which are not active ingredients, such as salts, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, flavouring agents, or colorants.

Salts, which are not phannaceutically acceptable, may be used for preparing pharmaceutically acceptable salts and are ed in the invention. Pharmaceutically acceptable salts of this kind comprise in a non limiting way those prepared from the following acids: hydrochloric, hydrobromic, ic, nitric, phosphoric, maleic, acetic, salicylic, citric, , c, succinic acids, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts, Suitable vatives for use in a pharmaceutical composition include benzalkonium chloride, chlorobutanol, paraben and thimerosal.

Suitable buffer substances for use in a pharmaceutical composition include acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.

The agents and compositions described herein may be administered via any conventional route, such as by parenteral administration including by injection or infilsion. stration is preferably parenterally, e.g. intravenously, intraarterially, subcutaneously, intradermally or intramuscularly.

W0 2016/110584 Pharmaceutical compositions suitable for parenteral administration usually comprise a sterile aqueous or non-aqueous preparation of the active compound, which is preferably isotonic to the blood of the recipient. Examples of compatible carriers/solvents/diluents are Ringer solution and isotonic sodium de solution. In addition, usually sterile, fixed oils are used as solution or suspension medium.

The agents and compositions described herein are administered in effective amounts. An “effective amount” refers to the amount, which achieves a desired reaction or a desired effect alone or together with fiirther doses. In the case of treatment of a particular disease or of a particular condition, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease or of a condition may also be delay of the onset or a prevention of the onset of said e or said condition. An effective amount of an agent or ition described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and , the on of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors.

Accordingly, the doses administered of the agents described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses ed by a ent, more localized route of administration) may be used.

As used herein, the term “kit of parts (in short: kit)” refers to an article of manufacture sing one or more containers and, optionally, a data carrier. Said one or more containers may be filled with one or more of the above mentioned means or reagents. Additional containers may be included in the kit that contain, e.g., ts, s and fiirther reagents. Said data carrier may be a non-electronical data carrier, e. g., a graphical data can-ier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD), a l versatile disk (DVD), a microchip or r semiconductor-based electronical data carrier. The access code may allow the access to a database, e.g., an intemet database, a centralized, or a decentralized database. Said data r may se ctions for the use of the binding agent, nucleic acid molecule, cell and/or pharmaceutical composition of the present invention.

W0 2016/110584 The agents and compositions described herein can be administered to patients, e.g., in vivo, to treat or t a variety of disorders such as those described herein.

The term “medicament”, as used herein, refers to a substance/composition used in therapy, i.e., in the ent of a disease.

According to the invention, the term “disease” refers to any ogical state, in particular cancer, infectious diseases, inflammatory diseases, lic diseases, autoimmune disorders, degenerative diseases, apoptosis-associated diseases and transplant rejections.

As used herein, the term “cancer” includes a e characterized by aberrantly regulated cellular growth, proliferation, differentiation, on, and/or migration. By “cancer cell” is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. The term “cancer” according to the invention comprises leukemias, mas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal , endometrial cancer, kidney , adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, inal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, te cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof. Examples thereof are lung carcinomas, ma.-.rna carcinomas, prostate carcinomas, colon carcinomas, renal cell omas, cervical carcinomas, or metastases of the cancer types or tumors described above.

The term “cancer” according to the invention also comprises cancer metastases. By "metastasis" is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, on of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, afier being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor, i.e. a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even afier the removal of the y tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the ion relates to "distant metastasis" which relates to a asis which is remote from the primary tumor and the al lymph node system.

W0 2016/110584 The term tious disease” refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent (e. g. common cold).

Examples of infectious diseases include viral infectious diseases, such as AIDS (HIV), tis A, B or C, herpes, herpes zoster (chicken-pox), German measles (rubella virus), yellow fever, dengue etc. flaviviruses, influenza viruses, hemorrhagic infectious diseases (Marburg or Ebola viruses), and severe acute respiratory syndrome (SARS), bacterial infectious es, such as Legionnaire's disease (Legionella), sexually itted diseases (e. g. chlamydia or gonorrhea), gastric ulcer (Helicobacter), cholera (Vibrio), tuberculosis, diphtheria, infections by E. coli, Staphylococci, Salmonella or Streptococci (tetanus); infections by protozoan ens such as malaria, sleeping sickness, leishmaniasis; toxoplasrnosis, i.e. infections by Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or fungal ions, which are caused, e.g., by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces itidis or Candida albicans.

The term “inflammatory disease” refers to any disease, which is characterized by or associated with high levels of inflammation in tissues, in particular connective tissues, or degeneration of these s. A chronic inflammatory disease is a medical condition which is characterized by persistent inflammation. Examples of (chronic) inflammatory diseases include celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), ble bowel disease, atherosclerosis, arthritis, ankylosing spondylitis, Crohn’s disease, colitis, chronic active hepatitis, dermatitis and psoriasis.

The term “metabolic e” refers to any e or disorder that disrupts normal lism. es e cystinosis, diabetes, idemia, hyperthyroidism, hypothyroidism, hyperlipidemia, hypolipidemia, galactosemia, r’s e, obesity and phenylketonuria.

The term “autoimmune disorder” refers to any disease/disorder in which the body produces immunogenic (i.e. immune system) response to some constituent of its own tissue. In other words, the immune system loses its ability to recognize some tissue or system Within the body as self and targets and attacks it as if it were foreign. Autoimmune diseases can be classified into those in which predominantly one organ is affected (eg. hemolytic anemia and anti-immune thyroiditis), and those in which the autoimmune disease process is diffused through many tissues (6.g. systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells ing the sheaths that surround the nerve fibers of the brain and spinal cord. This results in loss of coordination, weakness, and blurred . Autoimmune diseases are known in the art W0 20161110584 and include, for instance, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus matosus, celiac disease, Crohn‘s disease soriasis and the like. 2 colitis, diabetes, scleroderma, 7 The term erative disease” refers to any disease in which the function or ure of the affected tissues or organs will increasingly deteriorate over time. Examples include Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Huntington’s e, macular degeneration, multiple sclerosis, muscular dystrophy, Niemann Pick disease, osteoporosis and rheumatoid arthritis.

The term “apoptosis-associated diseases” refers to any disease in which alterations of apoptosis are involved. Examples include cancer, neurological disorders, such as Alzheimer’s e, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS) and stroke, heart diseases, such as ischemia reperfusion and c heart failure, infectious diseases and autoimmune diseases.

The term “transplant rejection” refers to the ion of a lanted tissue or organ by the recipient’s immune system, which may, ultimately, destroy the transplanted tissue or organ.

By ” is meant to administer a compound or composition or a combination of compounds or compositions to a subject in order to t or eliminate a disease, ing reducing the size of a tumor or the number of tumors in a subject; arrest or slow a disease in a subject; inhibit or slow the development of a new disease in a subject; decrease the frequency or severity of symptoms and/or recurrences in a subject who currently has or who usly has had a disease; and/or prolong, i.e. increase the lifespan ofthe subject.

In particular, the term “treatment of a disease” includes curing, shortening the duration, ameliorating, preventing, slowing down or ting progression or worsening, or preventing or ng the onset of a disease or the symptoms thereof.

The term “subjec ” means according to the invention a subject for ent, in particular a diseased subject (also referred to as “patient”), including human beings, man primates or other animals, in particular mammals, such as cows, horses, pigs, sheep, goats, dogs, cats, s, guinea pigs, hamsters or rodents, such as mice and rats. In a particularly preferred embodiment, the subject/patient is a human being.

