Antigen binding molecules comprising TNF family ligand trimers

Abstract

The present invention relates to a novel antigen binding molecule containing a TNF family ligand trimer, which comprises (a) at least one module capable of specifically binding to a target cell antigen and (b) a first and a second polypeptide connected to each other by a disulfide bond, characterized in that the first polypeptide comprises two extracellular domains or fragments thereof of a TNF ligand family member connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain or fragment thereof of the TNF ligand family member.

Description

[0001] This application is a divisional application of the invention application with the application date of November 13, 2015, the Chinese application number 201580073211.0, and the invention name “Antigen Binding Molecule Comprising a TNF Family Ligand Trimer”. Field of the Invention

[0002] The present invention relates to a novel antigen-binding molecule containing a TNF family ligand trimer, which comprises (a) at least one module capable of specifically binding to a target cell antigen and (b) a first and a second polypeptide linked to each other by a disulfide bond.

[0003] The feature of wherein said antigen binding molecules is that, said first polypeptide comprises two extracellular domains or its two fragments of tnf ligand family members connected to each other by a peptide linker, and said second polypeptide only comprises an extracellular domain or its fragment of said tnf ligand family members. The invention further relates to methods for producing these molecules and methods using them. Background of the Invention

[0004] Ligands that interact with the TNF (tumor necrosis factor) receptor superfamily of molecules play a key role in the organization and function of the immune system. While regulating normal functions such as immune responses, hematopoiesis, and morphogenesis, TNF family ligands (also known as cytokines) play a role in tumorigenesis, transplant rejection, septic shock, viral replication, bone resorption, rheumatoid arthritis, and diabetes (Aggarwal, 2003). The TNF ligand family includes 18 genes encoding 19 type II (i.e., intracellular N-terminus and extracellular C-terminus) transmembrane proteins, characterized by the presence of a conserved C-terminal domain that constitutes the "TNF homology domain" (THD). This domain is responsible for receptor binding and is therefore crucial for the biological activity of TNF ligand family members. The sequence identity between family members is approximately 20%-30% (Bodmer, 2002). Members of the TNF ligand family exert their biological functions by self-assembling non-covalent trimers (Banner et al., Cell 1993, 73, 431-445). Thus, TNF family ligands form trimers that are able to bind and activate the corresponding receptors of the TNFR superfamily.

[0005] TNF receptor superfamily member 4-1BB (CD137) is first identified as a molecule (Kwon and Weissman, 1989) induced by its expression via T cell activation. Subsequent studies have confirmed that 4-1BB is expressed in T and B lymphocytes (Snell et al., 2011; Zhang et al., 2010), NK cells (Lin et al., 2008), NKT- cells (Kim et al.), monocytes (Kienzle and vonKempis, 2000; Schwarz et al., 1995), neutrophils (Heinisch et al., 2000), mast cells (Nishimoto et al., 2005) and dendritic cells and cells of non-hematopoietic origin such as endothelial cells and smooth muscle cells (Broll et al., 2001; Olofsson et al., 2008). The expression of 4-1BB in different cell types can mostly be induced and driven by various stimulatory signals, such as T cell receptor (TCR) or B cell receptor triggering, as well as signal transduction induced by receptors of costimulatory molecules or proinflammatory cytokines (Diehl et al., 2002; von Kempis et al., 1997; Zhang et al., 2010).

[0006] The expression of the 4-1BB ligand (4-1BBL or CD137L) is more restricted, with expression observed on professional antigen-presenting cells (APCs) such as B cells, dendritic cells (DCs), and macrophages. Inducible expression of 4-1BBL is characteristic of T cells, including αβ and γδ T cell subsets, as well as endothelial cells (reviewed in Shao and Schwarz, 2011).

[0007] It is known that CD137 signaling stimulates IFNγ secretion and proliferation of NK cells (Buechele et al., 2012; Lin et al., 2008; Melero et al., 1998), as well as promotes DC activation (as manifested by increased survival and cytokine secretion) and upregulates co-stimulatory molecules (Choi et al., 2009; Futagawa et al., 2002; Wilcox et al., 2002). However, CD137 is best characterized as a co-stimulatory molecule that regulates TCR-induced activation of CD4+ and CD8+ subsets of T cells. In combination with TCR triggering, agonistic 4-1BB-specific antibodies enhance T cell proliferation, stimulate lymphokine secretion, and reduce the sensitivity of T lymphocytes to activation-induced cell death (reviewed by Snell et al., 2011).

[0008] Consistent with these co-stimulatory effects of 4-1BB antibodies on T cells in vitro, their administration to tumor-bearing mice resulted in effective anti-tumor effects in many experimental tumor models (Melero et al., 1997; Narazaki et al., 2010). However, 4-1BB is generally only shown to be effective as an anti-tumor agent when administered in combination with other immunomodulatory compounds (Curran et al., 2011; Guo et al., 2013; Morales-Kastresana et al., 2013; Teng et al., 2009; Wei et al., 2013), chemotherapeutic drugs (Ju et al., 2008; Kim et al., 2009), tumor-specific vaccination (Cuadros et al., 2005; Lee et al., 2011), or radiotherapy (Shi and Siemann, 2006). In vivo depletion experiments have shown that CD8+ T cells play the most critical role in the anti-tumor effects of 4-1BB-specific antibodies. However, depending on the tumor model or combination therapy including anti-4-1BB, the role of other cell types such as DCs, NK cells or CD4+ T cells has been reported (Melero et al. 1997; Murillo et al. 2009; Narazaki et al. 2010; Stagg et al. 2011).

[0009] In addition to the direct effects of 4-1BB agonists on different lymphocyte subsets, 4-1BB agonists can also induce the infiltration and retention of activated T cells in tumors through 4-1BB-mediated upregulation of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) on tumor vascular endothelium (Palazon et al., 2011).

[0010] 4-1BB triggering can also reverse the state of T cell anergy induced by exposure to soluble antigens, which may help to break immune tolerance in the tumor microenvironment or during chronic infection (Wilcox et al., 2004).

[0011] It seems that the immunomodulatory properties of 4-1BB agonistic antibodies in vivo require the presence of a wild-type Fc portion on the antibody molecule, suggesting that Fc-receptor binding is an important event required for the pharmacological activity of such agents, which are described as agonistic antibodies specific for other apoptosis-inducing or immunomodulatory members of the TNFR superfamily (Li and Ravetch, 2011; Teng et al., 2009). However, in mice, systemic administration of 4-1BB-specific agonistic antibodies with functionally active Fc domains also induced CD8+T cell expansion associated with hepatotoxicity (Dubrot et al., 2010), and in the absence of functional Fc receptors, hepatotoxicity was attenuated or significantly reduced. In a human clinical trial (ClinicalTrials.gov, NCT00309023), a 4-1BB agonistic antibody (BMS-663513) with Fc activity administered once every three weeks for 12 weeks induced stable disease in patients with melanoma, ovarian cancer, or renal cell carcinoma. However, the same antibody administered in another trial (NCT00612664) caused grade 4 hepatitis, leading to trial termination (Simeone and Ascierto, 2012).

[0012] Overall, the available preclinical and clinical data clearly demonstrate that there is a high clinical need for effective 4-1BB agonists. However, a new generation of drug candidates would not only effectively bind 4-1BB to the surface of hematopoietic and endothelial cells, but also achieve this binding through mechanisms other than Fc receptor binding to avoid uncontrolled side effects. The latter could be achieved through preferential binding and oligomerization of tumor-specific or tumor-associated modules.

[0013] Fusion proteins consisting of an extracellular domain of the 4-1BB ligand and a single-chain antibody fragment (Mueller et al., 2008; Hornig et al., 2012) or a single 4-1BB ligand fused to the heavy chain C-terminus (Zhang et al., 2007) have been produced. WO 2010 / 010051 discloses the production of a fusion protein consisting of three TNF ligand extracellular domains connected to each other and fused to an antibody module.

[0014] However, new antigen binding molecules are still needed, which will preferably be able to combine with a module that is combined with a tumor-specific or tumor-associated target and a module that can form a costimulatory TNF ligand trimer, and have enough pharmaceutically useful stability. Antigen binding molecules of the present invention include both, and they surprisingly provide trimeric and therefore biologically active TNF ligands, although one of the trimerized TNF ligand extracellular domains is located on another polypeptide relative to other two TNF ligand extracellular domains of the molecule. SUMMARY OF THE INVENTION

[0015] In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0016] (a) at least one module capable of specifically binding to a target cell antigen, and

[0017] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0018] The antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof.

[0019] In a specific aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0020] (a) at least one module capable of specifically binding to a target cell antigen,

[0021] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0022] wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof, and

[0023] (c) The Fc domain consists of a first and a second subunit that are capable of stably associating.

[0024] In a further aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0025] (a) at least one module capable of specifically binding to a target cell antigen, and

[0026] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0027] The antigen-binding molecule is characterized in that

[0028] (i) the first polypeptide contains a CH1 or CL domain, and the second polypeptide contains a CL or CH1 domain, respectively, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the CH1 or CL domain via a peptide linker, and wherein the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof linked to the CL or CH1 domain of the polypeptide via a peptide linker, or

[0029] (ii) the first polypeptide contains a CH3 domain, and the second polypeptide correspondingly contains a CH3 domain, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the C-terminus of the CH3 domain via a peptide linker, and wherein the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof linked to the C-terminus of the CH3 domain of the polypeptide via a peptide linker, or

[0030] (iii) the first polypeptide contains a VH-CL or VL-CH1 domain, and the second polypeptide contains a VL-CH1 domain or a VH-CL domain, respectively, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the VH or VL via a peptide linker, and wherein the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof linked to the VL or VH of the polypeptide via a peptide linker.

[0031] In a specific aspect, the TNF ligand family member is a member of a costimulatory human T cell activation. Therefore, the antigen binding molecule containing the TNF family ligand trimer comprises (a) at least one module that can specifically bind to a target cell antigen, and

[0032] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0033] Wherein the antigen binding molecule is characterized in that, the first polypeptide comprises two extracellular domains or two fragments thereof of a TNF ligand family member connected to each other by a peptide linker, and the second polypeptide only comprises one extracellular domain or a fragment thereof of the TNF ligand family member, wherein the TNF ligand family member co-stimulates human T cell activation. More specifically, the TNF ligand family member is selected from 4-1BBL and OX40L.

[0034] In one aspect, the TNF ligand family member is 4-1BBL.

[0035] In a further aspect, the extracellular domain of the TNF ligand family member comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 96, SEQ ID NO: 373, SEQ ID NO: 374 and SEQ ID NO: 375, in particular the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 96.

[0036] In another aspect, the extracellular domain of the TNF ligand family member or a fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 96, in particular an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 96. More specifically, the extracellular domain of the TNF ligand family member comprises an amino acid sequence of SEQ ID NO: 96.

[0037] In a further aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0038] (a) at least one module capable of specifically binding to a target cell antigen, and

[0039] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0040] The antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, and the second polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 3 and SEQ ID NO: 4.

[0041] In one aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0042] (a) at least one module capable of specifically binding to a target cell antigen, and

[0043] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0044] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6.

[0045] In a further aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0046] (a) at least one module capable of specifically binding to a target cell antigen, and

[0047] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0048] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 183.

[0049] In yet another aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0050] (a) at least one module capable of specifically binding to a target cell antigen, and

[0051] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0052] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO: 185.

[0053] In another aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0054] (a) at least one module capable of specifically binding to a target cell antigen,

[0055] (b) a first polypeptide comprising a CH1 or CL domain and a second polypeptide comprising a CL or CH1 domain, respectively, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof linked to each other and to the CH1 or CL domain via a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof linked to the CL or CH1 domain of the polypeptide via a peptide linker.

[0056] In one aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, comprising:

[0057] (a) at least one module capable of specifically binding to a target cell antigen,

[0058] (b) a first polypeptide comprising a CH1 domain and a second polypeptide comprising a CL domain, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the CH1 domain via a peptide linker, and the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof linked to the CL domain of the polypeptide via a peptide linker.

[0059] In another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, comprising:

[0060] (a) at least one module capable of specifically binding to a target cell antigen,

[0061] (b) a first polypeptide comprising a CL domain and a second polypeptide comprising a CH1 domain, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the CL domain via a peptide linker, and the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof linked to the CH1 domain of the polypeptide via a peptide linker.

[0062] In a further aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer as defined above, wherein the module capable of specifically binding to a target cell antigen is selected from an antibody, an antibody fragment and a scaffold antigen binding protein.

[0063] In one aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer as defined above, wherein the module capable of specifically binding to a target cell antigen is an antibody fragment.

[0064] Specifically, the module capable of specifically binding to a target cell antigen is selected from antibody fragments, Fab molecules, exchange Fab molecules, single-chain Fab molecules, Fv molecules, scFv molecules, single domain antibodies, aVH and scaffold antigen binding proteins.

[0065] In one aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer as defined above, wherein the module capable of specifically binding to a target cell antigen is a scaffold antigen binding protein.

[0066] In a specific aspect, the present invention relates to an antigen binding molecule comprising a TNF family ligand trimer as defined above, wherein the module capable of specifically binding to a target cell antigen is a Fab molecule capable of specifically binding to a target cell antigen.

[0067] The invention provides the antigen binding molecules containing TNF families ligand trimer, which include at least one module that can be specifically bound to a target cell antigen. In a specific aspect, the antigen binding molecules containing TNF families ligand trimer include a module that can be specifically bound to a target cell antigen. In another aspect, the invention provides the antigen binding molecules containing TNF families ligand trimer, which include two modules that can be specifically bound to a target cell antigen.

[0068] In another aspect, an antigen binding molecule containing a TNF family ligand trimer of the present invention is provided, wherein the target cell antigen is selected from fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), CD19, CD20 and CD33.

[0069] In a specific aspect, the target cell antigen is fibroblast activation protein (FAP).

[0070] In one aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer, wherein the module capable of specifically binding to FAP comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 100, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 101, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 102, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 103, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 104, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 105.

[0071] In one aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer, wherein the module capable of specifically binding to FAP comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0072] In a specific aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer, wherein the module capable of specifically binding to FAP comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 102, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 103, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 104, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 105.

[0073] In one aspect, an antigen binding molecule containing a TNF family ligand trimer as defined above is provided, wherein the module capable of specifically binding to FAP comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 17, or wherein the module capable of specifically binding to FAP comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 106 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 107.

[0074] In a further aspect, an antigen binding molecule containing a TNF family ligand trimer according to the present invention is provided, wherein a peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to the CH1 or CL domain of the heavy chain via a second peptide linker, and wherein one extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to the CL or CH1 domain on the light chain via a third peptide linker.

[0075] In a specific aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the peptide linker is (G4S)2, that is, a peptide linker with SEQ ID NO: 13. In one aspect, the first peptide linker is (G4S)2, the second peptide linker is GSPGSSSSGS (SEQ ID NO: 57), and the third peptide linker is (G4S)2. In another aspect, the first, second, and third peptide linkers are all (G4S)2.

[0076] The present invention further relates to an antigen binding molecule comprising a TNF family ligand trimer as defined above, which comprises an Fc domain consisting of a first and a second subunit capable of stably associating.

[0077] Specifically, the antigen-binding molecule of the present invention comprising a TNF family ligand trimer comprising (c) an Fc domain consisting of a first and a second subunit capable of stably combining further comprises (a) a Fab molecule capable of specifically binding to a target cell antigen, wherein the Fab heavy chain is fused at the C-terminus to the N-terminus of the CH2 domain in the Fc domain.

[0078] In a further aspect, the Fc domain is an IgG, specifically an IgG1 Fc domain or an IgG4 Fc domain. More specifically, the Fc domain is an IgG1 Fc domain. In a specific aspect, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

[0079] In another aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer as defined above, comprising

[0080] (c) an Fc domain composed of a first and a second subunit capable of stably associating, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor.

[0081] Specifically, the Fc domain comprises amino acid substitutions at positions 234 and 235 (EU numbering) and / or 329 (EU numbering) of the IgG heavy chain. More specifically, there is provided an antigen binding molecule containing a trimeric TNF family ligand according to the present invention, comprising an IgG1 Fc domain with amino acid substitutions L234A, L235A and P329G (EU numbering).

[0082] In a further aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer, wherein the antigen binding molecule comprises:

[0083] A first heavy chain and a first light chain, which together constitute a Fab molecule capable of specifically binding to a target cell antigen, a first peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker, the first peptide being fused at its C-terminus to the second heavy chain or light chain via a second peptide linker, and a second peptide comprising an extracellular domain of the TNF ligand family member, the second peptide being fused at its C-terminus to the second light chain or heavy chain, respectively, via a third peptide linker.

[0084] In another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein a first peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to a CH1 domain as part of the heavy chain via a second peptide linker, and a second peptide comprising one extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to a CL domain as part of the light chain via a third peptide linker.

[0085] In yet another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein a first peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to a CL domain as part of the heavy chain through a second peptide linker, and a second peptide comprising an extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to a CH1 domain as part of the light chain through a third peptide linker.

[0086] In a further aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, wherein a first peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to a VH domain as part of the heavy chain via a second peptide linker, and a second peptide comprising one extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to a VL domain as part of the light chain via a third peptide linker.

[0087] Further provided are antigen binding molecules containing a TNF family ligand trimer, wherein in the CL domain of the adjacent TNF ligand family member, the amino acid at position 123 (EU numbering) has been replaced with arginine (R), the amino acid at position 124 (EU numbering) has been replaced with lysine (K), and wherein in the CH1 domain of the adjacent TNF ligand family member, the amino acids at position 147 (EU numbering) and at position 213 (EU numbering) have been replaced with glutamic acid (E).

[0088] In a further aspect, an antigen binding molecule containing a TNF family ligand trimer as described above is provided, wherein the antigen binding molecule comprises:

[0089] (a) a first heavy chain and a first light chain, which together constitute a Fab molecule capable of specifically binding to a target cell antigen,

[0090] (b) a second heavy chain and a second light chain, wherein the second heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, and the second light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 3 and SEQ ID NO: 4.

[0091] In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, wherein the antigen binding molecule comprises:

[0092] (a) a Fab molecule capable of specifically binding to FAP, and

[0093] (b) a second heavy chain and a second light chain, wherein the second heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, and the second light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 3 and SEQ ID NO: 4.

[0094] In a specific aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, which comprises a module capable of specifically binding to FAP. In one aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises:

[0095] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 106 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 107,

[0096] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 108, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 139, and SEQ ID NO: 148, and

[0097] (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, and SEQ ID NO: 115.

[0098] In another aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, wherein the antigen binding molecule comprises:

[0099] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 106 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 107,

[0100] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 173, and

[0101] (iii) a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 174.

[0102] In yet another aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0103] (a) at least one module capable of specifically binding to a target cell antigen, and

[0104] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0105] The antigen binding molecule is characterized in that the first polypeptide contains a CH3 domain and the second polypeptide correspondingly contains a CH3 domain, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other and to the C-terminus of the CH3 domain via a peptide linker, and wherein the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof connected to the C-terminus of the CH3 domain of the polypeptide via a peptide linker.

[0106] Specifically, such an antigen-binding molecule containing a TNF family ligand trimer comprises two modules capable of specifically binding to target cell antigens.

[0107] In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, which comprises two modules capable of specifically binding to FAP. Specifically, an antigen binding molecule containing a TNF family ligand trimer as described above is provided, which comprises:

[0108] (i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 121, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 122, and two light chains comprising the amino acid sequence of SEQ ID NO: 19, or

[0109] (ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 123, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 124, and two light chains comprising the amino acid sequence of SEQ ID NO: 125, or

[0110] (iii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 126, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 127, and two light chains comprising the amino acid sequence of SEQ ID NO: 125.

[0111] In another specific aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer as described above, wherein the target cell antigen is CD19.

[0112] In one aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the module capable of specifically binding to CD19 comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 195 or SEQ ID NO: 252, (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 196 or SEQ ID NO: 253, and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 197 or SEQ ID NO: 254, the VL domain comprising (iv) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 198 or SEQ ID NO: 249, (v) a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 199 or SEQ ID NO: 250, and (vi) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 200 or SEQ ID NO: 251.

[0113] In a further aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the module capable of specifically binding to CD19 comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 201 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 202, or wherein the module capable of specifically binding to FAP comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 357 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 358.

[0114] In a specific aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises:

[0115] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 201 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 202, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 357 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 358,

[0116] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 108, SEQ ID NO: 111 and SEQ ID NO: 113, and

[0117] (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114.

