New Regulation 4-1BBL Trio contains antigen binding molecules

Abstract

The present invention relates to 4-1BBL trimer-containing antigen-binding molecules comprising at least one antigen-binding domain capable of specifically binding to PD-L1, and their use in the treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to 4-1BBL trimer-containing antigen binding molecules that comprise an antigen binding domain capable of specifically binding to PD-L1, and their use in the treatment of cancer. The present invention further relates to methods of making these molecules, and methods of using them. [Background technology]

[0002] 4-1BB (CD137), a member of the TNF receptor superfamily, was first identified as an inducible molecule expressed by activated T cells (Kwon and Weissman, 1989, Proc Natl Acad Sci USA 86, 1963-1967). Subsequent studies demonstrated that many other immune cells also express 4-1BB, including NK cells, B cells, NKT cells, monocytes, neutrophils, mast cells, dendritic cells (DCs), and cells of non-hematopoietic origin such as endothelial cells and smooth muscle cells (Vinay and Kwon, 2011, Cell Mol Immunol 8, 281-284). Expression of 4-1BB in various cell types is largely inducible and driven by various stimulatory signals, such as triggering of the T cell receptor (TCR) or B cell receptor, as well as signaling induced through costimulatory molecules or receptors of proinflammatory cytokines (Diehl et al., 2002, J Immunol 168, 3755-3762; Zhang et al., 2010, Clin Cancer Res 13, 2758-2767).

[0003] 4-1BB ligand (4-1BBL or CD137L) was identified in 1993 (Goodwin et al., 1993, Eur J Immunol 23, 2631-2641). Expression of 4-1BBL has been shown to be restricted on professional antigen-presenting cells (APCs) such as B cells, DCs and macrophages. Inducible expression of 4-1BBL is characteristic of T cells, including both αβ and γδ T cell subsets, as well as endothelial cells (Shao and Schwarz, 2011, J Leukoc Biol 89, 21-29).

[0004] Costimulation via the 4-1BB receptor (e.g., by 4-1BBL ligation) triggers multiple signaling cascades in T cells (CD4 + and CD8 + 4-1BB activates T cells (both IL-1 and IL-2 subsets) and potently enhances T cell activation (Bartkowiak and Curran, 2015). In combination with TCR triggering, agonistic 4-1BB-specific antibodies enhance T cell proliferation, stimulate lymphokine secretion, and reduce the susceptibility of T lymphocytes to activation-induced cell death (Snell et al., 2011, Immunol Rev 244, 197-217). This mechanism was further advanced as the first proof of concept in cancer immunotherapy. In preclinical models, administration of agonistic antibodies against 4-1BB in tumor-bearing mice resulted in potent antitumor effects (Melero et al., 1997, Nat Med 3, 682-685). Accumulating evidence subsequently demonstrated that 4-1BB typically exhibits efficacy as an antitumor agent only when administered in combination with other immunomodulatory compounds, chemotherapeutic agents, tumor-specific vaccination, or radiation therapy (Bartkowiak and Curran, 2015, Front Oncol 5, 117).

[0005] Signaling in the TNFR superfamily requires cross-linking of trimerized ligands to engage the receptor, as does the 4-1BB agonist antibody, which requires wild-type Fc binding (Li and Ravetch, 2011, Science 333, 1030-1034). However, systemic administration of a 4-1BB-specific agonist antibody with a functionally active Fc domain inhibits CD8 expression in mice, which is associated with hepatotoxicity that is reduced or significantly ameliorated in the absence of functional Fc receptors. + It resulted in an influx of T cells (Dubrot et al., 2010, Cancer Immunol Immunother 59, 1223-1233). In the clinic, an Fc-competent 4-1BB agonistic Ab (BMS-663513) (NCT00612664) caused grade 4 hepatitis, leading to termination of the trial (Simeone and Ascierto, 2012, J Immunotoxicol 9, 241-247). Thus, there is a need for effective and safe 4-1BB agonists.

[0006] Programmed death-ligand 1 (PD-L1) is a protein that has been implicated in suppressing immune system responses in chronic infection, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 regulates immune responses by binding to an inhibitory receptor known as programmed death 1 (PD-1), which is expressed on the surface of T cells, B cells, and monocytes. PD-L1 also negatively regulates T cell function through its interaction with another receptor, B7-1. Formation of PD-L1 / PD-1 and PD-L1 / B7-1 complexes negatively regulates T cell receptor signaling, subsequently resulting in downregulation of T cell activation and suppression of antitumor immune activity. Several PD-1 and PD-L1 antibodies are currently in clinical use for the treatment of various solid cancers and lymphomas, and blockade of the PD-1 pathway has been shown to induce impressive response rates across a broad range of tumor types. Meanwhile, the commercially available PD-L1 antibodies atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi) have been approved for different types of cancer, including urothelial carcinoma, non-small cell lung cancer, and Merkel cell carcinoma. Although immunotherapeutics targeting PD-1 or PD-L1 have made substantial clinical progress in cancer, a significant proportion of patients remain unresponsive to treatment. Therefore, there is still a need for novel drug candidates that combine PD-L1 with costimulatory targets to overcome immune resistance in the tumor environment. Summary of the Invention

[0007] The novel antigen-binding molecules of the present invention combine an anti-PD-L1 antigen-binding domain with a moiety capable of forming a costimulatory 4-1BBL trimer and sufficiently stable to be pharmacologic. The antigen-binding molecules of the present invention are trimers and thus provide biologically active human 4-1BB ligand, but one of the trimerized 4-1BBL ectodomains is located on a separate polypeptide from the other two 4-1BBL ectodomains of the molecule. The antigen-binding molecules of the present invention targeted by the anti-PD-L1 antigen-binding domain have increased activity against tumor sites and contain the natural human 4-1BB ligand, and therefore should have fewer safety issues compared to conventional 4-1BB agonist antibodies or more artificial fusion proteins.

[0008] In one aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule comprising: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen-binding molecule comprising:

[0009] In a particular aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule, wherein the ectodomain of 4-1BBL or a fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, in particular the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5.

[0010] In a further aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule, comprising: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, and the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:3 and SEQ ID NO:4; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen-binding molecule comprising:

[0011] In one embodiment, the Fc domain is an IgG, in particular an IgG1 Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is an IgG1 Fc domain. In a particular embodiment, the Fc domain comprises a modification that promotes the association of the first and second subunits of the Fc domain. In a particular embodiment, the present invention provides a 4-1BBL trimer-containing antigen binding molecule, in which the Fc domain comprises a knob-into-hole modification that promotes the association of the first and second subunits of the Fc domain. In a specific embodiment, the present invention provides a 4-1BBL trimer-containing antigen binding molecule, in which the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).

[0012] In another aspect, the present invention relates to a 4-1BBL trimer-containing antigen-binding molecule as defined herein before, comprising (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the Fc domain comprising one or more amino acid substitutions that reduce binding to Fc receptors, in particular to Fcγ receptors. In particular, the Fc domain comprises amino acid substitutions at positions 234 and 235 (EU numbering according to Kabat) and / or 329 (EU numbering according to Kabat) of the IgG heavy chain. In particular, a 4-1BBL trimer-containing antigen-binding molecule is provided, in which the Fc domain is an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and P329G (numbering according to Kabat EU index).

[0013] In one aspect, the 4-1BBL trimer-containing antigen binding molecule is one in which the antigen-binding domain capable of specifically binding to PD-L1 is a Fab molecule capable of specifically binding to PD-L1, hi another aspect, the antigen-binding domain capable of specifically binding to PD-L1 is a crossover Fab molecule or scFV molecule capable of specifically binding to PD-L1.

[0014] In one aspect, the invention provides a 4-1BBL trimer-containing antigen binding molecule as described herein above, wherein the 4-1BBL trimer-containing antigen binding molecule comprises one Fab domain capable of specifically binding to PD-L1, which means that it comprises monovalent binding to PD-L1.

[0015] In a further aspect, a 4-1BBL trimer-containing antigen binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to PD-L1 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15. H (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18.L PD-L1).

[0016] In a further aspect, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises a heavy chain variable region (V) 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: 19. H PD-L1), and a light chain variable region (V L In a specific embodiment, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises a heavy chain variable region (V H PD-L1), and a light chain variable region (V L PD-L1).

[0017] In a further aspect, a 4-1BBL trimer-containing antigen binding molecule is provided, wherein the antigen binding molecule comprises: a first heavy chain and a first light chain comprising a Fab molecule capable of specifically binding to PD-L1; a second heavy chain comprising a constant domain and two ectodomains of 4-1BBL or fragments thereof connected to each other by a first peptide linker and fused at its C-terminus to a second heavy or light chain by a second peptide linker; and a second light chain comprising a constant domain and one ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to a second light chain or heavy chain, respectively, by a third peptide linker; Includes. More specifically, a 4-1BBL trimer-containing antigen-binding molecule is provided, in which a first peptide comprising two ectodomains of 4-1BBL or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus to a CL domain that is a part of a heavy chain by a second peptide linker, and a second peptide comprising one ectodomain of 4-1BBL or a fragment thereof is fused at its C-terminus to a CH1 domain that is a part of a light chain by a third peptide linker.

[0018] In a particular embodiment, the present invention relates to a 4-1BBL trimer-containing antigen binding molecule as defined above, wherein the peptide linker is (G4S)2, i.e., the peptide linker of SEQ ID NO: 36. In one embodiment, the peptide linker in all cases is (G4S)2.

[0019] Further provided is a 4-1BBL trimer-containing antigen-binding molecule in which, in the CL domain adjacent to 4-1BBL, the amino acid at position 123 (EU numbering) is substituted with arginine (R) and the amino acid at position 124 (EU numbering) is substituted with lysine (K), and, in the CH1 domain adjacent to 4-1BBL, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are substituted with glutamic acid (E).

[0020] In another aspect, a 4-1BBL trimer-containing antigen binding molecule is provided, wherein the antigen binding molecule comprises: (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 19 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 20; (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27; and (iii) a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28. Includes.

[0021] In one particular embodiment, a 4-1BBL trimer-containing antigen binding molecule is provided, comprising a first heavy chain comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29, a first light chain comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 30, a second heavy chain comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21, and a second light chain comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. In a further specific aspect, a 4-1BBL trimer-containing antigen binding molecule is provided, comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29, a first light chain comprising the amino acid sequence of SEQ ID NO: 30, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a second light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0022] According to another aspect of the present invention, there is provided an isolated nucleic acid molecule encoding a 4-1BBL trimer-containing antigen binding molecule as defined hereinbefore. The present invention further provides a vector, particularly an expression vector, comprising the isolated nucleic acid of the present invention, and a host cell comprising the isolated nucleic acid or vector of the present invention. In some embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell.

[0023] In another aspect, a method for producing a 4-1BBL trimer-containing antigen binding molecule of the present invention is provided, comprising culturing a host cell of the present invention under conditions suitable for expression of the 4-1BBL trimer-containing antigen binding molecule, and isolating the 4-1BBL trimer-containing antigen binding molecule. The present invention also includes a 4-1BBL trimer-containing antigen binding molecule produced by the method of the present invention.

[0024] The present invention further provides a pharmaceutical composition comprising the 4-1BBL trimer-containing antigen binding molecule of the present invention and at least one pharma- ceutically acceptable additive. In another aspect, a pharmaceutical composition is provided comprising the 4-1BBL trimer-containing antigen binding molecule of the present invention and at least one pharma- ceutically acceptable additive, and further comprising an additional therapeutic agent, such as a chemotherapeutic agent and / or other agent for use in cancer immunotherapy. In a further aspect, a pharmaceutical composition is provided further comprising a T cell-activating anti-CD3 bispecific antibody.

[0025] The present invention also encompasses the 4-1BBL trimer-containing antigen binding molecule of the present invention or the pharmaceutical composition of the present invention for use as a medicament. In one aspect, the 4-1BBL trimer-containing antigen binding molecule of the present invention or the pharmaceutical composition of the present invention is provided for use in the treatment of a disease in an individual in need of such treatment. In a particular embodiment, the 4-1BBL trimer-containing antigen binding molecule of the present invention or the pharmaceutical composition of the present invention is provided for use in the treatment of cancer. In another aspect, the 4-1BBL trimer-containing antigen binding molecule of the present invention or the pharmaceutical composition of the present invention is provided for use in upregulating or extending cytotoxic T cell activity. In another aspect, the 4-1BBL trimer-containing antigen binding molecule of the present invention or the pharmaceutical composition of the present invention is provided for use in the treatment of cancer, in which the 4-1BBL trimer-containing antigen binding molecule is used in combination with another therapeutic agent, such as a chemotherapeutic agent and / or other agent for use in cancer immunotherapy, or a T cell-activating anti-CD3 bispecific antibody. In one embodiment, the other therapeutic agent is administered simultaneously with, prior to, or after the 4-1BBL trimer-containing antigen binding molecule.