W0 2016/110584 2016/050308 The agents and compositions provided herein may be used alone or in combination with therapeutic regimens.

Examples Example 1: Generation of agonistic anti~CD27, anti—CD134 and anti-CD137 antibodies 1.1 Immunization and collection of B-cell clones 9 s were immunized with a mixture of human CD27 (Enzo Life es, ALX031- 0000), human CD134 (0X40; AdipoGen, AG-40B-0014) and human CD137 (4-lBB; Enzo Life Sciences, ALX029-0000), wherein each protein was fused to the Fc portion of human IgGl. Blood was taken at 4 different time points and enriched for B-lymphocytes (also referred to as B-cells). Single B-cells were sorted by fluorescence-activated cell sorting (FACS) into wells of a l microtiter plate and ated for 8 days, releasing antibodies into the cultivation medium. The B-cell tants were removed and prepared for testing. 1.2 Screening for target-binding antibodies by ELISA In order to identify antibodies that bind to human CD27, CD134 and CD137, reSpectively, enzyme-linked immunosorbent assay (ELISA) was ished. For this purpose, CD27 protein (His-tagged, Sino Biological Inc. 10039-H08B1), CD134 protein (His-tagged, Sino Biological Inc. 10481-H08H) and CD137 protein (His-tagged, Sino Biological Inc. 10041-H08H), respectively, were coated onto microtiter plates. PBS containing 2% BSA and 0.05% Tween was used for blocking. After incubation with B-cell atants, bound antibodies were detected by using a peroxidase-labeled anti-rabbit—Fc antibody.

Commercially available antibodies were used as reference dies for assay development.

Figure 1A shows binding curves of reference anti-CD27 antibodies (Armenian hamster monoclonal anti-human CD27 clone LG7F9 and mouse onal anti-human CD27 clone 0323, both sed from eBiosciences) to recombinant protein. Figure 1B shows binding curves of a reference anti-CD134 antibody (mouse monoclonal anti-human CD134 clone Ber— ACT35, purchased from eBiosciences) to recombinant n. Figure 1C shows binding curves of reference anti-CD137 antibodies (mouse anti-human CD137 clone 4B4—1 from Biolegend and rat monoclonal anti-human CD137 from Biozol BZL06614) to recombinant protein. For all three W0 20161110584 ELISAs, OD 450 urn/620 nm values > 0,5 indicated g, and the maximum value was at about 3,6.

In total, 47.471 B-cells were , cultivated and screened for binding to CD27, CD134 and CD137 by ELISA. 5.096 antibodies were fied to bind to at least to one of the three targets. 1.3 Identification of cell-binding antibodies The 5.096 ELISA hits were then tested in a cellular binding assay for binding to cell surface expressed CD27, CD134 and CD137, respectively. For this purpose, following stable ectants of the HT1080 cell line were used: HT1080_CD27, HT1080_OX40 and HT1080_4-1BB. Cells were ated in RPMI 1640 GlutaMAX supplemented with 5% PCS, 100 IU/mL penicillin, and 100 ug/mL streptomycin. Cell surface expression ofTNF receptors on HTlOSO-transfectants was analyzed by FACS, n cells were stained using antibodies against CD27-PE (BD), OX40-PE (BD) and 4-1BB-PE (BD) (Figure 2).

In order to test cellular binding of the antibodies, HTlOSO cells overexpressing the respective target (1.000 cells per well of a 384-we11 microtiter plate) were incubated together with samples for 18 hours. After gentle g, an labeled detection antibody and Hoechst dye were added, and fluorescence intensity was read in the cell imager Celllnsight (Thermo). Data analysis was based on the calculation of the mean object spot average intensity. cially ble antibodies were used as reference antibodies for assay development.

Figure 3A shows binding curves of reference anti—CD27 antibodies (Armenian r monoclonal anti-human CD27 clone LG7F9) to HT1080_CD27 cells. Figure ""1 shows bindingVJ" curves of reference anti-CD134 antibodies (mouse monoclonal anti-human CD134 clone Ber- ACT35) to HT1080_OX40 cells. Figure 3C shows binding curves of reference anti-CD137 antibodies (mouse anti-human CD137 clone 4B4—1) to HT1080_4-1BB cells. For all three cell lines, a mean spot intensity > 25 indicated binding, and the maximum values were at about 300. 3.176 antibodies out of the 5.096 ELISA hits were identified to bind at least to one of the three cell surface expressed targets by the cellular binding assay. 1.4 Recombinant antibody production W0 10584 644 antibody hits, identified by ELISA and the cellular binding assay as described above, were selected. These hits were picked, their RNA was extracted, and sequencing was performed. 582 unique sequences were identified, and the corresponding variable regions of heavy and light chains were gene synthesized. The variable regions were cloned in front of human immunoglobulin constant parts (IgGl/K). HEK293 cell transient transfections protocols were executed by a Tecan Freedom Evo device on a Dionex Ultimate 3000 with a plate auto-sampler.

Alter normalization, 531 dies were available for confirmation of the r , screening data set , cellular binding) and for functional assays. 1.5 Analysis of recombinantly expressed antibodies In order to confirm the primary screening data set, all 531 recombinantly produced antibodies were retested (i) for their binding to the correSponding inant protein in the ELISA and (ii) for their g to the corresponding target expressed on the cell surface of HT1080 cells in the cellular binding assay (both assays as bed .

Furthermore, for the analysis of agonistic antibody functions, target-overexpressing HT1080 cells were used, wherein ation of the targets (CD27, CD134 and CD137, tively) induced IL-8 release from these cell lines. The recombinant CD27-, CD134- and CD137- overexpressing HT1 080 cells (5.000 cells per well of a 384-well microtiter plate) were incubated for 6 hours at 37°C with the antibody samples. IL-8 concentrations in the supematants were then determined by using an IL-8 ELISA kit (BD Bioscience). In a second ch, antibodies were cross-linked. Therefor, the target-overexpressing HTIOSO cells were incubated with a mixture of the antibody sample and a F(ab ’)2 fiagrnent goat anti-human IgG (Dianova). An agonistic reference anti-CD27 antibody {described in US 2013/0183316 A1 (sequences 3 and 4, hCD27.15)] was used for assay development. Figure 4 shows results of the IL—8 ELISA upon antibody ion with and without cross-linking. For all three IL—8 ELISAs, OD 450 nm/620 nm values > 0,5 indicated agonistic antibody activities, the maximum values being at about 3. 3O ther, 186 agonistic anti-CD27 antibodies, 92 agonistic anti-CD134 antibodies and 12 agonistic anti-CD137 antibodies were identified by the fianctional assay under cross-linking conditions. To some extent, antibodies already showed agonistic activity t cross-linking.

The data of the ELISAs, the cellular binding assays and the functional assays are summarized in Table 1 for the agonistic anti-CD27 antibodies, in Table 2 for the agonistic anti-CD134 antibodies and in Table 3 for the agonistic anti-CD137 antibodies.

