Antigen binding molecules comprising trimeric TNF family ligands

Abstract

The present invention relates to an antigen binding molecule comprising a trimer TNF family ligand. The present invention relates to a TNF family ligand trimer, which contains (a) at least one module capable of specifically binding to a target cell antigen, (b) comprising three polypeptides of the extracellular part of a TNF ligand family member (preferably OX40L or 4-1BBL) connected to each other by (c) a peptide linker (e.g., Fc domains). It is also claimed to generate methods for these molecules and methods using them.

Description

[0001] This application is a divisional application of the invention application with the application date of March 29, 2016, the Chinese application number 201680008222.5, and the invention name “Antigen binding molecules comprising trimeric TNF family ligands”. Field of the Invention

[0002] The present invention relates to novel antigen-binding molecules containing a TNF family ligand trimer, comprising (a) at least one module capable of specifically binding to a target cell antigen, (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof connected to each other by a peptide linker, and (c) an Fc domain consisting of a first and a second subunit capable of stably combining. The present invention further relates to methods for producing these molecules and methods of using them. Background of the Invention

[0003] 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). Their diverse and important roles in immunity make them of interest for the development of cancer immunotherapeutics (Bremer, 2013). 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 as self-assembled non-covalent trimers (Banner et al., 1993). Therefore, TNF family ligands form trimers that can bind to and activate the corresponding receptors of the TNFR superfamily. Some members of the TNF ligand family have a co-stimulatory effect on T cells, which means that they maintain the T cell response after initial T cell activation (Watts, 2005). 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT belong to this group of co-stimulatory TNF family ligands.

[0004] Among several co-stimulatory molecules, OX40 (CD134), a member of the tumor necrosis factor (TNF) receptor family, plays a key role in the survival and homeostasis of effector and memory T cells (Croft et al., 2009). OX40 (CD134) regulates immune responses to infection, tumors, and self-antigens, and its expression on the surface of T cells, NKT cells, NK cells, and neutrophils has been demonstrated (Baumann et al., 2004) and has been shown to be strictly inducible or strongly upregulated in response to a variety of stimulatory signals. In combination with T cell receptor triggering, OX40 engagement on T cells by their natural ligands or agonistic antibodies leads to synergistic activation of the PI3K and NFκB signaling pathways (Song et al., 2008). This, in turn, leads to enhanced proliferation, elevated cytokine receptor and cytokine production, and better survival of activated T cells.

[0005] TNF receptor superfamily member 4-1BB (CD137) was first identified as a molecule (Kwon and Weissman, 1989) induced by its expression via T cell activation. Subsequent studies 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 the 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 generally only shows its effectiveness 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 demonstrated 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 appears 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), which was attenuated or significantly reduced in the absence of functional Fc receptors. In a human clinical trial (ClinicalTrials.gov, NCT00309023), an Fc-active 4-1BB agonistic antibody (BMS-663513) 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 4-1BB ligand and a single-chain antibody fragment (Mueller et al., 2008; Hornig et al., 2012; Kermer et al., 2014) or a single 4-1BB ligand fused to the heavy chain C-terminus (Zhang et al., 2007) have been produced. EP 1 736 482A1 relates to recombinant 4-1BBL polypeptides comprising the entire extracellular domain of natural 4-1BBL and a cross-linkable peptide domain. 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 needed, which will preferably be able to combine with a module that is capable of binding to a tumor-specific or tumor-associated target and a module that can form a costimulatory TNF family ligand trimer, and possess enough pharmaceutically useful stability. The antigen binding molecules of the present invention include both, and therefore may be able to not only effectively trigger TNF receptors, but also very selectively trigger TNF receptors at the desired site, thereby alleviating side effects. SUMMARY OF THE INVENTION

[0015] 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,

[0017] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other (optionally via a peptide linker), and

[0018] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0019] In one 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 polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0022] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0023] In another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0024] (a) at least one module capable of specifically binding to a target cell antigen,

[0025] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0026] (c) an Fc domain consisting of a first and a second subunit capable of stably combining, wherein the polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof connected to each other by a peptide linker is fused to the N- or C-terminal amino acid of one of the two subunits of the Fc domain, optionally via a peptide linker.

[0027] In a particular aspect, the antigen binding molecules containing TNF families ligand trimer include (a) at least one module that can specifically bind to target cell antigens, (b) include three TNF ligand family members connected to each other by peptide linkers The extracellular domain or a polypeptide of its fragment, and (c) by the Fc domains that can stably constitute the first and second subunits of association, wherein the TNF ligand family members costimulate human T cell activation. Therefore, the TNF ligand family members are costimulatory TNF family ligands. Specifically, the costimulatory TNF family ligands are selected from 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT, more specifically, the costimulatory TNF family ligands are selected from 4-1BBL and OX40L.

[0028] In one aspect, the TNF ligand family member is 4-1BBL.

[0029] In a further aspect, the extracellular domain of the TNF ligand family member comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102, in particular an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 99.

[0030] In a specific aspect, the extracellular domain of a TNF ligand family member or a fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, in particular the amino acid sequence of SEQ ID NO: 1.

[0031] In yet another aspect, the antigen binding molecules containing a TNF family ligand trimer of the present invention comprise

[0032] (a) at least one module capable of specifically binding to a target cell antigen, and

[0033] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103, and

[0034] (c) an Fc domain composed of a first and a second subunit capable of stably associating. More specifically, the polypeptide comprises the amino acid sequence of SEQ ID NO: 5.

[0035] In another aspect, the TNF ligand family member is OX40L.

[0036] In a specific aspect, the extracellular domain of the TNF ligand family member comprises an amino acid sequence selected from SEQ ID NO: 6 and SEQ ID NO: 7, in particular the amino acid sequence of SEQ ID NO: 6.

[0037] In yet another aspect, the antigen binding molecules containing a TNF family ligand trimer of the present invention comprise

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

[0039] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, and

[0040] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0041] In one aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the polypeptide comprising the extracellular domain of three TNF ligand family members or a fragment thereof is fused to the N-terminal amino acid of one of the subunits of the Fc domain at the C-terminal amino acid. Specifically, an antigen binding molecule containing a TNF family ligand trimer is provided, which comprises

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

[0043] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0044] (c) an Fc domain composed of a first and a second subunit capable of stably associating,

[0045] The polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof is fused to the N-terminal amino acid of the CH2 domain in the Fc domain at the C-terminal amino acid.

[0046] In another aspect, the antigen binding molecule containing a TNF family ligand trimer is as follows, wherein the polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof is fused at the N-terminal amino acid to the C-terminal amino acid of one of the subunits of the Fc domain, for example, the polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof is fused at the N-terminal amino acid to the C-terminal amino acid of the CH3 domain in the Fc domain.

[0047] In another aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, comprising

[0048] (a) at least one module capable of specifically binding to a target cell antigen,

[0049] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0050] (c) an Fc domain composed of a first and a second subunit capable of stably associating,

[0051] The module capable of specifically binding to a target cell antigen is not fused to the polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof.

[0052] 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.

[0053] 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.

[0054] In another 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. Specifically, the antibody fragment is selected from Fab molecules, exchange Fab molecules, single-chain Fab molecules, Fv molecules, scFv molecules, single domain antibodies, and VH.

[0055] In yet another 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.

[0056] Thus, in yet another aspect, the present invention relates to an antigen binding molecule containing 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 fragment, a Fab molecule, an exchange Fab molecule, a single-chain Fab molecule, an Fv molecule, a scFv molecule, a single domain antibody, a VH and a scaffold antigen binding protein.

[0057] 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.

[0058] 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.

[0059] In a specific aspect, the target cell antigen is fibroblast activation protein (FAP). In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the module capable of specifically binding to FAP comprises a VH domain and a VL domain,

[0060] (a) the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or

[0061] (b) the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 104, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 105, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 106, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 108, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 109.

[0062] In one aspect, 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: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In another specific aspect, 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: 104, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 105, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 106, and the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 108, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 109.

[0063] In another 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 wherein the module capable of specifically binding to FAP comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 110 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 111.

[0064] In yet another aspect, target cell antigen is CEA.In one aspect, the invention provides the antigen binding molecules containing TNF families ligand trimer as described above, wherein the module that can specifically bind CEA includes VH domains and VL domains, the VH domains include (i) including SEQ ID NO:112 amino acid sequence CDR-H1, (ii) including SEQ ID NO:113 amino acid sequence CDR-H2, and (iii) including SEQ ID NO:114 amino acid sequence CDR-H3, the VL domains include (iv) including SEQ ID NO:115 amino acid sequence CDR-L1, (v) including SEQ ID NO:116 amino acid sequence CDR-L2, and (vi) including SEQ ID NO:117 amino acid sequence CDR-L3.Specifically, the module that can specifically bind CEA includes including SEQ ID NO:118 amino acid sequence variable heavy chain and including SEQ ID NO:119 amino acid sequence variable light chain.

[0065] In yet another aspect, the target cell antigen is CD19. In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the module capable of specifically binding to CD9 comprises a VH domain and a VL domain,

[0066] (a) the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125; or

[0067] (b) the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 129, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 130, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 131, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 132, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 133. Specifically, the module capable of specifically binding to CD19 comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 126 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 127, or wherein the module capable of specifically binding to FAP comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 134 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0068] In yet another aspect, an antigen binding molecule containing a TNF family ligand trimer as defined above is provided, wherein the Fc domain consisting of the first and second subunits capable of stably combining is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. Specifically, the Fc domain consisting of the first and second subunits capable of stably combining is an IgG1 Fc domain.

[0069] In another aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer as defined above, comprising

[0070] (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.

[0071] Specifically, the Fc domains are included in positions 234 and 235 (EU numbering) and / or 329 (EU numbering) amino acid replacements. More specifically, there is provided an antigen binding molecule containing a trimeric TNF family ligand according to the present invention, comprising an Fc domain with amino acid replacements L234A, L235A and P329G (EU numbering) in an IgG heavy chain.

[0072] In a specific aspect, the invention provides an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the Fc domain is an IgG1 Fc domain and comprises amino acid substitutions at positions 234 and 235 (EU numbering) and 329 (EU numbering) of the IgG heavy chain.

[0073] In another aspect, a monovalent antigen binding molecule comprising a TNF family ligand trimer as defined above is provided, which comprises a module capable of specifically binding to a target cell antigen.

[0074] Specifically, the antigen binding molecule containing a TNF family ligand trimer of the present invention, comprising (c) an Fc domain composed 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.

[0075] Furthermore, the present invention provides an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0076] (a) heavy and light chains, which together constitute the Fab molecule capable of specifically binding to the target cell antigen, and

[0077] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 15.

[0078] In another aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0079] (a) heavy and light chains, which together constitute the Fab molecule capable of specifically binding to the target cell antigen, and

[0080] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

[0081] In yet another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0082] (i) a first heavy chain comprising a VH domain of a Fab molecule capable of specifically binding to a target cell antigen,

[0083] (ii) a light chain comprising the VL domain of a Fab molecule capable of specifically binding to a target cell antigen, and

[0084] (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0085] In a specific aspect, the antigen binding molecule containing a trimer of TNF family ligands comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 16 or a VH domain comprising an amino acid sequence of SEQ ID NO: 110, (ii) a light chain comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 17 or a VL domain comprising an amino acid sequence of SEQ ID NO: 111, and (iii) a second heavy chain comprising an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8. In another aspect, the antigen binding molecule containing a trimer of TNF family ligands comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 118, (ii) a light chain comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 119, and (iii) a second heavy chain comprising an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8. In yet another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 126 or a VH domain comprising an amino acid sequence of SEQ ID NO: 134, (ii) a light chain comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 127 or a VL domain comprising an amino acid sequence of SEQ ID NO: 135, and (iii) a second heavy chain comprising an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0086] In another aspect, a bivalent antigen binding molecule containing a TNF family ligand trimer is provided, which comprises two modules capable of specifically binding to a target cell antigen.

[0087] In one aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0088] (i) a first heavy chain comprising a VH domain of a Fab molecule capable of specifically binding to a target cell antigen,

[0089] (ii) two light chains, each comprising a VL domain of a Fab molecule capable of specifically binding to a target cell antigen, and

[0090] (iii) a second heavy chain comprising the VH domain of a Fab molecule capable of specifically binding to a target cell antigen and the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103 or SEQ ID NO: 8.

[0091] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 or a VH domain comprising the amino acid sequence of SEQ ID NO: 110, (ii) two light chains, each comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17 or a VL domain comprising the amino acid sequence of SEQ ID NO: 111, and (iii) a second heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 or a VH domain comprising the amino acid sequence of SEQ ID NO: 110 and an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8. In another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 118, (ii) two light chains, each comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 119, and (iii) a second heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 118 and an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8. In yet another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 126 or a VH domain comprising an amino acid sequence of SEQ ID NO: 134, (ii) two light chains comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 127 or a VL domain comprising an amino acid sequence of SEQ ID NO: 135, and (iii) a second heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 126 or a VH domain comprising an amino acid sequence of SEQ ID NO: 134 and an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0092] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0093] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16,

[0094] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 17, and

[0095] (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0096] More specifically, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0097] (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18,

[0098] (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 19, and

[0099] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO:15.

[0100] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0101] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16,

[0102] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 17, and

[0103] (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

[0104] More specifically, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0105] (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18,

[0106] (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 19, and

[0107] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] The present invention also encompasses the antigen binding molecules containing TNF families ligand trimers of the present invention used as medicament, or pharmaceutical compositions of the present invention. In one aspect, there is provided an antigen binding molecule containing TNF families ligand trimers of the present invention for treating a disease in an individual in need, or pharmaceutical compositions of the present invention. In a specific embodiment, there is provided an antigen binding molecule containing TNF families ligand trimers of the present invention for treating cancer, or pharmaceutical compositions of the present invention.

[0112] 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

[0113] Figure 1 A and 1B show polypeptides comprising three extracellular domains of TNF ligand family members connected to each other by peptide linkers or their fragments. Figure (1A) shows a polypeptide comprising three extracellular domains of 4-1BBL connected to each other by peptide linkers, and Figure (1B) shows a polypeptide comprising three extracellular domains of OX40L connected to each other by peptide linkers.

[0114] Figure 2 A and 2B schematically illustrate variants (2A) and (2B) of the antigen binding molecules containing TNF ligand trimer of unit price of the present invention.Three TNF ligands are represented with the gray area with white dots.The white area with black dots represents the Fc part, and the thick black dot represents the knob-into-hole modification.