[0118] In another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises:

[0119] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 201 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 202, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 357 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 358,

[0120] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 173, and

[0121] (iii) a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 174.

[0122] In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, which comprises two modules capable of specifically binding to CD19. Specifically, there is provided an antigen binding molecule containing a TNF family ligand trimer according to any one of claims 1 to 14, 29, 30 and 32 to 34, wherein the antigen binding molecule comprises:

[0123] (i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 209, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 210, and two light chains comprising the amino acid sequence of SEQ ID NO: 206, or

[0124] (ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 213, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 214, and two light chains comprising the amino acid sequence of SEQ ID NO: 206, or

[0125] (iii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 309, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 310, and two light chains comprising the amino acid sequence of SEQ ID NO: 279, or (iv) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 313, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 314, and two light chains comprising the amino acid sequence of SEQ ID NO: 279.

[0126] In another specific aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer as described above, wherein the target cell antigen is CEA.

[0127] In one aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the module capable of specifically binding to CEA comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 321, (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 322, and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 323, the VL domain comprising (iv) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 324, (v) a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 325, and (vi) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 326.

[0128] In another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the module capable of specifically binding to CEA comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 329 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 330.

[0129] In one aspect, an antigen binding molecule containing a TNF family ligand trimer as described above is provided, wherein the antigen binding molecule comprises:

[0130] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 329 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 330,

[0131] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 108, SEQ ID NO: 111 and SEQ ID NO: 113, and

[0132] (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114.

[0133] In another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises:

[0134] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 329 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 330,

[0135] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 173, and

[0136] (iii) a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 174.

[0137] In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising two modules capable of specifically binding to CEA. Specifically, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises:

[0138] (i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 337, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 338, and two light chains comprising the amino acid sequence of SEQ ID NO: 334, or

[0139] (ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 341, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 342, and two light chains comprising the amino acid sequence of SEQ ID NO: 334.

[0140] In a further aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the TNF ligand family member is OX40L. In one aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer, wherein the extracellular domain of the TNF ligand family member comprises SEQ ID NO:53 or SEQ ID NO:54 amino acid sequence, especially SEQ ID NO:53 amino acid sequence.

[0141] In a further aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer according to any one of claims 1 to 5, 10 to 24, 29, 30, 32 to 34, 38 to 40, 44 and 45, comprising:

[0142] (a) at least one module capable of specifically binding to a target cell antigen, and

[0143] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0144] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 371 or SEQ ID: 372, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 54.

[0145] In another aspect, an antigen binding molecule comprising a TNF family ligand trimer is provided, wherein the target cell antigen is fibroblast activation protein (FAP), and wherein the module capable of specifically binding to FAP comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 100, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 101, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 102, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 103, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 104, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 105.

[0146] Specifically, provided are antigen binding molecules containing a TNF family ligand trimer as described herein, wherein the antigen binding molecule comprises:

[0147] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 106 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 107,

[0148] (ii) a second heavy chain comprising an amino acid sequence selected from SEQ ID NO: 355, and

[0149] (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 356.

[0150] According to another aspect of the present invention, there is provided a polynucleotide encoding the separation of the antigen binding molecules containing the TNF family ligand trimer as defined above. The present invention further provides a vector comprising the isolated polynucleotide of the present invention, especially an expression vector, and a host cell comprising the isolated polynucleotide of the present invention or a vector. In some embodiments, the host cell is a eukaryotic cell, especially a mammalian cell.

[0151] In another aspect, there is provided a method for producing an antigen binding molecule containing a TNF families ligand trimer of the present invention, the method comprising the following steps: (i) cultivating a host cell of the present invention under conditions suitable for expressing the antigen binding molecule, and (ii) reclaiming the antigen binding molecules. The present invention also encompasses the antigen binding molecules containing a TNF families ligand trimer produced by the method of the present invention.

[0152] The present invention further provides a pharmaceutical composition comprising the antigen binding molecule containing a TNF family ligand trimer of the present invention and at least one pharmaceutically acceptable excipient.

[0153] The present invention also encompasses the antigen binding molecules containing TNF families ligand trimers of the present invention or the pharmaceutical composition of the present invention used as medicament. In one aspect, there is provided an antigen binding molecule containing TNF families ligand trimers of the present invention or the pharmaceutical composition of the present invention for treating a disease in an individual in need. In a specific embodiment, there is provided an antigen binding molecule containing TNF families ligand trimers of the present invention or the pharmaceutical composition of the present invention for treating cancer.

[0154] Also provided is the use of an antigen binding molecule containing a TNF families ligand trimer of the present invention in the manufacture of a medicament for treating a disease in an individual in need, especially in the manufacture of a medicament for treating cancer, and a method for treating an individual disease, the method comprising administering a therapeutically effective amount of a pharmaceutically acceptable form of the antigen binding molecules containing a TNF families ligand trimer of the present invention to the individual. In a specific embodiment, the disease is cancer. In any of the above embodiments, the individual is preferably a mammal, especially a person. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] Figure 1 The components of the human 4-1BB ligand used to assemble the split trimer are shown. Figure (1A) shows a dimeric ligand fused to a human CH1 or CL domain or a VL or VH domain at the C-terminus, and Figure (1B) shows a monomeric ligand fused to a human CL or CH1 domain or a VL or VH domain. Figure (1C) shows a dimeric ligand fused to a human CH3 domain at the N-terminus, and Figure (1D) shows a monomeric ligand fused to a human CH3 domain at the N-terminus.

[0156] Figure 2 Antigen-binding molecule constructs 1.1 to 1.10 containing 4-1BBL trimers of the present invention are shown. The preparation and generation of these constructs are described in Example 1. The VH and VL domains are those of the anti-FAP antibody 28H1, with bold black dots representing knob-into-hole modifications. * Indicates amino acid modifications (so-called charged residues) in the CH1 and CL domains.

[0157] Figure 3 The components of the mouse 4-1BB parts for assembling the split trimer are shown. Figure (3A) shows the dimeric parts fused to the mouse CL domain at the C-terminus, and Figure (3B) shows the monomer parts fused to the mouse CH1 domain at the C-terminus. The components of the mouse 4-1BB parts for assembling the split trimer of FAP targeting are shown. Figure (3C) shows the antigen binding molecules containing the mouse 4-1BBL- trimer assembled as described in more detail in Example 1.3.

[0158] Figure 4 Antigen-binding molecule constructs 2.1 to 2.6 containing 4-1BBL trimers of the present invention are shown. The preparation and generation of these constructs are described in Example 2. The VH and VL domains are those of the anti-FAP antibody 4B9, with bold black dots representing knob-into-hole modifications. * Indicates amino acid modifications (so-called charged residues) in the CH1 and CL domains.

[0159] Figure 5 : Figure 5 A and Figure 5 B shows a "non-targeting" variant of constructs 1.1 and 1.2, which contains a DP47 Fab molecule instead of an anti-FAP Fab molecule. The molecules are designated Control A and Control B, respectively. The preparation method is described in Example 1.4. Figure 5 C is a diagram of the monomeric 4-1BB Fc(kih) construct prepared in Example 3.

[0160] Figure 6 It is related to Fc (kih) fusion antigen binding molecules (4-1BBL trimers separated by FAP, filled circles) containing 4-1BB ligand trimers for FAP targeting or DP-47 non-targeted Fc (kih) fusion antigen binding molecules (4-1BBL trimers separated by DP47, open circles) containing 4-1BB ligand trimers and the combination of resting (immature) or activated human PMBC. Specifically, the combination with resting (immature) or activated human CD8+T cells is shown in Figure (6A), the combination with resting (immature) or activated human CD4+T cells is shown in Figure (6B), and the combination with resting (immature) or activated human NK cells is shown in Figure (6C). The combination is shown as the median fluorescence intensity (MFI) of the anti-human IgG Fcγ specific goat IgG F(ab')2 fragment labeled with red macroalgae phycoerythrin (R-PE) as the second detection antibody. MFI was measured by flow cytometry and corrected for baseline by subtracting the MFI of the blank control.

[0161] Binding of different FAP-targeted or non-targeted split-trimeric human 4-1BB ligand Fc (kih) constructs to human 4-1BB-expressing T cells from PHA-L and human PBMCs preactivated with Proleukin and reactivated with anti-human CD3 / anti-human CD28 was shown. Figure 7A and 7B In. Binding was detected using R-phycoerythrin-fluorescent dye-conjugated anti-human IgG Fcγ-specific goat IgG F(ab')2 fragments. Shown is the median fluorescence intensity (MFI) relative to the concentration of the test constructs 1.1 to 1.10 of Example 1. For better display, the binding curve was divided into four different blots, where construct 1.1 (monovalent FAP-targeted split trimer human 4-1BB ligand Fc (kih)) and control B (monovalent non-targeted split trimer human 4-1BB ligand Fc (kih) with CH-CL cross and charged residues) were used as comparison curves. In CD3+CD8+T cells ( Figure 7A ) and CD3+CD4+T cells ( Figure 7B ). 4-1BB expression levels on CD8 T cells are generally higher than on CD4 T cells. All forms bind to human 4-1BB with very similar affinity.

[0162] Figure 8A and 8B Different FAP-targeted or non-targeted split-trimeric human 4-1BB ligand Fc (kih) constructs were shown to be fused to CD4+ or CD8+ T cells from fresh PBMCs ( Figure 8A ) or with PHA-L expressing human 4-1BB and with human PBMCs ( Figure 8B) combination. Anti-human IgGFc specific goat IgG F(ab')2 fragments conjugated with R-phycoerythrin-fluorescent dye are detected for binding. Shown is the median fluorescence intensity (MFI) of the concentration of test constructs 2.1, 2.3, 2.4, 2.5 and 2.6 relative to Example 2 and control molecules control B, control C, control E and control F. In order to better demonstrate, the binding curve is divided into two different traces, wherein construct 2.1 (human 4-1BB ligand Fc (kih) of the split trimer targeted by monovalent FAP) and control B (human 4-1BB ligand Fc (kih) of the split trimer with non-targeted monovalent CH-CL cross and charged residues) are used as comparison curves. Combination is monitored on CD45+CD3+CD8+T cells (bottom trace) and CD45+CD3+CD4+T cells (top trace). The 4-1BB expression level on CD8 T cells is generally higher than that on CD4 T cells. All constructs bound human 4-1BB with very similar affinities, whereas the bivalent construct 2.3 and its non-targeting control C showed lower MFIs. This may be due to steric hindrance of 4-1BB binding and / or reduced detection of the secondary detection antibody induced by the Fc-conjugated split 4-1BB ligand.

[0163] exist Figure 9 In, it is shown that Fc (kih) fused antigen binding molecules (4-1BBL trimers that FAP is split, filled circles) containing 4-1BB ligand trimers of FAP targetings or DP47 non-targeted Fc (kih) fused antigen binding molecules (4-1BBL trimers that DP47 is split, open circles) containing 4-1BB ligand trimers are combined with activated mouse splenocytes. Specifically, with the combination of activated mouse CD4+T cells are shown in Figure (9A), and with the combination of activated mouse CD8+T cells are shown in Figure (9B). Anti-mouse CD137 specific human IgG1 P329G LALA antibody (clone Lob12.3) is used as positive control (triangle). Characterize combination by drawing the MFI of the anti-human IgG Fc γ specific goat IgG F (ab ') 2 fragments of the R-PE labels used as the second detection antibody, and the MFI is relative to the concentration nM of the split 4-1BBL trimer construct tested. MFI was measured by flow cytometry and corrected for baseline by subtracting the MFI of the blank control.

[0164] Figure 10Shown are Fc (kih) fusion antigen binding molecules (filled circles: FAP-targeted fusion antigen binding molecule construct 1.1 of Fc (kih) containing 4-1BB ligand trimers, hollow circles: DP47 non-targeted Fc (kih) fusion antigen binding molecules containing 4-1BB ligand trimers control A) and human melanoma (10A) MV-3 ​​cell lines and (10B) WM-266-4 cell lines expressing fibroblast activation protein (FAP). Combination is characterized by drawing the MFI of anti-human IgG Fcγ-specific goat IgG F (ab') 2 fragments of R-PE labeled as the second detection antibody, which is expressed in nM relative to the concentration of the split 4-1BBL trimer construct tested. MFI is measured by flow cytometry, and baseline is corrected by subtracting the MFI of the blank control.

[0165] exist Figure 11A and 11B In Figure 2, different FAP-targeted or non-targeted split-trimeric human 4-1BB ligand Fc (kih) constructs were incubated with human melanoma MV-3 ​​cells expressing human FAP ( Figure 11A ) and / or NIH / 3T3-huFAP clone 39 transfected mouse embryonic fibroblasts ( Figure 11B ) binding. Binding was detected using R-phycoerythrin-fluorescent dye or fluorescein-fluorescent dye-conjugated anti-human IgG Fcγ-specific goat IgG F(ab')2 fragments. Shown are the median fluorescence intensity (MFI) relative to the test construct concentration. For better visualization, the binding curves were distributed to four ( Figure 11A ) or two blots ( Figure 11B ), while construct 1.1 (monovalent FAP-targeted split trimeric human 4-1BB ligand Fc (kih)) was used as a comparison curve. Except for the bivalent FAP-targeted constructs (constructs 1.5, 1.7, and 1.8), all constructs bind to human FAP with similar affinity. They show a tendency to have lower EC50 values ​​and lower median fluorescence intensity. This can be explained by their bivalent targeting (higher affinity, due to the occupancy of two epitopes, fewer molecules can bind simultaneously, resulting in lower MFI). Structural differences can also explain the differences between construct 1.8 (complete bivalent targeting) and constructs 1.5 and 1.7 (only partial bivalent targeting).

[0166] exist Figure 12A and 12B In Figure 2, different FAP-targeted or non-targeted split trimer human 4-1BB ligand Fc (kih) constructs 2.1, 2.3, 2.4, 2.5 and 2.6 were compared with human melanoma MV-3 ​​cells expressing human FAP ( Figure 12A) and WM-266-4 cells ( Figure 12B ). Binding was detected using an anti-human IgG Fcγ-specific goat IgG F(ab')2 fragment conjugated with R-phycoerythrin-fluorescent dye. Shown is the median fluorescence intensity (MFI) relative to the test construct concentration. For better display, the binding curve was distributed to two blots, and construct 2.1 (monovalent FAP-targeted split trimer human 4-1BB ligand Fc (kih)) was used as a comparison curve. Except for the bivalent FAP-targeted construct 2.3, all constructs bind to human FAP with similar affinity. It has a tendency to show a lower EC50 value and a lower median fluorescence intensity. This can be explained by its bivalent targeting, which results in higher affinity, but less occupancy or FAP molecules on the cell surface, resulting in a lower MFI.

[0167] Figure 13 Different FAP-targeted or non-targeted split-trimeric mouse 4-1BB ligand Fc (kih) constructs were shown to be incubated with CD4+ or CD8+ T cells from fresh splenocytes ( Figure 13 A) or mouse splenocytes activated with anti-mouse CD3 / anti-mouse CD28 monoclonal agonistic antibodies expressing mouse 4-1BB ( Figure 13 B). Anti-mouse IgG Fcγ-specific goat IgG F(ab')2 fragments conjugated with FITC-fluorescent dyes were used to detect binding. Shown is the median fluorescence intensity (MFI) relative to the test construct concentration. Binding was monitored on CD3+CD8+T cells (left blot) and CD3+CD4+T cells (right blot). The 4-1BB expression level on CD8 T cells is generally higher than that on CD4 T cells. All constructs bind to mouse 4-1BB with very similar affinities.

[0168] Binding of different FAP-targeted or non-targeted split-trimeric mouse 4-1BB ligand Fc (kih) constructs to tumor cells expressing human FAP was demonstrated. Figure 14 Binding was detected using FITC-fluorescent dye-conjugated anti-mouse IgG Fc-specific goat IgG F(ab')2 fragments. Shown are median fluorescence intensities (MFI) relative to the concentration of the test construct. Figure 14 A) and WM-266-4 cells ( Figure 14 B) Binding was monitored on the FAP-targeted split trimeric mouse 4-1BB ligand Fc (kih) constructs M.1 and M.2 bound to FAP with very similar affinities.

[0169] Figure 15The scheme of the general principle of the NFkB activity assay described in Example 6.1 using a reporter cell line is shown. An activation assay established with a HeLa reporter cell line expressing human 4-1BB is shown. Cross-linking of 4-1BB expressed on reporter cells induces NFκB activation and NFκB-mediated luciferase expression. After cell lysis, luciferase can catalyze the oxidation of luciferin to oxyluciferin. This chemical reaction is positively correlated with the intensity of NFκB-mediated luciferase expression and can be measured by luminescence intensity (the number of units of light released). The ratio of FAP-expressing tumor cells to the reporter cell line HeLa-huCD137-NFkB-luc is 5 to 1.

[0170] exist Figure 16 In the figure, it is shown that the activation of NFkB signal transduction pathway by Fc (kih) fusion antigen binding molecules (construct 1.1) containing 4-1BB ligand trimers targeted by FAP is strictly dependent on its combination with target cells expressing FAP. NFkB reporter HeLa cells expressing human CD137 are co-cultured with designated tumor cells expressing different levels of cell surface FAP. After culturing cells for 6 hours in the absence or presence of molecules containing 4-1BBL at a specified concentration, luciferase activity is assessed as described in Example 6.1. The solid circle refers to construct 1.1. The hollow circle refers to Fc (kih) fusion antigen binding molecules (control A) containing 4-1BB ligand trimers of DP47 non-targeting. Cell line NIH / 3T3-human FAP clone 39 is used as the target cell in graph (16A), and graph (16B) shows the activation of MV3 cell line as target cell, and graph (16C) uses WM-266-4 cell line as target cell. Activity was characterized by plotting the number of units of light released (URL) measured in 0.5 seconds relative to the nM concentration of the split 4-1BBL trimer construct tested. URL emission is due to luciferase-mediated oxidation of luciferin to oxyluciferin.

[0171] Figures 17A-17C The expression and activity of luciferase induced by NFκB activation measured by the assay method described in Example 6.1 are shown. The counts (CPS) of light released per second were measured at 0.5 seconds / well and the concentration of the human 4-1BB ligand Fc (kih) construct of the split trimer targeted or non-targeted FAP was plotted. HeLa reporter cells expressing human 4-1BB were grown in the absence of ( Figure 17A ) or in the presence of the human melanoma cell line MV-3 ​​( Figure 17B ) or WM-266-4( Figure 17C) were incubated for 6 hours. CPS was measured and the concentration of human 4-1BB ligand Fc (kih) constructs of different FAP-targeted or non-targeted split trimers was blotted. The cell ratio was 1 HeLa reporter cell expressing human 4-1BB to 5 tumor cells. For better display, the activation curve was divided into four different display blots, wherein construct 1.1 (human 4-1BB ligand Fc (kih) of the split trimer targeted by monovalent FAP) and control B (human 4-1BB ligand Fc (kih) of the split trimer of non-targeted monovalent with CH-CL cross and charged residues) were used as comparison curves. Figure 17A showed activation in the absence of tumor cells expressing cross-linked FAP, Figure 17B showed activation in the presence of MV-3 ​​tumor cells expressing cross-linked FAP, Figure 17C Activation is shown in the presence of WM-266-4 tumor cells expressing cross-linked FAP.

[0172] Figure 18 The luciferase expression and activity of the NFκB activation-induced luciferase measured for the construct of Example 2 are shown. The count (URL) of the light release is measured in 0.5 seconds / well, and the concentration of the human 4-1BB ligand Fc (kih) construct of the split trimer for the FAP targeting or non-targeting is plotted. The HeLa reporter cells expressing human 4-1BB are incubated for 6 hours in the absence or presence of human melanoma cell lines MV-3 ​​or WM-266-4 expressing cross-linked human FAP. The URL is measured and the concentration of human 4-1BB ligand Fc (kih) constructs 2.1, 2.3, 2.4, 2.5 and 2.6 of the split trimer for different FAP targeting or non-targeting and controls B, C, E and F are blotted. The cell ratio is 1 HeLa reporter cell expressing 4-1BB to 5 tumor cells. For better presentation, the activation curves were split into two different displayed blots with construct 2.1 (monovalent FAP-targeted split trimeric human 4-1BB ligand Fc (kih)).

[0173] exist Figure 19 In the present invention, an activation assay established with a T293-HEK reporter cell line expressing cynomolgus monkey 4-1BB is shown. Cross-linking of cynomolgus monkey 4-1BB expressed on reporter cells induced NFκB activation and NFκB-mediated luciferase expression. After cell lysis, luciferase can catalyze the oxidation of luciferin to oxyluciferin. This chemical reaction is positively correlated with the intensity of NFκB-mediated luciferase expression and can be measured by luminescence intensity (the number of units of light released).