W0 2016/110584 ECSO values of the agonistic on of anti-CD27, anti-CD134 and anti-CD137, respectively, upon cross-linking were determined by measuring IL-S release values of 12 different antibody concentrations applied in the above—described functional assay. The ECSO value was calculated using Xlfit-analysis tools (Fit Models 205). Resulting typical sigmoidal curve progression is exemplarily shown in Figure 5 for one anti-CD27 antibody (P018.SO9.A.A06; ECSO: 387,2 ng/mL), for one anti-CD134 antibody (P018.SO2.A.H03; EC50: 214,9 ng/mL) and for one anti- CD137 antibody (P018.SOS.A.EO3; ECSO: 4963,8 ng/mL). ECSO values of the dies are listed in Table 4 (anti-CD27), Table 5 (anti-CD134) and Table 6 (anti-CD137). ce information of the tested antibodies are listed in Table 7 CD27), Table 8 (anti- CD134) as well as Table 9 (anti-CD137), wherein the sequences of the CDR regions are given ing to the IMGT unique numbering scheme for V— and V-like—domains (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999)).

W0 2016/110584 Cellular g Functional assay assay Anti-C027 Antibody ID [abs. corn] Cross-linked by Fiab'n fragment lL-8 [abs. corn] .01.A.GO4 3,765 388 0 273 P018.S.04.A.D04 0 486 P018.S.01.A.GOZ 0 848I P018.S.07.A.BO4 ‘ 0 304 P018.S.06.A.303 424 P018.S.03.A.H04 0,211 .08.A.H02 0,328 P018.S.09.A.H03 0 578I P018.S.09.A.E06 307 018.8.09.A.C03 0,464 P018.S.07.A.F03 3,528 380 0,444 1,413 P018.S.03.A.C04 3,524 0 394 P018.S.09.A.E05 P018.S.O4.A.E02 m P018.S.01.A.BOS 37 m6 1,882 P013.S.04.A.B05 414 1,084 P018.S.01.A.GO3 P018.S.10.A.F05 P018.S.02.A.BOS 0 201 1,761 P018.S.04.A.GO4 3,479 P018.S.07.A.H02 3,471 0,107 P013.S.05.A.H02 3,469 336 0 736’ O 302 O 448 . . . 379 1,230 394 1,120 448 1,570 P018.S.06 A A03 387 W0 20161110584 'Po13.s.10.A.c06 P018.S.OZ.A.HOS —_“_— __mum P018.S.03.A.D02 PbNN I.”NN 9WHU'I .07.A.602 lp0| 9UItoM P018.S.03.A.F04 W0 20161110584 P018.S.06.A.E02 P018.S.10.A.EOI 227 0,257 P018.S.01,A.EO3 3,297 1,220 P018.S.01.A.H02 0,300 P018.S.01.A.C06 0,325 fw8 P018.S.05.A.F02 472 P018.S.03.A.E04 0,200 P018.S.04.A.A05 .07.A.C06 0,408 1,044 —P018.S.09.A.DOS 0,187 0,262 _m 0,444 _m- 0,264 0,123 0,394 1,971 P018.S.09.A.E03 3,272 334 0,099 1,545 P018.S.01.A.D02 3,258 323 0,675 1,667 P018.S.10.A.B06 0,455 P018.S.10.A.H02 1,573 .02.A.803 0,120 P018.S.01.A.C02 0,395 .O7.A.H05 —0,160 1.186 P018.S.01.A.A04 0,236 P018.S.01.A.C04 0,383 P018.S.02.A.F06 0,094 1,728 P018.S.06.A.H04 U)3 9.. 0, 173 .06.A.C02 P018.S.06.A.A02 0,737 P018,S.02.A.A01 0.400 P018.S.01.A.E05 0,209 P018.S.03.A.D03 3,216 0,281 P018.S.08.A.A03 W4N 0,276 P018.S.10.A.C03 0,079 P018.S.03.A.G03 P018.S.O7.A.E05 3,198 31 0,055 1,151 P018.S.03.A.D06 0,245 P018.S.01.A.C05 0,327 P018.S.02.A.GO4 3,188 0,205 1,674 P018.S.10.A.B01 0,080 1,037 P018.S.10.A.A06 -0, 130 P018.S.06.A.F04 0,678 P018.S.09.A.A05 1,953 P018.S.04.A.C05 3,173 0,382 P018.S.01.A.606 3,17 1 313 0,491 1,088- P018.S.06.A.F06 3,170 0,700 W0 20161110584 P018.S.09.A.A03 P018.S.06.A.E04 281 0,327 1,223 P018.S.06.A.FO3 3,157 P018.S.07.A.GO4 p\l PN\lb P01 .S.10.A.E04 P018.S.03.A.H02 0,130 1,071 P018.S.03.A.H03 iInNN P018.S.03.A,F03 P018.S.01.F\.A05 3,148 0,328 1,086 .06.A.C03 0,272 1,131 P018.S.10.A.C05 11 P018.S.01.A.D05 P018.S.03.A.BOS "m:- .10.A.A04 1,778 2,159 P018.S.05.A.C06 3,132 P018.S.02.A.GO3 P013.S.07.A.C02 P018.S.02.A.A06 0,299 1,476 P018.S.01.A.EOZ P018.S.09.A.F05 P018.S.09.A.GOS P018.$.03.A.D05 P018.S.10.A.BOZ ‘Op\10 1,907 P018.S.04.A.E05 P018.S.05.A.A05 0,324 1,582 P018.S.05.A.D03 9:pO01 1,278 P013.S.08.A.F01 P018.S.05.A.306 P018.S.03.A.DO4 3,098 P018.S.01.A.A06 P018.S.09.A.A06_mm“ P018.S.07.A.A02 3.081 314 m P018.S.O7.A.D05 _-z-m P018.S.05.A.E03 “ .10.A.E02 n— P018.S.03.A.GOS 0,031 P018.S.09.A.EOZ 0,172 P018.S.09.A.F06 0,530 P018.S.06.A.H05 .10.A.H05 P018.S.06.A.A05 P018.S.09.A.A02 P018.S.09.A.C02 P018.S.06.A.B01 0,106 1,176 P018.S.10.A.C01 P018.S.05.A.D06 W0 20161110584 P018.S.02.A.B01 m P018.s.1o.A.c02m P018.S.10.A.FO4 2,889 P018.S.10.A.A03 Cellular g Functional assay assay Anti-CD134 Antibody ID Cross-linked by F(ab')z fragment IL-S [abs. corn] 419 0,314 rrrrr P018.S.05.A.GO3 —-m- P0185 04 A A06 P013 5 04 A F06 2,358 402 2,071 P018 5 11 A C04m 406 0,253 P0185 12 A H05 . 527 QWJ:00 2,190 —m570 2,171 P018.S.12.A.F05 m —m537 —m 5)OJOJ0 —-m- ——1 898 P0188 12 A A01m 0,071 P018 5 11 A F04 m 486 0,568 P018.S 04 A 304 P0185 12 A cos m P0188 12 A 801m W0 10584 P018.S.12.A.AO6 P018.S.11.A.E02 2,652 525 0,214 V 21043 P018.S.11 A A03 "Ia-m P018.S.12.A.BOZ_“ 0,157 1,955 “mm-m“ _-m- P018 5 12 A E02 2,589 497 P018 5 11 A H04 P018.S.13 A 605 0,002 P018.S.12.A.A02 1,577 .