[0115] Representative examples of antigen binding molecules containing TNF ligand trimers of the present invention are as follows Figure 3AThe FAP-targeted trimeric 4-1BB ligand Fc (kih) P329G LALA fusion antigen binding molecule shown. Its preparation is described in Examples 1.1 and 1.3. Figure 3B A "non-targeting" variant (control 1) comprising a DP47 Fab molecule and a single-chain trimeric 4-1BB ligand Fc(kih)P329G LALA fusion antigen binding molecule prepared in Example 2 is shown. Figure 3C is a diagram of the monomeric 4-1BB Fc(kih) construct prepared in Example 4.

[0116] Another example of the antigen binding molecules containing TNF ligand trimer of the present invention is as follows Figure 4 A is a FAP-targeted trimeric OX40 ligand Fc(kih)P329G LALA fusion antigen binding molecule. Its preparation is described in Example 6. Figure 4 B shows a "non-targeting" variant comprising a DP47 Fab molecule and a single-chain trimeric OX40 ligand Fc(kih)P329GLALA fusion molecule prepared in Example 7. Figure 4 C is a diagram of the monomeric OX40 Fc(kih) construct prepared in Example 8.

[0117] Figure 5 Shown is the binding of a FAP-targeted Fc(kih) fusion molecule containing a 4-1BB ligand trimer (compound S1) to the recombinant 4-1BB Fc(kih) receptor assessed by surface plasmon resonance. Figure 5 In A, the assay setup is shown, with 4-1BB Fc(kih) immobilized on a SA chip. Figure 5 B shows binding to human 4-1BB Fc (kih), Figure 5 C represents the binding to cynomolgus monkey 4-1BB Fc (kih), Figure 5 D is binding to mouse 4-1BB Fc (kih).

[0118] Binding of the Fc(kih) fusion antigen binding molecule containing DP47 and a 4-1BB ligand trimer (control 1) to the recombinant 4-1BB Fc kih receptor is shown in Figure 2. Figure 6 As shown, 4-1BB Fc (kih) was immobilized on the SA chip. Figure 6 A shows binding to human 4-1BB Fc (kih), Figure 6 B is binding to cynomolgus monkey 4-1BB Fc (kih), Figure 6 C represents binding to mouse 4-1BB Fc (kih).

[0119] Figure 7The binding of recombinant human 4-1BB to a FAP-targeted Fc(kih) fusion antigen binding molecule (compound S1) containing a 4-1BB ligand trimer is demonstrated. Figure 7 The setup for affinity measurements is shown in A. Figure 7 B and 7C show the binding of human 4-1BBavi-His tag to FAP-targeted Fc(kih) fusion antigen binding molecule containing 4-1BB ligand trimer (FAP-targeted single-chain trimer 4-1BBL). Figure 7 D and 7E show the binding of the human 4-1BB avi-His tag to the Fc(kih) fusion antigen-binding molecule containing DP47 and a 4-1BB ligand trimer (a single-chain trimeric 4-1BBL molecule containing DP47).

[0120] Figure 8 Binding of a FAP-targeted Fc(kih) fusion antigen binding molecule (compound S10) containing an OX40 ligand trimer to the recombinant OX40 Fc(kih) receptor as assessed by surface plasmon resonance is shown. Figure 8 In A, the assay setup is shown, with OX40 Fc(kih) immobilized on a SA chip. Figure 8 B shows binding to human OX40 Fc (kih), Figure 8 C represents the binding to cynomolgus monkey OX40 Fc (kih), Figure 8 D is binding to murine OX40 Fc (kih).

[0121] exist Figure 9 In Figure 3, binding of an Fc(kih) fusion molecule containing an OX40 ligand trimer (control 3) containing DP47 to a recombinant OX40 Fc kih receptor was demonstrated. OX40 Fc(kih) was immobilized on an SA chip. Figure 9 A shows binding to human OX40 Fc (kih), Figure 9 B is binding to cynomolgus monkey OX40 Fc (kih), Figure 9 C represents binding to murine OX40 Fc (kih).

[0122] Figure 10 The binding of recombinant human OX40 to a FAP-targeted Fc(kih) fusion antigen binding molecule (compound S10) containing an OX40 ligand trimer is shown. Figure 10 The setup for affinity measurements is shown in A. Figure 10 In B, binding of human OX40 avi-His tag to FAP-targeted Fc(kih) fusion antigen-binding molecule containing OX40 ligand trimer (FAP-targeted single-chain OX40 ligand) is shown. Figure 10C shows the binding of human 4-1BB avi-His tag to Fc(kih) fusion molecule containing OX40 ligand trimer containing DP47 (single-chain trimer OX40L containing DP47).

[0123] 4-1BB and OX40 are not expressed in resting (naive) human PBMCs ( Figure 11 A, 11C, 11E and 11G, Figure 12 A and 12C). After human PBMC activation, 4-1BB and OX40 are expressed on CD4 + and CD8 + T cells, and human CD8 + T cells showed 10-fold higher upregulation of 4-1BB ( Figure 11 B, 11F, 11K and 11M and 12B), but upregulation was 4-fold lower than that of OX40 on CD4+ T cells ( Figure 11 As shown, FAP-targeted single-chain trimer 4-1BBL or OX40L Fc (kih) fusion antigen binding molecules (filled circles) and DP47-containing ("non-targeted") single-chain trimer 4-1BBL and OX40L (open circles, dotted lines) showed similar binding to activated CD4 + and CD8 + The combination of T cells, but not with immature human T cells.Therefore, it is unrelated to the combination of FAP or DP47, and both single-chain trimer 4-1BBL and single-chain trimer OX40L show similar combination with cells expressing human 4-1BB or OX40.In the case of cells expressing human 4-1BB or OX40, it is not possible to observe combination (resting immature cells). Combination is shown as the fluorescence intensity median (MFI) of the anti-human IgG Fcγ specific goat IgG F (ab') 2 fragments conjugated to red macroalgae phycoerythrin (R-PE) or fluorescein isothiocyanate (FITC) as the second detection antibody. MFI is measured by flow cytometry, and baseline is corrected by subtracting the MFI of blank control. The x-axis shows the concentration of single-chain trimer 4-1BBL or OX40L Fc (kih) fusion molecules containing FAP or DP47.

[0124] Figure 11 A to 11D relate to the binding of Fc (kih) fusion antigen binding molecules containing 4-1BB ligand trimers targeted by FAP (compound S1, solid circles) or Fc (kih) fusion molecules containing 4-1BB ligand trimers containing DP47 (control 1, open circles) to resting (naive) or activated human PMBCs. Specifically, the binding of resting (naive) or activated human CD8 +Binding of T cells is shown in the lower two panels to resting (naive) or activated human CD4 + T cell binding.

[0125] Figure 11 E to 11H show the binding of a monovalent targeted single-chain trimeric 4-1BB ligand Fc (kih) fusion antigen binding molecule to resting and activated human T cells. 4-1BB is not expressed on resting (naive) CD4 or CD8 T cells isolated from the blood of healthy donors. After activation, 4-1BB is expressed on CD4 + (See lower right image) and CD8 + (See upper right figure) T cells are upregulated, while CD8 + T cells show 4-1BB expression more than CD4 + T cells were 5-10 times higher (depending on the donor). As shown, all monovalent targeted single chain trimer 4-1BBL Fc (kih) fusion molecules were fused to 4-1BB + The combination of human T cells is similar, and has nothing to do with their targeting.The mean fluorescence intensity (MFI) of the anti-human IgG Fc γ fragment specific F (ab') 2 goat IgG fragment of R-PE conjugation is shown on the y-axis.The test concentration of single-chain trimer 4-1BB ligand Fc (kih) fused antigen binding molecules (compound S1, S3 and S5) of different targetings is indicated on the x-axis.These values ​​are baseline corrected by subtracting the MFI of blank control.

[0126] exist Figure 11 J to 11M show the binding of monovalent and bivalent targeted single-chain trimeric 4-1BB ligand Fc (kih) fusion antigen binding molecules to activated human T cells. After activation, 4-1BB is activated in CD4 + (left hand side) and CD8 + (right hand side) is upregulated on T cells. As shown, all monovalent and bivalent targeted single-chain trimer 4-1BBL Fc (kih) fusion antigen binding molecules bind to 4-1BB + The combination of human T cells is similar, and has nothing to do with their targeting.The mean fluorescence intensity (MFI) of the anti-human IgG Fc γ fragment specific F (ab') 2 goat IgG fragment of R-PE conjugation is shown on the y-axis.The test concentration of single-chain trimer 4-1BB ligand Fc (kih) fused antigen binding molecules (compound S1, S3, S2 and S4) of different targetings is indicated on the x-axis.These values ​​are baseline corrected by subtracting the MFI of blank control.

[0127] Figure 12A to 12D show the binding of FAP-targeted Fc(kih) fusion antigen binding molecules containing OX40 ligand trimer (compound S10, filled circles) or DP47-containing Fc(kih) fusion molecules containing OX40 ligand trimer (DP47 single-chain OX40L trimer, control 3, open circles) to resting (naive) or activated human PMBCs. Specifically, Figure 12 Figures A and 12B show the relationship between resting (naive) or activated human CD8 + T cell binding, respectively Figure 12 C and 12D show the relationship between resting (naive) or activated human CD4 + Binding of T cells. Binding of DP47 human IgG1 P329G LALA is represented by solid diamonds (negative control).

[0128] Figure 13 The results show that Fc(kih) fusion antigen binding molecules containing single-chain TNF ligand trimer (compounds S1 and S10, respectively) and transgenic mouse embryonic fibroblasts NIH / 3T3-huFAP clone 39 ( Figure 13 A) or human WM266-4 cell line expressing fibroblast activation protein (FAP) ( Figure 13 B) binding. These cells express high levels of human fibroblast activation protein (huFAP). Binding is shown as the median fluorescence intensity (MFI) of anti-human IgG Fcγ specific goat IgG F(ab')2 fragments labeled with fluorescein isothiocyanate (FITC) as the second detection antibody. MFI was determined by flow cytometry. The x-axis shows the concentration of single-chain trimer 4-1BBL or OX40L antigen binding molecules targeted by FAP or DP47 ("non-targeted"). Figure 13 As shown in A, only the FAP-targeted single-chain trimer 4-1BBL molecule (compound S1, solid circle) but not the DP47-containing single-chain trimer 4-1BBL antigen-binding molecule (open circle, dotted line) binds to NIH / 3T3-huFAP clone 39 cells. Figure 13 B shows that only FAP-targeted single-chain trimer OX40L antigen-binding molecules (compound S10, solid circles) but not DP47-containing single-chain trimer OX40 antigen-binding molecules (open circles, dotted lines) bind to WM266-4 cells. As a negative control, DP47 human IgG1 P329G LALA, represented by solid diamonds, also did not bind.

[0129] exist Figure 13In Figures C and 13D, binding of monovalent and bivalent FAP (4B9 or 28H1)-targeted single-chain trimeric 4-1BB ligand Fc (kih) fusion antigen binding molecules to FAP-expressing tumor cells is shown. As shown, the FAP (4B9)-targeted molecule showed the strongest binding to FAP with a lower EC 50 Using a strong FAP (4B9) binder, we see significant differences in MFI or EC between monovalent and bivalent targeting single-chain trimeric 4-1BB ligand (KIH) fusion antigen molecules (compounds S3 and S2, respectively). 50 There are only very minor differences. This is different for the weaker FAP binder 28H1 (compound S1). Here, bivalent binding (shown as bivalently bound FAP (28H1) targeted huIgG1 P329G LALA) results in a lower MFI. The DP47 non-targeted molecule does not bind to FAP. + Tumor cells are combined, so it is truly non-targeted.The mean fluorescence intensity (MFI) of the anti-human IgG Fc γ fragment specific F (ab') 2 goat IgG fragment of R-PE conjugation is shown on the y-axis.The test concentration of the single-chain trimer 4-1BB ligand Fc (kih) fused antigen binding molecule of different targetings is indicated on the x-axis.

[0130] Figure 13 E shows the combination of single-chain trimer 4-1BB ligand Fc (kih) fused antigen binding molecules and tumor cell lines expressing CEA for unit price and divalent CEA targetings.The combination of single-chain trimer 4-1BB ligand Fc (kih) fused antigen binding molecules (compound S4) for divalent CEA targetings produces a lower MFI than the combination of single-chain trimer 4-1BB ligand Fc (kih) fused antigen binding molecules (compound S5) for unit price CEA targetings.This can be explained by occupying two CEA molecules on the tumor cell surface at the same time.DP47 non-targeted molecules (compared to controls 1 and 2) are not combined with CEA+MKN45 tumor cells, so they are truly non-targeted.The mean fluorescence intensity (MFI) of anti-human IgG Fc γ fragment specific F (ab') 2 goat IgG fragments conjugated to PE is shown on the y-axis.The test concentrations of single-chain trimer 4-1BB ligand Fc (kih) fused antigen binding molecules for different targetings are indicated on the x-axis.

[0131] exist Figure 14In A and 14B, it is shown that Fc (kih) fused antigen binding molecules (compound S1, filled circles) containing 4-1BB ligand trimers of FAP targetings or Fc (kih) fused antigen binding molecules (control 1, open circles) containing 4-1BB ligand trimers containing DP47 are not combined with the mouse splenocytes expressing the activation of mouse 4-1BB. Anti-mouse CD137 specific human IgG1 P329G LALA antibody (clone Lob12.3) is used as positive control (triangle). Combination is characterized 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 primary 4-1BBL tripolymer antigen binding molecules tested. MFI is measured by flow cytometry, and baseline is corrected by subtracting the MFI of blank control.

[0132] exist Figure 15 The scheme in Figure 1 shows the general principle of the NFκB activity assay using the reporter cell line described in Example 11.1.2. The ratio of anti-hu IgG1 Fcγ-specific antibody to Fc(kih) fusion antigen binding molecule containing human 4-1BB ligand trimer is 1 to 4.