[0174] Figure 20NFκB activation-induced luciferase expression and activity are shown. The count (URL) of light release is measured at 0.5 seconds / well, and the concentration of human 4-1BB ligand Fc (kih) constructs for the FAP targeting or non-targeted split trimers used is plotted. The T293-HEK reporter cells expressing cynomolgus monkey 4-1BB are incubated for 6 hours in the absence or presence of human melanoma cell lines MV-3 ​​or WM-266-4 expressing cross-linked human FAP. URL is measured and the concentration of human 4-1BB ligand Fc (kih) constructs for the split trimers of different FAP targeting or non-targeted are blotted. The cell ratio is 1 T293-HEK reporter cell expressing 4-1BB to 5 MV-3 ​​or 2 WM-266-4 cells. In order to better show, the activation curve is divided into two different blots, wherein construct 2.1 is used as a comparative curve.

[0175] Figure 21 : This scheme illustrates the principle of the T cell activation assay described in Example 6.3. Shown is a schematic activation assay established with HLA-A2-NLV-specific CD8 T cells and NLV-pulsed HLA-A2+FAP+ human melanoma cell line MV-3 ​​in the presence of different titration concentrations of FAP-targeted or non-targeted split trimeric human 4-1BB ligand Fc (kih) constructs. The cells were incubated for 28 hours, with the last 4 hours incubated in the presence of Golgi-Stop containing monensin. The ratio of NLV-specific CD8 T cells to MV-3 ​​tumor cells was 1:8.

[0176] Figure 22 (22A-22E) and 23 (23A-23E) relate to activation assays performed with HLA-A2-NLV-specific CD8 T cells and NLV-pulsed HLA-A2 + FAP + human melanoma cell line MV-3 ​​in the presence of different FAP-targeted or non-targeted split trimeric human 4-1BB ligand Fc (kih) constructs at different titration concentrations prepared in Example 1. For better presentation, the expression curves were divided into several different display blots, where construct 1.1 (monovalent FAP-targeted split trimeric human 4-1BB ligand Fc (kih)) and control B (monovalent non-targeted split trimeric human 4-1BB ligand Fc (kih)) were used as comparison curves. Results were obtained in four independent similar experiments and showed that prolonged IFNγ secretion and CD137 expression by NLV-specific CD8+ T cells were strictly dependent on simultaneous T cell activation through recognition of the NLV-HLA-A2 complex (signal 1) and 4-1BB triggering through FAP-targeted split human 4-1BBL (signal 2). The effect of 4-1BB upregulation was shown in Figure 22 The effect of INFγ expression of CD8+ T cells is shown in the curve Figure 23 The frequency of positive cells as a percentage of the total CD8+ T cell population is always shown in the graph. All FAP-targeted variants induced similar improvements in the activation of NLV peptide-activated CD8 T cells. Figure 22 4-1BB upregulation (positive feedback loop) is shown in Figure 23 The middle shows the expression of IFNγ after 24 hours of stimulation. The difference between the curves is within the range of normal error and is not significant.

[0177] Figure 24 and 25 Refers to the activation assay performed with HLA-A2-NLV specific CD8 T cells and NLV pulsed HLA-A2+FAP+ human melanoma cell line MV-3 ​​in the presence of titrated concentrations of different FAP-targeted or non-targeted split trimeric human 4-1BB ligand Fc (kih) constructs of Example 2. For better display, the expression curves were divided into two different display blots, where construct 2.1 (monovalent FAP-targeted split trimeric human 4-1BB ligand Fc (kih)) and control B were used as comparison curves. All FAP-targeted split trimeric human 4-1BB ligand Fc (kih) constructs showed similar activation improvements of HLA-A2-NLV peptide-specific CD8 T cells, in Figure 24 4-1BB upregulation (positive feedback loop) is shown in Figure 25 The middle shows the expression of IFNγ after 24 hours of stimulation. The difference between the curves is within the range of normal error and is not significant.

[0178] Figure 26 : This scheme demonstrates the experiment described in Example 6.4.

[0179] Figure 27 Induction of CD8+ T cell proliferation is shown. Shown is the frequency of proliferating CD8+ T cells relative to the concentration of the construct tested.

[0180] Figure 28A Single-dose PK experiment in healthy NOG mice involving construct 1.2 and control B. Shown is the decline in construct concentration over time. Figure 28B Shown are the results of single-dose PK experiments of constructs 2.1, 2.3, control B, and control C in tumor-bearing NOG mice humanized with stem cells. Figure 28C A single-dose PK experiment involving comparison of constructs 2.1 and 2.3 in healthy NOG mice was performed.

[0181] Figure 29The components of the human 4-1BB ligands for assembling the split trimer are shown. Figure (29A) shows the dimeric ligands fused to the human CL domain with mutations E123R and Q124K (charged residues) at the C-terminus, and Figure (29B) shows the monomer 4-1BB ligands fused to the human CH1 domain with mutations K147E and K213E (charged residues). Components of human 4-1BB ligands (71-254) antigen binding molecules (construct 3.3) for assembling the split trimer targeted by divalent CD19 are shown. Figure (29C) shows the dimeric ligands fused to the C-terminus of human IgG1 Fc hole chains. Figure (29D) shows the monomeric ligands fused to the C-terminus of human IgG1 Fc section chains.

[0182] Figure 30 Show the antigen binding molecule constructs 3.1 to 3.6 of the 4-1BBL trimer containing CD19 targeting of the present invention.The preparation and generation of these constructs are described in Example 3.VH and VL domains are the domains of anti-CD19 antibody 8B8-018, and thick black dot represents that knob enters hole (knob-into-hole) and is modified.* represents that the amino acid modifications (so-called charged residues) in CH1 and CL domains.

[0183] exist Figure 31A The randomization strategy for the CDR regions of the parental clone 8B8 is shown in Figure 1. The variable domain of the parental clone 8B8 and the CDR regions (boxed) are shown according to Kabat numbering. (X) represents the randomized position. Figure 31B Shown is a schematic description of a library generation strategy. Shown is a pcr amplification and cloning strategy for generating libraries based on 8B8 with A) randomized CDR1 and CDR2 regions in the light and heavy chains or B) randomized CDR1 and CDR3 regions in the light chain and the CDR3 regions in the heavy chain. Indicated are various enzymes for cloning into phagemids.

[0184] Figure 32 An alignment of the parental anti-CD19 clone 8B8 and selected affinity-matured binders is shown. The sequences of clone 8B8 and all selected affinity-matured binders are shown. The CDRs of the heavy and light chains are boxed.

[0185] Figure 33 Figure 3 SPR analysis of the parental 8B8 clone and its affinity-matured variants. Sensorgrams of clone 8B8 and its affinity-matured derivatives, which lack the LCDR1 N27d and N28 hotspots, are shown.

[0186] Figure 34 Diagram illustrating the assay setup for measuring simultaneous binding of CD19-targeted split-trimeric 4-1BBL to hu4-1BB and huCD19 (Example 8.2).

[0187] Figure 35 The graph in shows the simultaneous binding of CD19-targeted trimeric 4-1BBL FC fusion antigen binding molecule constructs 3.1, 3.3, 3.4, 3.5, 3.6 and 4.4 (Analyte 1) to immobilized human 4-1BB and human CD19 (Analyte 2).

[0188] Figure 36A and 36B Shown are different CD19-targeted or non-targeted split trimer human 4-1BB ligand Fc (kih) constructs and PHB-L expressing 4-1BB CD4 and CD8 T cells and human PBMCs pre-activated with Proleukin and reactivated with anti-human CD3 / human CD28. Binding was detected using R-phycoerythrin-fluorescent dye-conjugated anti-human IgG Fcγ-specific goat IgG F(ab')2 fragments. Shown is the median fluorescence intensity (MFI) relative to the test construct concentration. For better display, the binding curve was divided into three different blots, in which construct 3.4 and control F (isotype control huIgG1P329G LALA) were used as comparative curves. In CD45+CD3+CD8+T cells ( Figure 36A ) and CD45+CD3+CD4+T cells ( Figure 36B ). 4-1BB expression levels on CD8 T cells are generally higher than on CD4 T cells. All constructs bind to human 4-1BB with very similar affinities.

[0189] Figures 37A-37D The human 4-1BB ligand Fc (kih) antigen binding molecules of the split trimer of CD19 targeting or non-targeting are shown to be combined with the B cell lymphoma cell lines expressing human CD19: diffuse large B cell non-Hodgkin's lymphoma SU-DHL-8 (37A), acute precursor B cell lymphoid leukemia Nalm6 (37B), diffuse large cell lymphoblastic lymphoma Toledo (37C) and diffuse large B cell lymphoma OCI-Ly18 (37D). The binding was detected by anti-human IgG Fcγ specific goat IgG F (ab') 2 fragment conjugated with R-phycoerythrin-fluorescent dye. What is shown is the median fluorescence intensity (MFI) relative to the concentration of the test construct. For better display, the binding curve is divided into three different blots, wherein construct 3.4 and control F (isotype control huIgG1 P329G LALA) are used as comparison curves. All constructs are bound to human CD19 with very similar affinities.

[0190] Figure 38Luciferase expression and activity of NFκB activation-induced human 4-1BB ligand Fc (kih) antigen binding molecules related to CD19-targeted or non-targeted split trimers. The number of units (URL) of light released was measured at 0.5 seconds per well, and the concentration of human 4-1BB ligand Fc (kih) constructs 3.1 and 3.3 and control molecules B and C for the CD19-targeted or non-targeted split trimers used was plotted. HeLa reporter cells expressing human 4-1BB were incubated for 7.5 hours in the absence or presence of SU-DHL-8 or Pfeiffer cells expressing cross-linked human CD19. URL was measured and the concentration of human 4-1BB ligand Fc (kih) constructs for the split trimers for different CD19-targeted or non-targeted splits was blotted. The cell ratio was 1 HeLa reporter cell expressing 4-1BB to 2.5 or 5 tumor cells.

[0191] Figure 39 The binding of different humanized variants of T84.66 IgG on human gastric adenocarcinoma cells expressing CEA is shown. Based on this data, humanized variant 1 was selected to be included in the trimeric human 4-1BB ligand Fc (kih) antigen binding molecules targeted by CEA.

[0192] Figure 40 Shown are antigen binding molecule constructs 5.1 to 5.6 of the 4-1BBL trimer containing CEA targetings of the present invention. The preparation and generation of these constructs are described in Example 11. VH and VL domains are the domains of anti-CEA antibody T84.66-LCHA, and thick black dots represent knob-into-hole modifications. * Represents amino acid modifications (so-called charged residues) in CH1 and CL domains.

[0193] Figure 41 : Figure 41 A shows a schematic depiction of human NA3B3A2-avi His, the antigen used to assess binding of CEA-targeted split-trimeric 4-1BBL Fc(kih) antigen binding molecules. Figure 41 B illustrates the assay setup for measuring simultaneous binding of CEA-targeted split-trimeric 4-1BBL to hu4-1BB and human NA3B3A2 (Example 12.1).

[0194] Figure 42 The graph in shows the simultaneous binding of CEA-targeted trimeric 4-1BBL Fc fusion antigen binding molecule constructs 5.4, 5.6, 5.7 and 5.8 (Analyte 1) to immobilized human 4-1BB and human NA3B3A2 (Analyte 2).

[0195] Binding of different CEA-targeted or non-targeted split-trimeric human 4-1BB ligand Fc (kih) constructs to PHA-L 4-1BB-expressing CD4 and CD8 T cells and human PBMCs preactivated with Proleukin and reactivated with anti-human CD3 / anti-human CD28 was shown. Figure 43 In. Anti-human IgG Fcγ-specific goat IgG F(ab')2 fragments conjugated with R-phycoerythrin-fluorescent dye were used to detect binding. Shown is the median fluorescence intensity (MFI) relative to the test construct concentration. For better display, the binding curve was divided into two different blots, in which construct 5.4 and control F (isotype control huIgG1P329G LALA) were used as comparison curves. Binding was monitored on CD45+CD3+CD8+T cells (bottom blot) and CD45+CD3+CD4+T cells (top blot). The 4-1BB expression level on CD8 T cells is generally higher than that on CD4 T cells. All constructs bind to human 4-1BB with very similar affinities.

[0196] Figure 44 The binding of 4-1BB ligand Fc (kih) constructs of CEA-targeted or non-targeted split trimers to human gastric cell line MKN-45 (left) and human colorectal adenocarcinoma cell line LS180 (right) expressing human CEA is shown. Binding is detected by anti-human IgG Fcγ-specific goat IgG F(ab')2 fragment conjugated with R-phycoerythrin-fluorescent dye. Shown is the median fluorescence intensity (MFI) relative to the concentration of the test construct.

[0197] Figure 45 luciferase expression and activity of NFκB activation-induced human 4-1BB ligand Fc (kih) antigen binding molecules related to CEA targeting or non-targeted split trimers. The number of units (URL) of light released was measured at 0.5 seconds / well, and the concentration of human 4-1BB ligand Fc (kih) constructs 5.4, 5.6, 5.7 and 5.8 of the CEA targeting or non-targeted split trimers and control molecules were blotted. The HeLa reporter cells expressing human 4-1BB were incubated for 6 hours in the absence or presence of human gastric cancer cell line MKN-45 expressing cross-linked human CEA. The cell ratio was 1 HeLa reporter cell expressing 4-1BB to 3 tumor cells.

[0198] Figure 46 :exist Figure 46 In A and 46B, the components used to assemble a monovalent FAP-targeted split trimeric human OX40 ligand (Construct 6.1) are shown. Figure 46 A involves a dimeric ligand fused to the human IgG1-CL domain, Figure 46B relates to a monomeric ligand fused to the human IgG1-CH1 domain. Figure 46 C shows the antigen-binding molecule construct 6.1 containing the OX40L trimer targeted by FAP. Figure 46 In D, DP47 "non-targeted" human IgG1 PGLALA is shown (control F).

[0199] Figure 47A Binding of FAP-targeted, split-trimeric human OX40L to FAP-positive WM-266-4 cells is shown. WM-266-4 cells express high levels of human fibroblast activation protein (huFAP). Only the FAP-targeted OX40 ligand Fc(kih) construct (filled squares), but not control 5 (filled diamonds), bound to WM-266-4 cells. Binding is shown as the median fluorescence intensity (MFI) of a fluorescein isothiocyanate (FITC)-labeled anti-human IgG Fcγ-specific goat IgG F(ab')2 fragment used as a secondary detection antibody. MFI was determined by flow cytometry. The x-axis shows the concentration of the antibody construct. Figure 47B Binding of the FAP-targeted OX4O ligand Fc(kih) construct to human FAP-human OX40-negative A549 NucLight Red cells is shown. The FAP-targeted OX4O ligand Fc(kih) construct showed no binding to OX40-negative, FAP-negative A549 tumor cells. Binding is shown as the median fluorescence intensity (MFI) of a FITC-labeled anti-human IgG Fcγ-specific goat IgG F(ab')2 fragment used as a secondary detection antibody. MFI was measured by flow cytometry and baseline corrected by subtracting the MFI of the blank control.

[0200] Figure 48 :exist Figure 48 In A, the combination of FAP-Ox40L and resting and activated human CD4 T cells is shown. Ox40 is not expressed on resting human CD4 T cells (left). In the absence of cells expressing human Ox40, no binding is observed (left figure). After activating human PBMC, Ox40 is upregulated on CD4+ T cells (right). FAP-Ox40L binds to activated CD4 T cells of Ox40+. Binding is shown as the median fluorescence intensity (MFI) of FITC-labeled anti-human IgG Fcγ-specific goat IgG F(ab')2 fragments used as the second detection antibody. MFI is measured by flow cytometry, and the baseline is corrected by subtracting the MFI of the blank control. The x-axis shows the concentration of the antibody construct. Figure 48B shows that Ox40 is not expressed on resting human CD8 T cells (left side). In the absence of cells expressing human Ox40, no binding was observed (left figure). After activating human PBMC, Ox40 was upregulated on CD8+T cells (right side). The expression of Ox40 on human CD8+T cells was lower than that on CD4+T cells, and varied between donors and time points. The expression of Ox40 on the depicted CD8 T cells was lower. FAp-Ox40L binds to the activated CD8 T cells of Ox40+. Binding was shown as the median fluorescence intensity (MFI) of the anti-human IgG Fcγ-specific goat IgG F(ab')2 fragment of FITC-labeled second detection antibody. MFI was measured by flow cytometry, and baseline was corrected by subtracting the MFI of the blank control. The x-axis shows the concentration of the antibody construct.

[0201] exist Figure 49 In the present invention, activation of the NFκB signaling pathway by a split trimeric human OX40L antigen binding molecule (FAP-OX40L) targeted by FAP in HeLa_hOx40_NFkB_Luc1 reporter cells is demonstrated. Activation is shown with (right graph) or without (left graph) cross-linking by a second antibody. Reporter cells were cultured for 5 hours in the presence of FAP-OX40L at a specified concentration with or without a 1:2 ratio of cross-linked second polyclonal anti-huIgG1 Fcγ-specific goat IgG IgGF(ab)2 fragment. Luciferase activity was assessed as described in Example 6.1. Activity was characterized by blotting the number of units of light released (URL) measured in 0.5 seconds relative to the nM concentration of the test construct. The emission of URL is due to the luciferase-mediated oxidation of luciferin to oxyluciferin.

[0202] Figure 50 : Activation of NFκB by FAP-OX40L in HeLa_hOx40_NFkB_Luc1 reporter cells in the presence of FAP-positive cells is shown in Figure 50 In A. Activation of the NFκB signaling pathway by FAP-OX40L in reporter cells in the presence of low FAP-expressing NIH-3T3 human FAP cells is shown (ratio of 3 FAP+ tumor cells to 1 reporter cell). NFκB-mediated luciferase activity is characterized by plotting the number of units of released light (URL) measured in 0.5 seconds relative to the nM concentration of the test compound. The emission of URL is due to the luciferase-mediated oxidation of luciferin to oxyluciferin. These values ​​are baseline corrected by subtracting the URL of the blank control. For better comparison, the area under the curve of the dose-response curve of each blot was quantified as a marker of the agonist ability of each construct. In Figure 50The comparison is shown in B. The areas were calculated using GraphPad Prism. The values ​​were baseline corrected by subtracting the values ​​of the blank control.

[0203] Figure 51 Shown is the costimulation (Example 15.5) of the resting human PBMC of the suboptimal TCR triggering of OX40 mediation. The super crosslinking of FAP-Ox40L of NIH / 3T3-huFAP clone 39 cells of the present invention strongly promotes the survival and proliferation of human CD4 and CD8 T cells. Show the event counts of viable CD4+ (left) and CD8+ (right) T cells. Subtract the baseline value of the sample containing only anti-human CD3 (clone V9, huIgG1), resting human PBMC and NIH / 3T3-huFAP clone 39. Therefore, the enhancing effect of OX40 costimulation is shown here, but it is not the effect of suboptimal anti-CD3 stimulation itself. In the figure at the bottom, the recovery-proliferation of the suboptimal TCR stimulation of resting human PBMC with cell surface-fixed FAP-Ox40L is shown. Detailed Description of the Invention

[0204] definition

[0205] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly used in the art to which the invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0206] As used herein, the term "antigen binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are antibodies, antibody fragments, and scaffold antigen binding proteins.

[0207] As used herein, the term "module capable of specific binding to target cell antigens" refers to a polypeptide molecule specifically bound to an antigenic determinant. In one aspect, an antigen binding module can activate signal transduction by its target cell antigen. In a specific aspect, an antigen binding module can guide the entity (e.g., TNF families ligand trimer) connected thereto to a target site, such as to a tumor cell or tumor stroma of a specific type with an antigenic determinant. The module capable of specific binding to target cell antigens includes antibodies and fragments thereof further defined herein. In addition, the module capable of specific binding to target cell antigens includes the scaffold antigen-binding proteins further defined herein, such as, based on the binding domains of the repeat proteins of design or the repeat domains of design (see, for example, WO 2002 / 020565).

[0208] With respect to antibodies or fragments thereof, the term "module capable of specific binding to a target cell antigen" refers to the portion of the molecule that comprises a region that specifically binds to part or all of the antigen and is complementary thereto. The module capable of specific antigen binding can be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). Specifically, the module capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0209] The term "antibody" is used herein in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0210] The term "monoclonal antibody" as used herein refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies comprising the group are identical and / or bind to the same epitope, except for possible variant antibodies, e.g., antibodies containing naturally occurring mutations or produced during the production of a monoclonal antibody preparation, which variants are typically present in small amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen.