[0026] Also provided are the use of the 4-1BBL trimer-containing antigen binding molecule of the present invention for the manufacture of a medicament for the treatment of a disease in an individual in need thereof, in particular for the manufacture of a medicament for the treatment of cancer, as well as a method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising a 4-1BBL trimer-containing antigen binding molecule disclosed herein in a pharma- ceutically acceptable form. In a particular embodiment, the disease is cancer. Further provided is the use of the 4-1BBL trimer-containing antigen binding molecule of the present invention for the manufacture of a medicament for the treatment of cancer, in which the 4-1BBL trimer-containing antigen binding molecule is used in combination with another therapeutic agent. Further provided is a method for treating an individual with cancer, comprising administering to a subject an effective amount of the 4-1BBL trimer-containing antigen binding molecule of the present invention. Also provided is a method of upregulating or prolonging cytotoxic T cell activity in an individual with cancer, comprising administering to the individual an effective amount of the 4-1BBL trimer-containing antigen binding molecule of the present invention or the pharmaceutical composition of the present invention. In any of the above embodiments, the individual is preferably a mammal, in particular a human. [Brief description of the drawings]

[0027] [Figure 1A-1B] Figure 1 shows the building blocks for the assembly of monovalent PD-L1-targeting split trimeric 4-1BB ligand Fc fusion antigen binding molecules. Figure 1A shows the dimeric 4-1BB ligand fused at its C-terminus to a human IgG1-CL domain with the mutations E123R and Q124K (charge variant), and Figure 1B shows the monomeric 4-1BB ligand fused at its C-terminus to a human IgG1-CH1 domain with the mutations K147E and K213E (charge variant). [Figure 2A-2C]Figure 2A shows a schematic structure of a monovalent PD-L1-targeting split trimeric 4-1BB ligand Fc(kih) fusion antigen binding molecule containing a CH-CL crossover with charged residues. The solid black dots represent knob-into-hole modifications. * represents amino acid modifications in the CH1 and CL domains (so-called charged variants). This molecule is designated PD-L1-4-1BBL. Figure 2B shows a schematic structure of a monovalent PD-L1 and bivalent 4-1BB (clone 20H4.9) targeting molecule in a further termed 2+1 format. The solid black dots represent knob-into-hole modifications. This molecule is designated 4-1BBxPD-L1 2+1. Figure 2C shows a schematic structure of a monovalent PD-L1 and 4-1BB (clone 20H4.9) targeting molecule in a further termed 1+1 format. The solid black dots represent knob-into-hole modifications. Therefore, this molecule is designated 4-1BBxPD-L1 1+1. [Figure 3A-3B] Figure 3A shows the SPR experimental set-up for simultaneous binding of PD-L1-targeting split trimeric 4-1BB ligand-containing antigen binding molecules of the invention. The simultaneous binding of PD-L1-4-1BBL (analyte 1) to immobilized human 4-1BB and human PD-L1-Fc (analyte 2) is shown in Figure 3B. [Figure 4A-4B] Figures 4A and 4B show the binding of PD-L1-targeting 4-1BB split trimeric ligand Fc fusion antigen binding molecule or 4-1BBxPD-L1 bispecific antibody to the parental cell line MKN45 (Figure 4A) or the PD-L1-expressing cell line MKN45-PD-L1 (Figure 4B) measured in two independent experiments. The concentration of PD-L1-4-1BBL or control molecule is blotted against the geometric mean of the fluorescence intensity of the PE-conjugated secondary detection antibody. All values ​​are baseline corrected by subtracting the baseline value of the blank control (e.g., detection antibody only secondary, no primary). Only PD-L1-4-1BBL or 4-1BB-PDL1 bispecific antibody efficiently binds to human PD-L1-expressing MKN45-huPD-L1 cells (Figure 4B), but not to the parental cell line MKN45 (Figure 4A). [Figure 5A-5C]Figures 5A, 5B, and 5C show NFκB-mediated luciferase expression activity in the 4-1BB expressing reporter cell line Jurkat-hu4-1BB-NFkB-luc2. To test the functionality of PD-L1-4-1BBL against a control, the molecule was incubated with the reporter cell line Jurkat-hu4-1BB-NFkB-luc2 at a 1:5 ratio for 6 hours in the absence or presence of MKN45 or the MKN45 cell line expressing human PD-L1. The cells were then washed, lysed, and incubated with luciferin in detection buffer. Luciferase-catalyzed oxidation of luciferin was detected via luminescence as units of light emitted (y-axis). The concentration of PD-L1-4-1BBL molecule or its control is blotted against units of light emitted (RLU) measured after 6 hours of incubation and addition of luciferase detection solution. All values ​​are baseline corrected by subtracting the baseline value of a blank control (eg, no antibody added). [Fig. 5D-5E] Figures 5D and 5E show NFκB-mediated luciferase expression activity in the 4-1BB expressing reporter cell line Jurkat-hu4-1BB-NFkB-luc2. To test the functionality of PD-L1-4-1BBL against the control, the molecule was incubated with the reporter cell line Jurkat-hu4-1BB-NFkB-luc2 at a 1:5 ratio in the absence or presence of MKN45 or the MKN45 cell line expressing human PD-L1 for 6 hours. The cells were then washed, lysed and incubated with luciferin in detection buffer. Luciferase-catalyzed oxidation of luciferin was detected via luminescence as units of light emitted (y-axis). The concentration of PD-L1-4-1BBL molecule or its control is blotted against units of light emitted (RLU) measured after 6 hours of incubation and addition of luciferase detection solution. All values ​​are baseline corrected by subtracting the baseline value of a blank control (eg, no antibody added). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] definition Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa.

[0029] As used herein, the term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, antibody fragments, and scaffold antigen-binding proteins.

[0030] The term "antigen-binding domain" refers to a part of an antigen-binding molecule that comprises an area that specifically binds to and is complementary to part or all of an antigen. If the antigen is large, the antigen-binding molecule may bind only to a specific part of the antigen, which part is called an epitope. An antigen-binding domain may, for example, be provided by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0031] As used herein, the term "antigen-binding domain capable of specifically binding to PD-L1" or "moiety capable of specifically binding to PD-L1" refers to a polypeptide molecule that specifically binds to PD-L1. In one aspect, the antigen-binding domain is capable of inhibiting signaling through PD-L1. In certain aspects, the antigen-binding domain is capable of directing the entity to which it is attached (e.g., a 4-1BBL trimer) to a target site, e.g., a particular type of tumor cell that bears PD-L1. Antigen-binding domains capable of specifically binding to PD-L1 include antibodies and fragments thereof as further defined herein. In the context of an antibody or fragment thereof, the term "moiety capable of specifically binding to PD-L1" refers to a portion of a molecule that comprises a region that specifically binds to and is complementary to part or all of an antigen. A moiety capable of specific antigen binding may be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). In particular, a moiety capable of specific antigen binding includes an antibody light chain variable region (VL), and an antibody heavy chain variable region (VH).

[0032] The term "antibody" herein is used in the broadest sense and encompasses a variety of 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.

[0033] The term "monoclonal antibody," as used herein, refers to antibodies obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for variant antibodies that may generally be present in minor amounts (e.g., including naturally occurring mutations or that arise during manufacture of the monoclonal antibody preparation). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0034] The term "monospecific" antibody, as used herein, 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 an antigen-binding molecule can specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants (particularly two antigenic determinants expressed on two distinct cells).

[0035] The term "valency," as used herein, refers to the presence of a particular number of binding sites in an antigen-binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule.

[0036] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a native antibody. "Native antibody" refers to a naturally occurring immunoglobulin molecule having a variety of structures. For example, a native IgG class antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called the variable heavy domain or the heavy chain variable domain) followed by three constant domains (CH1, CH2 and CH3) (also called the heavy chain constant domain). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called the variable light domain or the light chain variable domain) followed by a light chain constant domain (CL) (also called the light chain constant domain). The heavy chain of an antibody may be divided into one of five types called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), several of which may be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0037] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an 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 specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Plueckthun, 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 description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased half-life in vivo, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments that contain 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). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, Massachusetts; see, e.g., U.S. Pat. No. 6,248,516 B1).Antibody fragments may be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0038] Papain digestion of an intact antibody yields two identical antigen-binding fragments, called "Fab" fragments, each of which contains the heavy and light chain variable domains, as well as the constant domain 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 containing the VL domain and constant domain of the light chain (CL) and the VH domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy 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 ​​residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields a F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0039] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. Two possible chain compositions of crossover Fab molecules 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 have been exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1) and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). This crossover Fab molecule is called a cross-Fab (VLVH) On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule contains a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL) and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab molecule is called a cross-Fab (CLCH1)It is also called.

[0040] A "single-chain Fab fragment" or "scFab" is a polypeptide 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, said antibody domains and said linker having one of the following orders from N-terminus to C-terminus: (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 said linker is a polypeptide of at least 30 amino acids, preferably 32-50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules may be further stabilized by the creation of an interchain disulfide bond through the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat numbering).

[0041] A "crossover single chain Fab fragment" or "x-scFab" is a polypeptide 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, said antibody domains and said linker having one of the following orders from N-terminus to C-terminus: (a) VH-CL-linker-VL-CH1 and (b) VL-CH1-linker-VH-CL; VH and VL together form an antigen binding site that specifically binds to an antigen, and said linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules may be further stabilized by the creation of an interchain disulfide bond by the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to the Kabat numbering).

[0042] A "single-chain variable fragment (scFv)" is a fragment of an antibody heavy chain (V) linked together using a short linker peptide of 10 to about 25 amino acids. H) and light chain (V L ) variable region fusion proteins. The linker is usually glycine-rich for flexibility and serine or threonine-rich for solubility, and H N-terminus and V L or vice versa. This protein retains the specificity of the original antibody, although the constant regions have been removed and a linker has been introduced. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments contain a single polypeptide chain characterized by a VH domain (i.e. capable of assembling with a VL domain) or a VL domain (i.e. capable of assembling with a VH domain into a functional antigen-binding site), thereby conferring the antigen-binding properties of a full-length antibody.

[0043] An "antigen-binding molecule that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks the binding of the reference molecule to its antigen by 50% or more in a competitive assay; conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay.

[0044] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to the site on a polypeptide macromolecule to which an antigen-binding moiety binds (e.g., a contiguous stretch of amino acids or a conformational structure composed of different regions of non-contiguous amino acids) forming an antigen-binding moiety-antigen complex. 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 diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, proteins useful as antigens herein may be proteins in any naturally occurring form from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When a particular protein is referred to herein, the term encompasses the "full-length" unprocessed protein and any form of the protein obtained by processing within the cell. The term also encompasses naturally occurring protein variants, e.g., splice variants or allelic variants.

[0045] The term "capable of specifically binding to PD-L1" refers to an antigen-binding molecule that is capable of binding to PD-L1 with sufficient affinity such that the antigen-binding molecule is useful as a diagnostic and / or therapeutic agent in targeting PD-L1. Antigen-binding molecules include, but are not limited to, antibodies, multispecific antibodies, Fab molecules, crossover Fab molecules, single-chain Fab molecules, Fv molecules, scFv molecules, single domain antibodies, and fusion proteins. In one aspect, the extent of binding of an anti-PD-L1 antigen-binding molecule to an unrelated non-PD-L1 protein is less than about 10% of the binding of the antigen-binding molecule to PD-L1 as measured, for example, by surface plasmon resonance (SPR). In particular, an antigen-binding molecule capable of specifically binding to PD-L1 has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example 10-9 M~10 -13 Dissociation constant (K d ). In certain aspects, the anti-PD-L1 antigen binding molecule binds to PD-L1 from different species. In particular, the anti-PD-L1 antigen binding molecule binds to human and cynomolgus PD-L1.

[0046] "Specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques known in the art, such as surface plasmon resonance (SPR) techniques (analyzed by 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 extent of binding of the antigen-binding molecule to unrelated proteins is less than about 10% of the binding of the antigen-binding molecule to the antigen, for example, as measured by SPR. In certain embodiments, molecules that bind to an antigen have a dissociation constant (Kd) 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 less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M).

[0047] "Affinity" or "binding affinity" refers to the total strength of 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 specified, 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 binding partner Y can generally be expressed by the dissociation constant (Kd), which is determined by the dissociation rate constant and the association rate constant (k, respectively). off and k on) is the ratio of the rate constants. Thus, equivalent affinities may contain different rate constants, as long as the ratio of the rate constants is the same. Affinity can be measured by methods common in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0048] "Target cell antigen" as used herein means an antigenic determinant displayed on the surface of a target cell, e.g., a cell within a tumor, such as a T cell or B cell, a cancer cell, or a tumor stromal cell. In certain embodiments, the target cell antigen is an antigen on the surface of a cancer cell. In one embodiment, the target cell antigen is PD-L1.

[0049] The term "PD-L1", also known as CD274 or B7-H1, refers to any naturally occurring PD-L1 (particularly "human PD-L1") from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of fully human PD-L1 is set forth in UniProt (www.uniprot.org) Accession No. Q9NZQ7 (SEQ ID NO:37). The terms "anti-PD-L1 antibody" or "antibody that binds human PD-L1" or "antibody that specifically binds human PD-L1" or "agonist anti-PD-L1" refer to an antibody that specifically binds to human PD-L1, with a specific binding activity of 1.0×10 -8 KD value of 1.0×10 mol / L or less, in one embodiment, -9 It refers to an antibody that specifically binds to the human PD-L1 antigen with a binding affinity of KD value of 0.01 mol / L or less. Binding affinity is measured using standard binding assays such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden).