[0133] exist Figure 16 In, the activation of the NFκB signaling pathway in the reporter cell line of the single-chain trimer 4-1BBL antigen binding molecules (compound S1, solid circles) or the single-chain trimer 4-1BBL molecules (control 1, open circles, dotted lines) of the FAP targeting cross-linked by secondary antibodies is shown. In the presence of a 1 to 4 ratio of a specified concentration of antigen binding molecules containing single-chain 4-1BBL trimers and a cross-linked secondary polyclonal anti-huIgG1 Fcγ specific goat IgG F (ab) 2 fragment, reporter cells were cultured for 6 hours. Luciferase activity was assessed as described in Example 11.1.1. Activity was characterized by the trace of the nM concentration of the single-chain human 4-1BBL trimer antigen binding molecules measured in 0.5 seconds. The emission of URL is due to the oxidation of luciferin mediated by luciferase to oxyluciferin.

[0134] exist Figure 17 The protocol in Figure 1 shows the general principle of the NFκB-mediated luciferase activity assay described in Example 11.1.3 using HeLa-hu4-1BB-NFκB-luc clone 26 as the reporter cell line. FAP-expressing tumor cells were used at a ratio of 5 to 1 with reporter cells expressing human 4-1BB.

[0135] Figure 18A to 18D show the activation of the NFκB signaling pathway in reporter cells of FAP-targeted single-chain trimer 4-1BBL Fc (kih) fusion antigen binding molecules (compound S1, solid circles) cross-linked by FAP-expressing tumor cell lines. The data of uncross-linked single-chain trimer 4-1BBL molecules containing DP47 are shown as negative controls (control 1, open circles, dotted lines). In the stepwise addition of FAP-targeted or DP47-containing single-chain trimer 4-1BBL molecules and cross-linked FAP + In the presence of tumor cell line, reporter cells were cultured for 6 hours. The ratio of reporter cells to tumor cells expressing FAP was 1: 5. The luciferase activity mediated by NFκB was characterized by the trace of the nM concentration of the single-chain trimer 4-1BBL antigen binding molecules of the release light measured in 0.5 seconds (URL). The emission of URL is due to the oxidation of luciferin mediated by luciferase to oxyluciferin. These values ​​were baseline corrected by subtracting the URL of the blank control. Figure 18 A shows the activation of MV3 cell line as target cells. Figure 18 In B, the cell line NIH / 3T3-human FAP clone 39 was used as target cells. Figure 18 C shows the activation of WM-266-4 cell line as target cells, Figure 18 D shows the absence of FAP + Measurement data of targeted cells.

[0136] Figure 19 NK-κB activation mediated by cross-linking of 4-1BB activation by FAP targeting of single-chain trimer 4-1BB ligand Fc (kih) fusion antigen binding molecules is shown. Activation of the NFκB signaling pathway in HeLa-hu4-1BB-NFκB-luc clone 26 reporter cells of FAP-targeted single-chain trimer 4-1BB ligand Fc (kih) fusion molecules cross-linked by FAP-expressing tumor cell lines is shown. DP47 non-targeted single-chain trimer 4-1BB ligand Fc (kih) fusion molecules do not induce NK-κB activation (controls 1 and 2). Monovalent FAP-targeted single-chain trimer 4-1BB ligand Fc (kih) fusion antigen binding molecules (compounds S1 and S3) induce stronger NFκB activation than divalent FAP-targeted single-chain trimer 4-1BB ligand Fc (kih) fusion molecules (compound S2). In the stepwise addition of FAP-targeted or DP47-non-targeted single-chain trimer 4-1BB ligand Fc (kih) fusion antigen binding molecules and cross-linked FAP +In the presence of tumor cell line, HeLa-hu4-1BB-NF-κB-luc reporter cells were cultured for 6 hours. The ratio of reporter cells to tumor cells expressing FAP was 1: 5. The luciferase activity mediated by NFκB was characterized by the trace of the nM concentration of the single-chain trimer 4-1BB ligands of the light release units (URL) measured in 0.5 seconds. The emission of URL is due to the oxidation of luciferin mediated by luciferase to oxyluciferin. These values ​​were baseline corrected by subtracting the URL of the blank control. Figure 19 A shows the activation of MV3 cell line as target cells, Figure 19 B shows activation using the WM-266-4 cell line as target cells. Figure 19 C shows the absence of FAP + Measurement data of the presence of targeted cells.

[0137] exist Figure 20 , shows the activation of the NFκB signaling pathway in HeLa-hu4-1BB-NFκB-luc clone 26 reporter cells of CEA (sm9b)-targeted single-chain trimeric 4-1BB ligand Fc (kih) fusion antigen binding molecules (compounds S4 and S5) cross-linked by CEA-expressing tumor cell lines. DP47 non-targeted single-chain trimeric 4-1BB ligand Fc (kih) fusion molecules did not induce NK-κB activation (controls 1 and 2). The monovalent CEA (sm9b)-targeted single-chain trimeric 4-1BB ligand Fc (kih) fusion antigen molecule (compound S5) induced stronger N-κB activation than the divalent CEA (sm9b)-targeted single-chain trimeric 4-1BB ligand Fc (kih) fusion antigen binding molecule (compound S4). In the stepwise addition of CEA (sm9b)-targeted or DP47-non-targeted single-chain trimer 4-1BB ligand Fc (kih) fusion molecules and cross-linked CEA + In the presence of MKN45 tumor cell line, HeLa-hu4-1BB-NF-κB-luc reporter cells were cultured for 6 hours. The ratio of reporter cells to MKN45 tumor cells expressing CEA was 1: 5. The luciferase activity mediated by NFκB was characterized by the trace of the nM concentration of the single-chain trimer 4-1BB ligands measured in 0.5 seconds (URL) of the released light. The emission of URL is due to the oxidation of luciferin mediated by luciferase to oxyluciferin. These values ​​were baseline corrected by subtracting the URL of the blank control. Figure 20 A shows the activation of MKN45 cell line as target cells, Figure 20 B shows the absence of CEA + Measurement data of targeted cells.

[0138] Activation of the NFκB signaling pathway in HeLa-huOX40-NFκB-luc1 reporter cells by cross-linking of FAP-targeted single-chain trimer OX40L molecules (filled circles, compound S10) via the FAP-expressing WM266-4 tumor cell line. Figure 21 As shown in B. The uncrosslinked single-chain trimer OX40L molecule containing DP47 (open circle, dotted line, control 3) is shown as a negative control. + In the presence of tumor cell lines, reporter cells were cultured for 6 hours. The ratio of reporter cells to FAP-expressing tumor cells was 1:2.5. The NFκB-mediated luciferase activity was characterized by blotting the number of units of released light (URL) measured in 0.5 seconds relative to the nM concentration of the tested single-chain trimer OX40L antigen binding molecule. The emission of URL is due to the oxidation of luciferin mediated by luciferase to oxyluciferin. These values ​​were baseline corrected by subtracting the URL of the blank control. Figure 21 As shown in A, if there is no FAP + In the presence of target cells and therefore without cross-linking, the FAP-targeted single-chain trimeric OX40L molecule (compound S10, filled circles) and the DP47-containing single-chain trimeric OX40L antigen binding molecule (control 3) already induced a certain amount of NFκB activation.

[0139] Figure 22 The scheme in Figure 1 shows the antigen-specific CD8 + General principles of T cell activation assays.

[0140] Figure 23 and 24 The single-chain trimeric 4-1BBL antigen binding molecule was shown to be specific for NLV-specific CD8 + Effects of CD137 expression on T cells ( Figure 23 ) and NLV-specific CD8 + Effects of IFNγ secretion by T cells ( Figure 24 ). NLV-specific CD8 + Prolonged IFNγ secretion and CD137 expression by T cells are strictly dependent on the expression of NLV-HLA-A2 complex (Signal 1, Figure 23 A and 23B and Figure 24 A and 24B) and the recognition of single-chain trimeric 4-1BBL antigen-binding molecules by FAP targeting (signal 2, Figure 23 C to 23F and Figure 24C to 24F) simultaneously activates 4-1BB-triggered T cells. Solid circles: FAP-targeted single-chain trimer 4-1BBL molecules (compound S1); open circles: single-chain trimer 4-1BBL molecules containing DP47 (control 3). The effect of 4-1BB upregulation is shown in Figure 23 A to 23F, and the effect of INFγ expression on CD8+ T cells is shown in Figure 24 A to 24F. Total CD8 is always displayed + Median fluorescence intensity in the T cell population (left panel) and percent frequency of positive cells (right panel).

[0141] exist Figure 25 and 26 In the figure, NLV-specific CD8 + Prolonged IFNγ secretion and CD137 expression by T cells are strictly dependent on the expression of NLV-HLA-A2 complex (Signal 1, Figure 25 A and 25B and Figure 26 A and 26B and) and the recognition of the single-chain trimeric 4-1BB ligand by FAP targeting (signal 2, Figure 25 C to 25F and Figure 26 C to 26F) of 4-1BB-triggered T cells are activated simultaneously. The effect of 4-1BB upregulation is shown in Figure 25 A to 25F, while the effect of INFγ expression was shown in Figure 26 A to 26F. The median fluorescence intensity in the total CD8+ T cell population (left panel) and the percentage frequency of positive cells (right panel) are shown, both relative to the concentration of monovalent or bivalent FAP (28H1 or 4B9)-targeted or DP47-non-targeted single-chain trimeric 4-1BB ligand (kih) fusion antigen binding molecules.

[0142] Figure 27 A to 27F show that human CD4 + (left) and CD8 + In T cells (right), co-stimulation with trimeric single-chain OX40L molecules containing DP47 (open circles, dotted line, control 3) only slightly promoted proliferation and induced an enhanced activation phenotype (CD25 expression). Hypercrosslinking of FAP-targeted trimeric single-chain OX40L molecules by current NIH / 3T3-huFAP clone 39 cells strongly increased this effect (filled circles, compound S10). Event counts, percentages of proliferating (CFSE-low) cells, or in viable CD4 + and CD8 +MFI of CD25-PerCPCy5.5 on T cells. Baseline values ​​of blank samples containing only anti-human CD3 (clone V9, huIgG1), resting human PBMCs, and NIH / 3T3-huFAP clone 39 were subtracted from the test compound samples. Therefore, the enhancement effect of OX40L co-stimulation, but not the effect of suboptimal anti-CD3 stimulation itself, is shown here.

[0143] Figure 28 A shows a polypeptide comprising the three extracellular domains of 4-1BBL connected to each other via a peptide linker. Figure 28 B shows the Figure 28 Representative examples of the antigen binding molecules containing bivalent TNF ligand trimers of the polypeptide of the present invention of A. Three TNF ligands are represented by the gray area at the bottom. The white area with black dots represents the Fc part, and the thick black dots represent the knob-into-hole modification. Detailed Description of the Invention

[0144] definition

[0145] 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.

[0146] 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.

[0147] 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 particular 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 particular 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 domain (see, for example, WO 2002 / 020565) of the repeating domain of the repeating domain of design.

[0148] 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).

[0149] 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.

[0150] 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.

[0151] 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.

[0152] The term "valency" as used in this application refers to the presence of a specific number of binding sites in an antigen binding molecule. For this reason, the terms "divalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antigen binding molecule.

[0153] 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 natural antibodies. "Native antibodies" refer 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.

[0154] "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 may be bivalent or bispecific, see, e.g., 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 a modular heavy chain variable domain or all or a modular 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.

[0155] 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 comprising 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 having two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0156] 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, i.e. 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) .

[0157] "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 orders 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.

[0158] "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 the insertion of 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.

[0159] 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 L The antibody fragment is a fragment of a polypeptide that is cleaved from the C-terminus of the antibody 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 characteristics of a VH domain, i.e., capable of assembling together with a VL domain into a functional antigen-binding site, or characteristics of a VL domain, i.e., capable of assembling together with a VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0160] "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); novel antigen receptor β-lactamase variable domain (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).

[0161] 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 an antibody (e.g., a domain antibody). For further details, see Journal of Immunological Methods 248(1-2), 31-45(2001).

[0162] 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.

[0163] Affibodies are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. This 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. 2004, 17, 455-462 and EP 1641818 A1.

[0164] 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).

[0165] 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).

[0166] 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.

[0167] 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 antibody heavy chain from camelids (nanoantibodies 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.

[0168] 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 the 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.

[0169] 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).

[0170] 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 of engineered cysteine ​​knot peptide domains, see WO2008098796.

[0171] 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.

[0172] The term "antigen binding domain" refers to a module of an antigen binding molecule that comprises 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 module 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).

[0173] 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.

[0174] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific 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

[0175] "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).

[0176] 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 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).

[0177] The term "fibroblast activation protein (FAP)", also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any native 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: 21), or in 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: 22 and 23, respectively. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 126, SEQ ID NO: 24), or in 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: 25 and 26 show the amino acid and nucleotide sequences of the His-tagged mouse FAP ECD, respectively. SEQ ID NOs: 27 and 28 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.

[0178] 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: 29). 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, respiratory, and urogenital tracts, 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 makes CEA available for antibody binding 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(aSuppl.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.

[0179] 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).

[0180] The term "melanoma-associated chondroitin sulfate proteoglycan (MCSP)", also known as chondroitin sulfate proteoglycan 4 (CSPG4), refers to any naturally occurring MCSP 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 MCSP is shown in UniProt accession number Q6UVK1 (version 103, SEQ ID NO: 30). 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 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 EGFR is shown in UniProt accession number P00533 (version 211, SEQ ID NO: 31).

[0181] The term "CD19" refers to the B lymphocyte antigen CD19, also known 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: 32). The term encompasses "full-length" unprocessed human CD19 as well as 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. CD19 expression on plasma cells further suggests that it may be expressed in differentiated B-cell neoplasms 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.

[0182] 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 specified. The amino acid sequence of human CD20 is shown in Uniprot accession number P11836 (version 149, SEQ ID NO: 33). 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 specified. The amino acid sequence of human CD33 is shown in Uniprot Accession No. P20138 (version 157, SEQ ID NO: 34).

[0183] 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.

[0184] 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 the modules 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.

[0185] Table A. CDR Definitions 1

[0186]

[0187]

[0188] 1 The numbering of all CDR definitions in Table A follows the numbering convention proposed by Kabat et al. (see below).

[0189] 2 "AbM" with a lowercase "b" as used in Table A refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0190] 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.

[0191] In addition to the CDR1 in VH, CDRs generally include amino acid residues that form hypervariable loops. CDRs also include "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 (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al. above.

[0192] 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.

[0193] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The variable domain FR is typically 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.

[0194] 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.