P018.S.04.A.F04 0,000 Table 2. Binding and functional ty properties of agonistic anti-CD134 antibodies ar binding Functional. assay Anti-c0137 Antibody ID Cross-linked. by F(ab’)z fragment IL-8 [abs. corn] P018.S.13.A.A05 0,687 P018.S.14.A.H01 2 968I P018.S.08.A.E03 m 9 P018.S.13.A.GO4 0. 1 P018.S.13.A.A06 3 388l P018.s.os.A.A04m P018.S.14.A.D06 Table 3. Binding and onal activity properties of agonistic anti-CD137 antibodies Anti-C027 EC 50 [ng/mL] Antibody ID W0 10584 P0185. 10A. E04 233,6 P018.S,09.A.H06 329,1 P018.S.10.A.AO4 334,7 P018.S.10.A.C05 340, 1 Po1‘s.‘s.05.A. F05 P018.S.09.A.A06 P013..s.o4.A.Aos P018.S.10.A.C02 P018.S.07.A.GOZ P018.S.10.A.H01 P018.S.09.A.E05 P018.S.10.A.A03 P018.S.02.A.GO3 P018.S.09.A.DOS .0.6.A.A02 P018.$.10.A.E01 P018.S.07.A.CO4 P018.S.03.A.A02 834,9 Table 4. ECSO values of agonistic anti-CD27 antibodies Anti-(3313Al EC 50 [nglmL] Antibody ID mam-Bus P018.5.02.A.H03 ' 214,9 W0 20161110584 P018.S.05.A.GOS P018.S.12.A.F03 P018.S.11.A.A05 P018.S.12.A.DOS 2401,8 P018.S.12.A.DOG P018.S.05.A.F05 7119,8 Table 5. B050 values of agonistic anti-CD134 antibodies Anti-C0137 EC 50 [ng/mL] Antibody ID P018-5.08-A-503 P018.S.08.A.A04 5152,23 99185.1 .A.H01 5818.0 P018.S.14.A.A04 6789.0 P018.S.13.A.A06 7785,1 P018.S.14.A.DO6 0 P018.S.13.A.GO4 ,8 .