[0050] As used herein, "T cell antigen" refers to an antigenic determinant presented on the surface of T lymphocytes, particularly cytotoxic T lymphocytes.

[0051] As used herein, a "T cell activation therapeutic agent" refers to a therapeutic agent capable of inducing T cell activation in a subject, particularly a therapeutic agent designed to induce T cell activation in a subject. Examples of T cell activation therapeutic agents include bispecific antibodies that specifically bind to an activating T cell antigen, such as CD3, and a target cell antigen, such as CEA or the folate receptor.

[0052] As used herein, "activating T cell antigen" refers to an antigenic determinant expressed by T lymphocytes, particularly cytotoxic T lymphocytes, which can induce or enhance T cell activation upon interaction with an antigen-binding molecule. Specifically, the interaction of an antigen-binding molecule with a T cell activation antigen can induce T cell activation by triggering a cascade of signal transduction of the T cell receptor complex. An exemplary activating T cell antigen is CD3.

[0053] Unless otherwise indicated, the term "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed CD3 and any form of CD3 obtained by intracellular processing. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, in particular the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) Accession No. P07766 (Version 144), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also SEQ ID NO:59. The amino acid sequence of Macaca fascicularis CD3ε is shown in UniProt (www.uniprot.org) accession number Q95LI5. See also SEQ ID NO:60.

[0054] The term "variable domain" or "variable region" refers to the domain of an antibody heavy or light chain that is involved in binding of an antigen-binding molecule to an antigen. The variable domains of heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th, WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0055] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antigen-binding variable domain that are hypervariable in sequence and determine antigen-binding specificity, e.g., "complementarity determining regions" (CDRs). Generally, an antigen-binding domain contains six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) the hypervariable loops occurring 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)); (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0056] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that the designation of CDRs can be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature. Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. Those skilled in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence without reliance on experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise indicated, references to the numbering of specific amino acid residue positions in an antibody variable region are in accordance with the Kabat numbering system.

[0057] As used herein, the term "affinity maturation" in relation to an antigen-binding molecule (e.g., an antibody) refers to an antigen-binding molecule that is derived from a reference antigen-binding molecule, e.g., by mutation, binds to the same antigen, preferably the same epitope, as the reference antibody, and has a higher affinity for the antigen than 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, the affinity-matured antigen-binding molecule binds to the same epitope as the original reference antigen-binding molecule.

[0058] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains, FR1, FR2, FR3 and FR4. Thus, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0059] An "acceptor human framework" for the purposes of this specification is a framework that comprises 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 may comprise the same amino acid sequence or may 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.

[0060] The term "chimeric" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular origin or species, while the remainder of the heavy and / or light chain is derived from a different origin or species.

[0061] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0062] The term "constant region of human origin" or "human constant region" refers to the constant heavy chain region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4, and / or the constant light chain kappa or lambda region. Such constant regions are known in the art and described, for example, in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, numbering of amino acid residues in the constant region is according to the EU numbering system (also called the EU index of Kabat) as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0063] 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 comprises substantially all of at least one, typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to a non-human antibody and all or substantially all of the FRs corresponding to a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have been further modified or altered from those of the original antibody to create properties according to the invention, particularly in terms of C1q binding and / or Fc receptor (FcR) binding.

[0064] 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 a human cell, or of an antibody derived from a non-human source that utilizes the human antibody repertoire, or to sequences encoding other human antibodies. This definition of human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0065] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an antibody heavy chain that comprises at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, antibodies produced by a host cell may undergo post-translational truncation of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or may comprise a truncated variant of the full-length heavy chain. This is the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447) may or may not be present. The amino acid sequence of a heavy chain comprising an Fc region is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one embodiment, a heavy chain comprising an Fc region as specified herein and comprised in an antibody of the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment, a heavy chain comprising an Fc region as specified herein and comprised in an antibody of the invention comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The IgG Fc region comprises the IgG CH2 and IgG CH3 domains.The "CH2 domain" of a human IgG Fc region typically extends from about amino acid residue 231 to about amino acid residue 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 the stretch of residues from the C-terminus of the Fc region to the CH2 domain (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG). The CH3 region of the present invention may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "bulge" ("knob") introduced in one of its chains and a corresponding "cavity" ("hole") introduced in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains as described herein.

[0066] The "knob-into-hole" technique is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves promoting heterodimer formation and hindering homodimer formation by introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity at the interface of a second polypeptide, respectively, such that the protuberance can be located within the corresponding cavity. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine). The protuberance and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In a particular 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 a further particular embodiment, the subunit of the Fc domain that comprises the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc domain that comprises the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues allows the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Numbering follows 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.

[0067] "A region corresponding to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants having modifications that result in substitutions, additions, or deletions, but do not substantially reduce the ability of the immunoglobulin to mediate effector functions (e.g., antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantially impairing biological function. Such variants can be selected according to general rules known in the art to have minimal effect on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0068] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody and varies with 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 activation of B cells.

[0069] An "activating Fc receptor" is an Fc receptor that, following binding by the Fc region of an antibody, triggers signaling events that stimulate a receptor-bearing cell to carry out an effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0070] The term "TNF ligand family member" or "TNF family ligand" refers to a pro-inflammatory cytokine. Cytokines in general, and members of the TNF ligand family in particular, play an important role in stimulating and regulating 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 similarities. 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, known as the TNF homology domain (THD), has 20-30% amino acid identity among superfamily members and is responsible for binding to receptors. The TNF ectodomain also carries the TNF ligands that form trimeric complexes that are recognized by their specific receptors. Members of the TNF ligand family are lymphotoxin alpha (also known as LTA or TNFSF1), TNF (also known as TNFSF2), LT beta (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 (APO2L, CD253 or TNFSF1), and IL-1 (also known as TNFSF1). 0), 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 ectozyme A1), and EDA-A2 (also known as ectozyme A2).Unless otherwise specified, the term refers to natural TNF family ligands from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term "costimulatory TNF ligand family members", or "costimulatory TNF family ligands", refers to a subgroup of TNF ligand family members that can costimulate T cell proliferation and cytokine production. These TNF family ligands can costimulate TCR signals when interacting with their corresponding TNF receptors, and interaction with the receptors results in recruitment of TNFR-associated factors (TRAFs) that initiate a signaling cascade that leads to T cell activation. The costimulatory TNF family ligand is selected from the group consisting of 4-1BBL, OX40L, GITRL, CD70, CD30L, and LIGHT, and more particularly, the costimulatory TNF ligand family member is 4-1BBL.

[0071] As previously described herein, 4-1BBL is a type II transmembrane protein and a member of the TNF ligand family. It has been described that complete or full-length 4-1BBL, having the amino acid sequence of SEQ ID NO: 38, forms trimers on the surface of cells. Trimer formation is made possible by a specific motive in the ectodomain of 4-1BBL. Said motif is referred to herein as the "trimerization region." Amino acids 50-254 of the human 4-1BBL sequence (SEQ ID NO: 39) form the extracellular domain of 4-1BBL, but fragments thereof can also form trimers. In a particular embodiment of the invention, the term "ectodomain of 4-1BBL or a fragment thereof" refers to a polypeptide having an amino acid sequence selected from SEQ ID NO: 4 (amino acids 52-254 of human 4-1BBL), SEQ ID NO: 1 (amino acids 71-254 of human 4-1BBL), SEQ ID NO: 3 (amino acids 80-254 of human 4-1BBL) and SEQ ID NO: 2 (amino acids 85-254 of human 4-1BBL), or a polypeptide having an amino acid sequence selected from SEQ ID NO: 5 (amino acids 71-248 of human 4-1BBL), SEQ ID NO: 8 (amino acids 52-248 of human 4-1BBL), SEQ ID NO: 7 (amino acids 80-248 of human 4-1BBL) and SEQ ID NO: 6 (amino acids 85-248 of human 4-1BBL), but also other fragments of the ectodomain capable of trimerization are included herein.

[0072] An "ectodomain" is a domain of a membrane protein that extends into the extracellular space (i.e. the space outside a target cell). The ectodomain is the part of a protein that usually initiates contact with a surface resulting in signal transduction. Thus, the ectodomain of a TNF ligand family member as defined herein refers to the part of the TNF ligand protein that extends into the extracellular space (the extracellular domain), but also includes shorter parts or fragments thereof that are responsible for trimerization and binding to the corresponding TNF receptor. Thus, the term "ectodomain of a TNF ligand family member or a fragment thereof" refers to the extracellular domain of a TNF ligand family member that forms the extracellular domain or the part thereof that is still capable of binding to the receptor (receptor binding domain).

[0073] The term "peptide linker" refers to a peptide that includes one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n ,(SG4) n or G4 (SG4) n a peptide linker, wherein "n" is generally a number between 1 and 10, typically between 1 and 4, in particular 2, i.e. a peptide selected from the group consisting of GGGGS (SEQ ID NO:40), GGGGSGGGGS (SEQ ID NO:36), SGGGGSGGGG (SEQ ID NO:41), (G4S)3 or GGGGSGGGGSGGGGGS (SEQ ID NO:42), GGGGSGGGGSGGGG or G4(SG4)2 (SEQ ID NO:43), and (G4S)4 or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:44), but also including the sequences GSPGSSSSGS (SEQ ID NO:45), GSGSGSGS (SEQ ID NO:46), GSGSGNGS (SEQ ID NO:47), GGSGSGSG (SEQ ID NO:48), GGSGSG (SEQ ID NO:49), GGSG (SEQ ID NO:50), GGSGNGSG (SEQ ID NO:51), GGNGSGSG (SEQ ID NO:52) and GGNGSG (SEQ ID NO:53). Peptide linkers of particular interest are (G4S)1 or GGGGS (SEQ ID NO:40), (G4S)2 or GGGGSGGGGS (SEQ ID NO:36), (G4S)3 (SEQ ID NO:42) and (G4S) 4( Sequence number 44).

[0074] The term "amino acid", as used herein, refers to the group of naturally occurring carboxy α-amino acids including 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).

[0075] As used herein, a "fusion polypeptide" or "fusion protein" refers to a single chain polypeptide composed of an antibody fragment and a non-antibody derived peptide. In one embodiment, the fusion polypeptide is composed of one or two ectodomains of 4-1BBL, or a fragment thereof, fused to an antigen binding domain or part of the Fc portion. The fusion can occur by directly linking the N- or C-terminal amino acid of the antigen binding portion to the C- or N-terminal amino acid of the ectodomain of said 4-1BBL or fragment thereof via a peptide linker.

[0076] By "fused" or "connected" is meant that the components (e.g., the polypeptide and ectodomain of the TNF ligand family member) are linked by a peptide bond directly or via one or more peptide linkers.

[0077] "Percent (%) amino acid sequence identity" to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence when these sequences are aligned, gaps are introduced as necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be obtained using a variety of methods within the skill of the art, such as 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 sequence alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed in the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from its 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 do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be written as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identity matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0078] In certain embodiments, amino acid sequence variants of the TNF ligand trimer-containing antigen binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the TNF ligand trimer-containing antigen binding molecules. Amino acid sequence variants of the TNF ligand trimer-containing antigen binding molecules can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, e.g., antigen binding. Sites of interest for substitution mutagenesis include HVRs and frameworks (FRs). Conservative substitutions are provided in Table B under the heading of "preferred substitutions" and are further described below with reference to amino acid side chain classes (1)-(6). Amino acid substitutions can be introduced into the molecule of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0079] TIFF0007675083000001.tif176170

[0080] Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0081] Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0082] The term "amino acid sequence variant" includes substantial variants in which there are amino acid substitutions in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing will have modified (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antigen-binding molecule and / or will substantially retain specific biological properties of the parent antigen-binding molecule. Exemplary substitutional variants are affinity-matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, variant antigen-binding molecules in which one or more CDR residues have been mutated, phage-displayed, and screened for a specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions can be made within one or more CDRs, so long as these changes do not substantially reduce the binding ability of the antigen-binding molecule to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made within a CDR. A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced by 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 the amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antigen binding molecule complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they have the desired properties.

[0083] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is a 4-1BBL trimer-containing antigen-binding molecule with an N-terminal methionyl residue. Other insertion variants of the molecule include N- or C-terminal fusions to polypeptides that increase the serum half-life of the 4-1BBL trimer-containing antigen-binding molecule.

[0084] In certain embodiments, the 4-1BBL trimer-containing antigen binding molecules provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Glycosylation variants of the molecule can be conveniently obtained by altering the amino acid sequence to generate or remove one or more glycosylation sites. In cases where the 4-1BBL trimer-containing antigen binding molecule comprises an Fc region, the carbohydrate attached to the Fc region can be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc of the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of oligosaccharides in 4-1BBL trimer-containing antigen binding molecules can be made to generate variants with specific improved properties. In one aspect, variants of 4-1BBL trimer-containing antigen binding molecules are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. Such fucosylated variants may have improved ADCC function, see, for example, US Patent Application Publication No. 2003 / 0157108 (Presta, L.) or US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Further variants of the 4-1BBL trimer-containing antigen binding molecules of the present invention include those with bisected oligosaccharides, for example, those in which the biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, e.g., WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.) and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.).Variants are also provided that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved 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.).