[0195] 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 remaining modules of the heavy and / or light chain are derived from a different source or species.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] The term "Fc domain" or "Fc region" as used herein 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 IgG CH2 and IgG CH3 domains. The "CH2 domain" of a human IgG Fc region generally 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" comprises a residue segment from the C-terminus to the CH2 domain in the Fc region (i.e., from an amino acid residue at approximately position 341 to an amino acid residue at approximately position 447 of IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "protrusion" ("knot") introduced in one chain and a corresponding "cavity" ("hole") introduced in the other chain; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). As described herein, this variant CH3 domain can be used to promote the heterodimerization of two different 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) in the Fc region may or may not exist. 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.

[0200] " 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 a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a 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 generated 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.

[0201] "A region equivalent to an immunoglobulin Fc region" is intended to include naturally occurring allelic variants of the immunoglobulin Fc region, as well as variants that have produced 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)).

[0202] 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 receptor), and B cell activation.

[0203] 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 No. P08637, version 141).

[0204] The term "TNF ligand family member" or "TNF family ligand" refers to a proinflammatory cytokine. Cytokines, and 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.

[0205] The 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 natural 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 present invention, the TNF ligand family member is selected from 4-1BBL, OX40L, GITRL, CD70, CD30L, CD40L and LIGHT. In certain embodiments, the TNF ligand family member is selected from 4-1BBL and OX40L.

[0206] 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: 35), human TNF (UniProt Accession No. P01375, SEQ ID NO: 36), human lymphotoxin beta (UniProt Accession No. Q06643, SEQ ID NO: 37), human OX40L (UniProt Accession No. P23510, SEQ ID NO: 38), human CD40L (UniProt Accession No. P29965, SEQ ID NO: 39), human FasL (UniProt Accession No. P48023, SEQ ID NO: 40), human CD27L (UniProt Accession No. P32970, SEQ ID NO: 41), human CD30L (UniProt Accession No. P32971, SEQ ID NO: 42), 4-1BBL (UniProt Accession No. P41273, SEQ ID NO: 43). NO:43), TRAIL (UniProt Accession No. P50591, SEQ ID NO:44), RANKL (UniProt Accession No. 014788, SEQ ID NO:45), TWEAK (UniProt Accession No. 043508, SEQ ID NO:46), APRIL (UniProt Accession No. 075888, SEQ ID NO:47), BAFF (UniProt Accession No. Q9Y275, SEQ ID NO:48), LIGHT (UniProt Accession No. 043557, SEQ ID NO:49), TL1A (UniProt Accession No. 095150, SEQ ID NO:50), GITRL (UniProt Accession No. Q9UNG2, SEQ ID NO:51) and exoprotein A (UniProt Accession No. Q92838, SEQ ID NO:52).

[0207] " 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 protein module that causes contact with the surface, causes signal transduction. Therefore, the ectodomain of TNF ligand family members as defined herein refers to the module of the TNF ligand protein that extends into the extracellular space (extracellular domain), also includes the shorter part or its fragment that is responsible for trimerization and is combined with corresponding TNF receptor. Therefore, 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 module (receptor binding domain) that can still be combined with receptor.

[0208] 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 signaling cascade that leads to T cell activation. The co-stimulatory TNF family ligands are selected from 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT, more specifically, the co-stimulatory TNF ligand family members are selected from 4-1BBL and OX40L.

[0209] 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:43 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:53) 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), SEQ ID NO: 2 (amino acids 85-254 of human 4-1BBL), SEQ ID NO: 99 (amino acids 71-248 of human 4-1BBL), SEQ ID NO: 100 (amino acids 85-248 of human 4-1BBL), SEQ ID NO: 101 (amino acids 80-248 of human 4-1BBL) and SEQ ID NO: 102 (amino acids 52-254 of human 4-1BBL), but other fragments of the extracellular domain capable of trimerization are also included herein.

[0210] 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: 38. Amino acids 51-183 of the human OX40L sequence (SEQ ID NO: 6) 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: 6 (amino acids 51-183 of human OX40L) or SEQ ID NO: 7 (amino acids 52-183 of human OX40L), but other fragments of the extracellular domain capable of trimerization are also included herein.

[0211] The term "peptide linker" refers to a peptide comprising one or more amino acids. Peptide linkers comprise 1-44 amino acids, more particularly 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 2 to 4, in particular 2, i.e., these peptides are selected from GGGGS (SEQ ID NO: 136), GGGGSGGGGS (SEQ ID NO: 54), 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: 136), (G4S)2 or GGGGSGGGGS (SEQ ID NO: 54) and GSPGSSSSGS (SEQ ID NO: 57), more specifically (G4S)2 or GGGGSGGGGS (SEQ ID NO: 54) and GSPGSSSSGS (SEQ ID NO: 57).

[0212] 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).

[0213] 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.

[0214] 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.

[0215] "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 sequence identity, and without considering any conservative substitutions as part of sequence identity). Alignment for determining percentages of 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. in 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 contains relative to (with, or to) a given amino acid sequence B) is calculated as follows:

[0216] Multiply 100 by the fraction X / Y

[0217] 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.

[0218] In certain embodiments, amino acid sequence variants of antigen binding molecules containing TNF ligand trimers provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of antigen binding molecules containing TNF ligand trimers. Amino acid sequence variants of antigen binding molecules containing TNF ligand trimers 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 residues in the antibody amino acid sequence. Any combination of deletions, insertions and substitutions 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 "preferred substitutions" 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.

[0219] Table B

[0220]

[0221]

[0222] Amino acids can be grouped according to common side chain properties:

[0223] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0224] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0225] (3) Acidic: Asp, Glu;

[0226] (4) Basic: His, Lys, Arg;

[0227] (5) Residues that affect chain orientation: Gly, Pro;

[0228] (6) Aromatic: Trp, Tyr, Phe.

[0229] Non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0230] The term "amino acid sequence variant" includes substantial variants in which amino acid substitutions are present in one or more hypervariable region residues of a parent antigen binding molecule (e.g., humanized or human antibody). Typically, the resulting variants selected for further study will have changes (e.g., improvements) and / or will have certain biological properties of the parent antigen binding molecule substantially retained in terms of certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antigen binding molecule. Exemplary substitution variants are affinity matured antibodies, which can be readily 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 (e.g., 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 molecule to bind to antigens. For example, conservative changes (e.g., 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, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is 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.

[0231] 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.

[0232] 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 the 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 fucosylated variants can have enhanced ADCC function, see, for example, U.S. Patent Publication Nos.US 2003 / 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 oligosaccharide attached to the Fc district is bisected by GlcNAc. These variants may have reduced fucosylation and / or enhanced ADCC function, see, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 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.).

[0233] 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.

[0234] 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. Non-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.

[0235] 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.

[0236] 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.

[0237] With respect to "isolated" nucleic acid molecules or polynucleotides, it is meant a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a 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 normally contain polynucleotide molecules, but the polynucleotide molecules exist extrachromosomally or at a chromosomal position different from their 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, the polynucleotides or nucleic acids can be or can include regulatory elements, such as promoters, ribosome binding sites or transcription terminators.

[0238] 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 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, alternatively, multiple nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations 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 a group or multiple 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 invention can be routinely determined using known computer programs such as ALIGN-2, as discussed above for polypeptides.

[0239] 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.

[0240] 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.

[0241] 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 progeny of these cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that 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, to name a few, as well as cells included in transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] "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.

[0247] 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.

[0248] 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 the individual being treated, either for prevention or during clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and alleviation or improved 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.

[0249] 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 cancer, cutaneous or intraocular melanoma, 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, urinary tract cancer, ureteral cancer, ureteral carcinoma ... cancer of the kidney or ureter, renal cell carcinoma, renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, central nervous system (CNS) tumors, chordomas, brain stem gliomas, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas and Ewing's sarcoma, melanoma, multiple myeloma, B-cell cancers (lymphomas), 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.

[0250] Antigen binding molecules containing TNF family ligand trimers of the present invention

[0251] 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.

[0252] Thus, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer, comprising

[0253] (a) at least one module capable of specifically binding to a target cell antigen,

[0254] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other (optionally via a peptide linker), and

[0255] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0256] In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising

[0257] (a) at least one module capable of specifically binding to a target cell antigen,

[0258] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0259] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0260] In another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0261] (a) at least one module capable of specifically binding to a target cell antigen,

[0262] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0263] (c) an Fc domain consisting of a first and a second subunit capable of stably combining, wherein the polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof connected to each other by a peptide linker is fused to the N- or C-terminal amino acid of one of the two subunits of the Fc domain, optionally via a peptide linker.

[0264] In a particular aspect, the antigen binding molecules containing TNF families ligand trimer include (a) at least one module that can specifically bind to target cell antigens, (b) include three TNF ligand family members connected to each other by peptide linkers The extracellular domain or a polypeptide of its fragment, and (c) by the Fc domains that can stably constitute the first and second subunits of association, wherein the TNF ligand family members costimulate human T cell activation. Therefore, the TNF ligand family members are costimulatory TNF family ligands. Specifically, the costimulatory TNF family ligands are selected from 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT, more specifically, the costimulatory TNF family ligands are selected from 4-1BBL and OX40L.

[0265] In one aspect, the TNF ligand family member is 4-1BBL.

[0266] In a further aspect, the extracellular domain of the TNF ligand family member comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102, in particular an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 99.

[0267] In a specific aspect, the extracellular domain of a TNF ligand family member or a fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, in particular the amino acid sequence of SEQ ID NO: 1.

[0268] In yet another aspect, the antigen binding molecules containing a TNF family ligand trimer of the present invention comprise

[0269] (a) at least one module capable of specifically binding to a target cell antigen, and

[0270] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103, and

[0271] (c) an Fc domain composed of a first and a second subunit capable of stably associating. More specifically, the polypeptide comprises the amino acid sequence of SEQ ID NO: 5.

[0272] In another aspect, the TNF ligand family member is OX40L.

[0273] In another aspect, the extracellular domain of the TNF ligand family member comprises an amino acid sequence selected from SEQ ID NO: 6 and SEQ ID NO: 7, in particular the amino acid sequence of SEQ ID NO: 6.

[0274] In yet another aspect, the antigen binding molecules containing a TNF family ligand trimer of the present invention comprise

[0275] (a) at least one module capable of specifically binding to a target cell antigen, and

[0276] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, and

[0277] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0278] In one aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0279] (a) at least one module capable of specifically binding to a target cell antigen,

[0280] (b) a polypeptide comprising three ectodomains of TNF ligand family members or fragments thereof linked to each other, and

[0281] (c) an Fc domain consisting of a first and a second subunit capable of stably associating, wherein the extracellular domains of the three TNF ligand family members or fragments thereof are linked to each other directly or with a suitable chemical linker, such as a disulfide, ester or ether bridge.

[0282] In another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0283] (a) at least one module capable of specifically binding to a target cell antigen,

[0284] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0285] (c) an Fc domain consisting of a first and a second subunit capable of stably associating, wherein the extracellular domains of the three TNF ligand family members or fragments thereof are connected to each other by a peptide linker which is the same or different and is selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 54), SGGGGSGGGG (SEQ ID NO: 55) and GGGGSGGGGSGGGG (SEQ ID NO: 56), but also includes 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). Specifically, the peptide linker is identical and is selected from (G4S)2 (SEQ ID NO: 54) and GSPGSSSSGS (SEQ ID NO: 57). More specifically, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the extracellular domains of the three TNF ligand family members or fragments thereof are connected to each other via a (G4S)2 linker.

[0286] In another aspect, the antigen binding molecules containing TNF family ligand trimers of the present invention comprise

[0287] (a) at least one module capable of specifically binding to a target cell antigen,

[0288] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0289] (c) by being able to stablize the Fc domains that combine first and second subunit is constituted, the polypeptide of the extracellular domain comprising three tnf ligand family members or its fragment is fused to the N-terminal amino acid of one of the subunit of the Fc domains at C-terminal amino acid.In a particular aspect, the polypeptide of the extracellular domain comprising three tnf ligand family members or its fragment is fused to the N-terminal amino acid of the CH2 domains in the Fc domains at C-terminal amino acid.In yet another aspect, the polypeptide of the extracellular domain comprising three tnf ligand family members or its fragment is connected to the N-terminal amino acid of the Fc domains at C-terminal amino acid by a peptide linker.

[0290] In another aspect, the antigen binding molecules containing TNF family ligand trimers of the present invention comprise

[0291] (a) at least one module capable of specifically binding to a target cell antigen,

[0292] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0293] (c) by being able to stablize the Fc domains that combine first and second subunit is constituted, the polypeptide of the extracellular domain comprising three tnf ligand family members or its fragment is fused to the C-terminal amino acid of the Fc domains at N-terminal amino acid.In a particular aspect, the polypeptide of the extracellular domain comprising three tnf ligand family members or its fragment is fused to the C-terminal amino acid of the CH3 domains in the Fc domains at N-terminal amino acid.In another aspect, the polypeptide of the extracellular domain comprising three tnf ligand family members or its fragment is connected to the C-terminal amino acid of the Fc domains at N-terminal amino acid by a peptide linker.

[0294] In another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0295] (a) at least one module capable of specifically binding to a target cell antigen,

[0296] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0297] (c) an Fc domain composed of a first and a second subunit capable of stably associating,

[0298] The module capable of specifically binding to a target cell antigen is not fused to the polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof.

[0299] 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.

[0300] 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.

[0301] In another 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. Specifically, the antibody fragment is selected from Fab molecules, exchange Fab molecules, single-chain Fab molecules, Fv molecules, scFv molecules, single domain antibodies, and VH.

[0302] In yet another 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.

[0303] Thus, in yet another aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the module capable of specific binding to a target cell antigen is selected from an antibody fragment, a Fab molecule, an exchange Fab molecule, a single-chain Fab molecule, an Fv molecule, a scFv molecule, a single domain antibody, a VH and a scaffold antigen binding protein. Thus, in a specific aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising (a) at least one antibody fragment capable of specific binding to a target cell antigen, selected from a Fab molecule, an exchange Fab molecule, a single-chain Fab molecule, an Fv molecule, a scFv molecule, a single domain antibody, a VH and a scaffold antigen binding protein, (b) a polypeptide comprising three extracellular domains of a TNF ligand family member connected to each other by a peptide linker or a fragment thereof, and (c) an Fc domain consisting of a first and a second subunit capable of stabilizing the combination. In one aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (a) an antibody fragment capable of specifically binding to a target cell antigen, which is selected from Fab molecules, exchange Fab molecules, single-chain Fab molecules, Fv molecules, scFv molecules, single domain antibodies, VH and scaffold antigen binding proteins.