Table 6. ECSO values of agonistic anti—CD137 antibodies WO 10584 . SEQ ID NOs of CDRs SEQ “3 "05 0333:1444 °°mams Anti-CD27 Antibody ID HCDR1/HCDR2/HCDR3 LCDRI/LCDRZ/LCDR3 , ,4 373 / 374/ 375 ASL—1 P"18.5.01.A.A04 “2 376/377/378 379 I 380/ 381 A3273 1 P018.S.01.A.A05 3/4 382 / 383/ 384 -_—273 P018.S.01.A.A06 385/386/387 6 4444444// 391 / 392 / 393 ABZ7_4 P018 s 01 A 303. . . . 7/8 I 394/395 ,/ 396 I 397 / 39s / 399 I AB2 _S7 P018501A30. . . . 4 9/10 400/401/402 -__403 /404 / 405 7 6 P018.s,o1.A.305 11 12 406/407 / 408 409 /410 / 411 7 7 P018.S.01.A.C 1 14 412 / 413 I414 415 I416 / 417 AB27 8 P018. 5 0. 1.A.C04 15 16 " I 418 I419 / 420 421 /422 / 423 A327 9 P018. .01.A.C 17 / 18 444/4244/ 427 / 428 / 429 AB27 10 P018 5. .01.A.C06 19 20 - / 430/431/432 22:;121224: 2244:2122: " " A32 _167 P01850. . 1.14.504 31 / 32 463/464/465 . 466/467/468 469 [470 / 471 A827 17 P 1 . . 1.A.E 5 33 34 AB27 P° 85°1 6] 477 38 . . 1.A.EOG 35 36 473/479/480 A327 19 P 481/ 482/ 483 085°1 . . 1.A.F02 37 38 - 484/485/486 487 / 488 / 489 AB27_20 P013501AF05. . . . 39/ 40 490/491/492 W0 2016/110584 _538/ 539/540 AB27 29 P018.S.02.A.B01 541/ 542/543 _ 57 /58 — 544/ 545 I 546 547 I 548 I 549 ABZ7_30 P018.S.02.A.303 59/ 60 — 550/551 I 552 P018.S.02.A.805 553/ 554 I 555 7 I P018.S.02.A.601 ‘ 73/74 233/2323: ] 595 I 596 I 597 ABZ7_38 P018.S.02.A.603 75 I76 598 / 599/ 600 601 / 602 I 603 ABZ7~39 P018.S‘.02.A.GO4 77/ 78 , 604 I 605 I 606 AB27_40 .02.A.H05 607/ 608 I 609 79/80 610/ 611 I 612 1 613 / 514 I 615 P018.S.03-.A.A02 81 I' 82 616 I 617 / 618 ABZ7_42 619 I 620 / 621 P018.S.03.A.BOZ 83/ 84 622 I 623 I 624 AB27_43 625 / 626 I 627 P018.S.03.A..BDS 85/86 628 I 629 I 630 ABZ7_44 -- P018.S.03<A.C02. . . 631/632/633 87/88 634/ 635 I 636 AB27_45_P018.-S.03.A.C04 637/ 638 I 639 89/ 90 640/ 641 I 642 P018.S.03.A.GOZ- ——_-1Em_ W0 2016/1 10584 _——712/713/714 715 / 716 / 717 ABZ7~58 P018.S.03.A.G05 115 / 116 718 / 719 / 720 721/ 722 / 723 ABZ7_59 P018.S103.A.G(}6 117/ 118 724 /725/726 ABZ7_60 . 9018.S.03.A.!-!02 119/120 3/729 730/731/732 A327 61 P018'.S.03.A.HO3 733/734/735 121 122 - _—73677377733 ABZ7_62 P018.S.03.A.HO4 123 / 124 i ’1 740/. 741 - 742 , 743; 744 745 / 746 I 747 1182163 P018.S.03..A.H05 125/ 126 748 / 749 / 750 751/ 752 / 753 AB27_54 P0181S.041A.A01 127 / 128 754 / 755 / 756 760 / 761/ 762 763 l 764 I 755 A327_66 P018.S.04.A.B,01 131 / 132 766/767 / 763 769 / 77o / 771 A827 67 .04.A.BO3 133 134 - / 772 / 773 / 774 85:23:22 3:282:82: ABZ7_71 P018.S.04.A.D02 $333133: A827_72 P018.s.04.A.Do4 gag/Eggjggi 282; 811/ 812/ 813 A327 74 P018.S.04.A.E02 147 4 _ l1 8 814 / 815 /' 816 817/ 818 / 819 ABZ7_75 P018.S.O4.A.EO4 14g / 150 820/ 821 / 822 823/ 824 / 825 AB27 76 P018.S.04.A.EOS 151 152 829/830 / 831 ABZ7_77 P018.S.04.A.F04 153 / 154 832/ 833 / 834 835 / 836 / 837 ABZ7_78 P018.S.O4.A.GO4 155 / 156 838/839 / 840 841 / 842 I 843 A327 79 P018.S.04.A. 157 153 847 / 848 / 849 A827_80 P018.S.05.A.A02 850/ 851 / 852 853/ 854 / 855 A827 81 P018.S.05.A.A04 1 1 152 859/ 860/ 861 ABZ7_82 .05.A.A06 163 / 164 862/ 863 / 864 865 / 866 / 867 A827 83 P018.s.05.A.302 155 165 871 / 872 / 873 AB27_84 P018.S.OS.A.B06 167 / 168 874 / 875 / 876 877 /878 / 879 ABZ7_85 P018.S.05.A.C06 159 / 1.70 880/ 881 / 882 ABZ7_86 P018.S.05.A.D03 171 / 172 883 I 884/ 885 10584 ——886 / 887/888 889 / 890 I891 A32 _877 P018.S.05.A.DO4 173/ 174 892 i 893 {894 895 / 896/ 897 ABZ7_88 P018.S.05.A.DOG 175 / 176 898 / 899/900 A2w3 7 89 P01. 8.5.05.A.E03 177 I18_. 901 /902/903 904/905/906 907 / 908/909 A327 90 P 18.5. .A.E04 17 ‘ 9/180 910/ 911/ 912 913 /914 /915 ABZ7_91 P018.5.05.A. F02 181/132 ; 916/91?/918 mm . 3:343:93; 212292;; 2:333:32: 323222532: 943 / 944/ 945 A827_96 P018.s 06 A A03 ’ . ., . 191 , 192 946 / 947 / 948 949 / 950 /951 Aa27 97 P018.S.-06.A.A 50 _ 193/194 952 / 953/954, 32722322: 3:212:29: 3:212:72: 22:92:92: 985 / 986 / 987 ABZ7_103 .0018,5.06 A 004. . 205" 205 988 / 989/ 990 991/ 992/ 993 A 2_B 7 104 .06 .A. E01 207/208 /996 99? / 998 / 999 ABZ7_105 P018.S.06.A.E02 209/ 210 1000/ 1001/1002 1003/ 1004/ 1005 06 P018.S.06.A.E04 211 / 212 1006 I 1007 / 1008 13232312131: 13131319132; 131132232: 1322;133:132: 1222;132:232: 1323;132:022: 1322;132:1131; 1051/1052 / 1053 4 P018.S.07.A.AOS 227 I 228 1054/1055 / 1056 ABZ7_115 P018.5.07.A.303 229 / 230 1057 / 1058 / 1059 W0 2016/1 10584 —__— ABZ7__116 P018.S.07.A.BO4 231 / 232 I 10 AB27_118 .07.A.806 13;: / 10:: j 12;; ABZ7_121 P018.S.07.A.C06 241 / 242 :33: j :33: 5 :33: 1099/ 1100 / 1101 ABZ7_122 P018.S.07.A.D03 243 I 244 1102 / 1103/ 1104 1105 / 1106 / 1107 ABZ7_123 .07.A.DO4 245 / 246 1108 / 1109 / 1110 I ABZ7_124 , P018.S,07.A.DOS 1111/ 1112 / 1113 1 247/ 248 1114 / 1115 / 111 1117 / 1118 / 1119 A827_125 P018.S407.A.E03 249/250 1120 / 1121 / 1122 1123 / 1124/ 1125 AB27H126 P018.$.07.A.E05 251 I 252 1126 / 1127/ 1128 1129 / 1130 / 1131 AB27_‘127 P018.S.07.A.F03 253 / 254 1132 / 1133 / 1134 1135 / 1136 / 1137 ABZ7_128 P018.S.07.A.F04 255 / 256 1138 / 1139 / 1140 1141/ 1142 / 1143 ABZ7_129 P018.S.07.A.602 257/ 258 1144 I 1145 / 1146 ABZ7_130 P018.S.07.A.GO4 2 1147/ 1148/ 1149 59 / 260 1150/ 1151 / 1152 4927 131 02 I 1153 / 1154/ 1155 P018.S.O7.A.H 261 262 _ / 1156 / 1157 / 1153 1159 / 1160 / 1161 AB27 132 P018.5.07.A.H04 263 264 — I 1162 / 1163/ 1164 AB27 133 P018.S.07.A.H05 1165/ 1166 / 1167 265 266 - l 1168 l 1169 I 1170 1171 / 1172/ 1173 4927 134 P018.S.07.A.H06 267 / 268 - 1174/1175 /1176 1177 / 1178/ 1179 4327 135 P018.S.08.A.A01 269 - / 270 1180/ 1181/ 1182 1183 / 1184/ 1185 4927 136 .08.A.AO3 271 272 ‘ _ l 1186 I 1187/ 1188 4327 137 P018.S.08.A.DOI 1189/ 1190/ 1191 273 274 ‘ _ / 1192 / 1193 / 1194 1195/ 1196/ 1197 ABZ7_138 P018.S.08.A.DOZ 275 / 276 1198 / 1199/ 1200 1201 / 1202/ 1203 A827 139 P018‘S..08.A.E02 277 278 - I 1204/ 1205 / 1206 1207 / 1208/ 1209 4327 140 P018.5.08.A.F01 279 / 280 - - 211/1212 4927_141 P018.S.08-.A.GOZ 1213/ 1214/ 1215 281/282 1216 / 1217 / 1218 1219 l 1220/ 1221 4327_142 P018.S.08..A.H02 283 / 284 1222/ 1223/ 1224 1225 l 1226 l 1227 4827_143 P018.s.09.4.402 235 / 286 1228 / 1229/ 1230 ABZ7_144 P018.S.09.A1A03 287/ 288 1231 / 1232 / 1233 W0 20161110584 _——— A327_145 P018.S.09.A.AOS 289 / 290 - 11111111151111 1111111111111 | 1 11111111151111 A327_151 P018 s..09 A DO. » 1273/ 1274 / 1275 . 4 301/ 302.- 1276 / 1277/ 1278 A327 152 P018.S.09.A.D 1279 / 1280/ 1281 OS . ' 303/3 04 . 1282/1283/1284 11111111111111 AB27_155 P018.S.09.A.EOS 309 / 31? 1:335 1:321; / 1:3: 56 P018.S.O9.A.E0‘6 311/312 1:3: ; 33:; :33: '. ' AB27_157 P0185309.A.F05 4. » 1309/1310/1311 1312/1313/1314 AB27 158 P018.5 09..A.F06 315 / 31. 1315/ 1316 / 1317 ~ 6 1318/ 1319 / 1320 AB27 159 P018.S.09.A. .. 1321/ 1322 / 1323 - GOG 317 / 318 1324/ 1325 / 1326 AB27 160 P018 s 09. .A.H0 1327/ 1323 / 1329 . 3 31 v - 9 l 320 1330/ 1331 / 1332 ABZ7_161 .09.A.H04 1333 I 1334 / 1335 321 / 322 1336/ 1337/ 1338 A327 162 P018.S.09.A.H06 1339/ 1340 / 1341 - 323 I 324 1342/ 1343/ 1344 A327_163 11111111111111 AB27_164 11111111111111 ABZ7_165 11111111111111 ABZ7_166 11115111111111 AB27_167 11115111111111 AB27_168 P018.s.10.A.303 Egg/1:331:33 ABZ7_169 11115111111111 f ' ' ABZ7¢170 P018 S 10.A.C01 339 I 3401 1387/1388/ 1389 1390/ 1391/ 1392 AB27 171 .10.A.C02 4, 1393/ 1394/ 1395 - _ 3 1’ 342 1396/1397/ 1398 1399 / 1400 / 1401 AB27 172 P018 s. 10.4.003 4 344_ - ' 3 3’ 1402/1403 / 1404 73 P018.S.10.A.C05 mm 1405/ 1406 / 1407 W0 2016/110584 — 1408 / 1409/ 1410 1411 / 1412/ 1413 A327 174 P018.S.10.A.C06 347 348 . 1417 [1418/ 1419 A327 175 P018.S.10.A.E01 349 ’ 350 - 1420 / 1421 / 1422 1423 / 1424 / 1425 A327 176 P018.S.10.A.EOZ 351 352 1429 / 1430/ 1431 A327 177 P018.S.10.A.E04 353 354 1435 / 1436/ 1437 4327 178 P018.S.10.A.E06 355 356 1441 / 1442 / 1443 A327 179 P018.S,10.A.F01 57 358 1447 / 1448 / 1449 A327 180 .10.A.F04 359 360 1453 / 1454/ 1455 A327 181 P018.S.10.A.F05 361 / 362 _ 1456/ 1457/ 1458 1459/ 1460/ 1461 A327 182 P018.S.10.A.GU3 363 364 I ‘ I l 1462/ 1463/ 1464 1465 / 1466/ 1467 A327 183 P018.s..10.A.c-ios 365 366 1458/1469,1470 1471 / 1472/ 1473 A 7 1 4 P° 851 . .10.A.H01 367 368 14747147571476 1477/ 1473/ 1479 A327_185 301331041402 369/370 1480/ 1481/ 1482 -—-1483 / 1484 / 1485 AB27 186 P018.S.10.A.H05 371 372 14867148771488 Table 7. Sequence information of agonistic anti-CD27 antibodies ble domains and CDRs) "3 "05 °f CDRS SEQ ID NOs of Variable Domains Short iD Anti-C5134 Afiiibfidv i0 HCDRZ/HCDRB, EEO VHIVI.