[0085] In certain embodiments, it may be desirable to generate cysteine ​​engineered variants of the 4-1BBL trimer-containing antigen binding molecules of the invention, e.g., "thioMAbs" in which one or more residues of the molecule are replaced with cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the molecule. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antigen binding molecules may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0086] In certain aspects, the 4-1BBL trimer-containing antigen binding molecules provided herein may be further modified to contain additional non-proteinaceous moieties that are known and readily available in the art. Suitable sites for derivatization of antibodies 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 copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous during manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when more than one polymer is attached, the polymers may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used in therapy under defined conditions. In another aspect, conjugates of antibodies and non-proteinaceous moieties are provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam, NWet al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation may be of any wavelength, including but not limited to wavelengths that are not harmful to normal cells but that heat the non-proteinaceous moiety to a temperature that kills cells proximal to the antibody-non-proteinaceous moiety.

[0087] In another embodiment, an immunoconjugate of the 4-1BBL trimer-containing antigen binding molecule provided herein can be obtained. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0088] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by a base sequence, where the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands and both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein can include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and / or in vivo, for example in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, the mRNA may be chemically modified to increase the stability of the RNA vector and / or expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler ert al, Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356, or EP 2101823 B1).

[0089] By "isolated" nucleic acid molecule or polynucleotide is intended 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 isolated for the purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that differs from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, and positive and negative stranded forms, double stranded forms. Additionally, isolated polynucleotides or nucleic acids of the present invention include such molecules produced synthetically. In addition, the polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, a ribosome binding site, or a transcription terminator.

[0090] A nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention is intended to be identical to the reference sequence, except that the nucleotide sequence of the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence. Such modifications of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, and may be interspersed individually among the residues in the reference sequence, or may be interspersed in one or more contiguous groups within the reference sequence. In practical terms, whether a 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 those described above for polypeptides (e.g., ALIGN-2).

[0091] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide with a set of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, a chromosome, mitochondrial DNA, plastid DNA, a virus, or a nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In a particular embodiment, an expression cassette of the invention includes a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention, or a fragment thereof.

[0092] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and induce expression of a particular gene to which it is operably linked in a target cell. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it is introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows for stable transcription of large amounts of mRNA. Once inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antibody of the present invention or a fragment thereof.

[0093] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", including the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parent cell and may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell. A host cell is any type of cell line that can be used to generate the bispecific antigen-binding molecules of the invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, BHK cells, NS0 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, but also include transgenic animals, transgenic plants or cells contained in cultured plants or animal tissues, to name just a few.

[0094] An "effective amount" of an agent refers to the amount necessary to induce a certain physiological change in a cell or tissue to which the agent is administered.

[0095] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent will, for example, eliminate, reduce, delay, minimize, or prevent the deleterious effects of a disease.

[0096] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic 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). In particular, an individual or subject is a human.

[0097] The term "pharmaceutical composition" refers to a preparation in a form such that the biological activity of the active ingredient contained therein is effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0098] "Pharmaceutically acceptable additive" refers to an ingredient in a pharmaceutical composition other than an active ingredient, which is non-toxic to a subject. Pharmaceutically acceptable additives include, but are not limited to, buffers, stabilizers, or preservatives.

[0099] The term "package insert" is used to refer to instructions customarily included in the commercial packaging of a therapeutic product, which contains information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or precautions pertaining to the use of that therapeutic product.

[0100] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or ameliorating the disease state, and remission or improved prognosis. In some embodiments, the molecules of the invention are used to delay the onset of disease or slow the progression of disease.

[0101] As used herein, the term "cancer" includes lymphoma, carcinoma, lymphoma, blastoma, sarcoma, leukemia, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar 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, and the like. cancer), colorectal cancer (CRC), pancreatic cancer, breast cancer, triple-negative breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axis tumors , brain stem glioma, glioblastoma multiforme, astrocytoma, schwanomas, ependymomas, medulloblastoma, meningiomas, squamous cell carcinoma, pituitary adenoma and Ewing's sarcoma, melanoma, multiple myeloma, B-cell cancer (lymphoma), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

[0102] An "advanced" cancer is one that has spread outside the site or organ of origin, either by local invasion or metastasis. Thus, the term "advanced" cancer includes both locally advanced and metastatic disease.

[0103] A "recurrent" cancer is one that has regrown, either at the original site or at a distant site, after responding to initial therapy such as surgery. A "locally recurrent" cancer is one that returns after treatment in the same location as the previously treated cancer. An "operable" or "resectable" cancer is one that is confined to the organ of origin and is suitable for surgery (resection). A "non-resectable" or "unresectable" cancer cannot be removed (resected) by surgery.

[0104] 4-1BBL trimer-containing antigen-binding molecule of the present invention The present invention provides novel 4-1BBL trimer-containing antigen-binding molecules that have particularly advantageous properties such as productivity, stability, binding affinity, biological activity, targeting efficiency, reduced toxicity, and reduced immunogenicity.

[0105] In a first aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule, comprising: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen-binding molecule comprising:

[0106] In a further aspect, a 4-1BBL trimer-containing antigen binding molecule as defined hereinbefore, comprising (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked together by a disulfide bond, wherein the antigen-binding molecule comprises: (i) a first polypeptide comprising a CH1 or CL domain and a second polypeptide comprising a CL or CH1 domain, respectively, the second polypeptide being linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, the first polypeptide comprising two ectodomains of 4-1BBL or fragments thereof connected to each other and to the CH1 or CL domains by a peptide linker, and the second polypeptide comprising one ectodomain of said 4-1BBL or fragments thereof connected via a peptide linker to the CL or CH1 domain of said polypeptide; or (ii) the first polypeptide comprises a CH3 domain and the second polypeptide comprises a CH3 domain, the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other and to the C-terminus of the CH3 domain by a peptide linker, and the second polypeptide comprises only one ectodomain of 4-1BBL or fragment thereof connected via a peptide linker to the C-terminus of the CH3 domain of the polypeptide; or (iii) a first polypeptide comprises a VH-CL or VL-CH1 domain and a second polypeptide comprises a VL-CH1 domain or a VH-CL domain, respectively, the second polypeptide being linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, the first polypeptide comprising two ectodomains of 4-1BBL or fragments thereof connected to each other and to the VH or VL by a peptide linker, and the second polypeptide comprising an ectodomain or fragment thereof of one of said TNF ligand family members connected to the VL or VH of said polypeptide via a peptide linker. A first polypeptide and a second polypeptide, (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen binding molecule comprising:

[0107] In another embodiment, a 4-1BBL trimer-containing antigen binding molecule as defined hereinbefore, (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked together by a disulfide bond, wherein the antigen-binding molecule comprises: (i) a first polypeptide comprising a CH1 or CL domain and a second polypeptide comprising a CL or CH1 domain, respectively, the second polypeptide being linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, the first polypeptide comprising two ectodomains of 4-1BBL or fragments thereof connected to each other and to the CH1 or CL domains by a peptide linker, and the second polypeptide comprising one ectodomain of said 4-1BBL or fragments thereof connected via a peptide linker to the CL or CH1 domain of said polypeptide; or (ii) the first polypeptide comprises a CH3 domain and the second polypeptide comprises a CH3 domain, the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other and to the C-terminus of the CH3 domain by a peptide linker, and the second polypeptide comprises only one ectodomain of 4-1BBL or fragment thereof connected via a peptide linker to the C-terminus of the CH3 domain of the polypeptide; A first polypeptide and a second polypeptide, (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen binding molecule comprising:

[0108] In one embodiment, the ectodomain of 4-1BBL 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: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, in particular the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5. More particularly, the ectodomain of 4-1BBL comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5. Most particularly, the ectodomain of 4-1BBL comprises the amino acid sequence of SEQ ID NO: 5. In particular, a 4-1BBL trimer-containing antigen binding molecule as defined herein before is provided, in which all three ectodomains of 4-1BBL or fragments thereof are identical.

[0109] Thus, a 4-1BBL trimer-containing antigen binding molecule comprising: (a) at least one Fab molecule capable of specifically binding to PD-L1; and (b) a first polypeptide and a second polypeptide linked together by a disulfide bond, wherein the antigen-binding molecule comprises: (i) a first polypeptide comprising a CH1 or CL domain and a second polypeptide comprising a CL or CH1 domain, respectively, the second polypeptide being linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, the first polypeptide comprising two ectodomains of 4-1BBL comprising amino acid sequences 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:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, connected to each other and to the CH1 or CL domain by a peptide linker, and the second polypeptide comprising one ectodomain of said 4-1BBL comprising 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:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, connected via a peptide linker to the CL or CH1 domain of said polypeptide; or (ii) the first polypeptide comprises a CH3 domain and the second polypeptide comprises a CH3 domain, the first polypeptide comprises two ectodomains of 4-1BBL comprising amino acid sequences 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:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 connected to each other and to the C-terminus of the CH3 domain by a peptide linker, and the second polypeptide comprises only one ectodomain of 4-1BBL comprising 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:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 connected via a peptide linker to the C-terminus of the CH3 domain of the polypeptide; or (iii) a first polypeptide comprises a VH-CL or VL-CH1 domain, and a second polypeptide comprises a VL-CH1 domain or a VH-CL domain, respectively, the second polypeptide being linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, the first polypeptide comprises two ectodomains of 4-1BBL comprising amino acid sequences 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:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, connected to each other and to the VH or VL by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL comprising 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:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, connected to the VL or VH of the polypeptide via a peptide linker. A first polypeptide and a second polypeptide, (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen binding molecule comprising:

[0110] In a further aspect, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, and the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:3 and SEQ ID NO:4; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; Includes.

[0111] In one aspect, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 10 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 5; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; Includes.

[0112] In a further aspect, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO:9 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:1; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; Includes.

[0113] In another embodiment, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide comprising a CH1 or CL domain and a second polypeptide comprising a CL or CH1 domain, the second polypeptide being linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other and to the CH1 or CL domain by a peptide linker, and the second polypeptide comprises only one ectodomain of 4-1BBL or fragment thereof connected via a peptide linker to the CL or CH1 domain of the polypeptide; Includes.

[0114] In one embodiment, a 4-1BBL trimer-containing antigen binding molecule comprising: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide comprising a CH1 domain and a second polypeptide comprising a CL domain, the second polypeptide being linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other and to the CH1 domain by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof connected via a peptide linker to the CL domain of the polypeptide; The present invention provides a 4-1BBL trimer-containing antigen binding molecule comprising:

[0115] In another aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule, comprising: (a) an antigen-binding domain capable of specifically binding to PD-L1; and (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen-binding molecule comprising:

[0116] In yet another aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule, comprising: (a) more than one antigen-binding domain capable of specifically binding to PD-L1; and (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen-binding molecule comprising:

[0117] In one aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule comprising: (a) two antigen-binding domains capable of specifically binding to PD-L1; and (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen-binding molecule comprising:

[0118] In a further aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule as defined hereinbefore, wherein the antigen-binding domain capable of specifically binding to PD-L1 is selected from the group consisting of antibodies or antibody fragments.

[0119] In one aspect there is provided a 4-1BBL trimer-containing antigen binding molecule as defined herein before, wherein the antigen binding domain capable of specifically binding to PD-L1 is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, an Fv molecule, an scFv molecule, a single domain antibody, or aVH. In one aspect the antigen binding domain capable of specifically binding to PD-L1, a VH and a VL domain.

[0120] In a particular aspect, a 4-1BBL trimer-containing antigen binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to PD-L1 is a Fab molecule or a crossover Fab molecule capable of specifically binding to PD-L1. In particular, the antigen-binding domain capable of specifically binding to PD-L1 is a Fab capable of specifically binding to PD-L1.

[0121] In a further aspect, a 4-1BBL trimer-containing antigen binding molecule according to the present invention is provided, in which a peptide comprising two ectodomains of 4-1BBL or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus to the CH1 domain of the heavy chain by a second peptide linker, and one ectodomain of 4-1BBL or fragment thereof is fused at its C-terminus to the CL domain on the light chain by a third peptide linker.

[0122] In another aspect, a 4-1BBL trimer-containing antigen binding molecule according to the present invention is provided, in which a peptide comprising two ectodomains of 4-1BBL or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus to the CL domain of the heavy chain by a second peptide linker, and one ectodomain of 4-1BBL or fragment thereof is fused at its C-terminus to the CH1 domain on the light chain by a third peptide linker.

[0123] In a further aspect, the present invention relates to a 4-1BBL trimer-containing antigen binding molecule according to the present invention, in which a peptide comprising two ectodomains of 4-1BBL or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus to the CL domain of a light chain by a second peptide linker, and one ectodomain of 4-1BBL or a fragment thereof is fused at its C-terminus to the CH1 domain of a heavy chain by a third peptide linker.

[0124] In a particular embodiment, the present invention relates to a 4-1BBL trimer-containing antigen binding molecule as defined above, wherein the peptide linker is (G4S) 2. In one embodiment, the first peptide linker is (G4S) 2 (SEQ ID NO: 41), the second peptide linker is (G4S) 2 (SEQ ID NO: 41), and the third peptide linker is (G4S) 2 (SEQ ID NO: 41).