[0304] In a particular aspect, the present invention is relevant to the antigen binding molecules containing a TNF family ligand trimer as defined above, wherein the module capable of specific binding to a target cell antigen is a Fab molecule capable of specific binding to a target cell antigen. Thus, the present invention provides an antigen binding molecule containing a TNF family ligand trimer, comprising (a) at least one Fab molecule capable of specific binding to a target cell antigen, (b) comprising three TNF ligand family members connected to each other by a peptide linker The extracellular domain or a polypeptide of a fragment thereof, and (c) by the Fc domains that can stably constitute the first and second subunits of the combination.

[0305] 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.

[0306] In yet another aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the Fc domains consisting of the first and second subunits capable of stabilizing association are IgG Fc domains, particularly IgG1 Fc domains or IgG4 Fc domains. Specifically, the Fc domains consisting of the first and second subunits capable of stabilizing association are IgG1 Fc domains. In a particular aspect, the Fc domains include modifications that promote the association of the first and second subunits of Fc domains.

[0307] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0308] 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.

[0309] The Fc domain confers 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 target tissues. Thus, the Fc domain advantageously promotes the pharmacokinetic parameters (PK data) of the antigen binding molecules, such as clearance, volume of distribution, or elimination half-life (t 1 / 2). However, at the same time, the bispecific antibodies of the present invention may cause cells expressing Fc receptors to be undesirably targeted rather than preferred antigen-bearing cells. Therefore, in a specific aspect, the Fc domains of the antigen binding molecules containing TNF family ligand trimers of the present invention exhibit reduced binding affinity to Fc receptors and / or reduced effector functions compared to native IgG1 Fc domains. In one aspect, the Fc domains do not substantially bind to Fc receptors and / or do not induce effector functions. In a specific aspect, the Fc receptors are Fcγ receptors. In one aspect, the Fc receptors are human Fc receptors. In a specific aspect, the Fc receptors are activating human Fcγ receptors, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the Fc domains do 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.

[0310] 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.

[0311] In one aspect, the present invention relates to an antigen binding molecule containing a TNF family ligand trimer as defined above, comprising:

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

[0313] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0314] (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.

[0315] 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.

[0316] 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 ).

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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).

[0321] 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)).

[0322] In a specific aspect, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

[0323] Fc domain modifications that promote heterodimerization

[0324] The Fc domains comprise the different modules fused with one or another of the two subunits of the Fc domains, and the subunit is generally contained in two unequal polypeptide chains (" heavy chains "). 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 trimers in recombinant production, it is therefore advantageous to introduce the combined modification promoting the desired polypeptide in the Fc domains of the antigen binding molecules containing TNF families ligand trimers of the present invention.

[0325] Therefore, the Fc domains of the antigen binding molecules containing TNF families ligand trimers of the present invention include modifications that promote 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.

[0326] 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 containing a TNF family ligand trimer as described above, comprising an IgG molecule, wherein the Fc module of the first heavy chain comprises a first dimerization module, and the Fc module 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.

[0327] 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.

[0328] 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.

[0329] Protrusions and cavities can be produced by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis, or by peptide synthesis.

[0330] 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)).

[0331] 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.

[0332] Specific antigen-binding molecules containing TNF family ligand trimers

[0333] In another aspect, a monovalent antigen binding molecule comprising a TNF family ligand trimer as defined above is provided, which comprises a module capable of specifically binding to a target cell antigen.

[0334] Specifically, the antigen binding molecule containing a TNF family ligand trimer of the present invention, comprising (c) an Fc domain composed 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.

[0335] Furthermore, the present invention provides an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0336] (a) heavy and light chains, which together constitute the Fab molecule capable of specifically binding to the target cell antigen, and

[0337] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 15.

[0338] In yet another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0339] (i) a first heavy chain comprising a VH domain of a Fab molecule capable of specifically binding to a target cell antigen,

[0340] (ii) a light chain comprising the VL domain of a Fab molecule capable of specifically binding to a target cell antigen, and

[0341] (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0342] In a specific aspect, the antigen binding molecule containing a trimer of TNF family ligands comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 16 or a VH domain comprising an amino acid sequence of SEQ ID NO: 110, (ii) a light chain comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 17 or a VL domain comprising an amino acid sequence of SEQ ID NO: 111, and (iii) a second heavy chain comprising an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8. In another aspect, the antigen binding molecule containing a trimer of TNF family ligands comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 118, (ii) a light chain comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 119, and (iii) a second heavy chain comprising an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8. In yet another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 126 or a VH domain comprising an amino acid sequence of SEQ ID NO: 134, (ii) a light chain comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 127 or a VL domain comprising an amino acid sequence of SEQ ID NO: 135, and (iii) a second heavy chain comprising an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0343] In another aspect, a bivalent antigen binding molecule containing a TNF family ligand trimer is provided, which comprises two modules capable of specifically binding to a target cell antigen.

[0344] In one aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0345] (i) a first heavy chain comprising the VH domain or VL domain of a Fab molecule capable of specifically binding to a target cell antigen,

[0346] (ii) two light chains, each comprising a VL domain or a VH domain of a Fab molecule capable of specifically binding to a target cell antigen, and

[0347] (iii) a second heavy chain comprising the VH domain or VL domain of a Fab molecule capable of specifically binding to a target cell antigen and the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103 or SEQ ID NO: 8.

[0348] In one aspect, the antigen binding molecule containing a TNF family ligand trimer comprises

[0349] (i) a first heavy chain comprising a VH domain of a Fab molecule capable of specifically binding to a target cell antigen,

[0350] (ii) two light chains, each comprising a VL domain of a Fab molecule capable of specifically binding to a target cell antigen, and

[0351] (iii) a second heavy chain comprising the VH domain of a Fab molecule capable of specifically binding to a target cell antigen and the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103 or SEQ ID NO: 8.

[0352] In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 or a VH domain comprising the amino acid sequence of SEQ ID NO: 110, (ii) two light chains, each comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17 or a VL domain comprising the amino acid sequence of SEQ ID NO: 111, and (iii) a second heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 or a VH domain comprising the amino acid sequence of SEQ ID NO: 110 and an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8. In another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 118, (ii) two light chains, each comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 119, and (iii) a second heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 118 and an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8. In yet another aspect, the antigen binding molecule containing a TNF family ligand trimer comprises (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 126 or a VH domain comprising an amino acid sequence of SEQ ID NO: 134, (ii) two light chains comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 127 or a VL domain comprising an amino acid sequence of SEQ ID NO: 135, and (iii) a second heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 126 or a VH domain comprising an amino acid sequence of SEQ ID NO: 134 and an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0353] In one aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer according to the present invention, wherein the antigen binding molecule comprises

[0354] (a) a heavy chain and a light chain, the heavy chain comprising a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and the light chain comprising a VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and

[0355] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 15.

[0356] In another aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer according to the present invention, wherein the antigen binding molecule comprises

[0357] (a) a module capable of specifically binding to FAP, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a VL domain comprising the amino acid sequence of SEQ ID NO: 17, and

[0358] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 15.

[0359] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0360] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16,

[0361] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 17, and

[0362] (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0363] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0364] (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 16,

[0365] (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 17, and

[0366] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO:15.

[0367] In another aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0368] (a) heavy and light chains, which together constitute the Fab molecule capable of specifically binding to the target cell antigen, and

[0369] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

[0370] In one aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer according to the present invention, wherein the antigen binding molecule comprises

[0371] (a) a heavy chain and a light chain, the heavy chain comprising a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and the light chain comprising a VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and

[0372] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

[0373] In yet another aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer according to the present invention, wherein the antigen binding molecule comprises

[0374] (a) a module capable of specifically binding to FAP, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a VL domain comprising the amino acid sequence of SEQ ID NO: 17, and

[0375] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

[0376] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0377] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16,

[0378] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 17, and

[0379] (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

[0380] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0381] (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 16,

[0382] (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 17, and

[0383] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

[0384] Antigen-binding molecule containing a trimer of TNF family ligands, wherein the target cell antigen is FAP

[0385] In a specific aspect, the target cell antigen is fibroblast activation protein (FAP). In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the module capable of specifically binding to FAP comprises a VH domain and a VL domain,

[0386] (a) the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or

[0387] (b) the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 104, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 105, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 106, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 108, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 109.

[0388] In one aspect, 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: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In another specific aspect, 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: 104, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 105, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 106, and the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 108, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 109.

[0389] In yet 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: 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. In a 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 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: 16 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 17.

[0390] In another specific aspect, the antigen binding molecule containing a TNF family ligand trimer of the present invention comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In a specific aspect, the antigen binding molecule containing a TNF family ligand trimer comprises: a heavy chain consisting of the amino acid sequence of SEQ ID NO: 18 and a light chain consisting of the amino acid sequence of SEQ ID NO: 19.

[0391] 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: 110, 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: 111. In a 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: 110 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 111. 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: 110 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 111.

[0392] In one aspect, a monovalent antigen binding molecule containing a TNF family ligand trimer as described above is provided, which comprises a module capable of specifically binding to FAP.

[0393] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0394] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 18,

[0395] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 19, and

[0396] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO:15.

[0397] In another specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0398] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 140,

[0399] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 142, and

[0400] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO:15.

[0401] In another specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0402] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 18,

[0403] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 19, and

[0404] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

[0405] In another aspect, a bivalent antigen binding molecule containing a TNF family ligand trimer as described above is provided, which comprises two modules capable of specifically binding to FAP.

[0406] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0407] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 140,

[0408] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 142, and

[0409] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 141.

[0410] Antigen binding molecule containing a trimer of TNF family ligands, wherein the target cell antigen is CEA

[0411] In one aspect, target cell antigen is CEA.In one aspect, the invention provides the antigen binding molecules containing TNF families ligand trimer as described above, wherein the module that can specifically bind CEA includes VH domains and VL domains, the VH domains include SEQ ID NO (i) including:112 amino acid sequence CDR-H1, (ii) including SEQ ID NO:113 amino acid sequence CDR-H2, and (iii) including SEQ ID NO:114 amino acid sequence CDR-H3, the VL domains include SEQ ID NO (iv) including:115 amino acid sequence CDR-L1, (v) including SEQ ID NO:116 amino acid sequence CDR-L2, and (vi) including SEQ ID NO:117 amino acid sequence CDR-L3.

[0412] In yet 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: 118, 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: 119. In a specific aspect, the module capable of specifically binding to CEA comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 118 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 119. 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: 118 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 119.

[0413] In one aspect, a monovalent antigen binding molecule containing a TNF family ligand trimer as described above is provided, which comprises a module capable of specifically binding to CEA.

[0414] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0415] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 146,

[0416] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 148, and

[0417] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO:15.

[0418] In another aspect, a bivalent antigen binding molecule containing a TNF family ligand trimer as described above is provided, which comprises two modules capable of specifically binding to CEA.

[0419] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0420] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 146,

[0421] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 148, and

[0422] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 147.

[0423] Antigen-binding molecule containing a trimer of TNF family ligands, wherein the target cell antigen is CD19

[0424] In yet another aspect, the target cell antigen is CD19. In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer as described above, wherein the module capable of specifically binding to CD9 comprises a VH domain and a VL domain,

[0425] (a) the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125; or

[0426] (b) the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 129, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 130, and the VL domain comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 131, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 132, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0427] In yet another aspect, the module capable of specifically binding to CD19 comprises a heavy chain variable region comprising an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 126, and a light chain variable region comprising an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 127. In another aspect, the module capable of specifically binding to CD19 comprises a heavy chain variable region comprising an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 134, and a light chain variable region comprising an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 135. 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: 126 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 127. In one specific aspect, the module capable of specifically binding to FAP comprises a VH domain consisting of the amino acid sequence of SEQ ID NO: 118 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 119. In another specific aspect, the module capable of specifically binding to CD19 comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 134 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 135. In one specific aspect, the module capable of specifically binding to FAP comprises a VH domain consisting of the amino acid sequence of SEQ ID NO: 134 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 135.

[0428] In one aspect, a monovalent antigen binding molecule containing a TNF family ligand trimer as described above is provided, which comprises a module capable of specifically binding to CD19.

[0429] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0430] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 152,

[0431] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 154, and

[0432] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO:15.

[0433] In another specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0434] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 158,

[0435] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 160, and

[0436] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO:15.

[0437] In another aspect, a bivalent antigen binding molecule containing a TNF family ligand trimer as described above is provided, which comprises two modules capable of specifically binding to CD19.

[0438] In a specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0439] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 152,

[0440] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 154, and

[0441] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 153.

[0442] In another specific aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0443] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 158,

[0444] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 160, and

[0445] (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 159.

[0446] Polynucleotides

[0447] The present invention further provides isolated polynucleotides or fragments thereof encoding antigen binding molecules containing a TNF family ligand trimer as described herein.

[0448] 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 (such as two or more) polynucleotides of coexpression. The polypeptide encoded by the coexpressed polynucleotides can be combined to form a functional antigen binding molecules by, for example, a disulfide bond or other means. For example, the light chain module of an immunoglobulin can be encoded by a polynucleotide separated from the heavy chain module of an immunoglobulin. The heavy chain polypeptide will be combined with the light chain polypeptide to form an immunoglobulin when coexpressed.

[0449] In some aspects, the polynucleotide encoding of separation is as described herein according to the complete antigen binding molecules containing TNF family ligand trimer of the present invention.In other embodiments, the polynucleotide encoding of separation is included in the polypeptide in the antigen binding molecules containing TNF family ligand trimer of the present invention as described herein.

[0450] 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 three extracellular domains of a TNF ligand family member or a fragment thereof connected to each other by a peptide linker, and (c) a sequence encoding an Fc domain consisting of a first and a second subunit capable of stably combining.

[0451] 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 three extracellular domains of 4-1BB or fragments thereof connected to each other by a peptide linker, and (c) a sequence encoding an Fc domain consisting of a first and a second subunit capable of stably combining.