LCDR1/LCDR2/LCDR3 1673 / 1674 / 1575 A3134 1 P018.S.02.A.F01 1439 1490 ——_15767167771678 1679/ 1530/ 1681 A3134_2 P018.S.02.A.FO3 1491/ 1492 1682 / 1683 / 1684 1688 / 1689 /1690 1691/ 1592 I 1593 AB134_4 P018.S.03.A.E03 1495 / 1496 1694/ 1695 / 1696 1697 I 1698 / 1599 A3134_5 P018.S.04.A.A06 1497 / 1498 1700 / 1701 / 1702 1706 / 1707 / 1708 1709/ 1710/1711 A3134_7 P018.S.04.A.806 1501/ 1502 1712/ 1713/ 1714 1715/ 1716’ 1717 A3134 3 P018.S.O4.A.C06 1503/1504 7 1718 / 1719 / 1720 1721 / 1722 / 1723 A3134 9 .O4.A.D06 1505 1506 725/1726 1730/ 1731/ 1732 1733 / 1734/ 1735 A3134_11 P018.S.04.A.E06 1509 / 1510 1736/ 1737/ 1738 1739 / 174° / 1741 AB134_12 P018.S.04.A.F05 1511 / 1512 1742/ 1743 / 1744 W0 2016/1 10584 -— 1745/ 1746/ 1747 3134 13 P018.S.04.A.F06 1513/ 1514 1751] 1752/ 1753 B134 14 P018.S.04.A.GOS 1515 l 1516 1754/ 1755/ 1756 1757 / 759 A8134 15 P018.5704A Hos. . 1517 ‘ / 1518 1760/ 1761/1762 1763/ 1764/ 1765 A3134 16 P013.S.05.A.F05 1 20 " 519/15 1766 / 1767/ 1768 -——1769/ 1770/ 1771 A3134 17 P018.S.05.A.603 1 1 1522 , , PO18.5.05.A,605 ' 1523/1524 2; 1331;323:332 3:233:13: . 1799 / 1300/ 1301 A313 324 P018.S.11.A.A05 153 1 l 1532 1302/ 1303 / 1804 1847/ 1848/ 1849 A3134 30 P018.S.11.A.003 1547 I 1548 _ 1850 / 1851/ 1852 ABI34~35 P018.S.11.A.E04 1557/ 1558 AB134_36 P018.S.11.A.E05 1559 I 1560 “3134-40 AB134_41 P018‘S.11.A.F05 1559 /'1570 W0 2016/1 10584 1919 / 1920/ 1921 34 42 P018.S.11.A. 02 1571 1 7 7 , 1925 / 1926/1927 A3134 43 P018.S.11.A 603. 1573 / 1 745 1928/ 1929/ 1930 1931 / 1932 ,I 1933 3134 44 P018.5.11.A.Go4 1575 157 1937 x 1938 / 1939 A3134 45 P018511.A605. 1577 / 1578 1940 /1941 /1942 -— 1943 / 1944/ 1945 A3134 46 P018.S.11.A.H02 1579 / 1580 1946 /1947 [1948 . . 1949 I 1950 I 1951 A3134 47 P018.S.11.A.H04 1581 1582 ' ' _ / 1952 / 1953 / 1954 I 1957 A3134 48 1955/ 1956 .11.A.H05 1583 1584 ' 1958 / 1959 / 1960 -_—1961 / 1962 / 1963 A3134 49 P018.S.11.A,H06 1585 1586 , , 1967 / 1968 / 1969 A3134 50 P018.S.12.A.A 1 1587 1588 1970/ , 1973 / 1974 / 1975 A3134 51 P018.S.12.A.A0 2 1589 I 1590 1976 I 1977 / 1978 -_—. 1979/ 1980/ 1981 A3134 52 P018.S212.A.A03 1591 1 92 , , 1985 i 1986/ 1987 A3134 53 P018.S212.A.A 40 1593 1 4 59 _ l 1988/1989/ 1990 22:2222: 22222222252222 2222;222:2222: 2222222252222 2075/2076 / 2077 A3134 68 P018.S.12.A.D03 1623 1624 ‘ / 2078/2079 / 2080 A3134 69 P018.S212.A.D04 2081/ 2082 / 2083 1625 1626 2087/ 2088/ 2089 A3134 70 P018.S.12.A.DOS 1627 -— I 1628 220,291,212 W0 2016/110584 2016/050308 P018.S.12.A.E02 1633/ 1634 AB134 70~ - , "018. :2.- __ 2123/2124/2125 r >1 A505.3 540 ' ' 2126/2127/2128 I P018.S.12.A.E06 1641 / 1642 2129/ 2130 / 2131 2132 / 2133 / 2134 2135/ 2136/ 2137 2138/ 2139/ 2140 ' ' AB134_30 P018.S.12.A.F04 1547/1648 3:3; 3:3; :12: I AB134_81 P018.s.12.A.I=05 1649 / 1650 AB134_82 P018.S.12.A.F06 AB134_83 P018.s.12.A.601 AB134_84 P018.S.12.A.GOZ A3134_85 .12.A.G03 ABI34_86 P018.S.12.~A.G 40 ‘ 2183 / 2184 / 2185 1659 / 1660' " 2186 / 2187 / 2188 7 2189/ 2190 / 2191 1661 l 1662 2192/ 2193 / 2194 I | 33331333313333 AB134_90 P018.S.12.A.‘H04 ABI34_91 P018.S.12‘.A.H05 A8134_92 P018.S.13.A.»GO$' Table 8. Sequence information of agonistic anti-CD134 antibodies (variable domains and CDRs) SEQ '0 NOs 1:73?” ”mam. . SEQ ID NOs of cons Anti-C0137 Antibody ID HCDRI/HCDRZ/HCDR3 LCDRl/LCDRZ/LCDRS AB137_1 P018 s 03 A A0. 4 2249/ 2250 / 2251 . 2225/ 2226 2252 / 2253 / 2254 P018308 A E03 2227 2255/2256 / 2257 [2228 2258/2259/2260 A8137 3 P018.5.08.A.F03 2261/ 2262 / 2263 2229/ 223° 2264/2265 [2266 AB137_4 P018.S 13 A A05 m 2267/ 2268/2269 _——2270/ 2271/ 2272 ' 2273 / 2274/ 2275 A3137 5 P018.S.13.A.A06 2233 2234 -_ / 2276/ 2277/ 2278 22791228012281 P018.S.13.A.C02 2235 I 2236 2282 / 2233 / 2284 _ ._ , 2285 ,l 2286 / 2287 I 9018 S. . .13 .A.603 2237 / 2238 2288 / 2289 l 2290 -—_2291 / 2292 / 2293 A 137 8 P018.S.13.A.GO4 2239 2240 2294 / 2295 / 2296 I 2297 / 2298 / 2299 ”137-9 P018 s. .14.A.A 40 2241 I 2242 2300 / 2301 / 2302 2303/2304/2305 A3137w10 P013 S 14. . .A.D06 2243 l 2244 2306 / 2307/ 2308 l 2309/ 2310 / 2311 A31 7-113 ms 5 1. . 4.A 603. 224 224 2312 / 2313/ 2314 -——2315 I 2315 / 2317 37 12 P018.S.14.A.H01 2247 2248 2318/2319/2320 Table 9. Sequence information of agonistic anti-CD137 antibodies (variable domains and CDRs) Exam 1e 2: Bis ecific antibodies tar etin CD40 and CD27 CD40 and CD137 or CD40 and CD134 2.1 Reporter assay measuring trans-activation CD40 is inantly expressed on antigen-presenting cells (APCs), such as dendritic cells, whereas CD27, CD134 and CD137 are predominantly expressed on T cells. Thus, ific antibodies binding to CD40 and CD27, to CD40 and CD137 or to CD40 and CD134 are able to bind simultaneously to APCs and T cells expressing these receptors. Thereby, these bispecific antibodies are able (i) to mediate cell—to-cell interaction between APCs and T cells by or binding and (ii) to activate both indicated receptors at once, which is primarily induced by cross- linking and receptor ring upon cell-to-cell interaction and not necessarily dependent on agonistic activity of the parental monospecific bivalent antibodies. Thus, these trans-activating bispecific dies exert costimulatory activity in the context ofAPC : T cell interactions.