[0211] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. The term "bispecific" means that the antigen binding molecule can specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for different antigenic determinants. In certain embodiments, a bispecific antigen binding molecule can simultaneously bind to two antigenic determinants, especially two antigenic determinants expressed on two different cells.

[0212] The term "valence" as used in this application means the presence of a specific number of binding sites in an antigen binding molecule. For this reason, the terms "divalent," "tetravalent," and "hexavalent" mean the presence of two, four, and six binding sites, respectively, in an antigen binding molecule.

[0213] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to antibodies with a structure that is substantially similar to the structure of a natural antibody. "Native antibody" refers to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two light chains and two heavy chains bound by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also referred to as a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also referred to as a light chain constant region. Antibody heavy chains can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Antibody light chains can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0214] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, cited in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments having two antigen binding sites that can be bivalent or bispecific, see, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Hudson et al. also describe triabodies and tetrabodies in Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0215] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the variable regions of the heavy and light chains, as well as the constant domains of the light chain and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to an antibody fragment comprising a light chain fragment including the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains have free sulfhydryl groups. Pepsin treatment produces a F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0216] The term "crossover Fab fragment" or "xFab fragment" or "exchange Fab fragment" refers to a Fab fragment in which the variable regions or constant regions of the heavy and light chains are exchanged. Two different chain compositions of the exchange Fab molecule are possible and are included in the bispecific antibodies of the present invention: on the one hand, the variable regions of the Fab heavy and light chains are exchanged, that is, the exchange Fab molecule comprises a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Such an exchange Fab molecule is also called a CrossFab (VLVH) On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the exchange Fab molecule contains a peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH1). This exchange Fab molecule is also called CrossFab. (CLCH1) .

[0217] "Single-chain Fab fragments" or "scFab" are polypeptides consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide having at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering) to create interchain disulfide bonds.

[0218] "Exchange single-chain Fab fragments" or "x-scFab" are polypeptides consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL together form an antigen-binding site that specifically binds to an antigen, and wherein the linker is a polypeptide having at least 30 amino acids. In addition, these x-scFab molecules can be further stabilized by inserting cysteine ​​residues (e.g., position 44 in the variable light chain and position 100 in the variable light chain according to Kabat numbering) to create interchain disulfide bonds.

[0219] A "single-chain variable fragment (scFv)" is an antibody heavy chain variable region (V) joined by a short linker peptide of 10 to about 25 amino acids. H ) and light chain variable region (V L The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can be V H The N-terminus of V LThe antibody fragment is a fragment of a polypeptide having a C-terminus and a C-terminus, or vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original antibody. ScFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments comprise single-chain polypeptides having VH domain characteristics, i.e., the ability to assemble together with the VL domain into a functional antigen-binding site, or VL domain characteristics, i.e., the ability to assemble together with the VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0220] "Scaffold antigen binding proteins" are known in the art, for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen binding domains, see, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13:695-701 (2008). In one aspect of the present invention, the scaffold antigen binding protein is selected from CTLA-4 (Evibody), lipocalin (Anticalin), a molecule derived from protein A, such as the Z domain of protein A (Affibody), the A domain (Avimer / Maxibody), serum transferrin (trans-body); a designed ankyrin repeat protein (DARPin), a variable domain of an antibody light chain or heavy chain (single domain antibody, sdAb), a variable domain of an antibody heavy chain (nanoantibody, aVH), a V NAR fragment, fibronectin (AdNectin), C-type lectin domain (Tetranectin); variable domain of novel antigen receptor β-lactamase (V NAR fragments), human γ-crystallin or ubiquitin (Affilin molecule); Kunitz-type domains of human protease inhibitors, microbodies such as proteins from the knottin family, peptide aptamers and fibronectin (adnectin).

[0221] CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a CD28 family receptor expressed primarily on CD4+ T cells. Its extracellular domain has an Ig fold similar to the variable domain. The loops corresponding to the CDRs of antibodies can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also referred to as Evibodies (e.g., US7166697B1). The size of Evibodies is approximately the same as the isolated variable region of antibodies (e.g., domain antibodies). For further details, see Journal of Immunological Methods 248(1-2), 31-45(2001).

[0222] Lipocalins are a family of extracellular proteins that transport small, hydrophobic molecules such as steroids, bilirubin, retinoids, and lipids. They have a rigid β-sheet secondary structure with multiple loops at the open end of the pyramidal structure that can be engineered to bind to different target antigens. Anticalins range in size from 160 to 180 amino acids and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), US Pat. No. 7,250,297 B1, and US Pat. No. 2007,0224,633.

[0223] Affibodies are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. The domain consists of a three-helix bundle of approximately 58 amino acids. Libraries were generated by randomizing surface residues. For further details, see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP 1641818 A1.

[0224] Avimers are multidomain proteins derived from the A-domain scaffold family. The native domain, consisting of approximately 35 amino acids, adopts a defined disulfide bond structure. Diversity is generated by altering the natural variation exhibited by the A-domain family. For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).

[0225] Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind to different target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include Trans-bodies. For further details, see J. Biol. Chem 274, 24066-24073 (1999).

[0226] Designed ankyrin repeat proteins (DARPins) are derived from ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. By randomizing the residues in the first α-helix and β-turn of each repeat, they can be engineered to bind to different target antigens. Their binding interface can be increased by increasing the number of modules (affinity maturation). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100 (4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.

[0227] Single-domain antibodies are antibody fragments composed of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of the heavy chain of an antibody from camelids (nanobodies or V H H fragment). In addition, the term single-domain antibody includes the autonomous human heavy chain variable domain (aVH) or VH derived from sharks. NAR fragment.

[0228] Fibronectin is a scaffold that can be engineered to bind to an antigen. Adnectins consist of a backbone composed of the native amino acid sequence of the 10th domain of 15 repeating units of human fibronectin type III (FN3). The three loops at one end of the β-sandwich can be engineered to enable the adnectin to specifically recognize the therapeutic target of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764, and US6818418B1.

[0229] Peptide aptamers are combinatorial recognition molecules composed of a constant scaffold protein, usually thioredoxin (TrxA), containing a constrained variable peptide loop inserted in the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005).

[0230] Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length that contain 3-4 cysteine ​​bridges—examples of microproteins include KalataBI and conotoxins and cysteine ​​knot peptides (knottins). Microproteins have loops that can be engineered to include up to 25 amino acids without affecting the overall folding of the microprotein. For more details on engineered cysteine ​​knot peptide domains, see WO2008098796.

[0231] An "antigen-binding molecule that binds to the same epitope as a reference molecule" refers to an antigen-binding molecule that blocks 50% or more of the binding of the reference molecule to its antigen in a competition assay, whereas conversely, the reference molecule blocks 50% or more of the binding of the antigen-binding molecule to its antigen in a competition assay.

[0232] The term "antigen binding domain" refers to a portion of an antigen binding molecule that includes a region that specifically binds to part or all of an antigen and is complementary. In the case of a large antigen, an antigen binding molecule may only bind to a specific portion of the antigen, which is called an epitope. The antigen binding domain can be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0233] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to a site on the polypeptide macromolecule to which an antigen-binding moiety is combined to form an antigen-binding moiety-antigen complex (e.g., a continuous segment of amino acids or a conformational configuration consisting of different regions of discontinuous amino acids). Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other pathological cells, on the surface of immune cells, and are free in serum and / or in the extracellular matrix (ECM). Proteins used as antigens herein can be any native form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. In a specific embodiment, the antigen is a human protein. In the case of referring to a specific protein herein, the term includes "full-length" unprocessed protein and any form of protein produced by processing in cells. The term also encompasses naturally occurring variants of proteins, for example, splice variants or allelic variants.

[0234] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of antigen binding molecules to bind to specific antigens can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, for example, surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17,323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)). In one embodiment, the degree of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the antigen binding molecule's binding to the antigen, as measured by, for example, SPR. In certain embodiments, the molecule bound to the antigen has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or smaller, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 The dissociation constant (Kd) of the

[0235] "Affinity" or "binding affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Therefore, equivalent affinities can comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0236] As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, such as a cell in a tumor, such as a cancer cell or a tumor stromal cell. In certain embodiments, the target cell antigen is an antigen on the surface of a tumor cell. In one embodiment, the target cell antigen is selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), CD19, CD20, and CD33. Specifically, the target cell antigen is fibroblast activation protein (FAP).

[0237] The term "fibroblast activation protein (FAP)", also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any natural FAP of any vertebrate origin, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term includes "full-length" unprocessed FAP as well as any form of FAP produced by processing in cells. The term also encompasses naturally occurring variants of FAP, e.g., splice variants or allelic variants. In one embodiment, the antigen binding molecules of the present invention are capable of specifically binding to human, mouse and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 149, SEQ ID NO: 20) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid and nucleotide sequences of the His-tagged human FAP ECD are shown in SEQ ID NOs: 15 and 16, respectively. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 126, SEQ ID NO: 23), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. SEQ ID NOs: 24 and 25 show the amino acid and nucleotide sequences of the His-tagged mouse FAP ECD, respectively. SEQ ID NOs: 26 and 27 show the amino acid and nucleotide sequences of the His-tagged cynomolgus monkey FAP ECD, respectively. Preferably, the anti-FAP binding molecules of the present invention bind to the extracellular domain of FAP. Exemplary anti-FAP binding molecules are described in International Patent Application No. WO 2012 / 020006 A2.

[0238] The term "carcinoembryonic antigen (CEA)", also known as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), refers to any native CEA of any vertebrate origin, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The amino acid sequence of human CEA is shown in UniProt accession number P06731 (version 151, SEQ ID NO: 28). CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med., 121: 439-462, 1965; Berinstein NL, J Clin Oncol., 20: 2197-2207, 2002). Initially classified as a protein expressed only in fetal tissues, CEA has now been identified in several normal adult tissues. These tissues are primarily epithelial in origin, including cells of the gastrointestinal tract, respiratory tract, and urogenital tract, as well as cells of the colon, cervix, sweat glands, and prostate (Nap et al., Tumour Biol., 9(2-3):145-53, 1988; Nap et al., Cancer Res., 52(8):2329-23339, 1992). Tumors of epithelial origin and their metastases contain CEA as a tumor-associated antigen. Although the presence of CEA itself does not indicate transformation into cancer cells, the distribution of CEA is indicative. In normal tissues, CEA is usually expressed on the apical surface of cells ( S., Semin Cancer Biol. 9(2):67-81(1999)), making it difficult for antibodies in the bloodstream to access. In contrast to normal tissues, CEA tends to be expressed on the entire surface of cancer cells ( S., Semin Cancer Biol. 9(2):67-81(1999)). This change in expression pattern allows CEA to bind to antibodies in cancer cells. In addition, CEA expression is increased in cancer cells. In addition, increased CEA expression promotes increased cell-cell adhesion, which may lead to metastasis (Marshall J., Semin Oncol., 30(a Suppl. 8):30-6, 2003). The incidence of CEA expression is generally high in various tumor entities. According to published data, self-analysis in tissue samples confirms its high incidence, approximately 95% in colorectal cancer (CRC), 90% in pancreatic cancer, 80% in gastric cancer, 60% in non-small cell lung cancer (NSCLC, which co-expresses HER3), and 40% in breast cancer; low expression was found in small cell lung cancer and glioblastoma.

[0239] CEA is easily cleaved from the cell surface and shed from the tumor directly or through lymphatic vessels into the bloodstream. Due to this property, serum CEA levels have been used as a clinical marker for cancer diagnosis and screening for cancer recurrence, especially colorectal cancer (Goldenberg DM., The International Journal of Biological Markers, 7: 183-188, 1992; Chau I. et al., J Clin Oncol., 22: 1420-1429, 2004; Flamini et al., Clin Cancer Res; 12(23): 6985-6988, 2006).

[0240] The term "melanoma-associated chondroitin sulfate proteoglycan (MCSP)", also known as chondroitin sulfate proteoglycan 4 (CSPG4), refers to any naturally occurring MCSP of any vertebrate origin, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The amino acid sequence of human MCSP is shown in UniProt accession number Q6UVK1 (version 103, SEQ ID NO: 29). The term "epidermal growth factor receptor (EGFR)", also known as proto-oncogene c-ErbB-1 or receptor tyrosine protein kinase erbB-1, refers to any naturally occurring EGFR of any vertebrate origin, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The amino acid sequence of human EGFR is shown in UniProt accession number P00533 (version 211, SEQ ID NO: 30).

[0241] The term "CD19" refers to the B lymphocyte antigen CD19, also referred to as the B lymphocyte surface antigen B4 or the T cell surface antigen Leu-12, including any natural CD19 of any vertebrate origin, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The amino acid sequence of human CD19 is shown in Uniprot accession number P15391 (version 160, SEQ ID NO: 31). The term encompasses "full-length" unprocessed human CD19 and any form of human CD19 produced by processing in cells, as long as the antibodies reported herein bind to them. CD19 is a structurally different cell surface receptor expressed on the surface of human B cells, including but not limited to pre-B cells, early developing B cells (i.e., immature B cells), mature B cells and malignant B cells that are terminally differentiated into plasma cells. CD19 is expressed by most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B-cell chronic lymphocytic leukemias (CLL), prolymphocytic leukemias, hairy cell leukemias, common acute lymphoblastic leukemias, and some unmarked acute lymphoblastic leukemias. The expression of CD19 on plasma cells further suggests that it may be expressed in differentiated B-cell tumors such as multiple myeloma. Therefore, the CD19 antigen is a target for immunotherapy for the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0242] The term "CD20" refers to the B lymphocyte antigen CD20, also known as transmembrane 4-domain subfamily A member 1 (MS4A1), B lymphocyte surface antigen B1, or leukocyte surface antigen Leu-16, including any native CD20 from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The amino acid sequence of human CD20 is shown in Uniprot accession number P11836 (version 149, SEQ ID NO: 32). The term "CD33" refers to the myeloid cell surface antigen CD33, also known as SIGLEC3 or gp67, including any native CD33 from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The amino acid sequence of human CD33 is shown in Uniprot Accession No. P20138 (version 157, SEQ ID NO: 33).

[0243] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain involved in binding an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0244] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is highly variable in sequence and / or forms structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs), the latter of which have the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) Hypervariable regions (HVRs) are also called complementarity determining regions (CDRs), and these terms are used interchangeably herein to refer to portions of the variable region that form the antigen binding region. This particular region has been described by Kabat et al. in US Patent. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al. in J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared to each other. However, the application of any definition referring to the CDR of an antibody or its variants is expected to be within the scope of the terms defined and used herein. Appropriate amino acid residues encompassing the CDRs defined in each of the references cited above are listed in Table A below for comparison. The exact number of residues encompassing a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

[0245] Table A: CDR Definition 1

[0246] CDR Kabat Chothia <![CDATA[AbM 2 ]]> <![CDATA[V H CDR1]]> 31-35 26-32 26-35 <![CDATA[V H CDR2]]> 50-65 52-58 50-58 <![CDATA[V H CDR3]]> 95-102 95-102 95-102 <![CDATA[V L CDR1]]> 24-34 26-32 24-34 <![CDATA[V L CDR2]]> 50-56 50-52 50-56 <![CDATA[V L CDR3]]> 89-97 91-96 89-97

[0247] 1 The numbering of all CDR definitions in Table A follows the numbering convention proposed by Kabat et al. (see below).

[0248] 2 "AbM" with a lowercase "b" as used in Table A refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0249] Kabat et al. also defined a numbering system that can be applied to the variable region sequence of any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al. in US Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise indicated, reference to specific amino acid residue position numbers in antibody variable regions is based on the Kabat numbering system.

[0250] In addition to the CDR1 in VH, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also comprise "specificity determination residues" or "SDRs" that are residues that contact antigens. SDRs are included in CDR regions referred to as abbreviated CDRs (or a-CDRs). Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0251] As used herein, in the context of antigen binding molecules (e.g., antibodies), the term "affinity maturation" refers to an antigen binding molecule derived from a reference antigen binding molecule, for example by mutation, that binds to the same antigen as the reference antibody, preferably to the same epitope; and has a higher affinity for the antigen than for the reference antigen binding molecule. Affinity maturation generally involves modification of one or more amino acid residues in one or more CDRs of the antigen binding molecule. Typically, affinity-matured antigen binding molecules bind to the same epitope as the initial reference antigen binding molecule.

[0252] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0253] For the purposes of this paper, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0254] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0255] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0256] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of the variable domains in at least one (typically two) variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or altered based on the constant region of the original antibody to produce properties according to the present invention, particularly those antibodies with respect to C1q binding and / or Fc receptor (FcR) binding.

[0257] A "human" antibody is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0258] As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an antibody heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises the IgG CH2 and IgG CH3 domains. The "CH2 domain" of a human IgG Fc region typically extends from an amino acid residue at approximately position 231 to an amino acid residue at approximately position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" is comprised of a residue segment from the C-terminus of the Fc region to the CH2 domain (i.e., from an amino acid residue at approximately position 341 of IgG to an amino acid residue at approximately position 447). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "knob" ("knob") introduced in one of its chains and a corresponding "cavity" ("hole") introduced in the other of its chains; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). As described herein, such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0259] " Knot into hole " technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001). Generally, the method includes introducing a protrusion (" knot ") at the interface of the first polypeptide and introducing a corresponding cavity (" hole ") in the interface of the second polypeptide, so that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. Protrusions are constructed by replacing the small amino acid side chains at the interface of the first polypeptide with larger side chains (such as tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (such as alanine or threonine), a compensatory cavity with the same or similar size as the protrusion is produced in the interface of the second polypeptide. Protrusions and cavities can be produced by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit of the Fc domain comprising the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Numbering is according to the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0260] "A region equivalent to an immunoglobulin Fc region" is intended to include naturally occurring allelic variants of the immunoglobulin Fc region, and variants that have changes that produce substitutions, additions, or deletions but do not substantially reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular toxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the immunoglobulin Fc region without substantial loss of biological function. These variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, for example, Bowie, JU et al., Science 247: 1306-10 (1990)).

[0261] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0262] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc region of an antibody, elicits a signaling event that stimulates cells bearing the receptor to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activating Fc receptor is human FcγRIIIa (see UniProt accession number P08637, version 141).

[0263] The term "TNF ligand family member" or "TNF family ligand" refers to a proinflammatory cytokine. Cytokines, in particular members of the TNF ligand family, generally play a key role in the stimulation and coordination of the immune system. Currently, 19 cytokines have been identified as members of the TNF (tumor necrosis factor) ligand superfamily based on sequence, function and structural similarity. All of these ligands are type II transmembrane proteins with a C-terminal extracellular domain (ectodomain), an N-terminal intracellular domain and a single transmembrane domain. The C-terminal extracellular domain, called the TNF homology domain (THD), has 20-30% amino acid identity between superfamily members and is responsible for binding to the receptor. The TNF ectodomain is also responsible for the formation of a trimeric complex of TNF ligands that is recognized by its specific receptor.

[0264] A member of the TNF ligand family is selected from the group consisting of lymphotoxin alpha (also known as LTA or TNFSF1), TNF (also known as TNFSF2), LT (also known as TNFSF3), OX40L (also known as TNFSF4), CD40L (also known as CD154 or TNFSF5), FasL (also known as CD95L, CD178 or TNFSF6), CD27L (also known as CD70 or TNFSF7), CD30L (also known as CD153 or TNFSF8), 4-1BBL (also known as TNFSF9), TRAIL (also known as APO2 L, CD253 or TNFSF10), RANKL (also known as CD254 or TNFSF11), TWEAK (also known as TNFSF12), APRIL (also known as CD256 or TNFSF13), BAFF (also known as CD257 or TNFSF13B), LIGHT (also known as CD258 or TNFSF14), TL1A (also known as VEGI or TNFSF15), GITRL (also known as TNFSF18), EDA-A1 (also known as exoprotein A1) and EDA-A2 (also known as exoprotein A2). The term refers to any naturally occurring TNF family ligand of any vertebrate origin, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. In a specific embodiment of the invention, the TNF ligand family member is selected from OX40L, FasL, CD27L, TRAIL, 4-1BBL, CD40L and GITRL. In certain embodiments, the TNF ligand family member is selected from 4-1BBL and OX40L.