[0125] In another embodiment, the 4-1BBL trimer-containing antigen binding molecule as defined herein above comprises an Fc domain composed of a first subunit and a second subunit capable of stable association.

[0126] In particular, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises: (a) a Fab molecule capable of specifically binding to PD-L1, wherein the Fab heavy chain is fused at its C-terminus to the N-terminus of a CH2 domain in an Fc domain; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association.

[0127] In a further embodiment, the Fc domain is an IgG, in particular an IgG1 Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is an IgG1 Fc domain. In a particular embodiment, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

[0128] Fc domain modifications that reduce Fc receptor binding and / or effector function The Fc domain of the 4-1BBL trimer-containing antigen-binding molecule of the present invention is composed of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, with each subunit containing the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other.

[0129] The Fc domain provides the antigen-binding molecules of the present invention with desirable pharmacokinetic properties, including a long serum half-life that contributes to good accumulation in target tissues, and a desirable tissue-blood distribution ratio. At the same time, however, the Fc region may cause undesirable targeting of the bispecific antibodies of the present invention to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Thus, in certain embodiments, the Fc domain of the 4-1BBL trimer-containing antigen-binding molecules of the present invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to the native IgG1 Fc domain. In one embodiment, the Fc does not substantially bind to the Fc receptor and / or does not induce effector function. In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, and most specifically human FcγRIIIa. In one embodiment, the Fc domain does not induce effector functions, including, but not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced maturation of dendritic cells, or reduced T cell priming.

[0130] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of a 4-1BBL trimer-containing antigen binding molecule provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0131] In a particular aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule, comprising: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the Fc domain comprising one or more amino acid substitutions that reduce binding to an Fc receptor, in particular to an Fcγ receptor; The present invention provides a 4-1BBL trimer-containing antigen-binding molecule comprising:

[0132] In one aspect, the Fc domain of the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In particular, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331 and P329 (EU numbering). In particular, the Fc domain comprises amino acid substitutions at positions 234 and 235 (EU numbering) and / or 329 (EU numbering) of the IgG heavy chain. More particularly, a trimeric TNF family ligand-containing antigen binding molecule according to the present invention is provided, comprising an Fc domain having amino acid substitutions L234A, L235A and P329G ("P329G LALA", EU numbering) in the IgG heavy chain. The amino acid substitutions L234A and L235A refer to the so-called LALA mutation. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor binding in human IgG1 Fc domains and is described in International Patent Application WO 2012 / 130831(A1), which also describes methods for preparing such mutant Fc domains and for determining their 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.

[0133] Fc domains with reduced Fc receptor binding and / or effector function also include those with 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 variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 have been substituted with alanine (U.S. Patent No. 7,332,581).

[0134] In another embodiment, the Fc domain is an IgG4 Fc domain. IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to IgG1 antibodies. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising the amino acid substitutions L235E and S228P and P329G (EU numbering). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are also described in WO 2012 / 130831.

[0135] Variant 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-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be confirmed, for example, by screening.

[0136] Binding to Fc receptors can be easily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare) and Fc receptors that can be obtained by recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of an Fc domain or a cell-activating bispecific antigen-binding molecule comprising an Fc domain to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor).

[0137] The effector function of the Fc domain or the bispecific antibody of the present invention comprising an 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 the ADCC activity of a molecule 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, non-radioactive assay methods may be used (e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0138] In some embodiments, binding of the Fc domain to complement components, particularly binding to C1q, is reduced. Thus, in some embodiments where the Fc domain is modified to reduce effector function, said reduced effector function comprises reduced CDC. To determine whether a bispecific antibody of the invention is capable of binding C1q and thus has CDC activity, a C1q binding assay can be performed. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may 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)).

[0139] In certain aspects, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

[0140] Fc domain modifications that promote heterodimerization In one aspect, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association. Thus, they typically contain different portions fused to one or the other of the two subunits of the Fc domain contained in two non-identical polypeptide chains ("heavy chains"). Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Therefore, in order to improve the yield and purity of 4-1BBL trimer-containing antigen-binding molecules in recombinant production, it would be advantageous to introduce modifications to the Fc domain of the 4-1BBL trimer-containing antigen-binding molecules of the present invention that promote the association of the desired polypeptides.

[0141] Thus, the Fc domain of the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises a modification that promotes the association of the first and second subunits of the Fc domain. The longest protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, the modification is specifically in the CH3 domain of the Fc domain.

[0142] In a particular embodiment, the modification is a so-called "knob-into-hole" modification, which comprises a "knob" modification of one of the two subunits of the Fc domain and a "hole" modification of the other of the two subunits of the Fc domain. Thus, in a particular embodiment, the present invention relates to a 4-1BBL trimer-containing antigen binding molecule as described herein, comprising an IgG molecule, in which the Fc portion of the first heavy chain comprises a first dimerization module and the Fc portion of the second heavy chain comprises a second dimerization module that allows heterodimerization of the two heavy chains of the IgG molecule, and according to the knob-into-hole, the first dimerization module comprises a knob and the second dimerization module comprises a hole.

[0143] Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method involves promoting heterodimer formation and hindering homodimer formation by introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity at the interface of a second polypeptide, respectively, such that the protuberance can be located within the corresponding cavity. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine).

[0144] Thus, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the 4-1BBL trimer-containing antigen binding molecule of the present invention, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit that can be positioned within a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc region, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit into which the protuberance within the CH3 domain of the first subunit can be positioned.

[0145] The protuberances and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0146] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced by 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 by a valine residue (Y407V). More particularly, in the second subunit of the Fc domain, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A). More particularly, in the first subunit of the Fc domain, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues leads to the formation of disulfide bridges between the two subunits of the Fc domain, which further stabilize the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0147] In an alternative embodiment, the modification that promotes the association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, for example as described in PCT Publication WO 2009 / 089004. Generally, this method involves the replacement of one or more amino acid residues at the interface of the two Fc domain subunits with a charged amino acid residue such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favorable.

[0148] Modifications within the CH1 / CL domain To further improve correct pairing, 4-1BBL trimer-containing antigen binding molecules can contain differently charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the crossed or non-crossed CH1 and CL domains. In a particular embodiment, the present invention relates to a 4-1BBL trimer-containing antigen binding molecule in which, in one of the CL domains, the amino acid at position 123 (EU numbering) is substituted with arginine (R), the amino acid at position 124 (EU numbering) is substituted with lysine (K), and, in one of the CH1 domains, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are substituted with glutamic acid (E).

[0149] More specifically, the present invention relates to a 4-1BBL trimer-containing antigen binding molecule in which, in the CL domain adjacent to the TNF ligand family member, the amino acid at position 123 (EU numbering) is substituted with arginine (R) and the amino acid at position 124 (EU numbering) is substituted with lysine (K), and, in the CH1 domain adjacent to the TNF ligand family member, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are substituted with glutamic acid (E).

[0150] Thus, in a particular embodiment, a 4-1BBL trimer-containing antigen binding molecule comprising: (a) an antigen-binding domain capable of specifically binding to PD-L1; (b) a first polypeptide comprising a CL domain comprising the amino acid mutations E123R and Q124K, and a second polypeptide comprising a CH1 domain comprising the amino acid mutations K147E and K213E, the second polypeptide being linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, the antigen-binding molecule being characterized in that the first polypeptide comprises two ectodomains of 4-1BBL or fragments thereof connected to each other and to the CL domain by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL or fragments thereof connected via a peptide linker to the CH1 domain of said polypeptide; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association; The present invention provides a 4-1BBL trimer-containing antigen binding molecule comprising:

[0151] In one aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule in which the amino acid at position 123 (EU numbering) in the CL domain adjacent to the TNF ligand family member is substituted with arginine (R), the amino acid at position 124 (EU numbering) is substituted with lysine (K), and the amino acid at position 147 (EU numbering) and the amino acid at position 213 (EU numbering) in the CH1 domain adjacent to the TNF ligand family member are substituted with glutamic acid (E). These modifications result in so-called charged residues, which have advantageous properties that avoid undesirable effects such as mispairing.

[0152] In particular, the CL domain contains the amino acid mutations E123R and Q124K, and the CH1 domain contains the amino acid mutations K147E and K213E.

[0153] Antigen-binding molecules containing specific 4-1BBL trimers The present invention provides a 4-1BBL trimer-containing antigen binding molecule comprising an antigen-binding domain capable of specifically binding to PD-L1. In a particular embodiment, the 4-1BBL trimer-containing antigen binding molecule comprises one moiety capable of specifically binding to PD-L1, meaning that the 4-1BBL trimer-containing antigen binding molecule is monovalent. In another embodiment, the present invention provides a 4-1BBL trimer-containing antigen binding molecule comprising two moieties capable of specifically binding to PD-L1, meaning that the 4-1BBL trimer-containing antigen binding molecule is bivalent.

[0154] In one aspect, the invention provides a 4-1BBL trimer-containing antigen binding molecule, wherein the antigen-binding domain capable of specifically binding to PD-L1 comprises: (i) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15. H (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. L PD-L1) Includes.

[0155] In one aspect, the invention provides a 4-1BBL trimer-containing antigen binding molecule, wherein the antigen-binding domain capable of specifically binding to PD-L1 comprises a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL domain comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18.

[0156] In a further aspect, the antigen binding domain capable of specifically binding to PD-L1 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: 19, 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:20.

[0157] In a further aspect, the invention provides a 4-1BBL trimer-containing antigen binding molecule, wherein the antigen-binding domain capable of specifically binding to PD-L1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 19 and a VL domain comprising the amino acid sequence of SEQ ID NO: 20.

[0158] In a further aspect, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises (i) a first heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 19 and a first light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 20, (ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 27, and (iii) a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28.

[0159] In a specific embodiment, the 4-1BBL trimer-containing antigen binding molecule of the present invention comprises: (a) an antigen-binding domain capable of specifically binding to PD-L1, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20; and (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 10 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 5; Includes.

[0160] In a particular embodiment, a 4-1BBL trimer-containing antigen binding molecule is provided, wherein the antigen binding molecule comprises a first heavy chain comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:29, a first light chain comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:30, a second heavy chain comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:21, and a second light chain comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:22.

[0161] In another aspect, the present invention provides a 4-1BBL trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29, a first light chain comprising the amino acid sequence of SEQ ID NO: 30, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a second light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0162] Polynucleotides The present invention further provides isolated nucleic acid molecules encoding the 4-1BBL trimer-containing antigen binding molecules described herein or fragments thereof.

[0163] The isolated polynucleotides encoding the 4-1BBL trimer-containing antigen-binding molecules of the present invention may be expressed as a single polynucleotide encoding the complete antigen-binding molecule, or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides expressed together may associate, for example, via disulfide bonds or other means, to form a functional antigen-binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a separate polynucleotide from the heavy chain portion of an immunoglobulin. When co-expressed, the heavy chain polypeptides associate with the light chain polypeptides to form an immunoglobulin.

[0164] In some embodiments, the isolated nucleic acid molecule encodes the entire 4-1BBL trimer-containing antigen binding molecule according to the present invention described herein. In particular, the isolated polynucleotide encodes a polypeptide contained in the 4-1BBL trimer-containing antigen binding molecule according to the present invention described herein.

[0165] In one aspect, the invention relates to an isolated nucleic acid molecule encoding a 4-1BBL trimer-containing antigen binding molecule, the isolated nucleic acid molecule comprising: (a) a sequence encoding an antigen-binding domain capable of specifically binding to PD-L1; (b) a sequence encoding a polypeptide comprising two ectodomains of 4-1BBL or fragments thereof connected together by a peptide linker; and (c) a sequence encoding a polypeptide comprising one ectodomain of said 4-1BBL or fragment thereof.

[0166] In another aspect, there is provided an isolated polynucleotide encoding a 4-1BB ligand trimer-containing antigen binding molecule, the isolated polynucleotide comprising: (a) a sequence encoding a portion capable of specifically binding to PD-L1; (b) a sequence encoding a polypeptide comprising two ectodomains of 4-1BBL or two fragments thereof connected together by a peptide linker; and (c) a sequence encoding a polypeptide comprising one ectodomain of 4-1BBL or a fragment thereof.

[0167] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the invention may be single-stranded or double-stranded.