A corresponding reporter assay system was established. To that end, HEK293_NFK_gfp_luc cells were stably transduced with human ”INF-receptors, CD27, CD40, CD134 and CD137, respectively. HEK293_NFK_gfp_luc B/293/GFP-LucTM Transcriptional Reporter Cell Line obtained from System ences ) is a reporter cell line which allows to monitor the NF- KB pathway by the detection of GFP fluorescence as well as luciferase activity for quantitative transcription activation reporter assays. onally, K562 cells were stably uced with human TNF-receptors, CD27, CD40, CD134 and CD137, as well, in order to generate cell lines W0 2016/1 10584 2016/050308 mediating trans-presentation of the indicated receptors. Cell surface expression of the receptors on HEK293_NFK_gfp_luc cells and K562 cells was analyzed by flow cytometry depicted in Figure 6A and Figure 6B.

The er assay measuring trans-activation was set up as follows: HEK293_NFK_gfpdluc cells expressing one of the 4 indicated TNF-receptors were seeded in multi-weli culture .

Bispecific antibodies and corresponding monospecific control antibodies were added in concentrations from 100 ng/ml to 10.000 ng/ml. K562 cells expressing the second TNF-receptor or wildtype K562 (K562_wt) were added to ted wells. Thus, the bispecific antibodies are able to bind to the first "INF-receptor on the _NFK_gfp_~luc cell line and, at the same time, to the second ceptor on the K562 ceil line mediating trans-presentation. Only or activation of the first TNF receptor on HEK293_NFK_gfp_luc cells is measured by luciferase activity d upon NF-KB -signaling. Thus, biSpecific antibodies targeting CD40 and CD27, CD40 and CD137 or CD40 and CD134 were analyzed by tw0 reporter assays: (i) the first assay measuring CD27—, CD134— or CD137—activation upon cross—presentation by K562_CD40 and (ii) the second assay measuring CD40-activation upon cross-presentation by K562_CD27, K562_CD134 or D137, respectively. 2.2 Analysis of CD40xCDl37 bispecific antibodies Bispecific antibodies binding with one arm to CD40 and with the second arm to CD137 were produced and analyzed by the two Trans-reporter assays using HEK293_NFK_CD40wgfp_luc + K562_CD137 and HEK293_NFK_CD137_gfp_luc + K562_CD40. Two different bispecific antibodies as well as the control antibodies, the parental ecific nt anti-CD40 and anti-CD137 antibodies and corresponding monovalent antibodies with one irrelevant arm, were tested (Figure 7A and 7B). All antibodies were tested under the indicated trans—presentation conditions using K562 cells expressing the second TNF-receptor and for control without trans- presentation using K562_wt. None of the l antibodies, neither the parental monospecific bivalent antibodies nor the monovalent antibodies with one irrelevant arm, induced luciferase activity in HEK293_NFK“CD40__gfp_1uc cells or in HEK293_NFK_CD137_gfp_Iuc cells. Only the two bispecific CD40XCD137 antibodies resulted in luciferase activity upon addition of antibodies (100 ng/ml and higher trations) to _NFK_CD40_gfp_luc or HEK293gNFK_CDl37Mgfpkluc cells under trans-presentation conditions. Thus, even if the parental monOSpecific, bivalent anti—CD40 and anti-CD137 IgGl antibodies do not show W0 2016/110584 2016/050308 agonistic activity, the bispecifie CD40xCD137 antibody is able to induce activation of both receptors resulting in intracellular signaling and NF-KB pathway activation. 2.3 Analysis of D27 ific antibodies A bispecific antibody binding with one arm to CD40 and with the second arm to CD27 was produced and analyzed by the two Trans-reporter assays using HEK293_NFK_CD40__gfi9_luc + KS62_CD27 and HEK293_NFK_CD27_gfp_luc + K562_CD40. The bispecific antibody as well as the control antibodies, the parental monospecific bivalent anti-CD40 and anti-CD27 antibodies and ponding monovalent antibodies with one irrelevant arm, were tested (Figure 8): All antibodies were tested under the indicated trans-presentation conditions using K562 cells expressing the second "INF-receptor and for control without trans-presentation using K562_wt. The bispecific antibody (100 ng/ml and higher concentrations) induced strong luciferase activity in HEK293_NFK_CD40wgfp_luc and in HEK293_NFK_CD27_gfp_luc cell lines, respectively, under presentation conditions. Thus, the bispecific CD40xCD27 antibody is able to induce activation of both receptors resulting in strong intracellular signaling and NF-KB pathway activation. 2.4 Analysis of CD40xCD134 bispecific antibodies Bispecific antibodies binding with one arm to CD40 and with the second arm to CD134 were produced and ed by the two reporter assays using HEK293_NFK_CD40_gfp_luc + K562_CD134 and HEK293fiNFK_CD134_gfp_luc + K562_CD40. Two different bispecific antibodies as well as the control dies, the parental monospecifie nt anti-CD40 and anti-CD134 antibodies and the ponding monovalent antibodies with one vant arm, were tested e 9A and 98). All antibodies were tested under the indicated trans- presentation conditions using K562 cells expressing the second TNF-receptor and for control without trans-presentation using K562_wt. The parental monospecific bivalent anti-CD134 antibodies induced only very weak luciferase activity. However, the bispecific antibodies (100 ng/ml and higher concentrations) induced strong luciferase activity in HEK293_NFK_CD40_ fp_1uc and in HEK293_NFK_CD134_gfp_”luc cell lines, respectively, under trans-presentation conditions. Thus, even if the parental ecific, bivalent anti-CD40 and anti-CD134 IgG1 antibodies show only slight agonistic activity the bispecific CD40xCD134 antibody is able to induce activation of both receptors resulting in strong intracellular signaling and NF-KB y activation.