[0265] More information on TNF ligand family members, in particular sequences, can be obtained from publicly accessible databases such as Uniprot (www.uniprot.org). For example, human TNF ligands have the following amino acid sequences: human lymphotoxin alpha (UniProt Accession No. P01374, SEQ ID NO: 34), human TNF (UniProt Accession No. P01375, SEQ ID NO: 35), human lymphotoxin beta (UniProt Accession No. Q06643, SEQ ID NO: 36), human OX40L (UniProt Accession No. P23510, SEQ ID NO: 37), human CD40L (UniProt Accession No. P29965, SEQ ID NO: 38), human FasL (UniProt Accession No. P48023, SEQ ID NO: 39), human CD27L (UniProt Accession No. P32970, SEQ ID NO: 40), human CD30L (UniProt Accession No. P32971, SEQ ID NO: 41), 4-1BBL (UniProt Accession No. P41273, SEQ ID NO: 42), human TNF ligands have the following amino acid sequences: human lymphotoxin alpha (UniProt Accession No. P01374, SEQ ID NO: 34), human TNF (UniProt Accession No. P01375, SEQ ID NO: 35), human lymphotoxin beta (UniProt Accession No. Q06643, SEQ ID NO: 36), human OX40L (UniProt Accession No. P23510, SEQ ID NO: 37), human CD40L (UniProt Accession No. P29965, SEQ ID NO: 38), human FasL (UniProt Accession No. P48023, SEQ ID NO: 39), human CD27L (UniProt Accession No. P32970, SEQ ID NO NO:42), TRAIL (UniProt Accession No. P50591, SEQ ID NO:43), RANKL (UniProt Accession No. 014788, SEQ ID NO:44), TWEAK (UniProt Accession No. 043508, SEQ ID NO:45), APRIL (UniProt Accession No. 075888, SEQ ID NO:46), BAFF (UniProt Accession No. Q9Y275, SEQ ID NO:47), LIGHT (UniProt Accession No. 043557, SEQ ID NO:48), TL1A (UniProt Accession No. 095150, SEQ ID NO:49), GITRL (UniProt Accession No. Q9UNG2, SEQ ID NO:50) and exoprotein A (UniProt Accession No. Q92838, SEQ ID NO:51).

[0266] " ectodomain " is the domain of the membrane protein that extends to the extracellular space (i.e., the space outside the target cell). Extracellular domain is typically the part that causes contact with the surface in protein, causing signal transduction. Therefore, the ectodomain of TNF ligand family members as defined herein refers to the part (ectodomain) that extends into the extracellular space in TNF ligand protein, also includes the shorter part or its fragment that is responsible for trimerization and is combined with corresponding TNF receptor. Therefore, the term " ectodomain of TNF ligand family members or its fragment " refers to the ectodomain of the TNF ligand family members forming extracellular domain or its part (receptor binding domain) that can still be combined with receptor.

[0267] The term "co-stimulatory TNF ligand family member" or "co-stimulatory TNF family ligand" refers to a subtype of a TNF ligand family member that can co-stimulate the proliferation of T cells and cytokine production. These TNF family ligands can co-stimulate TCR signals when interacting with their corresponding TNF receptors, and the interaction with their receptors leads to the recruitment of TNFR-associated factors (TRAFs), which trigger a signal cascade that leads to T cell activation. Co-stimulatory TNF family ligands are selected from 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT, more specifically, co-stimulatory TNF ligand family members are selected from 4-1BBL and OX40L.

[0268] As mentioned above, 4-1BBL is a type II transmembrane protein and a member of the TNF ligand family. It has been described that the complete or full-length 4-1BBL with the amino acid sequence SEQ ID NO:42 that forms a trimer on the cell surface. Trimer formation is started by the specific motif of the extracellular domain of 4-1BBL. The motif is named "trimerization region" in this article. The amino acid 50-254 (SEQ ID NO:52) of people 4-1BBL sequence forms the extracellular domain of 4-1BBL, but even if its fragment can also form a trimer. In a specific embodiment of the present invention, the term "extracellular domain of 4-1BBL or a fragment thereof" refers to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 (amino acids 52-254 of human 4-1BBL), SEQ ID NO: 1 (amino acids 71-254 of human 4-1BBL), SEQ ID NO: 3 (amino acids 80-254 of human 4-1BBL) and SEQ ID NO: 2 (amino acids 85-254 of human 4-1BBL), or a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 96 (amino acids 71-248 of human 4-1BBL), SEQ ID NO: 375 (amino acids 52-248 of human 4-1BBL), SEQ ID NO: 374 (amino acids 80-248 of human 4-1BBL) and SEQ ID NO: 373 (amino acids 85-248 of human 4-1BBL), but other fragments of the extracellular domain capable of trimerization are also included herein.

[0269] As previously described, OX40L is another type II transmembrane protein and another member of the TNF ligand family. The complete or full-length human OX40L has the amino acid sequence of SEQ ID NO: 37. Amino acids 51-183 of the human OX40L sequence (SEQ ID NO: 53) form the extracellular domain of OX40L, but even fragments thereof are capable of forming trimers. In specific embodiments of the present invention, the term "extracellular domain of OX40L or a fragment thereof" refers to a polypeptide having an amino acid sequence selected from SEQ ID NO: 53 (amino acids 51-183 of human OX40L) or SEQ ID NO: 54 (amino acids 52-183 of human OX40L), but other fragments of the extracellular domain capable of trimerization are also included herein.

[0270] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, (G4S) n 、(SG4) n or G4 (SG4) n Peptide linkers, wherein "n" is generally a number between 1 and 10, typically 1 to 4, and in particular 2, i.e., these peptides are selected from GGGGS (SEQ ID NO: 128), GGGGSGGGGS (SEQ ID NO: 13), SGGGGSGGGG (SEQ ID NO: 55) and GGGGSGGGGSGGGG (SEQ ID NO: 56), but also include the sequence GSPGSSSSGS (SEQ ID NO: 57), GSGSGSGS (SEQ ID NO: 58), GSGSGNGS (SEQ ID NO: 59), GGSGSGSG (SEQ ID NO: 60), GGSGSG (SEQ ID NO: 61), GGSG (SEQ ID NO: 62), GGSGNGSG (SEQ ID NO: 63), GGNGSGSG (SEQ ID NO: 64) and GGNGSG (SEQ ID NO: 65). Peptide linkers of particular interest are (G4S)1 or GGGGS (SEQ ID NO: 128), (G4S)2 or GGGGSGGGGS (SEQ ID NO: 13) and GSPGSSSSGS (SEQ ID NO: 57), more specifically (G4S)2 or GGGGSGGGGS (SEQ ID NO: 13) and GSPGSSSSGS (SEQ ID NO: 57).

[0271] The term "amino acid" as used in this application refers to the group of naturally occurring carboxy α-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0272] As used herein, "single-chain fusion protein" refers to a single-chain polypeptide consisting of one or two extracellular domains of the TNF ligand family member fused to a portion of an antigen binding moiety or Fc portion. Fusion can be performed by directly connecting the N-terminal or C-terminal amino acid of the antigen binding moiety to the C-terminal or N-terminal amino acid of the extracellular domain of the TNF ligand family member via a peptide linker.

[0273] By "fused" or "linked" is meant that the components (eg, a polypeptide and the extracellular domain of the TNF ligand family member) are linked directly by a peptide bond or via one or more peptide linkers.

[0274] "Percentage (%) of amino acid sequence identity relative to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after alignment and introduction of gaps (if necessary, to achieve maximum percentage sequence identity, and without considering any conservative substitutions as part of sequence identity). Alignment for determining percentage amino acid sequence identity can be achieved with various methods well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN.SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. However, for the purposes of this article, amino acid sequence identity % values ​​were generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, and the source code has been filed with the U.S. Copyright Office (US Copyright Office, Washington DC, 20559) along with user files, and is registered in the U.S. Copyright Office with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not changed. When ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A relative to (with, or to) a given amino acid sequence B (or alternatively, the % amino acid sequence identity that a given amino acid sequence A has or comprises relative to (with, or to) a given amino acid sequence B) is calculated as follows:

[0275] Multiply 100 by the fraction X / Y

[0276] wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in a program alignment of A and B, and wherein Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein were obtained as described in the preceding paragraph using the ALIGN-2 computer program.

[0277] In certain embodiments, amino acid sequence variants of the antigen binding molecules containing TNF ligand trimer provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen binding molecules containing TNF ligand trimer. The amino acid sequence variants of the antigen binding molecules containing TNF ligand trimer can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the molecule or by peptide synthesis. These modifications include, for example, deletions and / or insertions and / or substitutions of the residues in the antibody amino acid sequence. Any combination of deletion, insertion and substitution can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding. The site of interest of substitution-type mutagenesis includes HVR and framework (FR). Conservative substitutions are provided under the heading "preferably substituted" in Table B, and are further described hereinafter with reference to amino acid side chain categories (1) to (6). Amino acid substitutions can be introduced into the molecule of interest, and products with desired activity can be screened out, such as retained / enhanced antigen binding, reduced immunogenicity, or enhanced ADCC or CDC.

[0278] Table B

[0279]

[0280]

[0281] Amino acids can be grouped according to common side chain properties:

[0282] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0283] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0284] (3) Acidic: Asp, Glu;

[0285] (4) Basic: His, Lys, Arg;

[0286] (5) Residues that affect chain orientation: Gly, Pro;

[0287] (6) Aromatic: Trp, Tyr, Phe.

[0288] Non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0289] The term "amino acid sequence variant" includes a substantial variant in which amino acid substitutions are present in one or more hypervariable region residues of a parent antigen binding molecule (such as a humanized or human antibody). Typically, the resulting variant selected for further study will have a change (such as improvement) and / or some biological properties of the parent antigen binding molecule substantially retained in terms of certain biological properties (such as, increased affinity, reduced immunogenicity) relative to the parent antigen binding molecule. Exemplary substitution variants are affinity matured antibodies, which can be easily produced using, for example, affinity maturation techniques based on phage display such as those described herein. In short, one or more HVR residues are mutated, and the variant antigen binding molecules are displayed on phage and screened for specific biological activity (such as binding affinity). In certain embodiments, substitutions, insertions or deletions can be carried out in one or more HVRs, as long as these changes do not substantially reduce the ability of the antigen binding molecules to bind antigens. For example, conservative changes (such as conservative substitutions provided herein) that do not substantially reduce binding affinity can be made in HVR. A useful method for identifying antibody residues or regions that may be targeted for mutagenesis is referred to as "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or a group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity for the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antigen binding molecule complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they possess desired characteristics.

[0290] Amino acid sequence insertion includes length from one residue to the amino and / or carboxyl terminal fusion of the polypeptide containing one hundred or more residues, and insertion between the sequence of single or multiple amino acid residues.The example of terminal insertion includes the antigen binding molecules containing TNF families ligand trimer with N-terminal methionyl residues.Other insertion variants of molecule include fusion with the N-terminal or C-terminal of polypeptide, to increase the serum half-life of the antigen binding molecules containing TNF ligand trimer.

[0291] In certain embodiments, the antigen binding molecules containing TNF families ligand trimer provided herein are changed to increase or reduce the degree to which the antibody is glycosylated. By changing the amino acid sequence to produce or remove one or more glycosylation sites, the glycosylation variants of the molecule can be easily obtained. When the antigen binding molecules containing TNF families ligand trimer include Fc district, the carbohydrate attached thereto may be changed. The natural antibodies produced by mammalian cells generally include the biantennary oligosaccharides of side chains, which are generally connected to the Asn297 of the CH2 domains in the Fc district by an N bond. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose attached to the GlcNAc in the " stem " of biantennary oligosaccharide structure. In some embodiments, oligosaccharide modification can be carried out in the antigen binding molecules containing TNF families ligand trimer, to produce variants with some improved properties. In one aspect, there is provided a variant of the antigen binding molecules containing TNF families ligand trimer with a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc district. Such fucosylation variants can have enhanced ADCC function, see, for example, U.S. Patent Publication Nos.US2003 / 0157108 (Presta, L.) or US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Other variants of the antigen binding molecules containing TNF families ligand trimer of the present invention include those with bisected oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc district are bisected by GlcNAc. These variants can have reduced fucosylation and / or enhanced ADCC function, see, for example, WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have enhanced CDC function and are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0292] In certain embodiments, it may be desirable to produce a cysteine ​​engineered variant of an antigen binding molecule containing a TNF families ligand trimer of the present invention, such as "thioMAb", wherein one or more residues of the molecule are replaced by cysteine ​​residues. In a specific embodiment, the substituted residue appears in an accessible position of the molecule. By replacing those residues with cysteine, reactive thiol groups are thus positioned at accessible sites of the antibody, and can be used to conjugate the antibody with other modules (such as drug modules or linker-drug modules) to produce immunoconjugates. In certain embodiments, any one or more of the following residues can be replaced by cysteine: V205 (Kabat numbering) of a light chain; A118 (EU numbering) of a heavy chain; and S400 (EU numbering) in a heavy chain Fc region. Cysteine ​​engineered antigen binding molecules can be produced according to the description in, for example, U.S. Patent No. 7,521,541.

[0293] In some aspects, the antigen binding molecules of the ligand trimer containing TNF families provided herein can be further modified to contain other non-protein modules known in the art and readily available. Modules suitable for antibody derivatization include but are not limited to water-soluble polymers. The limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polyoxypropylene / ethylene oxide copolymer, polyoxyethylated polyol (such as glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. Polymer can have any molecular weight and can be side chain or non-branched. In one embodiment, the polymer of the present invention can be attached to an antibody. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers for derivatization can be determined based on the following considerations, including but not limited to, the specific properties or functions of the antibody to be improved, whether the bispecific antibody derivative is used for treatment under limited conditions, etc. In another aspect, there is provided a conjugate of an antibody and a non-protein module that can be selectively heated by exposure to radiation. In one embodiment, the non-protein module is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). Radiation can have any wavelength, and includes but is not limited to a wavelength that does not damage ordinary cells, but the wavelength heats the non-protein module to the temperature of the cells near the antibody-non-protein module that kills.

[0294] In another aspect, immunoconjugates of the antigen binding molecules containing TNF family ligand trimers provided herein can be obtained."Immunoconjugates" are antibodies conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0295] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide can contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0296] About " isolated " nucleic acid molecule or polynucleotide, it is meant the nucleic acid molecule, DNA or RNA that has been removed from its natural environment. For example, the recombinant polynucleotide encoding the polypeptide contained in the vector is considered to be isolated for the purposes of the present invention. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially purified) polynucleotides in solution form. Isolated polynucleotides include polynucleotide molecules contained in cells that usually contain polynucleotide molecules, but polynucleotide molecules exist outside the chromosome or exist at a chromosomal position different from its natural chromosomal position. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention also include such molecules produced synthetically. In addition, polynucleotides or nucleic acids can be or can include regulatory elements, such as promoters, ribosome binding sites or transcription terminators.

[0297] By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to 5 point mutations per every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or, in the reference sequence, multiple nucleotides up to 5% of the total nucleotides may be inserted into the reference sequence. These changes in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed independently between residues in the reference sequence or in one or more consecutive groups within the reference sequence. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be routinely determined using known computer programs such as ALIGN-2, as discussed above for polypeptides.

[0298] The term "expression cassette" refers to a recombinant or synthetically produced polynucleotide having a series of specific nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, in addition to other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0299] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene operably associated therewith into a target cell and to direct the expression of the specific gene. The term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which it has been introduced. The expression vectors of the present invention include expression cassettes. The expression vectors allow the transcription of large amounts of stable mRNA. Once the expression vector enters the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cell's transcription and / or translation machinery. In one embodiment, the expression vector of the present invention includes an expression cassette encoding a polynucleotide sequence of a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0300] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of these cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parental cells in terms of nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the original transformed cells are included herein. Host cells are any type of cell system that can be used to produce the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells and plant cells, only a few of which are listed here, as well as cells included in transgenic animals, transgenic plants or cultured plants or animal tissues.

[0301] An "effective amount" of an agent is that amount necessary to cause a physiological change in the cells or tissues to which it is administered.

[0302] A "therapeutically effective amount" of a pharmaceutical agent (e.g., a pharmaceutical composition) refers to an amount effective to achieve the desired therapeutic or preventive result, at dosages and for periods of time. For example, a therapeutically effective amount of an agent eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0303] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, the individual or subject is a human.

[0304] The term "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the preparation is administered.

[0305] "Pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, or preservatives.

[0306] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0307] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in a treated individual, either for prevention or during clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the molecules of the invention are used to delay the development of a disease or slow the progression of a disease.

[0308] As used herein, the term "cancer" refers to a proliferative disease, such as lymphoma, carcinoma, lymphoma, blastoma, sarcoma, leukemia, lymphocytic leukemia, lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck tumors, melanoma of the skin or in the eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colorectal cancer (CRC), pancreatic cancer, breast cancer, triple-negative breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma. , urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, central nervous system (CNS) tumors, chordoma, brain stem glioma, glioblastoma multiforme, astrocytoma, neurilemoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing's sarcoma, melanoma, multiple myeloma, B cell cancer (lymphoma), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, including refractory forms of any of the foregoing cancers, or a combination of one or more of the foregoing cancers.

[0309] Antigen binding molecules containing TNF family ligand trimers of the present invention

[0310] The present invention provides novel TNF family ligand trimer-containing antigen binding molecules with particularly advantageous properties, such as manufacturability, stability, binding affinity, biological activity, targeting efficiency, and reduced toxicity.

[0311] In a first aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0312] (a) at least one module capable of specifically binding to a target cell antigen, and

[0313] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0314] The antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof.

[0315] In a specific aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0316] (a) at least one module capable of specifically binding to a target cell antigen,

[0317] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0318] wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof, and

[0319] (c) The Fc domain consists of a first and a second subunit that are capable of stably associating.

[0320] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (a) at least one module capable of specifically binding to a target cell antigen, and

[0321] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0322] The antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof, wherein the TNF ligand family member co-stimulates human T cell activation.

[0323] In another specific aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (a) at least one module capable of specifically binding to a target cell antigen, and

[0324] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0325] The antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof, wherein the extracellular domains of the TNF ligand family members are identical in all cases.

[0326] In a further aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer of claim 1, comprising:

[0327] (a) at least one module capable of specifically binding to a target cell antigen, and

[0328] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0329] The antigen-binding molecule is characterized in that

[0330] (i) the first polypeptide contains a CH1 or CL domain, and the second polypeptide contains a CL or CH1 domain, respectively, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the CH1 or CL domain via a peptide linker, and wherein the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof linked to the CL or CH1 domain of the polypeptide via a peptide linker, or

[0331] (ii) the first polypeptide contains a CH3 domain, and the second polypeptide correspondingly contains a CH3 domain, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the C-terminus of the CH3 domain via a peptide linker, and wherein the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof linked to the C-terminus of the CH3 domain of the polypeptide via a peptide linker, or

[0332] (iii) the first polypeptide contains a VH-CL or VL-CH1 domain, and the second polypeptide contains a VL-CH1 domain or a VH-CL domain, respectively, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the VH or VL via a peptide linker, and wherein the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof linked to the VL or VH of the polypeptide via a peptide linker.

[0333] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer comprises a TNF ligand family member that co-stimulates human T cell activation, which is selected from 4-1BBL and OX40L. More specifically, the TNF ligand family member is 4-1BBL.

[0334] In another aspect, the extracellular domain of the TNF ligand family member comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 96, SEQ ID NO: 373, SEQ ID NO: 374, and SEQ ID NO: 375, in particular an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 96. In one aspect, the extracellular domain of the TNF ligand family member or a fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 96, in particular an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 96. In a specific aspect, the extracellular domain of the TNF ligand family member or a fragment thereof comprises an amino acid sequence of SEQ ID NO: 96.

[0335] In a further aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0336] (a) at least one module capable of specifically binding to a target cell antigen, and

[0337] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0338] The antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, and the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 3, and SEQ ID NO: 4. In a specific aspect, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 96.

[0339] In one aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0340] (a) at least one module capable of specifically binding to a target cell antigen, and

[0341] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0342] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6.

[0343] In a further aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0344] (a) at least one module capable of specifically binding to a target cell antigen, and

[0345] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0346] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 183.

[0347] In yet another aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0348] (a) at least one module capable of specifically binding to a target cell antigen, and

[0349] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0350] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 184 or SEQ ID NO: 185.

[0351] In another aspect, the TNF ligand family member is OX40L. In a specific aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the extracellular domain of the TNF ligand family member comprises SEQ ID NO: 53 or SEQ ID NO: 54 amino acid sequence, especially SEQ ID NO: 53 amino acid sequence.