[0452] In yet another aspect, the present invention relates to an isolated polynucleotide comprising a sequence encoding a polypeptide comprising three 4-1BBL fragments, wherein the 4-1BBL fragment comprises an amino acid sequence having at least about 90%, 95%, 98% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 or SEQ ID NO: 102. In one aspect, an isolated polynucleotide comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 is provided.

[0453] In another aspect, an isolated polynucleotide encoding an antigen-binding molecule containing an OX40 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 three extracellular domains of OX40L or fragments thereof connected to each other by a peptide linker, and (c) a sequence encoding an Fc domain composed of a first and a second subunit capable of stably associating.

[0454] In yet another aspect, the present invention relates to an isolated polynucleotide comprising a sequence encoding a polypeptide comprising three OX40L fragments comprising an amino acid sequence at least about 90%, 95%, 98%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7. In one aspect, an isolated polynucleotide comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 is provided.

[0455] In another aspect, there is provided 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) encoding a polypeptide comprising an extracellular domain of three TNF ligand family members connected to each other by a peptide linker or a fragment thereof and fused to the N-terminal amino acid of the CH2 domain in the Fc domain at the C-terminal amino acid, (c) encoding a sequence of the Fc domain consisting of the first and second subunits capable of stabilizing the combination. In yet another aspect, there is provided 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) encoding a polypeptide comprising an extracellular domain of three TNF ligand family members connected to each other by a peptide linker or a fragment thereof and fused to the C-terminal amino acid of the CH3 domain in the Fc domain at the N-terminal amino acid, and (c) encoding a sequence of the Fc domain consisting of the first and second subunits capable of stabilizing the combination.

[0456] In one aspect, the invention relates to an isolated polynucleotide comprising a sequence encoding an IgG1 Fc domain. In a specific aspect, the isolated polynucleotide comprises a sequence encoding an Fc domain having an amino acid substitution at position 234, 235 and / or 329 (EU numbering).

[0457] In another aspect, the present invention relates to an isolated polynucleotide comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 66. In yet another aspect, an isolated polynucleotide comprising a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 87 is provided.

[0458] In yet another aspect, the present invention relates to isolated polynucleotides, which encode antigen binding molecules containing a TNF family ligand trimer, wherein the polynucleotides include sequences encoding heavy and light chains, both of which constitute Fab molecules capable of specifically binding to target cell antigens, and the nucleic acid sequence of SEQ ID NO: 66. In yet another aspect, the present invention relates to isolated polynucleotides, which encode antigen binding molecules containing a TNF family ligand trimer, wherein the polynucleotides include sequences encoding heavy and light chains, both of which constitute Fab molecules capable of specifically binding to target cell antigens, and the nucleic acid sequence of SEQ ID NO: 87.

[0459] In one aspect, an isolated polynucleotide encoding an antigen binding molecule containing a 4-1BBL trimer is provided, wherein the polynucleotide comprises a sequence encoding the following: (a) a heavy chain and a light chain, the heavy chain comprising a VH domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, the light chain comprising a VL domain, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0460] In another aspect, an isolated polynucleotide encoding an antigen binding molecule containing a 4-1BBL trimer is provided, wherein the polynucleotide comprises a sequence encoding the following: (a) a heavy chain and a light chain, the heavy chain comprising a VH domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 104, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 105, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 106, the light chain comprising a VL domain, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 108, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 109, and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0461] In yet another aspect, an isolated polynucleotide encoding an antigen binding molecule containing a 4-1BBL trimer is provided, wherein the polynucleotide comprises a sequence encoding: (a) a heavy chain and a light chain, the heavy chain comprising a VH domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 112, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 113, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 114, the light chain comprising a VL domain, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 115, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 117, and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0462] In yet another aspect, an isolated polynucleotide encoding an antigen binding molecule containing a 4-1BBL trimer is provided, wherein the polynucleotide comprises a sequence encoding: (a) a heavy chain and a light chain, the heavy chain comprising a VH domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122, the light chain comprising a VL domain, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125, and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0463] In yet another aspect, an isolated polynucleotide encoding an antigen binding molecule containing a 4-1BBL trimer is provided, wherein the polynucleotide comprises a sequence encoding: (a) a heavy chain and a light chain, the heavy chain comprising a VH domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 129, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 130, the light chain comprising a VL domain, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 131, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 132, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 133, and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0464] In another aspect, the present invention provides an isolated polynucleotide encoding an antigen-binding molecule containing a 4-1BBL trimer, wherein the polynucleotide comprises a sequence encoding the following: (a) a module capable of specifically binding to FAP, which comprises a VH domain consisting of the amino acid sequence of SEQ ID NO: 16 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 17, and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0465] In a specific aspect, the present invention provides an isolated polynucleotide encoding an antigen binding molecule containing a 4-1BBL trimer, wherein the polynucleotide comprises a sequence encoding: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 16, (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 15.

[0466] In another aspect, the present invention provides an isolated polynucleotide encoding an antigen-binding molecule containing a 4-1BBL trimer, wherein the polynucleotide comprises a sequence encoding the following: (a) a module capable of specifically binding to FAP, which comprises a VH domain consisting of the amino acid sequence of SEQ ID NO: 110 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 111, and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0467] In a specific aspect, the present invention provides an isolated polynucleotide encoding a 4-1BBL trimeric antigen binding molecule, wherein the polynucleotide comprises a sequence encoding: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 110, (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 111, and (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 15.

[0468] In another aspect, an isolated polynucleotide encoding an antigen binding molecule containing an OX40L trimer is provided, wherein the polynucleotide comprises a sequence encoding: (a) a heavy chain and a light chain, the heavy chain comprising a VH domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, the light chain comprising a VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 8.

[0469] In another aspect, the present invention provides an isolated polynucleotide encoding an antigen-binding molecule containing an OX40L trimer, wherein the polynucleotide comprises a sequence encoding: (a) a module capable of specifically binding to FAP, which comprises a VH domain consisting of the amino acid sequence of SEQ ID NO: 16 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 17, and (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 8.

[0470] In a specific aspect, the invention provides an isolated polynucleotide encoding an antigen binding molecule containing an OX40L trimer, wherein the polynucleotide comprises a sequence encoding: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 16, (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

[0471] In certain embodiments, 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.

[0472] Recombination methods

[0473] 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:A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989) in technology. Expression vector can be a part for a plasmid, a virus, or can be a nucleic acid fragment. Expression vector includes encoding an antigen binding molecule containing a TNF families ligand trimer or a polynucleotide thereof (i.e., coding region) and promoter and / or other transcription or translation control elements to be cloned into an expression cassette therein in an operably associated manner. As used herein, " coding region " is a nucleic acid module consisting of codons 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.

[0474] 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).

[0475] 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.

[0476] 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.

[0477] 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).

[0478] 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.

[0479] 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).

[0480] 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 comprising three extracellular domains of TNF ligand family members or its fragment, and Fc domains) are not all fused to each other genetically. Polypeptide is designed so that its components (three extracellular domains of TNF ligand family members or its fragment and other components such as CH2 and CH3 in Fc domains) 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.

[0481] 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).

[0482] 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.

[0483] 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 epitope mapping combined with 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, fixed antigen is incubated in a solution comprising a first labeled antigen binding molecule and a second unlabeled antigen binding molecule that are bound to the antigen, and the second unlabeled antigen binding molecule is tested for the ability of the first antigen binding molecule to compete in binding to the antigen. The second antigen binding molecule may be present in a hybridoma supernatant. As a control, 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 allowing the first antibody to bind to the antigen, excess unbound antibody is removed, and the amount of the label combined with the fixed antigen is measured. If the amount of the label combined with the fixed antigen in the test sample is significantly reduced relative to the control sample, it is shown that the second antigen binding molecule competes with the first antigen binding molecule to bind to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0484] 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.

[0485] Assay

[0486] 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.

[0487] 1. Affinity Determination

[0488] 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 .

[0489] 2. Binding Assays and Other Assays

[0490] 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.

[0491] In yet another 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.

[0492] 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).

[0493] 3. Activity Assay

[0494] 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.

[0495] 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.

[0496] Pharmaceutical compositions, formulations and routes of administration

[0497] In yet another aspect, the invention provides pharmaceutical compositions 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.

[0498] 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.

[0499] 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.

[0500] 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 shaped articles, such as films or microcapsules. In particular embodiments, the absorption of the injectable composition can be prolonged by using an agent that delays absorption in the composition, such as aluminum monostearate, gelatin, or a combination thereof.

[0501] Exemplary pharmaceutically acceptable excipients herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc. Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0502] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation including a histidine-acetate buffer.

[0503] In addition to the aforementioned compositions, the fusion protein can also be formulated as a depot preparation. Such long-acting formulations can be given by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Therefore, for example, the fusion protein can be formulated with a suitable polymeric material or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or be formulated as a slightly soluble derivative, such as a slightly soluble salt.

[0504] The pharmaceutical composition comprising fusion protein of the present invention can be manufactured by conventional mixing, dissolving, emulsifying, encapsulating, embedding or lyophilizing process. One or more physiologically acceptable carriers, diluents, excipients or adjuvants that are conducive to processing protein into pharmaceutically usable preparations can be used to prepare pharmaceutical compositions in a conventional manner. Suitable preparatons depend on the selected route of administration.

[0505] The antigen binding molecules containing the TNF family ligand trimer can be formulated into compositions in the form of free acid or base, neutral or salt. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These salts include acid addition salts, such as those formed by the free amino groups of the protein composition or with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine or procaine. Compared with the corresponding free base form, the solubility of pharmaceutical salts in aqueous solvents and other protic solvents is higher.

[0506] When required for the particular indication being treated, the composition herein may also contain more than one active ingredient, preferably, the active ingredients have complementary activities that do not adversely affect each other. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

[0507] Formulations for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0508] Methods of treatment and compositions

[0509] Any of the antigen binding molecules comprising a TNF family ligand trimer provided herein can be used in therapeutic methods.

[0510] For use in therapeutic methods, the antigen binding molecules containing a TNF family ligand trimer of the present invention can be formulated, administered, and applied in a manner consistent with good medical practice. Factors considered in this case include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the medicament, the method of administration, the dosage regimen, and other factors known to practitioners.

[0511] In one aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer of the present invention for use as a medicament. In another aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer of the present invention for use in treating a disease, especially in cancer treatment. In some aspects, there is provided an antigen binding molecule containing a TNF family ligand trimer of the present invention for use in a method of treating a disease. In one aspect, the present invention provides an antigen binding molecule containing a TNF family ligand trimer as described herein for treating a disease in an individual in need. In some aspects, the present invention provides an antigen binding molecule containing a TNF family ligand trimer used in a method for treating an individual with a disease, the method including administering a therapeutically effective amount of fusion protein to an individual. In some aspects, the disease to be treated is cancer. The example of cancer includes solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B cell lymphoma, B cell leukemia, non-Hodgkin lymphoma and acute lymphoblastic leukemia. Therefore, there is provided an antigen binding molecule containing a TNF families ligand trimer as described herein for use in cancer treatment. Needing the experimenter, patient or " individuality " for treatment is typically a mammal, more specifically a person.

[0512] In yet another aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described herein for treating infectious diseases, especially for treating viral infections. In yet another aspect, there is provided an antigen binding molecule containing a TNF family ligand trimer as described herein for treating autoimmune diseases such as lupus.

[0513] In one aspect, provided is an antigen binding molecule comprising a TNF family ligand trimer for treating head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small cell lung cancer (NSCLC) and mesothelioma according to the present invention, wherein the target cell antigen is FAP.

[0514] In yet another aspect, the present invention relates to the use of an antigen binding molecule containing a TNF family ligand trimer in the manufacture or preparation of a medicament for treating a disease in an individual in need thereof. In one aspect, the medicament is used for use in a method for treating a disease, and the method includes administering a therapeutically effective amount of the medicament to an individual suffering from a disease. In certain embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Therefore, in one aspect, the present invention relates to the use of an antigen binding molecule containing a TNF family ligand trimer of the present invention in the manufacture or preparation of a medicament for treating cancer. Examples of cancer include solid tumors, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B cell lymphoma, B cell leukemia, non-Hodgkin's lymphoma and acute lymphoblastic leukemia. Other cell proliferation disorders that can be treated using the antigen binding molecules containing TNF families ligand trimers of the present invention include but are not limited to tumors located in the abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary glands, testicles, ovaries, thymus glands, thyroid glands), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvic cavity, skin, soft tissue, spleen, chest and urogenital system. Precancerous conditions or lesions and cancer metastasis are also included. In certain embodiments, cancer is selected from renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. Technicians can recognize that in some cases, the antigen binding molecules containing TNF families ligand trimers may not provide a cure, and may only provide partial benefits. In some respects, the physiological changes with some benefits are also considered to be useful for treatment. Therefore, in some respects, the amount of the antigen binding molecules containing TNF families ligand trimers providing physiological changes is considered to be "effective amount" or "therapeutically effective amount."

[0515] In another aspect, the present invention relates to the use of an antigen binding molecule containing a TNF family ligand trimer as described herein in the manufacture or preparation of a medicament for treating infectious diseases, especially viral infections or autoimmune diseases such as lupus.

[0516] In yet another aspect, the present invention provides a method for treating a disease in an individual, the method comprising administering to the individual a therapeutically effective amount of an antigen binding molecule containing a TNF family ligand trimer of the present invention. In one aspect, a composition comprising a fusion protein of the present invention in a pharmaceutically acceptable form is administered to the individual. In some aspects, the disease to be treated is a proliferative disorder. In a specific aspect, the disease is cancer. In another aspect, the disease is an infectious disease or an autoimmune disease. In some aspects, the method further comprises administering to the individual at least one other therapeutic agent of a therapeutically effective amount, for example, if the disease to be treated is cancer, it is an anticancer agent. According to "individual" of any of the above embodiments, it can be a mammal, preferably a human.

[0517] In order to prevent or treat disease, the appropriate dosage of the TNF families ligand trimer antigen binding molecules of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, route of administration, patient's weight, the type of fusion protein, the severity of the disease and the course of disease, whether to give fusion protein for prevention or treatment purposes, previous or simultaneous therapeutic intervention, patient's clinical history and the reaction to fusion protein and the decision of the attending physician. In any case, the physician responsible for administration will determine the concentration of active ingredient in the composition and the appropriate dosage of individual subjects. Various dosage regimens are contemplated herein, including but not limited to single or multiple administration, bolus administration and pulse infusion at various time points.