[0168] Recombination Method The 4-1BBL trimer-containing antigen-binding molecules of the present invention may be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the 4-1BBL trimer-containing antigen-binding molecules or polypeptide fragments thereof are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell, for example as described above. Such polynucleotides can be easily isolated and sequenced using standard procedures. In one aspect of the present invention, a vector, preferably an expression vector, is provided, comprising one or more polynucleotides of the present invention. Methods well known to those skilled in the art can be used to construct expression vectors comprising the coding sequence of the 4-1BBL trimer-containing antigen-binding molecules together with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in 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). An expression vector may be part of a plasmid, a virus, or a nucleic acid fragment. An expression vector includes an expression cassette into which a polynucleotide encoding a 4-1BBL trimer-containing antigen-binding molecule or a polypeptide fragment thereof (i.e., a coding region) is cloned in operable association with a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, but is considered to be part of the coding region (if present).However, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct, such as on a single vector, or in separate polynucleotide constructs, such as on separate (different) vectors. Furthermore, any vector may contain a single coding region or may contain two or more coding regions, for example, a vector of the invention may encode one or more polypeptides that are post-translationally or co-translationally separated into a final protein by proteolytic cleavage. In addition, a vector, polynucleotide or nucleic acid of the invention may encode a heterologous coding region, either fused or unfused to a polynucleotide encoding a 4-1BBL trimer-containing antigen-binding molecule of the invention or a polynucleotide encoding a polypeptide fragment thereof or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as, for example, secretory signal peptides or heterologous functional domains. An operably linked state is when a coding region for a gene product, such as a polypeptide, is linked to one or more control sequences such that expression of the gene product is under the influence or control of the control sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably linked" if induction of promoter function results in transcription of an mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region is said to be operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in a given cell. In addition to promoters, other transcriptional regulatory elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably linked to the polynucleotide to direct cell-specific transcription.

[0169] Suitable promoters and other transcriptional regulatory regions are disclosed herein. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, such as, but are not limited to, promoters and enhancer segments from cytomegalovirus (e.g., immediate early promoter and intron A), Simian Virus 40 (e.g., immediate early promoter), and retroviruses (e.g., Rous sarcoma virus, etc.). Other transcriptional regulatory regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone and rabbit a-globin, and other sequences capable of controlling gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline promoter-inducible). Similarly, various translational regulatory elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translational initiation and termination codons, and elements derived from viral systems (particularly, including internal ribosome entry sites, or IRES, also called CITE sequences). The expression cassette may also contain other features, such as an origin of replication and / or chromosomal integration elements, such as the long terminal repeats (LTRs) of retroviruses or the inverted terminal repeats (ITRs) of adeno-associated viruses (AAV).

[0170] The polynucleotide and nucleic acid coding regions of the present invention can be linked to additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides of the present invention. For example, if secretion of a 4-1BBL trimer-containing antigen-binding molecule or a polypeptide fragment thereof is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid encoding the 4-1BBL trimer-containing antigen-binding molecule of the present invention, or a polypeptide fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein when export transport of the growing protein chain across the rough endoplasmic reticulum is initiated. Those skilled in the art know that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce a secretory or "mature" form of the polypeptide. In certain embodiments, a native signal peptide (e.g., an immunoglobulin heavy or light chain signal peptide) or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide and is operably linked to that sequence is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0171] DNA encoding short protein sequences that can be used to facilitate subsequent purification or to aid in labeling of the fusion protein (e.g., histidine tags) can be included in the polynucleotide encoding the 4-1BBL trimer-containing antigen-binding molecule of the present invention or a polypeptide fragment thereof, either at or at both ends.

[0172] In a further aspect of the present invention, a host cell is provided comprising one or more polynucleotides of the present invention. In a particular embodiment, a host cell is provided comprising one or more vectors of the present invention. The polynucleotides and vectors may incorporate any of the features described herein in connection with the polynucleotides and vectors, respectively, alone or in combination. In one aspect, the host cell comprises a vector comprising a polynucleotide encoding (a part of) the 4-1BBL trimer-containing antigen binding molecule of the present invention (e.g., transformed or transfected with such a vector). As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce the fusion protein of the present invention or a fragment thereof. Suitable host cells for replicating and supporting the expression of antigen binding molecules are well known in the art. Such cells may be transfected or transduced, where appropriate, with a particular expression vector, and large amounts of the cells containing the vector can be grown to inoculate a large-scale fermenter, to obtain sufficient quantities of the antigen binding molecule for clinical use. Suitable host cells include prokaryotic microorganisms (e.g., E. coli) or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, the polypeptide may be produced in bacteria, especially if glycosylation is not required. After expression, the polypeptide may be isolated from the bacterial cell paste in an appropriate fraction and may be further purified. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce polypeptides with partially or completely human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006).

[0173] Suitable host cells for the expression of (glycosylated) polypeptides can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that may be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized 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 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7), human embryonic kidney lines (e.g., 293 cells or 293T cells as described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as 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 liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (e.g., Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC5 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)), myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0.For a review of certain mammalian host cells suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, 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 just a few, but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocytic cell (e.g., Y0, NS0, Sp20 cell). Standard techniques for expressing foreign genes in these systems are known in the art. Cells that express a polypeptide containing an immunoglobulin heavy or light chain can be engineered to also express the other of the immunoglobulin chains, such that the expressed product is an immunoglobulin having both a heavy and a light chain.

[0174] In one aspect, a method for producing a 4-1BBL trimer-containing antigen binding molecule of the present invention, or a polypeptide fragment thereof, is provided, comprising culturing a host cell comprising a polynucleotide encoding a 4-1BBL trimer-containing antigen binding molecule of the present invention, or a polypeptide fragment thereof, as provided herein, under conditions suitable for expressing the 4-1BBL trimer-containing antigen binding molecule of the present invention, or a polypeptide fragment thereof, and recovering the 4-1BBL trimer-containing antigen binding molecule of the present invention, or a polypeptide fragment thereof, from the host cell (or host cell culture medium).

[0175] In the 4-1BBL trimer-containing antigen binding molecule of the present invention, the components (at least one portion capable of specifically binding to a target cell antigen, one polypeptide comprising two ectodomains of 4-1BBL or fragments thereof, and one ectodomain of said 4-1BBL or fragments thereof) are not genetically fused to each other. The polypeptide is designed such that its components (two ectodomains of a TNF ligand family member or fragment thereof, and other components such as CH or CL) are fused to each other directly or via a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and tested for effectiveness. Examples of linker sequences between different components of the antigen binding molecule of the present invention are found in the sequences provided herein. If necessary, additional sequences, such as endopeptidase recognition sequences, can also be included to incorporate cleavage sites for separating the individual components of the fusion protein.

[0176] In certain embodiments, the portion capable of specifically binding to a target cell antigen (e.g., a Fab fragment) that forms part of an antigen-binding molecule comprises at least an immunoglobulin variable region capable of binding to an antigen. The variable region may form part of or be derived from natural or non-naturally occurring antibodies and fragments thereof. Methods for producing polyclonal and monoclonal antibodies are 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, produced recombinantly (e.g., as described in U.S. Pat. No. 4,186,567), or obtained, for example, by screening combinatorial libraries containing variable heavy and variable light chains (see, for example, U.S. Pat. No. 5,969,108 to McCafferty).

[0177] Immunoglobulins of any animal species can be used in the present invention. Non-limiting immunoglobulins useful in the present invention can be of murine, primate, or human origin. If the fusion protein is intended for human use, chimeric forms of immunoglobulins may be used in which the constant regions of the immunoglobulin are of human origin. 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 to Winter). Humanization may be achieved by a variety of methods, including, but not limited to, (a) grafting the CDRs of a non-human (e.g., a donor antibody) onto human (e.g., a recipient antibody) framework and constant regions with or without retaining key framework residues (e.g., those important for maintaining good antigen binding affinity or antibody function); (b) grafting only the non-human specificity determining regions (SDRs or a-CDRs; residues important for antibody-antigen interaction) onto human framework and constant regions; or (c) grafting the entire non-human variable domain but "cloaking" it with a human-like segment by replacement of surface residues.Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are described, for example, in 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 "resurfacing"); 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 a "guided selection" approach to FR shuffling). Particular immunoglobulins according to the invention are human immunoglobulins. Human antibodies and human variable regions can be produced using a variety of techniques known in the art. 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 may form part of or be derived from human monoclonal antibodies produced by hybridoma technology (see, e.g., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions may also be prepared by administering an immunogen to transgenic animals that have been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen 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., ed., 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 either single-chain Fv (scFv) fragments or Fab fragments.

[0178] In certain embodiments, the portion of the antigen-binding molecule of the invention capable of specifically binding to PD-L1 (e.g., a Fab fragment) is engineered to have enhanced binding affinity, for example, according to the methods disclosed in PCT Publication WO 2012 / 020006 (see Examples on affinity maturation) or US Patent Publication No. 2004 / 0132066. The binding ability of the antigen-binding molecule of the invention to a particular antigenic determinant can be measured either by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance techniques (Liljeblad, et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays can be used to identify antigen-binding molecules that compete with a reference antibody for binding to a particular antigen. In certain embodiments, such a competing antigen-binding molecule binds to the same epitope (e.g., a linear epitope or a conformational epitope) bound by the reference antigen-binding molecule. Detailed exemplary methods for mapping the epitopes bound by antigen-binding molecules are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In an exemplary competitive assay, immobilized antigen is incubated in a solution containing a first labeled antigen-binding molecule that binds to the antigen and a second unlabeled antigen-binding molecule that is being tested for its ability to compete with the first antigen-binding molecule for binding to the antigen. The second antigen-binding molecule may be present in the hybridoma supernatant. As a control, immobilized antigen is incubated in a solution containing the first labeled antigen-binding molecule but not the second unlabeled antigen-binding molecule. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed, and the amount of label bound to the immobilized antigen is measured.If the amount of label bound to the immobilized antigen is substantially reduced in the test sample compared to the control sample, this indicates that the second antigen-binding molecule competes with the first antigen-binding molecule for binding to the antigen.See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0179] The 4-1BBL trimer-containing antigen-binding molecules of the invention prepared as described herein 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 used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the 4-1BBL trimer-containing antigen-binding molecule binds can be used. For example, a matrix containing protein A or protein G can be used to affinity chromatographically purify the fusion proteins of the invention. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the antigen-binding molecule, essentially as described in the Examples. The purity of the 4-1BBL trimer-containing antigen-binding molecule or a fragment thereof can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, etc. For example, 4-1BBL trimer-containing antigen binding molecules expressed as described in the Examples were shown to be intact and properly assembled, as shown by reducing and non-reducing SDS-PAGE.

[0180] Assay The antigen-binding molecules provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. Biological activity can include, for example, the ability to enhance the activation and / or proliferation of various immune cells, particularly T cells. For example, they enhance the secretion of immunomodulatory cytokines. Other immunomodulatory cytokines that are or can be enhanced are, for example, IL2, granzyme B, etc. Biological activity can also include cynomolgus binding cross-reactivity, as well as binding to different cell types. Antigen-binding molecules having such biological activity in vivo and / or in vitro are also provided.

[0181] 1. Affinity Assay The affinity of the 4-1BBL trimer-containing antigen binding molecules provided herein for 4-1BB (CD137) can be determined by surface plasmon resonance (SPR) using standard equipment, such as a BIAcore instrument (GE Healthcare), and a receptor or target protein, such as that available by recombinant expression, according to the methods described in the Examples. The affinity of the 4-1BBL trimer-containing antigen binding molecules for PD-L1 can also be measured by surface plasmon resonance (SPR) using standard instrumentation, such as a BIAcore instrument (GE Healthcare), and a receptor or target protein, such as that available by recombinant expression. Specific examples and exemplary embodiments for measuring binding affinity are described in Example 4. According to one embodiment, K D is measured by surface plasmon resonance at 25° C. using a BIACORE® T100 machine (GE Healthcare).

[0182] 2. Binding and other assays The binding of the 4-1BBL trimer-containing antigen binding molecules provided herein to cells expressing the corresponding receptor can be assessed using a cell line expressing a particular receptor or target antigen, for example by flow cytometry (FACS). In one embodiment, fresh peripheral blood mononuclear cells (PBMCs) expressing 4-1BB can be used for binding assays. These cells are used immediately after isolation (naive PMBCs) or after stimulation (activated PMBCs). In another embodiment, activated mouse splenocytes (expressing 4-1BB) can be used to demonstrate the binding of the 4-1BBL trimer-containing antigen binding molecules of the present invention to 4-1BB-expressing cells.

[0183] In a further embodiment, cell lines expressing PD-L1 were used to demonstrate binding of the antigen binding molecule to this target cell antigen.

[0184] In another aspect, a competition assay can be used to identify antigen-binding molecules that compete with a specific antibody or antigen-binding molecule for binding to PD-L1 or 4-1BB, respectively. In certain embodiments, such competing antigen-binding molecules bind to the same epitope (e.g., a linear or conformational epitope) bound by a specific anti-PD-L1 antibody or a specific anti-4-1BB antibody. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0185] 3. Activity Assay In one aspect, an assay is provided for identifying 4-1BBL trimer-containing antigen binding molecules that bind to PD-L1 and 4-1BB and have biological activity. Biological activity can include, for example, agonistic signaling via 4-1BB to cells expressing PD-L1. Also provided are 4-1BBL trimer-containing antigen binding molecules identified by the assay as having such biological activity in vitro.

[0186] In a particular embodiment, the 4-1BBL trimer-containing antigen binding molecules of the present invention are tested for such biological activity. Assays for detecting the biological activity of the molecules of the present invention are described in Example 3. In addition, assays for detecting cell lysis (e.g., by measuring LDH release), induced apoptosis kinetics (e.g., by measuring caspase 3 / 7 activity), or apoptosis (e.g., using the TUNEL assay) are well known in the art. Furthermore, the biological activity of such complexes can be evaluated by assessing their effect on the survival, proliferation and lymphokine secretion of various lymphocyte subsets, such as NK cells, NKT cells or δγT cells, or by assessing their ability to modulate the phenotype and function of antigen-presenting cells, such as dendritic cells, monocytes / macrophages or B cells.