[0352] In one aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0353] (a) at least one module capable of specifically binding to a target cell antigen, and

[0354] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0355] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 371 or SEQ ID: 372, and the second polypeptide correspondingly comprises the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 54.

[0356] In one aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0357] (a) at least one module capable of specifically binding to a target cell antigen,

[0358] (b) a first polypeptide comprising a CH1 or CL domain and a second polypeptide comprising a CL or CH1 domain, respectively, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the CH1 or CL domain via a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof linked to the CL or CH1 domain of the polypeptide via a peptide linker.

[0359] In one aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, comprising:

[0360] (a) at least one module capable of specifically binding to a target cell antigen,

[0361] (b) a first polypeptide comprising a CH1 domain and a second polypeptide comprising a CL domain, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the CH1 domain via a peptide linker, and the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof linked to the CL domain of the polypeptide via a peptide linker.

[0362] In another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, comprising:

[0363] (a) at least one module capable of specifically binding to a target cell antigen,

[0364] (b) a first polypeptide comprising a CL domain and a second polypeptide comprising a CH1 domain, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof linked to each other and to the CL domain via a peptide linker, and the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof linked to the CH1 domain of the polypeptide via a peptide linker.

[0365] In another aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0366] (a) a module capable of specifically binding to a target cell antigen, and

[0367] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0368] The antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof.

[0369] In yet another aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0370] (a) one or more modules capable of specifically binding to target cell antigens, and

[0371] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0372] The antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof connected to the polypeptide via a peptide linker.

[0373] In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0374] (a) two modules capable of specifically binding to target cell antigens, and

[0375] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0376] The antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof.

[0377] In a specific aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, comprising:

[0378] (a) at least one module capable of specifically binding to a target cell antigen, and

[0379] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0380] The antigen binding molecule is characterized in that the first polypeptide contains a CH3 domain, the second polypeptide correspondingly contains a CH3 domain, and the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other and to the C-terminus of the CH3 domain via a peptide linker, and the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof connected to the C-terminus of the CH3 domain of the polypeptide via a peptide linker. Specifically, such an antigen binding molecule containing a TNF family ligand trimer comprises two modules capable of specifically binding to a target cell antigen.

[0381] In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0382] (a) two modules capable of specifically binding to target cell antigens, and

[0383] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0384] The antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof, wherein the two modules capable of specifically binding to a target cell antigen bind to two different target cell antigens.

[0385] In a further aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer as defined above, wherein the module capable of specifically binding to a target cell antigen is selected from an antibody, an antibody fragment and a scaffold antigen binding protein.

[0386] In one aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the module that can be specifically bound to a target cell antigen is selected from antibody fragments, Fab molecules, exchange Fab molecules, single-chain Fab molecules, Fv molecules, scFv molecules, single domain antibodies, aVH and scaffold antigen binding proteins. In one aspect, the module that can be specifically bound to a target cell antigen is aVH or scaffold antigen binding proteins. In one aspect, the module that can be specifically bound to a target cell antigen is a scaffold antigen binding proteins that can be specifically bound to a target cell antigen.

[0387] Specifically, the antigen-binding molecule containing a TNF family ligand trimer comprises one or two modules capable of specifically binding to target cell antigens.

[0388] In a particular aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the module capable of specifically binding to a target cell antigen is a Fab molecule or an exchange Fab molecule capable of specifically binding to a target cell antigen. Specifically, the module capable of specifically binding to a target cell antigen is a Fab capable of specifically binding to a target cell antigen.

[0389] In addition, provided is an antigen binding molecule containing a TNF family ligand trimer as described herein, wherein the target cell antigen is selected from fibroblast activation protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20 and CD33.

[0390] In a further aspect, an antigen binding molecule containing a TNF family ligand trimer according to the present invention is provided, wherein a peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to the CH1 domain of the heavy chain via a second peptide linker, and wherein one extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to the CL domain on the light chain via a third peptide linker.

[0391] In another aspect, an antigen binding molecule containing a TNF family ligand trimer according to the present invention is provided, wherein a peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to the CL domain of the heavy chain via a second peptide linker, and wherein one extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to the CH1 domain on the light chain via a third peptide linker.

[0392] In a further aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer according to the present invention, wherein a peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to the CL domain of the light chain via a second peptide linker, and wherein one extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to the CH1 domain of the heavy chain via a third peptide linker.

[0393] In a specific aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the peptide linker is (G4S)2. In one aspect, the first peptide linker is (G4S)2(SEQ ID NO:13), the second peptide linker is GSPGSSSSGS(SEQ ID NO:57), and the third peptide linker is (G4S)2(SEQ ID NO:13). Specifically, the present invention relates to an antigen binding molecule containing a TNF ligand trimer as described above, wherein the first peptide linker is (G4S)2(SEQ ID NO:13), the second peptide linker is GSPGSSSSGS(SEQ ID NO:13), and the third peptide linker is (G4S)2(SEQ ID NO:13).

[0394] In another aspect, the antigen binding molecule comprising a TNF family ligand trimer as defined above comprises an Fc domain consisting of a first and a second subunit capable of stably associating.

[0395] Specifically, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises (a) a Fab molecule capable of specifically binding to a target cell antigen, wherein the Fab heavy chain is fused at the C-terminus to the N-terminus of the CH2 domain in the Fc domain, and (c) an Fc domain composed of a first and a second subunit capable of stably combining.

[0396] In a further aspect, the Fc domain is an IgG, specifically an IgG1 Fc domain or an IgG4 Fc domain. More specifically, the Fc domain is an IgG1 Fc domain. In a specific aspect, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

[0397] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0398] The Fc domains of the antigen binding molecules containing TNF families ligand trimers of the present invention are composed of a pair of polypeptide chains comprising the heavy chain domains of immunoglobulin molecules. For example, the Fc domains of immunoglobulin G (IgG) molecules are dimers, each of which subunits comprises IgG heavy chain constant domains CH2 and CH3. The two subunits of the Fc domains can stably unite with each other.

[0399] The Fc domain gives favorable pharmacokinetic properties to the antigen binding molecules of the present invention, including a long serum half-life and a favorable tissue-blood distribution ratio that contribute to good accumulation in the target tissue. However, at the same time, the bispecific antibodies of the present invention may cause cells expressing Fc receptors to be unexpectedly targeted rather than preferred antigen-bearing cells. Therefore, in a specific aspect, compared with the natural IgG1 Fc domain, the Fc domain of the antigen binding molecules containing the TNF family ligand trimer of the present invention exhibits reduced binding affinity to Fc receptors and / or reduced effector functions. In one aspect, the Fc domain is substantially not bound to the Fc receptors and / or does not induce effector functions. In a specific aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the Fc domain does not induce effector functions. The reduced effector function may include, but is not limited to, one or more of: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), decreased cytokine secretion, decreased immune complex-mediated antigen uptake by antigen-presenting cells, decreased binding to NK cells, decreased binding to macrophages, decreased binding to monocytes, decreased binding to polymorphonuclear cells, decreased direct signaling-induced apoptosis, decreased dendritic cell maturation, or decreased T cell priming.

[0400] In certain aspects, one or more amino acid modifications can be introduced into the Fc region of the antigen binding molecules containing the TNF family ligand trimer provided herein to produce Fc region variants. Fc region variants can be included in a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0401] In a specific aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0402] (a) at least one module capable of specifically binding to a target cell antigen,

[0403] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0404] wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof, and

[0405] (c) an Fc domain composed of a first and a second subunit capable of stably associating, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor.

[0406] In one aspect, the Fc domains of the antigen binding molecules containing TNF families ligand trimers of the present invention include one or more amino acid mutations that reduce the binding affinity of Fc domains to Fc receptors and / or effector functions. Typically, identical one or more amino acid mutations are present in each of two subunits of the Fc domains. Specifically, the Fc domains are included in amino acid replacements at positions E233, L234, L235, N297, P331, and P329 (EU numbering). Specifically, the Fc domains are included in amino acid replacements at positions 234 and 235 (EU numbering) and / or 329 (EU numbering) of IgG heavy chains. More specifically, there is provided an antigen binding molecule containing trimeric TNF families ligands according to the present invention, comprising the Fc domains with amino acid replacements L234A, L235A, and P329G (" P329G LALA ", EU numbering) in IgG heavy chains. Amino acid replacements L234A and L235A refer to so-called LALA mutations. The amino acid substitution "P329G LALA" combination almost completely eliminates Fcγ receptor binding of the human IgG1 Fc domain and is described in International Patent Application Publication No. WO 2012 / 130831 A1, which also describes methods for preparing this mutant Fc domain and methods for determining its properties such as Fc receptor binding or effector function. "EU numbering" refers to the numbering according to the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0407] Fc domains with reduced Fc receptor binding and / or effector function also include Fc domains having substitutions at one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include Fc mutants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are replaced with alanine ( U.S. Patent No. 7,332,581 ).

[0408] In another aspect, the Fc domain is an IgG4 Fc domain. Compared with IgG1 antibodies, IgG4 antibodies exhibit reduced binding affinity and reduced effector functions to Fc receptors. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), specifically an amino acid substitution S228P. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution L235E and S228P and P329G (EU numbering). Such IgG4 Fc domain mutants and Fc gamma receptor binding properties thereof are also described in WO 2012 / 130831.

[0409] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes can be verified by, for example, sequencing.

[0410] Binding to Fc receptors can be easily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of the Fc domain or the cell-activating bispecific antigen binding molecules comprising the Fc domain to the Fc receptor can be assessed using a cell line known to express a specific Fc receptor, such as human NK cells expressing FcγIIIa receptors.

[0411] The effector functions of the Fc domain or the bispecific antibodies of the present invention comprising the Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al., Proc. Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, a non-radioactive assay method (see, for example, ACTI TMnon-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for this assay include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0412] In some embodiments, the binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some embodiments in which the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. C1q binding assays can be performed to determine whether the bispecific antibodies of the present invention are able to bind to C1q and therefore have CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0413] In a specific aspect, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

[0414] Fc domain modifications that promote heterodimerization

[0415] In one aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0416] (a) at least one module capable of specifically binding to a target cell antigen,

[0417] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0418] wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises only one extracellular domain of the TNF ligand family member or a fragment thereof, and

[0419] (c) by being able to stablize the Fc domains that the first and second subunits of association are constituted, wherein Fc domains include one or more reductions and Fc acceptors, especially Fc γ receptors that bind amino acid replacement.Therefore, they include the different modules that one or another fusion is carried out with two subunits of Fc domains, and described subunit is generally contained in two unequal polypeptide chains (" heavy chain ").The recombinant co-expression of these polypeptides and subsequent dimerization result in some possible combinations of two polypeptides.In order to improve the productivity and the purity of the antigen binding molecules containing TNF families ligand trimer in recombinant production, therefore in the Fc domains of the antigen binding molecules containing TNF families ligand trimer of the present invention, introducing the modification that promotes the combination of desired polypeptide will be advantageous.

[0420] Therefore, the Fc domains of the antigen binding molecules containing TNF families ligand trimers of the present invention include the modification promoting the union of the first and second subunits of the Fc domains. The site of the most extensive protein-protein interaction between the two subunits of human IgG Fc domains is in the CH3 domains of the Fc domains. Therefore, the modification is specifically in the CH3 domains of the Fc domains.

[0421] In one specific aspect, the modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Thus, in a specific aspect, the present invention relates to an antigen-binding molecule comprising a TNF family ligand trimer as described above, comprising an IgG molecule, wherein the Fc portion of the first heavy chain comprises a first dimerization module and the Fc portion of the second heavy chain comprises a second dimerization module that allows heterodimerization of the two heavy chains of the IgG molecule; according to the knob-into-hole technique, the first dimerization module comprises a knob and the second dimerization module comprises a hole.

[0422] The "knot into cavity" technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al. Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method comprises introducing a protrusion ("knot") at the interface of a first polypeptide and introducing a corresponding cavity ("cavity") into the interface of a second polypeptide such that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. Protrusions are constructed by replacing small amino acid side chains at the interface of a first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is generated at the interface of the second polypeptide.

[0423] Therefore, in a specific aspect, in the CH3 domain of the first subunit of the Fc domain of the antigen-binding molecule containing a TNF family ligand trimer of the present invention, the amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned in a cavity in the CH3 domain of the second subunit; and in the CH3 domain of the second subunit of the Fc domain, the amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit in which the protrusion in the CH3 domain of the first subunit can be positioned.

[0424] Protrusions and cavities can be produced by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis, or by peptide synthesis.

[0425] In a specific aspect, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). More specifically, in the second subunit of the Fc domain, the threonine residue at position 366 is additionally replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A). More specifically, in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced with a cysteine ​​residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domain. Disulfide bridges further stabilize the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0426] In an alternative aspect, modifications that promote association of the first and second subunits of the Fc domain include modifications that mediate electrostatic manipulation effects, for example, as described in PCT Publication No. WO 2009 / 089004. Generally, this approach involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues such that homodimer formation is electrostatically unfavorable but heterodimerization is electrostatically favorable.

[0427] Modification of the CH1 / CL domain

[0428] In order to further improve correct pairing, the antigen binding molecules containing the TNF family ligand trimer can include different charged amino acid substitutions (so-called " charged residues "). These modifications are introduced into cross or non-cross CH1 and CL domains. In a particular aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, wherein the amino acid at position 123 (EU numbering) in one of the CL domains has been replaced with arginine (R), the amino acid at position 124 (EU numbering) has been replaced with lysine (K), wherein the amino acid at position 147 (EU numbering) and at position 213 (EU numbering) in one of the CH1 domains has been replaced with glutamic acid (E).

[0429] More specifically, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, wherein in the CL domain of an adjacent TNF ligand family member, the amino acid at position 123 (EU numbering) has been replaced with arginine (R), the amino acid at position 124 (EU numbering) has been replaced with lysine (K), and wherein in the CH1 domain of an adjacent TNF ligand family member, the amino acids at position 147 (EU numbering) and at position 213 (EU numbering) have been replaced with glutamic acid (E).

[0430] Specific antigen-binding molecules containing TNF family ligand trimers

[0431] In another aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, wherein the antigen binding molecule comprises:

[0432] A first heavy chain and a first light chain, which together constitute a Fab molecule capable of specifically binding to a target cell antigen, a first peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker, the first peptide being fused at its C-terminus to the second heavy chain or light chain via a second peptide linker, and a second peptide comprising an extracellular domain of the TNF ligand family member, the second peptide being fused at its C-terminus to the second light chain or heavy chain, respectively, via a third peptide linker.

[0433] In a further aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein a first peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to a CH1 domain as part of the heavy chain via a second peptide linker, and a second peptide comprising one extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to a CL domain as part of the light chain via a third peptide linker.

[0434] In yet another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein a first peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to a CL domain as part of the heavy chain through a second peptide linker, and a second peptide comprising an extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to a CH1 domain as part of the light chain through a third peptide linker.

[0435] In a further aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein a first peptide comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker is fused at its C-terminus to a VH domain as part of the heavy chain via a second peptide linker, and a second peptide comprising one extracellular domain of the TNF ligand family member or a fragment thereof is fused at its C-terminus to a VL domain as part of the light chain via a third peptide linker.

[0436] In one aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer according to claim 21, wherein in the CL domain of the adjacent TNF ligand family member, the amino acid at position 123 (EU numbering) has been replaced with arginine (R), the amino acid at position 124 (EU numbering) has been replaced with lysine (K), and wherein in the CH1 domain of the adjacent TNF ligand family member, the amino acid at position 147 (EU numbering) and at position 213 (EU numbering) has been replaced with glutamic acid (E). These modifications produce so-called charged residues with advantageous properties to avoid undesirable consequences, such as mispairing.

[0437] In addition, provided is an antigen binding molecule containing a TNF family ligand trimer as described herein, wherein the target cell antigen is selected from fibroblast activation protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20 and CD33.

[0438] Antigen-binding molecule containing a trimer of TNF family ligands, wherein the target cell antigen is FAP

[0439] In a specific aspect, an antigen binding molecule comprising a TNF family ligand trimer is provided, wherein the target cell antigen is fibroblast activation protein (FAP).

[0440] In one aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer, wherein the module capable of specifically binding to FAP comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 100, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 101, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 102, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 103, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 104, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 105.

[0441] In a specific aspect, an antigen binding molecule containing a TNF family ligand trimer of the present invention is provided, wherein the module capable of specifically binding to a target cell antigen is a Fab molecule capable of specifically binding to FAP and comprises a VH domain and a VL domain, wherein the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0442] In another aspect, an antigen binding molecule containing a TNF family ligand trimer of the present invention is provided, wherein the module capable of specifically binding to a target cell antigen is a Fab molecule capable of specifically binding to FAP and comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 102, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 103, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 104, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 105.

[0443] In a further aspect, the module capable of specifically binding to FAP comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0444] In another aspect, the module capable of specifically binding to FAP comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 106, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 107.

[0445] In one aspect, the module capable of specifically binding to FAP comprises: a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 17, or a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 106 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 107.

[0446] In one specific aspect, the module capable of specifically binding to FAP comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17. In another specific aspect, the module capable of specifically binding to FAP comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 106 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 107. In a specific aspect, the module capable of specifically binding to FAP comprises: a VH domain consisting of the amino acid sequence of SEQ ID NO: 106 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 107.

[0447] In a further aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0448] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and

[0449] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0450] The antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, and the second polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 3 and SEQ ID NO: 4.

[0451] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0452] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and

[0453] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0454] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 96.

[0455] In another aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0456] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 106 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 107, and

[0457] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0458] The antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, and the second polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 3 and SEQ ID NO: 4.

[0459] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0460] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 106 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 107, and

[0461] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0462] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 96.

[0463] In another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises: a first heavy chain and a first light chain, both of which constitute a Fab molecule capable of specifically binding to a target cell antigen,

[0464] a second heavy chain and a second light chain, wherein the second heavy chain comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker, and the second heavy chain is fused at its C-terminus to the CH1 domain via a second peptide linker,

[0465] The second light chain comprises an extracellular domain of the TNF ligand family member or a fragment thereof, the second light chain is fused to the CL domain at its C-terminus via a third peptide linker, and the antigen binding molecule comprises:

[0466] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 106 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 107,

[0467] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 108, SEQ ID NO: 111 and SEQ ID NO: 113, and

[0468] (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114.

[0469] In another specific aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises: a first heavy chain and a first light chain, both of which constitute a Fab molecule capable of specifically binding to a target cell antigen,

[0470] a second heavy chain and a second light chain, wherein the second heavy chain comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker, and the second heavy chain is fused at its C-terminus to the CL domain via a second peptide linker,

[0471] The second light chain comprises an extracellular domain of the TNF ligand family member or a fragment thereof, the second light chain is fused to the CH1 domain at its C-terminus via a third peptide linker, and wherein the molecule comprises:

[0472] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 106 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 107,

[0473] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 173, and

[0474] (iii) a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 174.

[0475] More specifically, provided is an antigen binding molecule comprising a TNF family ligand trimer, comprising:

[0476] (a) a first heavy chain and a first light chain, which together constitute a Fab molecule capable of specifically binding to a target cell antigen, wherein the first heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 106, and the first light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 107, and

[0477] (b) a second heavy chain and a second light chain, the second heavy chain comprising two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker, the second heavy chain fused to a CL domain at its C-terminus via a second peptide linker, the second light chain comprising one extracellular domain of the TNF ligand family member or a fragment thereof, the second light chain fused to a CH1 domain at its C-terminus via a third peptide linker, wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 173, and the second light chain comprises the amino acid sequence of SEQ ID NO: 120 or SEQ ID NO: 174. Specifically, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 119, and the second light chain comprises the amino acid sequence of SEQ ID NO: 120.

[0478] In addition, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0479] (a) at least one module capable of specifically binding to a target cell antigen, and

[0480] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0481] The antigen binding molecule is characterized in that the first polypeptide contains a CH3 domain and the second polypeptide correspondingly contains a CH3 domain, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other and to the C-terminus of the CH3 domain via a peptide linker, and wherein the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof connected to the C-terminus of the CH3 domain of the polypeptide via a peptide linker.

[0482] In a specific aspect, such an antigen binding molecule containing a TNF family ligand trimer comprises two modules capable of specifically binding to a target cell antigen.

[0483] More specifically, such an antigen binding molecule containing a TNF family ligand trimer comprises:

[0484] (i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 121, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 122, and two light chains comprising the amino acid sequence of SEQ ID NO: 19, or

[0485] (ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 123, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 124, and two light chains comprising the amino acid sequence of SEQ ID NO: 125, or

[0486] (iii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 126, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 127, and two light chains comprising the amino acid sequence of SEQ ID NO: 125.