[0518] The antigen binding molecules containing TNF families ligand trimers are suitably administered to the patient in a one-time or series of treatment modes. Depending on the type and severity of the disease, the antigen binding molecules containing TNF families ligand trimers of about 1 μg / kg to 15mg / kg (e.g., 0.1mg / kg-10mg / kg) can be the initial candidate dose administered to the patient, such as by one or more separate administrations, or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may be in the range of about 1 μg / kg to 100mg / kg or higher. For repeated administration for several days or longer, according to the state of an illness, treatment will generally continue until the suppression of desired disease symptoms occurs. An exemplary dosage of fusion protein will be in the range of about 0.005mg / kg to about 10mg / kg. In other examples, dosage can also include each administration from about 1 μ g / kg body weight, about 5 μ g / kg body weight, about 10 μ g / kg body weight, about 50 μ g / kg body weight, about 100 μ g / kg body weight, about 200 μ g / kg body weight, about 350 μ g / kg body weight, about 500 μ g / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight, about 500 mg / kg body weight, to about 1000 mg / kg body weight or more, and any scope derivable therein. In the example of the derivable range of numerals listed in this article, it is possible to use based on the above-mentioned numerals with scopes such as about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 μ g / kg body weight to about 500 mg / kg body weight. Therefore, one or more dosages of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such dosage can be administered intermittently, for example, weekly or once every three weeks (for example, so that the patient receives about 2 to about 20, or for example, about 6 dosages of fusion protein). An initial higher loading dose can be administered, followed by one or more lower dosages. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0519] The antigen binding molecules containing TNF families ligand trimer of the present invention are generally used in an amount that effectively achieves the desired purpose. In order to be used for treating or preventing disease conditions, the antigen binding molecules containing TNF families ligand trimer of the present invention or its pharmaceutical composition are applied or applied in a therapeutically effective amount. The determination of a therapeutically effective amount is fully within the capabilities of those skilled in the art, especially in the case of the detailed disclosure provided herein.

[0520] For systemic administration, the therapeutically effective dose can be initially estimated based on in vitro assays (e.g., cell culture assays). A dose can then be formulated in animal models to achieve the IC50, including that determined in cell culture. 50 Such information can be used to more accurately determine useful doses in humans.

[0521] Initial dosages can also be estimated based on in vivo data (eg, animal models) using techniques well known in the art. Based on animal data, one of ordinary skill in the art can readily optimize dosing for humans.

[0522] Dosage and interval can be adjusted separately to provide the plasma level of the antigen binding molecules containing TNF families ligand trimer that is enough to maintain therapeutic effect.Conventionally the patient dosage of administration by injection is generally about 0.1 to 50mg / kg / days, generally about 0.5 to 1mg / kg / days.Can realize the effective plasma level of treatment by applying multiple dosages every day.Can, for example, measure the level in plasma by HPLC.

[0523] In the case of local administration or selective absorption, the effective local concentration of the antigen binding molecule containing the TNF family ligand trimer may be independent of the plasma concentration. Those skilled in the art will be able to optimize the therapeutically effective local dose without undue experimentation.

[0524] The therapeutically effective dose of the antigen binding molecules containing TNF family ligand trimers described herein will generally provide therapeutic benefits without causing substantial toxicity. The toxicity and therapeutic effect of the fusion protein can be determined by standard drug procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 / ED 50 . Antigen binding molecules containing TNF family ligand trimers that exhibit a large therapeutic index are preferred. In one embodiment, the antigen binding molecules containing TNF family ligand trimers according to the present invention exhibit a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating dosage ranges suitable for use in humans. The dose is preferably within a circulating concentration range that includes ED with little or no toxicity. 50The dosage may vary within this range depending on a variety of factors, such as the dosage form employed, the route of administration used, the condition of the subject, etc. The exact formulation, route of administration, and dosage can be selected by the individual physician taking into account the patient's condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, which is incorporated herein by reference in its entirety).

[0525] The attending physician of treating the patient with the fusion protein of the present invention knows how and when to stop, interrupt or adjust administration due to toxicity, organ dysfunction etc. On the contrary, if the clinical response is insufficient (excluding toxicity), the attending physician also will know to adjust the treatment to a higher level. In the management of interested disorders, the size of the administered dosage will change along with the severity of the disease to be treated, route of administration etc. The severity of the disease can be evaluated, for example, in part, by standard prognostic evaluation methods. In addition, dosage and dosage frequency will also change according to age, body weight and reaction of individual patients.

[0526] Other medications and treatments

[0527] The antigen binding molecules containing TNF family ligand trimers of the present invention can be co-administered with one or more other agents in treatment. For example, the fusion protein of the present invention can be co-administered with at least one other therapeutic agent. The term "therapeutic agent" encompasses any agent that can be used to treat the symptoms or diseases in an individual needing such treatment. This additional therapeutic agent may include any active ingredient that is suitable for the specific indication being treated, and preferably, the active ingredient has complementary activities that have no adverse effects on each other. In certain embodiments, the additional therapeutic agent is another anticancer agent.

[0528] Such other agents are suitably combined in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of the fusion protein used, the type of disorder or treatment, and other factors discussed above. Usually with the same dosage and route of administration as described herein, or about 1% to 99% of the dosage described herein, or with any dosage determined to be appropriate in experience / clinically and using the antigen binding molecules containing the TNF family ligand trimer by any approach.

[0529] Such combination therapies described above encompass both combined administration (wherein two or more therapeutic agents are included in the same or separate compositions) and separate administration. In the case of separate administration, the administration of the antigen binding molecules of the present invention containing a TNF family ligand trimer can occur before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant.

[0530] Products

[0531] In another aspect of the present invention, there is provided an article containing a material for treating, preventing and / or diagnosing the above-mentioned disorder. The article includes a container and a label or a package insert on the container or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Containers can be formed from various materials such as glass or plastic. The container holds the composition itself or together with another composition for effectively treating, preventing and / or diagnosing a disease, and can have a sterile access port (for example, the container can be an intravenous solution bag or a bottle with a stopper that can be pierced by a hypodermic needle). In the composition, at least one activating agent is an antigen binding molecule containing a TNF ligand trimer of the present invention.

[0532] The label or package insert indicates that the composition is used to treat the selected condition. Moreover, the product may include (a) a first container containing a composition, wherein the composition comprises an antigen binding molecule containing a TNF ligand trimer of the present invention; and (b) a second container containing a composition, wherein the composition comprises another cytotoxic therapeutic agent or other therapeutic agent. The product in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a specific condition.

[0533] Alternatively or additionally, the article of manufacture may further include a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other items desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0534] Table C (sequence):

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546] List specific embodiments of the present invention below:

[0547] An antigen-binding molecule containing a TNF family ligand trimer, comprising

[0548] (a) at least one module capable of specifically binding to a target cell antigen,

[0549] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other, optionally via a peptide linker, and

[0550] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0551] The antigen binding molecule containing a TNF family ligand trimer comprises

[0552] (a) at least one module capable of specifically binding to a target cell antigen,

[0553] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0554] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0555] The antigen binding molecule containing a TNF family ligand trimer as described above, comprising

[0556] (a) at least one module capable of specifically binding to a target cell antigen,

[0557] (b) a polypeptide comprising three extracellular domains of TNF ligand family members or fragments thereof linked to each other via a peptide linker, and

[0558] (c) an Fc domain consisting of a first and a second subunit capable of stably combining, wherein the polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof connected to each other by a peptide linker is fused to the N- or C-terminal amino acid of one of the two subunits of the Fc domain, optionally via a peptide linker.

[0559] An antigen binding molecule comprising a TNF family ligand trimer as described above, wherein the TNF ligand family member co-stimulates human T cell activation.

[0560] The antigen binding molecule comprising a TNF family ligand trimer as described above, wherein the TNF ligand family member is selected from 4-1BBL and OX40L.

[0561] The antigen binding molecule comprising a TNF family ligand trimer as described above, wherein the TNF ligand family member is 4-1BBL.

[0562] An antigen binding molecule containing a TNF family ligand trimer as described above, wherein 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: 99, SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102, in particular an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 99.

[0563] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the extracellular domain of the TNF ligand family member comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, in particular the amino acid sequence of SEQ ID NO: 1.

[0564] The antigen binding molecule containing a TNF family ligand trimer as described above, comprising

[0565] (a) at least one module capable of specifically binding to a target cell antigen, and

[0566] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103, and

[0567] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0568] The antigen binding molecule containing a TNF family ligand trimer as described above, comprising

[0569] (a) at least one module capable of specifically binding to a target cell antigen, and

[0570] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5, and

[0571] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0572] The antigen binding molecule comprising a TNF family ligand trimer as described above, wherein the TNF ligand family member is OX40L.

[0573] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the extracellular domain of the TNF ligand family member comprises an amino acid sequence selected from SEQ ID NO: 6 and SEQ ID NO: 7, in particular the amino acid sequence of SEQ ID NO: 6.

[0574] The antigen binding molecule containing a TNF family ligand trimer as described above, comprising

[0575] (a) at least one module capable of specifically binding to a target cell antigen, and

[0576] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, and

[0577] (c) An Fc domain composed of a first and a second subunit capable of stably associating.

[0578] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof is fused to the N-terminal amino acid of one of the subunits of the Fc domain at the C-terminal amino acid.

[0579] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the polypeptide comprising the extracellular domains of three TNF ligand family members or fragments thereof is fused to the C-terminal amino acid of one of the subunits of the Fc domain at the N-terminal amino acid.

[0580] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the module capable of specifically binding to a target cell antigen is selected from an antibody fragment, a Fab molecule, a crossover Fab molecule, a single-chain Fab molecule, an Fv molecule, a scFv molecule, a single domain antibody, a VH and a scaffold antigen binding protein.

[0581] The antigen-binding molecule containing a TNF family ligand trimer as described 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.

[0582] The antigen-binding molecule comprising a TNF family ligand trimer as described above, 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.

[0583] The antigen-binding molecule comprising a TNF family ligand trimer as described above, wherein the target cell antigen is fibroblast activation protein (FAP).

[0584] An antigen binding molecule containing a TNF family ligand trimer as described above, 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: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0585] An antigen binding molecule containing a TNF family ligand trimer as described above, 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: 104, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 105, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 106, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 108, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 109.

[0586] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the module capable of specific 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 specific binding to FAP comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 110 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 111.

[0587] The antigen-binding molecule comprising a TNF family ligand trimer according to any one of claims 1 to 15, wherein the target cell antigen is CEA.

[0588] An antigen binding molecule containing a TNF family ligand trimer as defined above, 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 the amino acid sequence of SEQ ID NO: 112, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 113, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 114, the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 115, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 117.

[0589] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the module capable of specifically binding to CEA comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 118 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 119.

[0590] The antigen binding molecule comprising a TNF family ligand trimer as defined above, wherein the target cell antigen is CD19.

[0591] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the module capable of specifically binding to CD19 comprises

[0592] (a) a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122, and the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125, or

[0593] (b) a VH domain and a VL domain, the VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 129, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 130, and the VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 131, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 132, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0594] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the module capable of specific binding to CD19 comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 126 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 127, or wherein the module capable of specific binding to FAP comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 134 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0595] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the Fc domain is IgG, in particular an IgG1 Fc domain or an IgG4 Fc domain.

[0596] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the Fc domain is an IgG1 Fc domain and comprises amino acid substitutions at positions 234 and 235 (EU numbering) and / or 329 (EU numbering) of the IgG heavy chain.

[0597] An antigen-binding molecule comprising a TNF family ligand trimer as defined above, comprising a module capable of specifically binding to a target cell antigen.

[0598] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0599] (i) a first heavy chain comprising a VH domain of a Fab molecule capable of specifically binding to a target cell antigen,

[0600] (ii) a light chain comprising the VL domain of a Fab molecule capable of specifically binding to a target cell antigen, and

[0601] (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0602] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0603] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 or a VH domain comprising the amino acid sequence of SEQ ID NO: 110,

[0604] (ii) a light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17 or a VL domain comprising the amino acid sequence of SEQ ID NO: 111, and

[0605] (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0606] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0607] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 118,

[0608] (ii) a light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 119, and

[0609] (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0610] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0611] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 126 or a VH domain comprising the amino acid sequence of SEQ ID NO: 134,

[0612] (ii) a light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 127 or a VL domain comprising the amino acid sequence of SEQ ID NO: 135, and

[0613] (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0614] An antigen binding molecule comprising a TNF family ligand trimer as defined above, comprising two modules capable of specifically binding to a target cell antigen.

[0615] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0616] (i) a first heavy chain comprising a VH domain of a Fab molecule capable of specifically binding to a target cell antigen,

[0617] (ii) two light chains, each comprising a VL domain of a Fab molecule capable of specifically binding to a target cell antigen, and

[0618] (iii) a second heavy chain comprising the VH domain of a Fab molecule capable of specifically binding to a target cell antigen and the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103 or SEQ ID NO: 8.

[0619] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0620] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 or a VH domain comprising the amino acid sequence of SEQ ID NO: 110,

[0621] (ii) two light chains, each comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 17 or a VL domain comprising the amino acid sequence of SEQ ID NO: 111, and

[0622] (iii) a second heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 16 or a VH domain comprising the amino acid sequence of SEQ ID NO: 110 and an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0623] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0624] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 118,

[0625] (ii) two light chains, each comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 119, and

[0626] (iii) a second heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 118 and the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0627] An antigen binding molecule containing a TNF family ligand trimer as defined above, wherein the antigen binding molecule comprises

[0628] (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 126 or a VH domain comprising the amino acid sequence of SEQ ID NO: 134,

[0629] (ii) two light chains comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 127 or a VL domain comprising the amino acid sequence of SEQ ID NO: 135, and

[0630] (iii) a second heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 126 or a VH domain comprising the amino acid sequence of SEQ ID NO: 134 and an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 or SEQ ID NO: 8.

[0631] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0632] (a) heavy and light chains, which together constitute the Fab molecule capable of specifically binding to the target cell antigen, and

[0633] (b) A fusion protein comprising an Fc domain and a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0634] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0635] (a) heavy and light chains, which together constitute the Fab molecule capable of specifically binding to the target cell antigen, and

[0636] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 15.

[0637] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0638] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16,

[0639] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 17, and

[0640] (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0641] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0642] (a) heavy and light chains, which together constitute the Fab molecule capable of specifically binding to the target cell antigen, and

[0643] (b) A fusion protein comprising an Fc domain and a polypeptide comprising the amino acid sequence of SEQ ID NO: 8.