[0187] Pharmaceutical Compositions, Formulations, and Routes of Administration In a further aspect, the present invention provides a pharmaceutical composition comprising any of the 4-1BBL trimer-containing antigen binding molecules provided herein, for example, for use in any of the following therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the 4-1BBL trimer-containing antigen binding molecules provided herein and at least one pharma- ceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises any of the 4-1BBL trimer-containing antigen binding molecules provided herein and at least one additional therapeutic agent, for example, as described below.

[0188] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more 4-1BBL trimer-containing antigen-binding molecules dissolved or dispersed in a pharma- ceutically acceptable excipient. The phrase "pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the doses and concentrations employed, i.e., do not produce side effects, allergies or other adverse reactions when administered as appropriate to an animal, e.g., a human. The preparation of pharmaceutical compositions containing at least one 4-1BBL trimer-containing antigen-binding molecule, and optionally additional active ingredients, will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference. In particular, the compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonicity agents, salts, stabilizers, and combinations thereof, as would be known to one of skill in the art.

[0189] Parenteral compositions include those designed to be administered by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the 4-1BBL trimer-containing antigen-binding molecule of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solutions may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the fusion protein may be in powder form for constitution before use with a suitable vehicle, e.g., sterile pyrogen-free water. Sterile injectable solutions are prepared by incorporating the fusion protein of the present invention in the required amount in the appropriate solvent, with various other ingredients as listed below, as required. Sterility can be readily achieved, for example, by filtration through a sterile filtration membrane. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and / or other ingredients. In the case of sterile powder for preparing sterile injectable solution, suspension or emulsion, the preferred preparation method is vacuum drying or freeze-drying technology, which obtains a powder of active ingredient and any additional desired ingredient from a liquid medium that has already been sterile filtered.The liquid medium should be appropriately buffered if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose before injection.The composition must be stable under the manufacturing and storage conditions and must be protected from the contaminating action of microorganisms such as bacteria and fungi.It is understood that endotoxin contamination should be kept to a minimum at a safe level, for example, less than 0.5ng / mg protein.Suitable pharma- ceutically acceptable excipients include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride, phenol, butyl, 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; protein The suspension may include a substance 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 non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension (e.g. sodium carboxymethylcellulose, sorbitol, dextran, etc.). Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds, allowing for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.

[0190] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, for example, films or microcapsules. In certain embodiments, sustained absorption of the injectable compositions may be brought about by the use in the compositions of agents that delay absorption (e.g., aluminum monostearate, gelatin, or a combination thereof).

[0191] Exemplary pharma- ceutically acceptable excipients of the present invention further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

[0192] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine acetate buffer.

[0193] In addition to the above compositions, the fusion protein may be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the fusion protein may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as a poorly soluble derivative, for example, as a poorly soluble salt.

[0194] Pharmaceutical compositions, including pharmaceutical compositions containing the fusion protein of the present invention, can be prepared by common mixing, dissolving, emulsifying, encapsulating, entrapment or lyophilization processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, additives, or auxiliary agents that facilitate the processing of the protein into a pharma-ceutically usable preparation. Appropriate formulations vary depending on the route of administration selected.

[0195] The 4-1BBL trimer-containing antigen-binding molecule can be formulated into a composition in the form of a free acid or base, neutral or salt. A pharmaceutically acceptable salt is one that substantially retains the biological activity of the free acid or base. Pharmaceutically acceptable salts include acid addition salts, for example, those formed with free amino groups of a proteinaceous composition, or those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or those formed 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, potassium, ammonium, calcium, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0196] The compositions of the present invention may contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0197] In one embodiment, a pharmaceutical composition may comprise any of the 4-1BBL trimer-containing antigen binding molecules provided herein and at least one additional therapeutic agent. In one embodiment, a pharmaceutical composition may comprise any of the 4-1BBL trimer-containing antigen binding molecules provided herein and a T cell-activating anti-CD3 bispecific antibody. In one embodiment, the T cell-activating anti-CD3 bispecific antibody comprises a first antigen-binding domain that binds to CD3 and a second antigen-binding domain that binds to a tumor-associated antigen.

[0198] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0199] Therapeutic Methods and Compositions Any of the 4-1BBL trimer-containing antigen binding molecules provided herein can be used in therapeutic methods.

[0200] For use in therapeutic methods, the 4-1BBL trimer-containing antigen binding molecules of the present invention can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the drug, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0201] In one aspect, a 4-1BBL trimer-containing antigen binding molecule of the invention is provided for use as a medicament. In a further aspect, a 4-1BBL trimer-containing antigen binding molecule of the invention is provided for use in the treatment of a disease, in particular for use in the treatment of cancer. In a particular aspect, a 4-1BBL trimer-containing antigen binding molecule of the invention is provided for use in a method of treatment. In one aspect, the invention provides a 4-1BBL trimer-containing antigen binding molecule as described herein for use in the treatment of a disease in an individual in need of such treatment. In a particular aspect, the invention provides a 4-1BBL trimer-containing antigen binding molecule for use in a method of treating an individual with a disease, comprising administering to the individual a therapeutically effective amount of a fusion protein. In a particular aspect, the disease to be treated is cancer. Examples of cancer include breast cancer, ovarian cancer, gastric cancer, bladder cancer, salivary gland cancer, endometrial cancer, pancreatic cancer and non-small cell lung cancer (NSCLC). In one aspect, the cancer is a solid tumor. In some aspects, the cancer is already an advanced cancer. Thus, there is provided a 4-1BBL trimer-containing antigen binding molecule as described herein for use in the treatment of these cancers. The subject, patient or "individual" in need of treatment is typically a mammal, more particularly a human.

[0202] In another aspect, the 4-1BBL trimer-containing antigen binding molecule described herein is provided for use in the treatment of infectious diseases, particularly viral infections. In a further aspect, the 4-1BBL trimer-containing antigen binding molecule described herein is provided for use in the treatment of autoimmune diseases (such as lupus disease).

[0203] In a further aspect, the present invention relates to the use of a 4-1BBL trimer-containing antigen binding molecule in the manufacture or preparation of a medicament for the treatment of a disease in an individual in need of such treatment. In one aspect, the medicament is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the medicament to an individual having the disease. In a particular embodiment, the disease to be treated is a proliferative disorder, in particular cancer. Thus, in one aspect, the present invention relates to the use of a 4-1BBL trimer-containing antigen binding molecule of the present invention in the manufacture or preparation of a medicament for the treatment of cancer, in particular cancer. Examples of cancer include breast cancer, ovarian cancer, gastric cancer, bladder cancer, salivary gland cancer, endometrial cancer, pancreatic cancer and non-small cell lung cancer (NSCLC). One skilled in the art can readily recognize that in some cases, a 4-1BBL trimer-containing antigen binding molecule may not provide a cure, but may provide a partial benefit. In some aspects, physiological changes with some benefit are also considered therapeutically beneficial. Thus, in some embodiments, the amount of 4-1BBL trimer-containing antigen binding molecule that produces a physiological change is considered an "effective amount" or a "therapeutically effective amount."

[0204] In a further aspect, the present invention provides a method for treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a 4-1BBL trimer-containing antigen binding molecule of the present invention. In one aspect, a composition comprising a fusion protein of the present invention in a pharma- ceutically acceptable form is administered to said individual. In a particular aspect, the disease to be treated is a proliferative disorder. In a particular aspect, the disease is cancer. In a particular aspect, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent (e.g., an anti-cancer agent when the disease to be treated is cancer). The "individual" according to any of the above embodiments may be a mammal, preferably a human.

[0205] For the prevention or treatment of a disease, the appropriate dosage of the 4-1BBL trimer-containing antigen binding molecule of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will be determined by the type of disease being treated, the route of administration, the patient's weight, the type of fusion protein, the severity and course of the disease, whether the antigen binding molecule is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's medical history and response to the fusion protein, and the discretion of the attending physician. The practitioner responsible for administration will in any case determine the concentration of active ingredient in the composition and the appropriate dose for the individual subject. Various administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0206] The 4-1BBL trimer-containing antigen-binding molecule is suitably administered to the patient in a single dose or over a series of treatments. Depending on the type and severity of the disease, for example, whether by single or multiple separate administrations or by continuous infusion, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the 4-1BBL trimer-containing antigen-binding molecule may be an initial candidate dose for administration to the patient. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. For repeated administration over several days or more, depending on the condition, treatment will generally be continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the fusion protein would be in the range of about 0.005 mg / kg to about 10 mg / kg. In other examples, the dose may also include about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 350 μg / kg body weight, about 500 μg / 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, or any derivable range therebetween, per administration. In examples of ranges derivable from the numbers listed herein, ranges such as about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 μg / kg body weight to about 500 mg / kg body weight, etc., may be administered based on the above numbers. Thus, one or more doses 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 doses may be administered intermittently, for example weekly or every three weeks (e.g., the patient receives from about 2 to about 20, or for example about 6 doses of the fusion protein). An initial high loading dose may be followed by one or more smaller doses. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0207] The 4-1BBL trimer-containing antigen binding molecules of the present invention are generally used in an amount effective to achieve the intended purpose. For use in treating or preventing disease symptoms, the 4-1BBL trimer-containing antigen binding molecules of the present invention or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0208] For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. The IC 50 A dose may be formulated in animal models to achieve a circulating concentration range including, but not limited to, 100 mg / kg / day. Such information can be used to more accurately determine useful doses in humans.

[0209] Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques well known in the art. Those skilled in the art can readily optimize human dosing based on animal data.

[0210] The amount and interval of administration are adjusted individually to provide plasma levels of the 4-1BBL trimer-containing antigen-binding molecule sufficient to maintain therapeutic efficacy. Usual patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.

[0211] In the case of local administration or selective uptake, the effective local concentration of the 4-1BBL trimer-containing antigen-binding molecule may not be related to the plasma concentration. Those skilled in the art will be able to optimize the therapeutically effective local dose without undue experimentation.

[0212] The therapeutically effective dose of the 4-1BBL trimer-containing antigen binding molecules described herein generally provides a therapeutic effect without causing substantial toxicity. The toxicity and therapeutic effect of the fusion protein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal studies, LD 50 (the dose at which 50% of the population is lethal) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 Therapeutic indices can be expressed as a ratio of ED to ED. 4-1BBL trimer-containing antigen binding molecules exhibiting large therapeutic indices are preferred. In one embodiment, the 4-1BBL trimer-containing antigen binding molecules of the present invention exhibit a large therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating a dosage range suitable for human use. Dosages are preferably administered at or above the ED with little or no toxicity. 50 The blood concentration range includes: 100-200 mg / kg / day, 100-200 mg / kg / day, 100-300 mg / kg / day, 100-400 mg / kg / day, 100-500 mg / kg / day, 100-600 mg / kg / day, 100-700 mg / kg / day, 100-800 mg / kg / day, 100-900 mg / kg / day, 100-1000 mg / kg / day, 100-1000 mg / kg / day, 100-1000 mg / kg / day, 100-1000 mg / kg / day, 100-2000 mg / kg / day, 100-3000 mg / kg / day, 100-4000 mg / kg / day, 100-5000 mg / kg / day, 100-6000 mg / kg / day, 100-7000 mg / kg / day, 100-8000 mg / kg / day, 100-1 ...

[0213] The attending physician of a patient treated with the fusion protein of the invention will know how and when to discontinue, interrupt or adjust dosing due to toxicity, organ failure, etc. Conversely, the attending physician will also know to adjust treatment levels upwards (preemptively excluding toxicity) if the clinical response is inadequate. The magnitude of the dose in the management of the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Furthermore, the dose and perhaps the dosing frequency will also vary according to the age, weight and response of the individual patient.

[0214] Other Medications and Treatments The 4-1BBL trimer-containing antigen binding molecule of the present invention can be administered in combination with one or more other agents in a therapeutic regimen. For example, the fusion protein of the present invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent that can be administered to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredient suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is another anti-cancer agent.

[0215] Such other drugs are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other drugs depends on the amount of 4-1BBL trimer-containing antigen binding molecule used, the type of disorder or treatment, and other factors described above. In general, the 4-1BBL trimer-containing antigen binding molecule is used in the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and route determined to be empirically / clinically appropriate.

[0216] Such combination therapy as described above includes combined administration (where two or more therapeutic agents are contained in the same or separate compositions) and separate administration, in which case administration of the 4-1BBL trimer-containing antigen binding molecule of the present invention may occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant.

[0217] Thus, in one aspect, there is provided a 4-1BBL trimer-containing antigen binding molecule as described herein for use in the treatment of cancer, wherein the 4-1BBL trimer-containing antigen binding molecule is used in combination with a T cell activating anti-CD3 bispecific antibody. In one aspect, the anti-TA / anti-CD3 antibody comprises a first antigen binding domain that binds to CD3 and a second antigen binding domain that binds to a tumor-associated antigen.

[0218] In a further embodiment, the 4-1BBL trimer-containing antigen binding molecule is used in combination with a T cell-activating anti-CD3 bispecific antibody, which is administered simultaneously with, prior to, or after the 4-1BBL trimer-containing antigen binding molecule.