[0487] In a further aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, wherein the antigen binding molecule is selected from:

[0488] a) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 18, a first light chain comprising the amino acid sequence of SEQ ID NO: 19, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 14, and a second light chain comprising the amino acid sequence of SEQ ID NO: 15;

[0489] b) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 18, a first light chain comprising the amino acid sequence of SEQ ID NO: 19, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 115, and a second light chain comprising the amino acid sequence of SEQ ID NO: 116;

[0490] c) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 135, a first light chain comprising the amino acid sequence of SEQ ID NO: 136, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 108, and a second light chain comprising the amino acid sequence of SEQ ID NO: 109;

[0491] d) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 18, a first light chain comprising the amino acid sequence of SEQ ID NO: 19, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 139, and a second light chain comprising the amino acid sequence of SEQ ID NO: 140;

[0492] e) a molecule comprising two light chains comprising the amino acid sequence of SEQ ID NO: 19, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 121, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 122;

[0493] f) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 18, a first light chain comprising the amino acid sequence of SEQ ID NO: 19, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 108, and a second light chain comprising the amino acid sequence of SEQ ID NO: 110;

[0494] g) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 145, a first light chain comprising the amino acid sequence of SEQ ID NO: 19, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 115, and a second light chain comprising the amino acid sequence of SEQ ID NO: 116;

[0495] h) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 18, a first light chain comprising the amino acid sequence of SEQ ID NO: 19, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 148, and a second light chain comprising the amino acid sequence of SEQ ID NO: 149;

[0496] i) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 18, a first light chain comprising the amino acid sequence of SEQ ID NO: 19, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 111, and a second light chain comprising the amino acid sequence of SEQ ID NO: 112; and

[0497] j) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 18, a first light chain comprising the amino acid sequence of SEQ ID NO: 19, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 113, and a second light chain comprising the amino acid sequence of SEQ ID NO: 114;

[0498] In another aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, wherein the antigen binding molecule is selected from:

[0499] a) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 164, a first light chain comprising the amino acid sequence of SEQ ID NO: 125, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 115, and a second light chain comprising the amino acid sequence of SEQ ID NO: 116;

[0500] b) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 164, a first light chain comprising the amino acid sequence of SEQ ID NO: 125, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 117, and a second light chain comprising the amino acid sequence of SEQ ID NO: 118;

[0501] c) a molecule comprising two light chains comprising the amino acid sequence of SEQ ID NO: 125, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 123, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 124;

[0502] d) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 164, a first light chain comprising the amino acid sequence of SEQ ID NO: 125, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO: 120;

[0503] e) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 164, a first light chain comprising the amino acid sequence of SEQ ID NO: 125, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 173, and a second light chain comprising the amino acid sequence of SEQ ID NO: 174; and

[0504] f) a molecule comprising two light chains comprising the amino acid sequence of SEQ ID NO: 125, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 126, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 127.

[0505] Specifically, the present invention provides an antigen binding molecule containing a ligand trimer of the TNF family, the antigen binding molecule comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 164, a first light chain comprising the amino acid sequence of SEQ ID NO: 125, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO: 120.

[0506] In another aspect, an antigen binding molecule comprising a TNF family ligand trimer is provided, wherein the TNF ligand family member is OX40L and wherein the target cell antigen is fibroblast activation protein (FAP), and wherein the module capable of specifically binding to FAP comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 100, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 101, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 102, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 103, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 104, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 105.

[0507] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer comprises:

[0508] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 106 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 107,

[0509] (ii) a second heavy chain comprising an amino acid sequence selected from SEQ ID NO: 355, and

[0510] (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 356.

[0511] Antigen binding molecule containing a trimer of TNF family ligands, wherein the target cell antigen is CD19

[0512] In a specific aspect, an antigen binding molecule comprising a TNF family ligand trimer is provided, wherein the target cell antigen is CD19.

[0513] In one aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer, wherein the module capable of specifically binding to CD19 comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 195 or SEQ ID NO: 252, (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 196 or SEQ ID NO: 253, and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 197 or SEQ ID NO: 254, the VL domain comprising (iv) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 198 or SEQ ID NO: 249, (v) a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 199 or SEQ ID NO: 250, and (vi) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 200 or SEQ ID NO: 251.

[0514] In a specific aspect, an antigen binding molecule containing a TNF family ligand trimer of the present invention is provided, wherein the module capable of specifically binding to a target cell antigen is a Fab molecule capable of specifically binding to CD19 and comprises a VH domain and a VL domain, wherein the VH domain comprises (i) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 195, (ii) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 196, and (iii) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 197, and the VL domain comprises (iv) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 198, (v) a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 199, and (vi) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 200.

[0515] In a further aspect, an antigen binding molecule containing a TNF family ligand trimer of the present invention is provided, wherein the module capable of specifically binding to a target cell antigen is a Fab molecule capable of specifically binding to CD19 and comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 252, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 253, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 254, and the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 249, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 250, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 251.

[0516] In another aspect, the module capable of specifically binding to CD19 comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 201, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 202.

[0517] In a further aspect, the module capable of specifically binding to CD19 comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 357, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 358.

[0518] In one aspect, the module capable of specifically binding to CD19 comprises: a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 201 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 202, or wherein the module capable of specifically binding to CD19 comprises: a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 357 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 358.

[0519] In one specific aspect, the module capable of specifically binding to CD19 comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 201 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 202. In another specific aspect, the module capable of specifically binding to CD19 comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 357 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 358.

[0520] In another aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0521] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 201 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 202, and

[0522] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0523] The antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, and the second polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 3 and SEQ ID NO: 4.

[0524] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0525] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 201 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 202, and

[0526] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0527] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 96.

[0528] In another aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0529] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 357 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 358, and

[0530] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0531] The antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, and the second polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 3 and SEQ ID NO: 4.

[0532] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0533] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 357 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 358, and

[0534] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0535] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 96.

[0536] In another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises: a first heavy chain and a first light chain, both of which constitute a Fab molecule capable of specifically binding to a target cell antigen,

[0537] a second heavy chain and a second light chain, wherein the second heavy chain comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker, and the second heavy chain is fused at its C-terminus to the CH1 domain via a second peptide linker,

[0538] The second light chain comprises an extracellular domain of the TNF ligand family member or a fragment thereof, the second light chain is fused to the CL domain at its C-terminus via a third peptide linker, and the antigen binding molecule comprises:

[0539] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 201 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 202, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 357 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 358,

[0540] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 108, SEQ ID NO: 111 and SEQ ID NO: 113, and

[0541] (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114.

[0542] In another specific aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises: a first heavy chain and a first light chain, both of which constitute a Fab molecule capable of specifically binding to a target cell antigen,

[0543] a second heavy chain and a second light chain, wherein the second heavy chain comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker, and the second heavy chain is fused at its C-terminus to the CL domain via a second peptide linker,

[0544] The second light chain comprises an extracellular domain of the TNF ligand family member or a fragment thereof, the second light chain is fused to the CH1 domain at its C-terminus via a third peptide linker, and wherein the molecule comprises:

[0545] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 201 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 202, or a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 357 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 358,

[0546] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 173, and

[0547] (iii) a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 174.

[0548] In addition, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0549] (a) at least one module capable of specifically binding to a target cell antigen, and

[0550] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0551] The antigen binding molecule is characterized in that the first polypeptide contains a CH3 domain and the second polypeptide correspondingly contains a CH3 domain, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other and to the C-terminus of the CH3 domain via a peptide linker, and wherein the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof connected to the C-terminus of the CH3 domain of the polypeptide via a peptide linker.

[0552] In a specific aspect, such an antigen binding molecule containing a TNF family ligand trimer comprises two modules capable of specifically binding to a target cell antigen.

[0553] More specifically, such an antigen binding molecule containing a TNF family ligand trimer comprises:

[0554] (i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 209, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 210, and two light chains comprising the amino acid sequence of SEQ ID NO: 206, or

[0555] (ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 213, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 214, and two light chains comprising the amino acid sequence of SEQ ID NO: 206, or

[0556] (iii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 309, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 310, and two light chains comprising the amino acid sequence of SEQ ID NO: 279, or

[0557] (iv) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 313, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 314, and two light chains comprising the amino acid sequence of SEQ ID NO: 279.

[0558] In a further aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, wherein the antigen binding molecule is selected from:

[0559] a) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 205, a first light chain comprising the amino acid sequence of SEQ ID NO: 206, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 115, and a second light chain comprising the amino acid sequence of SEQ ID NO: 116;

[0560] b) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 205, a first light chain comprising the amino acid sequence of SEQ ID NO: 206, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 117, and a second light chain comprising the amino acid sequence of SEQ ID NO: 118;

[0561] c) a molecule comprising two light chains comprising the amino acid sequence of SEQ ID NO: 206, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 209, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 210;

[0562] d) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 205, a first light chain comprising the amino acid sequence of SEQ ID NO: 206, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO: 120;

[0563] e) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 205, a first light chain comprising the amino acid sequence of SEQ ID NO: 206, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 173, and a second light chain comprising the amino acid sequence of SEQ ID NO: 174; and

[0564] f) a molecule comprising two light chains comprising the amino acid sequence of SEQ ID NO: 206, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 213, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 214.

[0565] Specifically, the present invention provides an antigen binding molecule containing a ligand trimer of the TNF family, the antigen binding molecule comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 205, a first light chain comprising the amino acid sequence of SEQ ID NO: 206, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO: 120.

[0566] In another aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, wherein the antigen binding molecule is selected from:

[0567] a) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 357, a first light chain comprising the amino acid sequence of SEQ ID NO: 358, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 115, and a second light chain comprising the amino acid sequence of SEQ ID NO: 116;

[0568] b) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 357, a first light chain comprising the amino acid sequence of SEQ ID NO: 358, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 117, and a second light chain comprising the amino acid sequence of SEQ ID NO: 118;

[0569] c) a molecule comprising two light chains comprising the amino acid sequence of SEQ ID NO: 358, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 209, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 210;

[0570] d) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 357, a first light chain comprising the amino acid sequence of SEQ ID NO: 358, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO: 120;

[0571] e) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 357, a first light chain comprising the amino acid sequence of SEQ ID NO: 358, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 173, and a second light chain comprising the amino acid sequence of SEQ ID NO: 174; and

[0572] f) a molecule comprising two light chains comprising the amino acid sequence of SEQ ID NO: 358, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 213, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 214.

[0573] Specifically, the present invention provides an antigen binding molecule containing a ligand trimer of the TNF family, the antigen binding molecule comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 357, a first light chain comprising the amino acid sequence of SEQ ID NO: 358, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO: 120.

[0574] Antigen binding molecule containing a trimer of TNF family ligands, wherein the target cell antigen is CEA

[0575] In a specific aspect, an antigen binding molecule comprising a TNF family ligand trimer is provided, wherein the target cell antigen is CEA.

[0576] In one aspect, the present invention provides an antigen binding molecule comprising a TNF family ligand trimer, wherein the module capable of specifically binding to CD19 comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 321, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 322, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 323, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 324, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 325, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 326.

[0577] In a specific aspect, an antigen binding molecule containing a TNF family ligand trimer of the present invention is provided, wherein the module capable of specifically binding to a target cell antigen is a Fab molecule capable of specifically binding to CEA and comprises a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 321, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 322, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 323, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 324, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 325, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 326.

[0578] In another aspect, the module capable of specifically binding to CEA comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 327, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 328.

[0579] In one aspect, the module capable of specifically binding to CEA comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 327 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 328.

[0580] In another aspect, the module capable of specifically binding to CEA comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 329, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 330.

[0581] In one aspect, the module capable of specifically binding to CEA comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 329 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 330.

[0582] In another aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0583] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 329 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 330, and

[0584] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0585] The antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, and the second polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 96, SEQ ID NO: 3 and SEQ ID NO: 4.

[0586] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises:

[0587] (a) at least one module capable of specifically binding to a target cell antigen, the module comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 329 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 330, and

[0588] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0589] The antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 96.

[0590] In another aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises: a first heavy chain and a first light chain, both of which constitute a Fab molecule capable of specifically binding to a target cell antigen,

[0591] a second heavy chain and a second light chain, wherein the second heavy chain comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker, and the second heavy chain is fused at its C-terminus to the CH1 domain via a second peptide linker,

[0592] The second light chain comprises an extracellular domain of the TNF ligand family member or a fragment thereof, the second light chain is fused to the CL domain at its C-terminus via a third peptide linker, and the antigen binding molecule comprises:

[0593] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 329 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 330,

[0594] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 108, SEQ ID NO: 111 and SEQ ID NO: 113, and

[0595] (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114.

[0596] In another specific aspect, an antigen binding molecule containing a TNF family ligand trimer is provided, wherein the antigen binding molecule comprises: a first heavy chain and a first light chain, both of which constitute a Fab molecule capable of specifically binding to a target cell antigen,

[0597] a second heavy chain and a second light chain, wherein the second heavy chain comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a first peptide linker, and the second heavy chain is fused at its C-terminus to the CL domain via a second peptide linker,

[0598] The second light chain comprises an extracellular domain of the TNF ligand family member or a fragment thereof, the second light chain is fused to the CH1 domain at its C-terminus via a third peptide linker, and wherein the molecule comprises:

[0599] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 329 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 330,

[0600] (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 173, and

[0601] (iii) a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 174.

[0602] In addition, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising:

[0603] (a) at least one module capable of specifically binding to a target cell antigen, and

[0604] (b) a first and a second polypeptide linked to each other via a disulfide bond,

[0605] The antigen binding molecule is characterized in that the first polypeptide contains a CH3 domain and the second polypeptide correspondingly contains a CH3 domain, and wherein the first polypeptide comprises two extracellular domains of a TNF ligand family member or a fragment thereof connected to each other and to the C-terminus of the CH3 domain via a peptide linker, and wherein the second polypeptide comprises one extracellular domain of the TNF ligand family member or a fragment thereof connected to the C-terminus of the CH3 domain of the polypeptide via a peptide linker.

[0606] In a specific aspect, such an antigen binding molecule containing a TNF family ligand trimer comprises two modules capable of specifically binding to a target cell antigen.

[0607] More specifically, such an antigen binding molecule containing a TNF family ligand trimer comprises:

[0608] (i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 337, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 338, and two light chains comprising the amino acid sequence of SEQ ID NO: 334, or

[0609] (ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 341, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 342, and two light chains comprising the amino acid sequence of SEQ ID NO: 334.

[0610] In a further aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, wherein the antigen binding molecule is selected from:

[0611] a) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 333, a first light chain comprising the amino acid sequence of SEQ ID NO: 334, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 115, and a second light chain comprising the amino acid sequence of SEQ ID NO: 116;

[0612] b) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 333, a first light chain comprising the amino acid sequence of SEQ ID NO: 334, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 117, and a second light chain comprising the amino acid sequence of SEQ ID NO: 118;

[0613] c) a molecule comprising two light chains comprising the amino acid sequence of SEQ ID NO: 334, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 337, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 338;

[0614] d) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 333, a first light chain comprising the amino acid sequence of SEQ ID NO: 334, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO: 120;

[0615] e) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 333, a first light chain comprising the amino acid sequence of SEQ ID NO: 334, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 173, and a second light chain comprising the amino acid sequence of SEQ ID NO: 174; and

[0616] f) a molecule comprising two light chains comprising the amino acid sequence of SEQ ID NO: 334, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 341, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 342.

[0617] Specifically, the present invention provides an antigen binding molecule containing a ligand trimer of the TNF family, the antigen binding molecule comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 333, a first light chain comprising the amino acid sequence of SEQ ID NO: 334, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO: 120.

[0618] Polynucleotides

[0619] The present invention further provides isolated polynucleotides or fragments thereof encoding antigen binding molecules containing a TNF family ligand trimer as described herein.

[0620] The polynucleotides encoding the separation of the antigen binding molecules containing the TNF ligand trimer of the present invention can be expressed as a single polynucleotide encoding the whole antigen binding molecules or a plurality of (e.g., two or more) polynucleotides of coexpression. The polypeptide encoded by the coexpressed polynucleotides can be combined to form a functional antigen binding molecule by, for example, a disulfide bond or other means. For example, the light chain portion of an immunoglobulin can be encoded by a polynucleotide separated from the heavy chain portion of an immunoglobulin. The heavy chain polypeptide will be combined with the light chain polypeptide to form an immunoglobulin when coexpressed.

[0621] In some respects, the polynucleotide encoding of separation is as described herein according to the complete antigen binding molecules containing TNF families ligand trimer of the present invention. Specifically, the polynucleotide encoding of separation is included in the polypeptide in the antigen binding molecules containing TNF families ligand trimer of the present invention as described herein.

[0622] In one aspect, the present invention relates to an isolated polynucleotide encoding an antigen binding molecule containing a TNF family ligand trimer, wherein the polynucleotide comprises (a) a sequence encoding a module capable of specifically binding to a target cell antigen, (b) a sequence encoding a polypeptide comprising two extracellular domains of a TNF ligand family member or two fragments thereof connected to each other by a peptide linker, and (c) a sequence encoding a polypeptide comprising one extracellular domain of the TNF ligand family member or a fragment thereof.

[0623] In another aspect, an isolated polynucleotide encoding an antigen-binding molecule containing a 4-1BB ligand trimer is provided, wherein the polynucleotide comprises (a) a sequence encoding a module capable of specifically binding to a target cell antigen, (b) a sequence encoding a polypeptide comprising two extracellular domains of 4-1BB or two fragments thereof connected to each other by a peptide linker, and (c) a sequence encoding a polypeptide comprising one extracellular domain of 4-1BB or a fragment thereof.

[0624] In a further aspect, the present invention relates to an isolated polynucleotide comprising a sequence encoding a polypeptide comprising two 4-1BBL fragments, wherein the two 4-1BBL fragments comprise an amino acid sequence that is at least about 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 96, and a sequence encoding a polypeptide comprising one 4-1BBL fragment, wherein the one 4-1BBL fragment comprises an amino acid sequence that is at least about 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 96.

[0625] Furthermore, provided are isolated polynucleotides encoding antigen-binding molecules comprising an OX40 ligand trimer, wherein the polynucleotide comprises (a) a sequence encoding a module capable of specifically binding to a target cell antigen, (b) a sequence encoding a polypeptide comprising two extracellular domains of OX40L or two fragments thereof linked to each other by a peptide linker, and (c) a sequence encoding a polypeptide comprising one extracellular domain of OX40L or a fragment thereof.

[0626] In another aspect, the present invention relates to an isolated polynucleotide comprising a sequence encoding a polypeptide comprising two 4-1BBL fragments, wherein the two 4-1BBL fragments comprise an amino acid sequence that is at least about 90%, 95%, 98% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54, and a sequence encoding a polypeptide comprising one 4-1BBL fragment comprising an amino acid sequence that is at least about 90%, 95%, 98% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54.

[0627] In a further aspect, the present invention relates to polynucleotides comprising a sequence that is at least about 90%, 95%, 98% or 100% identical to the specific cDNA sequences disclosed herein. In a specific aspect, the present invention relates to polynucleotides comprising a sequence that is identical to one of the specific cDNA sequences disclosed herein.

[0628] In other aspects, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 97, 98, 99, 183, 184, or 185. In a further aspect, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 14, 15, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 173, or 174.

[0629] In still other aspects, the nucleic acid molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 66, 67, 68, 69, 129, 130, 131, 132, 133, 134, 137, 138, 141, 142, 143, 144, 146, 147, 150, 151, 152, 153, 162, 163, 165, 166, 167, 168, 169, 170, 171, 172, 175, 176, 177, 178, 203, 204, 207, 208 8, 211, 212, 215, 216, 273, 274, 277, 278, 281, 282, 285, 286, 289, 290, 293, 294, 297, 298, 301, 302, 305, 307, 308, 311, 312, 315, 316, 331, 332, 335, 336, 339, 340, 343, 344, 347, 348, 353, or 354.