[0644] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0645] (a) heavy and light chains, which together constitute the Fab molecule capable of specifically binding to the target cell antigen, and

[0646] (b) A fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

[0647] The antigen binding molecule containing a TNF family ligand trimer as described above, wherein the antigen binding molecule comprises

[0648] (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 16,

[0649] (ii) a variable light chain comprising the amino acid sequence of SEQ ID NO: 17, and

[0650] (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

[0651] An isolated polynucleotide encoding an antigen binding molecule comprising a TNF family ligand trimer as described above.

[0652] - A vector, in particular an expression vector, comprising the isolated polynucleotide as described above.

[0653] - A host cell comprising the isolated polynucleotide as described above or the vector as described above.

[0654] A method for producing an antigen binding molecule containing a TNF family ligand trimer as described above, comprising the following steps:

[0655] (i) culturing the host cell as described above under conditions suitable for expressing the antigen-binding molecule, and

[0656] (ii) recovering the antigen-binding molecule.

[0657] A pharmaceutical composition comprising the antigen-binding molecule containing a TNF family ligand trimer as described above and at least one pharmaceutically acceptable excipient.

[0658] The antigen-binding molecule comprising a TNF family ligand trimer as described above, or the pharmaceutical composition as described above, for use as a medicament.

[0659] The antigen binding molecule comprising a TNF family ligand trimer as described above, or the pharmaceutical composition as described above, for use in treating cancer.

[0660] Use of the antigen-binding molecule containing a TNF family ligand trimer as described above in the manufacture of a medicament for treating cancer.

[0661] A method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising an antigen binding molecule containing a TNF family ligand trimer as described above in a pharmaceutically acceptable form.

[0662] - A method for treating a disease as described above, wherein the disease is cancer. Example

[0663] The following are examples of methods and compositions of the present invention.It will be understood that various other embodiments may be practiced, given the general description provided above.

[0664] Recombinant DNA technology

[0665] DNA was manipulated using standard methods, such as those described by Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. For general information on the nucleotide sequences of human immunoglobulin light and heavy chains, see Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242.

[0666] DNA sequencing

[0667] The DNA sequence was determined by double-strand sequencing.

[0668] gene synthesis

[0669] Use appropriate template to generate by PCR or synthesize desired gene segment by automatic gene synthesis from synthetic oligonucleotide and PCR product by Geneart AG (Regensburg, Germany).In the case of no available exact gene sequence, oligonucleotide primers were designed based on the sequence from the nearest homologue, and the gene was isolated from the RNA derived from the appropriate tissue by RT-PCR. The gene segment flanked by a single restriction endonuclease cleavage site was cloned into a standard cloning / sequencing vector. Plasmid DNA was purified from the transformed bacteria and concentration was determined by ultraviolet spectroscopy. The DNA sequence of the subcloned gene fragment was confirmed by DNA sequencing. The gene segment was designed with suitable restriction sites to allow subcloning into the corresponding expression vector. All constructs were designed to have a 5' end DNA sequence encoding a leader peptide, which targets proteins secreted in eukaryotic cells.

[0670] Cell culture technology

[0671] Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc. were used.

[0672] Protein purification

[0673] With reference to standard scheme, protein is purified from the cell culture supernatant of filtration.In brief, antibody is applied to protein A sepharose column (GE Healthcare) and washed with PBS.At pH 2.8, realize the elution of antibody, then neutralize sample immediately.By size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20mM histidine, 150mM NaCl (pH 6.0) from monomeric antibody separation aggregated protein.Merge monomeric antibody fraction, use for example MILLIPORE Amicon Ultra (30MWCO) centrifugal concentrator to concentrate (if necessary), at-20 ℃ or-80 ℃ freezing and store.Provide partial sample for example for carrying out protein analysis and analytical characterization by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry subsequently.

[0674] SDS-PAGE

[0675] Use according to the manufacturer's instructions Pre-Cast gel system (Invitrogen). Specifically, 10% or 4-12% Bis-TRIS Pre-Cast Gel (pH 6.4) and MES (reducing gel, with Antioxidant running buffer additive) or MOPS (non-reducing gel) running buffer.

[0676] Analytical size exclusion chromatography

[0677] The antibody of embodiment 1 is purified by size exclusion chromatography (SEC).Size exclusion chromatography (SEC) is carried out by HPLC chromatography, is used to measure the aggregation and oligomeric state of antibody.In brief, the antibody of protein A purification is applied to 300mM NaCl, 50mM KH on Agilent HPLC 1100 system PO / K HPO On the Tosoh TSKgel G3000SW post in (pH 7.5), or on the Superdex 200 post (GE Healthcare) in 2 x PBS on Dionex HPLC system.By the integration of UV absorbance and peak area, the protein of wash-out is quantitatively measured.With BioRad gel filtration standard 151-1901 as standard.

[0678] Mass spectrometry

[0679] This section describes the characterization of multispecific antibodies with VH / VL exchange (VH / VL CrossMabs), with an emphasis on their correct assembly. The expected primary structure was analyzed by electrospray ionization mass spectrometry (ESI-MS) of deglycosylated intact CrossMabs and deglycosylated / plasmin-digested or alternatively deglycosylated / limited LysC-digested CrossMabs.

[0680] VH / VL CrossMabs were deglycosylated with N-glycosidase F in phosphate or Tris buffer at a protein concentration of 1 mg / ml for 17 hours at 37°C. 100 μg of deglycosylated VH / VLCrossMabs were digested with plasmin or restricted LysC (Roche) for 120 hours at room temperature and 40 minutes at 37°C, respectively. Before mass spectrometry analysis, the sample was desalted on a Sephadex G25 column (GE Healthcare) by HPLC. Total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

[0681] Example 1

[0682] Preparation and purification of targeted Fc fusion antigen binding molecules containing single-chain 4-1BB ligand trimers

[0683] 1.1 Monovalent FAP (28H1)-targeted Fc (kih) fusion antigen binding molecule containing a single-chain 4-1BB ligand trimer (Compound S1)

[0684] According to UniProt accession number P41273 (SEQ ID NO: 43), a DNA sequence encoding a portion of the extracellular domain of human 4-1BB ligand (amino acids 71-254 or 71-248) was synthesized.

[0685] A polypeptide comprising three extracellular domains of the 4-1BB ligand, each separated by a (G4S)2 linker, was cloned (SEQ ID NO: 54). Figure 1 Description in A: human 4-1BB ligand, (G4S)2 linker, human 4-1BB ligand, (G4S)2 linker, human 4-1BB ligand.

[0686] The polypeptide encoding the single-chain trimeric 4-1BB ligand was subcloned in frame with the human IgG1 heavy chain CH2 and CH3 domains on the knob using a linker with SEQ ID NO: 57 (GSPGSSSSGS) (Merchant, Zhu et al., 1998).

[0687] The variable regions of the heavy and light chain DNA sequences encoding a specific binder to fibroblast activation protein (FAP) (clone 28H1) were subcloned in frame with either the constant heavy chain of IgG1 (Carter, 2001) or the constant light chain of human IgG1. The generation and preparation of FAP binders are described in WO 2012 / 020006 A2, which is incorporated herein by reference.

[0688] According to the method described in International Patent Application Publication No. WO 2012 / 130831 A1, Pro329Gly, Leu234Ala, and Leu235Ala mutations were introduced into the constant regions of the knob and hole heavy chains to abolish binding to Fcγ receptors.

[0689] The combination of a single-chain ligand-Fc segment containing S354C / T366W mutations with a targeted anti-FAP-Fc segment containing Y349C / T366S / L368A / Y407V mutations and an anti-FAP light chain allows for the generation of heterodimers comprising a single-chain 4-1BB ligand trimer and a FAP-binding Fab ( Figure 3A ).

[0690] Table 1 shows the cDNA and amino acid sequences of the Fc(kih) fusion antigen binding molecule containing a single-chain 4-1BB ligand trimer targeted by FAP(28H1).

[0691] Table 1: Sequence of FAP (28H1)-targeted Fc (kih) fusion molecule containing human 4-1BB ligand trimer (FAP single chain 4-1BBL trimer) (Compound S1)

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699] The coding sequence for the targeted Fc(kih) fusion antigen binding molecule containing a single-chain TNF ligand trimer was cloned into a plasmid vector driving expression of the insert from the MPSV promoter and containing a synthetic polyA sequence at the 3' end of the CDS. In addition, the vector contained an EBV OriP sequence for episomal maintenance of the plasmid.

[0700] By using polyethyleneimine to co-transfect HEK293-EBNA cells with mammalian expression vectors to produce targeted Fc (kih) fusion antigen binding molecules containing single-chain 4-1BBL. Cells were transfected with the corresponding expression vectors at a 1:1:1 ratio ("carrier section chain":"carrier hole chain":"carrier light chain").

[0701] For production in 500 mL shake flasks, 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection, cells were centrifuged at 210 x g for 5 minutes, and the supernatant was replaced with prewarmed CD CHO medium. The expression vector was mixed in 20 mL of CD CHO medium to a final DNA concentration of 200 μg. After adding 540 μL of PEI, the solution was vortexed for 15 seconds and incubated at room temperature for 10 minutes. The cells were then mixed with the DNA / PEI solution, transferred to a 500 mL shake flask, and incubated at 37°C in an incubator with a 5% CO2 atmosphere for 3 hours. Following incubation, 160 mL of F17 medium was added, and the cells were cultured for 24 hours. One day after transfection, 1 mM valproic acid and 7% feed containing supplements were added. After 7 days of culture, the supernatant was collected by centrifugation at 210 x g for 15 minutes. The solution was sterile filtered (0.22 μm filter), supplemented with sodium azide to a final concentration of 0.01% (w / v), and maintained at 4°C.

[0702] The Fc (kih) fused antigen binding molecules containing 4-1BBL trimers of targeting are purified from cell culture supernatant by affinity chromatography using protein A, and then size exclusion chromatography is performed. For affinity chromatography, supernatant is loaded onto HiTrap protein A HP posts (CV=5mL, GE Healthcare), and balanced with a buffer (pH 7.5) containing 20mM sodium phosphate, 20mM sodium citrate, 0.5M sodium chloride, 0.01% (v / v) Tween-20. Unbound protein is removed by washing with the same buffer solution of at least 10 column volumes. With more than 20 column volumes to 100% buffer (pH 2.5) containing 20mM sodium citrate, 0.5M sodium chloride, 0.01% (v / v) Tween 20, the protein bound by linear pH gradient elution is utilized. The column was then washed with 10 column volumes of 20 mM sodium citrate, 0.5 M sodium chloride, 0.01% (v / v) Tween 20 buffer (pH 2.5).

[0703] The pH of the collected fractions was adjusted by adding 1...

Claims

1. An antigen-binding molecule containing a 4-1BBL trimer, comprising: (a) an Fc domain consisting of a first and a second subunit capable of stably associating, wherein the Fc domain is an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and P329G according to the EU numbering system, (b) a Fab molecule capable of specifically binding to a target cell antigen, wherein the Fab heavy chain is fused at its C-terminus to the N-terminus of the CH2 domain in the Fc domain, wherein the target cell antigen is CD19, wherein the Fab molecule capable of specifically binding to CD19 comprises a VH domain and a VL domain, (1) the VH domain comprises (i) CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 120, (ii) CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 121, and (iii) CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 122, and the VL domain comprises (iv) CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 123, (v) CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 124, and (vi) CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 125; or (2) the VH domain comprises (i) CDR-H1 as shown in the amino acid sequence of SEQ ID NO: 128, (ii) CDR-H2 as shown in the amino acid sequence of SEQ ID NO: 129, and (iii) CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 130, the VL domain comprises (iv) CDR-L1 as shown in the amino acid sequence of SEQ ID NO: 131, (v) CDR-L2 as shown in the amino acid sequence of SEQ ID NO: 132, and (vi) CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 133, and (c) a polypeptide comprising three extracellular domains of 4-1BBL as shown in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103, wherein the polypeptide comprising three extracellular domains of 4-1BBL is fused at its C-terminal amino acid to the N-terminal amino acid of another CH2 domain of the Fc domain.

2. The antigen binding molecule containing the 4-1BBL trimer of claim 1, wherein the Fab molecule capable of specifically binding to CD19 comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 126 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 127, or wherein the Fab molecule capable of specifically binding to CD19 comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 134 and a variable light chain comprising the amino acid sequence of SEQ ID NO:

135.

3. The antigen-binding molecule containing a 4-1BBL trimer according to claim 1 or 2, wherein the antigen-binding molecule comprises (i) a first heavy chain comprising a VH domain of a Fab molecule capable of specifically binding to a target cell antigen, (ii) a light chain comprising the VL domain of a Fab molecule capable of specifically binding to a target cell antigen, and (iii) a second heavy chain comprising a polypeptide comprising three extracellular domains of 4-1BBL.

4. The antigen-binding molecule containing a 4-1BBL trimer according to claim 1 or 2, wherein the antigen-binding molecule comprises (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 126 or a VH domain comprising the amino acid sequence of SEQ ID NO: 134, (ii) a light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 127 or a VL domain comprising the amino acid sequence of SEQ ID NO: 135, and (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:

103.

5. The antigen-binding molecule comprising a 4-1BBL trimer according to claim 1, wherein the antigen binding molecule comprises (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 152, (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 154, and (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO: 15, or wherein the antigen binding molecule comprises (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 158, (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 160, and (iii) a fusion protein comprising the amino acid sequence of SEQ ID NO:

15. 6 . An isolated polynucleotide encoding the antigen-binding molecule containing a 4-1BBL trimer according to claim 1 .

7. A host cell comprising the isolated polynucleotide of claim 6.

8. A method for producing an antigen binding molecule containing a 4-1BBL trimer according to any one of claims 1 to 5, comprising the following steps: (i) culturing the host cell of claim 7 under conditions suitable for expressing the antigen-binding molecule, and (ii) recovering the antigen-binding molecule. 9 . An antigen-binding molecule comprising a 4-1BBL trimer, produced by the method of claim 8 . 10 . A pharmaceutical composition comprising the antigen-binding molecule containing a 4-1BBL trimer according to any one of claims 1 to 5 and 9 and at least one pharmaceutically acceptable excipient.

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