W0 2016/1 10584 Example 3: Bispecific antibodies targeting CD134 and CD27: CD134 and CD137 or CD27 and CD137 3.1 Reporter assay measuring cis—activation CD27, CD134 and CD137 are inantly expressed on T cells. While CD27 is constitutivcly expressed and CD27 expression density only varies upon T cell activation, C0134 and CD137 expression is only induced upon T cell activation. Thereby, bispecific antibodies binding to CD134 and CD27, to CD134 and CD137 or to CD27 and CD137 most probably target two receptors expressed on the same T Cell. However, the activity of these bispecific antibodies depends on the co-expression of the two receptors and thereby on the tion status of the T cells. Activation of these TNF-receptors is dependent on receptor cross-linking and receptor clustering, respectively, which can be achieved by binding agents simultaneously g to different receptors. If T cells co-express the indicated receptors, these bispecific antibodies can act as costimulatory agents mediating receptor-induced signaling in T cells upon binding of two different "INF-receptors on the same T cell.

A corresponding reporter assay system was established using TNF-receptor double transfectants.

To that end, HEK293_NFK_gfp_luc cells were stably transduced with two of the indicated human TNF-receptors, CD27, CD134 and CD137, respectively. Cell surface expression of the ors was analyzed by flow try ed in Figure 10A and Figure 193. 3.2 Analysis of CD134xCD27 bispecific dies A bispecific antibody binding with one arm to CD134 and with the second arm to CD27 was produced and analyzed by a Cis-reporter assay using HEK293_NFK_CD27_CD134_gfp_"luc cells. The bispecific antibody as well as the control antibodies, the parental monospecific bivalent anti-CD134 and anti-CD27 antibodies and the corresponding lent antibodies with one irrelevant arm, were tested (Figure 11). The bispecific antibody d the strongest luciferase activity in HEK293_NFK_CD27_CD134_gfp_1uc cells. Thus, the bispecific CD134xCD27 antibody is able to induce a strong activation of cells expressing both ors resulting in strong NF-KB ing. 3 .3 Analysis ofCDl 34XCD 137 bispecific antibodies W0 2016/110584 Bispecific antibodies binding with one arm to CD134 and with the second arm to CD137 were produced and analyzed by a Cis-reporter assay using the HEK293_NFK_CD134_CD137_gfp_luc cell line. Two different bispecific antibodies as well as the control antibodies, the al monospecific bivalent anti-CD134 and D137 antibodies and corresponding monovalent antibodies with one irrelevant arm, were tested (Figure 12A and 128). None of the control antibodies, r the parental monospecific bivalent antibodies nor the monovalent antibodies with one irrelevant arm, induced luciferase ty in the HEK293_NFK_CD134_CD137mgfp_luc. Only the bispecific CD134xCDl37 antibody resulted in luciferase activity. Thus, even if the parental monospecific, bivalent anti-CD134 and anti- CD137 IgG1 antibodies do not show any tic activity, the bispecific CD134xCD137 antibody is able to induce activation in the cell line sing both receptors resulting in NF-K‘B pathway activation. 3.3 Analysis of CD27xCD137 bispecific antibodies Bispecific antibodies binding with one arm to CD137 and with the second arm to CD27 were produced and analyzed by a Cis«reporter assay using HEK293_NFK_CD27_CD137_gfp_1uc cells. Two different ific antibodies as well as the control antibodies, the parental ecific bivalent anti—CD137 and anti-CD27 antibodies and corresponding monospecific antibodies with one irrelevant arm, were tested (Figure 13A and 133). In this case, the parental menospecific nt anti-CD27 dies d luciferase activity, whereas the parental monospecific bivalent anti-CD137 resulted only in extremely weak maximum activation signals.

Both control monovalent antibodies consisting of one irrelevant arm d very weak activation of HEK293_NFK_CD27_CD137_gfp__1uc cells. However, the bispecific antibodies induced stronger luciferase activity in HEK293_NFK__CD27_CD137_gfpwluc cells than the control antibodies with one irrelevant arm.

Claims (10)

Claims

1. A bispecific antibody sing two binding domains, wherein a first binding domain binds to CD40 and the second binding domain binds to 4-1BB (CD137), , wherein the first g domain binding to CD40 is selected from a first group ting of: (a) the heavy chain complementarity determining region HCDR1 having the amino acid sequence of SEQ ID NO: 2323, the HCDR2 having the amino acid sequence of SEQ ID NO: 2324 and the HCDR3 having the amino acid sequence of SEQ ID NO: 2325, and the light chain complementarity determining region LCDR1 having the amino acid ce of SEQ ID NO: 2326, the LCDR2 having the amino acid sequence of SEQ ID NO: 2327 and the LCDR3 having the amino acid sequence of SEQ ID NO: 2328; and (b) the heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 2321 and the light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 2322; and wherein the second binding domain binding to 4-1BB (CD137) is selected from a second group consisting of: (a) the heavy chain complementarity determining region HCDR1 having the amino acid sequence of SEQ ID NO: 2297, the HCDR2 having the amino acid sequence of SEQ ID NO: 2298 and the HCDR3 having the amino acid sequence of SEQ ID NO: 2299, and the light chain complementarity determining region LCDR1 having the amino acid sequence of SEQ ID NO: 2300, the LCDR2 having the amino acid sequence of SEQ ID NO: 2301 and the LCDR3 having the amino acid ce of SEQ ID NO: 2302; (b) the heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 2241 and the light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 2242; (c) the heavy chain complementarity determining region HCDR1 having the amino acid sequence of SEQ ID NO: 2255, the HCDR2 having the amino acid sequence of SEQ ID NO: 2256 and the HCDR3 having the amino acid sequence of SEQ ID NO: 2257, and the light chain complementarity determining region LCDR1 having the amino acid sequence of SEQ ID NO: 2258, the LCDR2 having the amino acid sequence of SEQ ID NO: 2259 and the LCDR3 having the amino acid sequence of SEQ ID NO: 2260; (d) the heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 2227 and the light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 2228; (e) the heavy chain complementarity determining region HCDR1 having the amino acid sequence of SEQ ID NO: 2315, the HCDR2 having the amino acid sequence of SEQ ID NO: 2316 and the HCDR3 having the amino acid sequence of SEQ ID NO: 2317, and the light chain complementarity determining region LCDR1 having the amino acid sequence of SEQ ID NO: 2318, the LCDR2 having the amino acid ce of SEQ ID NO: 2319 and the LCDR3 having the amino acid sequence of SEQ ID NO: 2320; and (f) the heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 2247 and the light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 2248.

2. The bispecific antibody according to claim 1 , which is an agonistic binding agent.

3. The bispecific antibody ing to claim 1 or 2, wherein said antibody is in the format of a full-length antibody or an antibody fragment.

4. A nucleic acid molecule encoding a bispecific antibody according to any one of claims 1 to 3.

5. A vector sing, the nucleic acid molecule according to claim 4.

6. A cell sing a nucleic acid molecule according to claim 4 or the vector according to claim 5, wherein the cell is not a cell in a human.

7. A pharmaceutical composition comprising, as an active agent, a bispecific antibody ing to any one of claims 1 to 3, a c acid molecule according to claim 4, or a cell according to claim 6.

8. The ceutical composition according to claim 7, further comprising a pharmaceutically acceptable carrier and/or excipient.

9. A kit sing a ific antibody according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4, a vector according to claim 5, a cell according to claim 6, or a pharmaceutical composition according to claim 7 or 8.

10. Use of a bispecific antibody according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4, a vector according to claim 5, a cell according to claim 6, or a pharmaceutical composition according to claim 7or 8 for use as a medicament. WO 10584