[0630] In some aspects, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0631] Recombination methods

[0632] The antigen binding molecules containing TNF families ligand trimer of the present invention can be obtained, for example, by solid-state peptide synthesis (such as Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more coding antigen binding molecules or its polypeptide fragment containing TNF families ligand trimer, for example, as described above, are separated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such polynucleotides can be easily separated using conventional procedures and sequenced. In one aspect of the invention, a vector comprising one or more polynucleotides of the present invention is provided, preferably an expression vector. Methods well known to those skilled in the art can be used to construct an expression vector comprising the coding sequence of the antigen binding molecules (fragment) containing TNF families ligand trimer and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, synthetic technology and in vivo recombination / genetic recombination. See, for example, Maniatis et al., MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989). The expression vector can be a part of a plasmid, a virus, or can be a nucleic acid fragment. The expression vector includes a polynucleotide (i.e., coding region) encoding an antigen binding molecule or its polypeptide fragment containing a TNF families ligand trimer and promoter and / or other transcription or translation control elements cloned into an expression cassette in an operably associated manner. As used herein, "coding region" is the part of a nucleic acid composed of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, it can be considered part of the coding region if present, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc. are not part of the coding region. Two or more coding regions can be present in a single polynucleotide construct, for example, on a single vector, or in separate polynucleotide constructs, for example, on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can contain two or more coding regions, for example, a vector of the invention can encode one or more polypeptides that are separated into final proteins by proteolytic cleavage post-translationally or co-translationally.In addition, the vector, polynucleotide or nucleic acid of the present invention can encode a heterologous coding region, which is fused or unfused with the polynucleotide encoding the antigen binding molecules containing the TNF family ligand trimer of the present invention or its polypeptide fragment or its variant or derivative. Heterologous coding regions include but are not limited to special elements or motifs, such as secretory signal peptides or heterologous functional domains. Operationally associated refers to such a way that the coding region of a gene product, such as a polypeptide, is associated with one or more regulatory sequences so that the expression of the gene product is carried out under the influence or control of the regulatory sequences. If the induction of promoter function leads to the transcription of the mRNA encoding the desired gene product, and if the connection properties between the two DNA fragments do not interfere with the ability of the expression control sequence to guide the expression of the gene product or do not interfere with the ability of the DNA template to be transcribed, the two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated". Therefore, if the promoter can affect the transcription of the nucleic acid encoding the polypeptide, the promoter region is operably associated with the nucleic acid. A promoter can be a cell-specific promoter that instructs substantial DNA transcription only in predetermined cells. In addition to a promoter, other transcriptional control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with a polynucleotide to direct cell-specific transcription.

[0633] Suitable promoters and other transcription control regions are disclosed herein. Many transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoters and enhancer segments from cytomegalovirus (e.g., immediate early promoter, associated with intron A), simian virus 40 (e.g., early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit α-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, many translation control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites or IRES, also known as CITE sequences). The expression cassette may also include other features such as an origin of replication, and / or chromosomal integration elements such as retroviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0634] The polynucleotides and nucleic acid coding regions of the present invention can be associated with coding regions encoding additional secretory peptides or signal peptides, and the secretory peptides or signal peptides guide the secretion of the polypeptides encoded by the polynucleotides of the present invention. For example, if it is desired to secrete an antigen binding molecule containing a TNF family ligand trimer or its polypeptide fragments, the DNA encoding the signal sequence can be located upstream of the nucleic acid encoding the antigen binding molecule containing a TNF family ligand trimer of the present invention or its polypeptide fragments. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence, which is cut from the mature protein after the initial growth protein chain is exported through the rough endoplasmic reticulum. Those of ordinary skill in the art know that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cut from the translated polypeptide to produce secretion or "mature" form of the polypeptide. In certain embodiments, a natural signal peptide such as an immunoglobulin heavy chain or light chain signal peptide or a functional derivative of the sequence is used, and the derivative retains the ability to guide the secretion of the polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide or its functional derivative can be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0635] DNA encoding a short protein sequence that can be used to facilitate subsequent purification (e.g., histidine tag) or assist in labeling the fusion protein can be included within or at the end of a polynucleotide encoding the antigen binding molecule containing a TNF family ligand trimer of the present invention or a polypeptide fragment thereof.

[0636] In another aspect of the present invention, a host cell comprising one or more polynucleotides of the present invention is provided. In certain embodiments, a host cell comprising one or more vectors of the present invention is provided. Polynucleotides and vectors can be incorporated into any feature described herein singly or in combination with respect to nucleotides and vectors, respectively. In one aspect, the host cell comprises (e.g., transformed or transfected) a vector comprising a polynucleotide encoding (a portion of) an antigen binding molecule containing a TNF family ligand trimer of the present invention. As used herein, the term "host cell" refers to a cell system that can be engineered to produce any type of fusion protein of the present invention or its fragment. Host cells suitable for replication and support of antigen binding molecule expression are well known in the art. Such cells can be suitably transfected or transduced using specific expression vectors, and a large number of cells containing vectors can be cultivated for inoculating large-scale fermentation tanks to obtain sufficient amounts of antigen binding molecules for clinical use. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, especially when glycosylation is not required, polypeptides can be produced in bacteria. After expression, the polypeptide can be isolated from the bacterial paste as a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006).

[0637] Suitable host cells for expressing (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TMVertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT060562); TRI cells (as described in Mather et al., Annals NY Acad Sci 383, 44-68 (1982)); MRC 5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few, as well as cells within transgenic animals, transgenic plants or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, for example a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (for example a Y0, NS0, Sp20 cell). Standard techniques for expressing foreign genes in these systems are known in the art. Cellular engineering of a polypeptide comprising an immunoglobulin heavy chain or light chain can be used to express another immunoglobulin chain so that the expressed product is an immunoglobulin with both a heavy chain and a light chain.

[0638] In one aspect, a method for producing an antigen binding molecule containing a TNF family ligand trimer of the present invention or a polypeptide fragment thereof is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding an antigen binding molecule containing a TNF family ligand trimer of the present invention or a polypeptide fragment thereof provided herein under conditions suitable for expressing the antigen binding molecule containing a TNF family ligand trimer of the present invention or a polypeptide fragment thereof, and recovering the antigen binding molecule containing a TNF family ligand trimer of the present invention or a polypeptide fragment thereof from the host cell (or host cell culture medium).

[0639] In the antigen binding molecules containing TNF families ligand trimer of the present invention, multiple components (at least one module that can be specifically bound to a target cell antigen, a polypeptide comprises two extracellular domains of TNF ligand family members or a fragment thereof, a polypeptide comprises an extracellular domain of the TNF families ligand family members or a fragment thereof) are not fused to each other genetically. Polypeptide is designed so that its components (two extracellular domains of TNF ligand family members or its fragment and other compositions such as CH or CL) are fused to each other directly or by a linker sequence. The composition and length of the joint can be measured according to methods well known in the art, and its effect can be tested. The example of the linker sequence between the different components of the antigen binding molecules of the present invention is shown in the sequence provided herein. If necessary, other sequences can also be included, such as endopeptidase recognition sequences, so that each component of the fusion protein is separated by cleavage site.

[0640] In certain embodiments, the module (such as Fab fragment) that can be specifically bound to the target cell antigen that forms a part of the antigen binding molecules at least includes an immunoglobulin variable region that can be bound to the antigen. The variable region can form a part of a natural or non-natural antibody and its fragment and is derived from it. The method for producing polyclonal antibodies and monoclonal antibodies is well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (for example, as described in U.S. Patent No. 4,186,567), or can be obtained by, for example, screening a combinatorial library comprising variable heavy chain and variable light chain (see, for example, U.S. Patent No. 5,969,108, which is granted to McCafferty).

[0641] Immunoglobulins of any animal species can be used in the present invention. Non-limiting immunoglobulins that can be used in the present invention can be of mouse, primate, or human origin. If the fusion protein is intended for human use, a chimeric form of the immunoglobulin can be used, in which the constant region of the immunoglobulin is from a human. Humanized or fully human forms of immunoglobulins can also be prepared according to methods well known in the art (see, for example, U.S. Patent No. 5,565,332 granted to Winter). Humanization can be achieved by various methods, including but not limited to (a) transplanting non-human (e.g., donor antibody) CDRs to a human (e.g., acceptor antibody) framework and a constant region with or without key framework residues (e.g., residues important for retaining good antigen binding affinity or antibody function), (b) only transplanting non-human specific determining regions (SDRs or α-CDRs; residues critical for antibody-antigen interactions) to a human framework and constant region, or (c) transplanting the entire non-human variable domain, but "masking" it with segments similar to those of a human by replacing surface residues. Humanized antibodies and methods for their preparation are reviewed in, e.g., Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and further described in, e.g., Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing "surfacing"); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describing the "guided selection" approach for FR shuffling).The specific immunoglobulin according to the present invention is a human immunoglobulin. Various techniques known in the art can be used to produce human antibodies and human variable regions. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form a part of the human monoclonal antibody prepared by the hybridoma method and can be derived therefrom (see, for example, Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see, e.g., Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variable regions can also be produced by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see, e.g., Hoogenboom et al., in Methods in Molecular Biology 178, 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments.

[0642] In some aspects, according to the method disclosed in for example PCT Publication WO 2012 / 020006 (referring to the example about affinity maturation) or U.S. Patent Application Publication No.2004 / 0132066, the module (such as Fab fragment) that can be specifically combined with target cell antigen included in the antigen binding molecules of the present invention is engineered, to have the binding affinity of enhancement. The ability of antigen binding molecules of the present invention to be combined with specific antigen determinants can be measured by enzyme-linked immunosorbent assay (ELISA) or other technologies familiar to those skilled in the art, for example, surface plasmon resonance technology (Liljeblad et al., Glyco J 17,323-329 (2000))) and traditional binding assay (Heeley, Endocr Res 28,217-229 (2002)). Competition assays can be used to identify the antigen binding molecules that compete with reference antibodies for binding to specific antigens. In certain embodiments, the epi-position (such as linear or conformational epitope) that this competitive antigen binding molecule is combined is identical with that combined with reference antigen binding molecules. Detailed exemplary methods for mapping the epitope bound by antigen binding molecules are provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular Biology vol.66 (Humana Press, Totowa, NJ). In an exemplary competitive assay, the fixed antigen is incubated in a solution comprising a first labeled antigen binding molecule and a second unlabeled antigen binding molecule that binds to the antigen, and the ability of the second unlabeled antigen binding molecule to compete with the first antigen binding molecule for binding to the antigen is tested. The second antigen binding molecule may be present in a hybridoma supernatant. As a control, the fixed antigen is incubated in a solution comprising a first labeled antigen binding molecule but not comprising a second unlabeled antigen binding molecule. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed, and the amount of the label bound to the fixed antigen is measured. If the amount of the label bound to the fixed antigen in the test sample is significantly reduced relative to the control sample, it indicates that the second antigen binding molecule competes with the first antigen binding molecule for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0643] The antigen binding molecules containing TNF ligand trimer of the present invention as described in this article can be purified by techniques known in the art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying specific proteins will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens combined with the antigen binding molecules containing TNF ligand trimer can be used. For example, for the affinity chromatography purification of the fusion protein of the present invention, a matrix with protein A or protein G can be used. Protein A or G affinity chromatography and size exclusion chromatography can be used to separate the antigen binding molecules as described in the examples. The purity of the antigen binding molecules containing TNF ligand trimer or its fragment can be measured by any of the various analytical methods known to all, including gel electrophoresis, high pressure liquid chromatography, etc. For example, as confirmed by reducing and non-reducing SDS-PAGE, the antigen binding molecules containing TNF ligand trimer expressed as described in the examples are shown to be complete and correctly assembled.

[0644] Assay

[0645] The antigen binding molecules provided herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activities by various assays known in the art.

[0646] 1. Affinity Determination

[0647] According to the method by surface plasmon resonance (SPR) proposed in the embodiment, using standard instruments such as BIAcore instruments (GE Healthcare) and can be obtained by such as recombinant expression receptor or target protein, it is possible to measure the affinity of the antigen binding molecules containing TNF families ligand trimer provided in this article and corresponding TNF receptor. By surface plasmon resonance (SPR), using standard instruments such as BIAcore instruments (GE Healthcare) and can be obtained by such as recombinant expression receptor or target protein, it is also possible to measure the affinity of the antigen binding molecules containing TNF families ligand trimer and target cell antigen. Specific illustrative and exemplary embodiments for measuring binding affinity are described in Example 4. According to one aspect, using K was measured by surface plasmon resonance using a T100 instrument (GE Healthcare) at 25 °C. D .

[0648] 2. Binding Assays and Other Assays

[0649] For example, by flow cytometry (FACS), using the cell line expressing specific receptors or target antigen, the combination of the antigen binding molecules containing TNF families ligand trimer provided in this article and corresponding receptor expressing cells can be evaluated. In one aspect, in conjunction with determination, fresh peripheral blood mononuclear cells (PBMC) expressing TNF receptor are used. After separation (naive PMBC) or stimulation (activated PMBC) directly use these cells. In yet another aspect, activated mouse splenocytes (expressing TNF receptor molecules) are used to prove the combination of the antigen binding molecules containing TNF families ligand trimer of the present invention and corresponding TNF receptor expressing cells.

[0650] In a further aspect, cancer cell lines expressing target cell antigens (eg, FAP) are used to demonstrate binding of the antigen binding molecule to the target cell antigen.

[0651] In another aspect, competition assays can be used to identify antigen binding molecules that compete with specific antibodies or antigen binding molecules for binding to a target or TNF receptor, respectively. In certain embodiments, the epitope (e.g., linear or conformational epitope) bound by such a competitive antigen binding molecule is the same as that bound by a specific anti-target antibody or a specific anti-TNF receptor antibody. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0652] 3. Activity Assay

[0653] In one aspect, there is provided an assay for identifying an antigen binding molecule containing a TNF families ligand trimer that is combined with a specific target cell antigen and a specific TNF receptor with biological activity. Biological activity can include, for example, excitatory signal transduction of the TNF receptor on a cell expressing a target cell antigen. There is also provided an antigen binding molecule containing a TNF families ligand trimer that is identified as having such in vitro biological activity by an assay.

[0654] In some aspects, the present invention has been tested for this biological activity of the antigen binding molecules containing TNF families ligand trimers. The assay method for detecting the biological activity of molecules of the present invention is those described in Example 6. In addition, it is well known in the art to measure the assay method for detecting cell lysis (for example, by measuring LDH release), the apoptosis kinetics of induction (for example, by measuring caspase 3 / 7 activity) or apoptosis (for example, using TUNEL to measure). In addition, the impact of the survival, proliferation and lymphokine secretion of various lymphocyte subsets such as NK cells, NKT cells or γ δ T cells can be assessed by assessing the compound, or assessing the phenotype and function of the antigen presenting cells such as dendritic cells, monocytes / macrophages or B cells to assess the biological activity of such compounds.

[0655] Pharmaceutical compositions, formulations and routes of administration

[0656] In a further aspect, the present invention provides a pharmaceutical composition comprising any provided herein antigen binding molecules containing TNF families ligand trimers, for example, for use in any of the following methods of treatment. In one embodiment, the pharmaceutical composition comprises any provided herein antigen binding molecules containing TNF families ligand trimers and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises any provided herein antigen binding molecules containing TNF families ligand trimers and at least one other therapeutic agent, such as described below.

[0657] The pharmaceutical composition of the present invention comprises a therapeutically effective amount of one or more antigen binding molecules containing a TNF family ligand trimer dissolved or dispersed in a pharmaceutically acceptable excipient. The phrase "pharmaceutical or pharmaceutically acceptable" refers to a molecular entity and composition that is generally non-toxic to the recipient at the dosage and concentration employed, i.e., does not produce adverse, allergic or other adverse reactions when appropriately applied to animals, such as humans. According to the present disclosure, the preparation of a pharmaceutical composition comprising at least one antigen binding molecule containing a TNF family ligand trimer and optionally another active ingredient will be known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. Specifically, the composition is a lyophilized formulation or an aqueous solution. As used herein, as known to those of ordinary skill in the art, "pharmaceutically acceptable excipient" includes any one and all of solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers, and combinations thereof.

[0658] Parenteral compositions include those designed for administration by injection, such as subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. In order to inject, the antigen binding molecules containing TNF families ligand trimers of the present invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks solution, Ringer's solution or normal saline buffer. The solution can contain a preparaton, such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the fusion protein can be in powder form with a suitable carrier (such as sterile pyrogen-free water) structure before use. As needed, a sterile injection solution is prepared by incorporating the fusion protein of the present invention in an appropriate solvent into a variety of other ingredients listed below in the amount required. Sterility can be easily achieved by filtering, for example, through a sterile filtration membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier, which contains a basic dispersion medium and / or other components. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered liquid medium. The liquid medium should be appropriately buffered if necessary and the liquid diluent should first be rendered isotonic with sufficient saline or glucose before injection. The composition must be stable under the conditions of manufacture and storage and remain resistant to the contaminating effects of antimicrobials such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a minimum at a safe level, for example, below 0.5 ng / mg protein. Suitable pharmaceutically acceptable excipients include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, Such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a highly concentrated solution. In addition, suspensions of these active compounds may be prepared as appropriate oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl cleats or triglycerides, or liposomes.

[0659] Active ingredients can be embedded in prepared microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and polymethyl methacrylate microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in coarse emulsions, for example, by coacervation techniques or by interfacial polymerization. Such technology is disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which are in the form of ...

Claims

1. An antigen-binding molecule containing a 4-1BB ligand (4-1BBL) trimer, comprising: (a) at least one Fab molecule capable of specifically binding to carcinoembryonic antigen (CEA), the Fab molecule capable of specifically binding to CEA comprising a VH domain and a VL domain, the VH domain comprising (i) CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 321, (ii) CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 322, and (iii) CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 323, the VL domain comprising (iv) CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 324, (v) CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 325, and (vi) CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 326, (b) a first polypeptide and a second polypeptide linked to each other via a disulfide bond, wherein the first polypeptide contains a first heavy chain constant (CH1) or light chain constant (CL) domain, and the second polypeptide contains a CL or CH1 domain, respectively, wherein the second polypeptide is linked to the first polypeptide via a disulfide bond between the CH1 and CL domains, and wherein the first polypeptide comprises two extracellular domains of 4-1BBL linked to the CH1 or CL domain and to each other via a peptide linker, and the second polypeptide comprises an extracellular domain of the 4-1BBL linked to the CL or CH1 domain of the polypeptide via a peptide linker, wherein the extracellular domain of 4-1BBL consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 96, and (c) An Fc domain composed of a first subunit and a second subunit capable of stably associating. 2 . The antigen-binding molecule comprising a 4-1BBL trimer according to claim 1 , wherein the 4-1BBL co-stimulates human T cell activation. 3 . The antigen binding molecule containing the 4-1BBL trimer according to claim 1 , wherein the extracellular domain of 4-1BBL consists of the amino acid sequence of SEQ ID NO:

96.

4. The antigen-binding molecule containing a 4-1BBL trimer according to claim 1 or 2, wherein The first polypeptide comprises the amino acid sequence of SEQ ID NO: 5, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or The first polypeptide comprises the amino acid sequence of SEQ ID NO:97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

96.

5. The antigen binding molecule containing a 4-1BBL trimer according to claim 1 or 2, wherein the Fab molecule capable of specifically binding to CEA comprises: a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 329 and a variable light chain comprising the amino acid sequence of SEQ ID NO:

330. The 4-1BBL trimer-containing antigen-binding molecule according to claim 1 or 2, wherein the Fc domain is an IgG Fc domain. The 4-1BBL trimer-containing antigen-binding molecule of claim 6 , wherein the Fc domain is an IgG1 Fc domain or an IgG4 Fc domain.

8. The 4-1BBL trimer-containing antigen binding molecule of claim 1 or 2, wherein the Fc domain is an IgG1 Fc domain comprising amino acid substitutions at positions 234 and 235 and / or 329 according to EU numbering.

9. The antigen binding molecule containing the 4-1BBL trimer of claim 1 or 2, wherein the antigen binding molecule comprises: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 333, a first light chain comprising the amino acid sequence of SEQ ID NO: 334, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO:

120. 10 . An isolated polynucleotide encoding the antigen-binding molecule containing a 4-1BBL trimer according to claim 1 .

11. A vector comprising the isolated polynucleotide of claim 10.

12. The vector of claim 11, wherein the vector is an expression vector.

13. A host cell comprising the isolated polynucleotide of claim 10 or the vector of claim 11 or 12.

14. A method for producing an antigen binding molecule containing a 4-1BBL trimer according to any one of claims 1 to 9, comprising the following steps: (i) culturing the host cell of claim 13 under conditions suitable for expressing the antigen-binding molecule, and (ii) recovering the antigen-binding molecule. 15 . An antigen-binding molecule comprising a 4-1BBL trimer, which is produced by the method of claim 14 . 16 . A pharmaceutical composition comprising the antigen-binding molecule containing a 4-1BBL trimer according to any one of claims 1 to 9 and 15 and at least one pharmaceutically acceptable excipient.

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