[0219] In a further aspect, the use of a 4-1BBL trimer-containing antigen binding molecule for the manufacture of a medicament for the treatment of cancer is provided, wherein the 4-1BBL trimer-containing antigen binding molecule is used in combination with a T cell-activating anti-CD3 bispecific antibody. Examples of cancer include breast cancer, ovarian cancer, gastric cancer, bladder cancer, salivary gland cancer, endometrial cancer, pancreatic cancer and non-small cell lung cancer (NSCLC).

[0220] In a further aspect, the present invention provides a method for treating cancer in an individual, comprising administering to said individual a therapeutically effective amount of a 4-1BBL trimer-containing antigen binding molecule of the present invention and an effective amount of a T cell activating anti-CD3 bispecific antibody. Examples of cancer include breast cancer, ovarian cancer, gastric cancer, bladder cancer, salivary gland cancer, endometrial cancer, pancreatic cancer and non-small cell lung cancer (NSCLC).

[0221] manufactured goods In another aspect of the present invention, an article of manufacture is provided that contains a substance useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The article of manufacture comprises a container and a label or package insert attached to or associated with the container. Examples of suitable containers include bottles, vials, syringes, IV infusion bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a compound, alone or in combination with other compositions, that is effective for the treatment, prevention, and / or diagnosis of a condition, and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a 4-1BBL trimer-containing antigen-binding molecule of the present invention.

[0222] The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may comprise (a) a first container in which a composition comprising a 4-1BBL trimer-containing antigen binding molecule of the present invention is contained; and (b) a second container in which a composition comprising an additional cytotoxic or other therapeutic agent is contained. The article of manufacture in this embodiment of the present invention may further comprise a package insert indicating that the composition can be used to treat a particular condition.

[0223] Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer (e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution). It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0224] TIFF0007675083000002.tif253170TIFF0007675083000003.tif254170TIFF0007675083000004.tif251170TIFF00076750830 00005.tif251170TIFF0007675083000006.tif251170TIFF0007675083000007.tif253170TIFF0007675083000008.tif213170

[0225] General information relating to the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of antibody chains are numbered and referenced according to the EU numbering system by Kabat (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) as defined above. EXAMPLES

[0226] The following are examples of the methods and compositions of the present invention. Given the general description above, it will be understood that various other embodiments may be practiced.

[0227] Recombinant DNA Technology Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No91-3242.

[0228] DNA sequencing The DNA sequence was determined by double-stranded sequencing.

[0229] Gene synthesis The desired gene segments were generated by PCR using appropriate templates or synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products by Geneart AG (Regensburg, Germany). In cases where the exact gene sequence was not available, oligonucleotide primers were designed based on the sequence of the closest homologue and the gene was isolated by RT-PCR from RNA derived from the appropriate tissue. Gene segments flanked by single restriction endonuclease cleavage sites were cloned into standard cloning / sequencing vectors. Plasmid DNA was purified from transformed bacteria and the concentration was measured by UV spectroscopy. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene segments were designed with appropriate restriction sites to allow subcloning into the respective expression vectors. All constructs were designed to contain a 5'-terminal DNA sequence encoding a leader peptide that targets the protein for secretion in eukaryotic cells.

[0230] Cell culture technology Standard cell culture techniques were used 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.

[0231] Protein purification Proteins were purified from filtered cell culture supernatants with reference to standard protocols. Briefly, antibodies were applied to a Protein A Sepharose column (GE healthcare) and washed with PBS. Elution of antibodies was achieved at pH 2.8, immediately followed by neutralization of the samples. Aggregated proteins were separated from monomeric antibodies by size-exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM histidine, 150 mM NaCl (pH 6.0). Monomeric antibody fractions were pooled and concentrated (if necessary) using, for example, a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, and stored frozen at -20°C or -80°C. A portion of these samples was submitted for subsequent protein analysis and analytical characterization, for example by SDS-PAGE, size-exclusion chromatography (SEC) or mass spectrometry.

[0232] SDS-PAGE The NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions, specifically, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and NuPAGE® MES (reducing gels, containing NuPAGE® antioxidant running buffer additive) or MOPS (non-reducing gels) running buffer.

[0233] Analytical Size Exclusion Chromatography Size exclusion chromatography (SEC) to determine the aggregation and oligomeric state of the antibodies was performed by HPLC chromatography. Briefly, Protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2PO4 / K2HPO4 (pH 7.5) on an Agilent HPLC 1100 system or to a Superdex 200 column in 2xPBS on a Dionex HPLC-System (GE Healthcare). Eluted proteins were quantified by UV absorbance and peak area integration. BioRad Gel Filtration Standard 151-1901 served as a standard.

[0234] Example 1 Generation and production of PD-L1-targeting 4-1BB ligand trimer-containing antigen-binding molecules 1.1. Generation and Production of PD-L1-Targeted 4-1BB Ligand Trimeric-Containing Antigen Binding Molecules The variable regions of the heavy and light chain DNA sequences encoding the antigen-binding domain specific for PD-L1 were subcloned in frame with either the Hall constant heavy chain or the human IgG1 constant light chain.

[0235] A DNA sequence encoding a portion of the ectodomain (amino acids 71-248) of the human 4-1BB ligand was synthesized according to the P41273 sequence in the Uniprot database.

[0236] A polypeptide containing the two ectodomains of 4-1BB ligand, separated by a (G4S)2 linker and fused to a human IgG1-CL domain, was cloned as shown in Figure 1A: human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand, (G4S)2 connector, human CL.

[0237] A polypeptide containing one ectodomain of the 4-1BB ligand fused to a human IgG1-CH domain was cloned as shown in FIG. 1B: human 4-1BB ligand, (G4S)2 connector, human CH.

[0238] The following mutations were introduced into the cross CH-CL to improve correct pairing: In the human CL domain fused to the dimeric 4-1BB ligand, the mutations E123R and Q124K were introduced. In the human CH1 domain fused to the monomeric 4-1BB ligand, the mutations K147E and K213E were cloned as described in International Patent Application WO 2015 / 150447.

[0239] The variable regions of the heavy and light chain DNA sequences encoding the antigen-binding domain capable of specifically binding to PD-L1 were subcloned in frame with either the Hall constant heavy chain or the human IgG1 constant light chain. The anti-PD-L1 clone (clone YW243.55.S70) is disclosed in WO 2010 / 077634.

[0240] In the Fc domain, P329G, L234A and L235A mutations were introduced into the constant regions of the knob and hole heavy chains to abolish binding to Fcγ receptors according to the methods described in International Patent Application WO 2012 / 130831. The combination of a dimeric ligand-Fc knob chain containing S354C / T366W mutations, a monomeric CH1 fusion, a targeted anti-PD-L1 Fc hole chain containing Y349C / T366S / L368A / Y407V mutations and an anti-PD-L1 light chain allowed the generation of a heterodimer containing an assembled trimeric 4-1BB ligand and a PD-L1-binding Fab (Figure 2).

[0241] Table 1 shows the amino acid sequence of the monovalent anti-PD-L1 split trimeric 4-1BB ligand Fc(kih) fusion antigen binding molecule containing the CH1-CL crossover and charged residues from the CH1 and CL domains fused to 4-1BBL. This molecule is designated PD-L1-4-1BBL.

[0242] TIFF0007675083000009.tif172170

[0243] Table 2 shows the amino acid sequence of the non-targeting control molecule DP47 split trimer 4-1BB ligand Fc(kih) fusion antigen binding molecule.

[0244] TIFF0007675083000010.tif107170

[0245] Bispecific constructs were produced by co-transfecting HEK293-EBNA cells with mammalian expression vectors using polyethylenimine. Cells were transfected with the corresponding expression vectors in a 1:1:1:1 ratio ("vector 4-1BBL Fc-knob chain":"vector 4-1BBL light chain":"vector Fc-hole chain":"vector light chain").

[0246] Production was performed in shake flasks using HEK293EBNA cells. Antibodies and bispecific antibodies were produced by transient transfection of HEK293 EBNA cells or CHO EBNA cells. Cells were centrifuged and the medium was replaced with pre-warmed CD CHO medium (Thermo Fisher, Cat. No. 10743029). Expression vectors were mixed in CD CHO medium, PEI (polyethyleneimine, Polysciences, Inc, Cat. No. 23966-1) was added, the solution was vortexed and incubated at room temperature for 10 minutes. Cells (2 Mio / mL) were then mixed with the vector / PEI solution, transferred to flasks and incubated at 37°C for 3 hours in a shaking incubator with a 5% CO2 atmosphere. After incubation, Excel medium containing supplements (80% of the total volume) was added (W. Zhou and A. Kantardjieff, Mammalian Cell Cultures for Biologics Manufacturing, DOI:10.1007 / 978-3-642-54050-9;2014). One day after transfection, supplements (Feed, 12% of the total volume) were added. After 7 days, cell supernatants were harvested by centrifugation and subsequent filtration (0.2 μm filter), and the harvested supernatants were purified by standard methods as described below.

[0247] Proteins were purified from filtered cell culture supernatants with reference to standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants by Protein A affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, and samples were immediately pH-neutralized. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (item number: UFC903096) and aggregated proteins were separated from monomeric proteins by size-exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0248] The concentration of purified proteins was determined by measuring the absorbance at 280 nm using the mass attenuation coefficient calculated based on the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of reducing agents using a LabChip GXII (Perkin Elmer). Aggregate content measurements were performed by HPLC chromatography at 25°C using analytical size exclusion columns (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7, or 200 mM KH2PO4, 250 mM KCl, pH 6.2, respectively).

[0249] Table 3 summarizes the yield and final monomer content of PD-L1-targeting 4-1BB ligand trimer-containing antigen binding molecules.

[0250] TIFF0007675083000011.tif18170

[0251] 1.2.4-Generation and production of bispecific antibodies with bivalent binding to 1BB and monovalent binding to PD-L1 For comparison, bispecific agonist 4-1BB antibodies with bivalent or monovalent binding to 4-1BB and monovalent binding to PD-L1 were also prepared.

[0252] Bispecific agonist 4-1BBxPD-L1 antibodies with bivalent binding to 4-1BB and monovalent binding to PD-L1 have been produced in the so-called head-to-head (H2H) 2+1 format, as described in WO 2020 / 007817(A1).

[0253] The first heavy chain HC1 of the construct was composed of the following components: VHCH1 of an anti-4-1BB binder (clone 20H4.9), followed by an Fc hole. The second heavy chain HC2 was composed of VLCH1 of an anti-PD-L1 binder (clone YW243.55.S70 in a cross-Fab format), followed by VHCH1 of an anti-4-1BB (clone 20H4.9) and an Fc knob. The PD-L1 binder YW243.55.S70 is described in WO 2010 / 077634. ​​For the 4-1BB binder, the VH and VL sequences of clone 20H4.9 were obtained according to US Patent No. 7,288,638 B2 or US Patent No. 7,659,384 B2. Combining the two heavy chains allowed the generation of a heterodimer that contained one PD-L1-binding cross-Fab and two 4-1BB-binding Fabs (Figure 2B). Another heterodimer with monovalent binding to 4-1BB was constructed from the VHCH1 of an anti-4-1BB binder (clone 20H4.9) followed by a first heavy chain HC1 containing an Fc hole, and the VLCH1 of an anti-PD-L1 binder (clone YW243.55.S70 in a cross-Fab format) followed by a second heavy chain HC2 containing an Fc knob (Figure 2C).

[0254] To improve correct pairing, the following mutations were introduced into the CH-CL of the anti-4-1BB Fab molecule: E123R and Q124K in CL, and K147E and K213E in CH1. The second light chain LC2 of the anti-PD-L1 binder is composed of VHCL (cross-Fab). The knob-into-hole technology was applied to allow the generation of heterodimers by introducing Y349C / T366S / L368A / Y407V mutations into the first heavy chain HC1 (Fc hole heavy chain) and S354C / T366W into the second heavy chain HC2 (Fc knob heavy chain).

[0255] Furthermore, Pro329Gly, Leu234Ala and Leu235Ala mutations were introduced into the constant regions of the knob and hole heavy chains to abolish binding to Fcγ receptors, according to the method described in International Patent Application WO 2012 / 130831(A1).

[0256] The 4-1BBxPD-L1 antibody in a 2+1 format comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:54, a heavy chain comprising the amino acid sequence of SEQ ID NO:55, two light chains each comprising the amino acid sequence of SEQ ID NO:56, and a light chain comprising the amino acid sequence of SEQ ID NO:57.

[0257] The 4-1BBxPD-L1 antibody in a 1+1 format comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:54, a heavy chain comprising the amino acid sequence of SEQ ID NO:58, a light chain comprising the amino acid sequence of SEQ ID NO:56, and a light chain comprising the amino acid sequence of SEQ ID NO:57.

[0258] Example 2 Functional characterization of PD-L1-targeting 4-1BB ligand trimer-containing antigen-binding molecules by surface plasmon resonance Preparation of 4-1BB Fc(kih) fusion molecule A DNA sequence encoding the ectodomain of human 4-1BB (amino acids 24-186 of human 4-1BB from Q07011, SEQ ID NO: 33) was subcloned in frame with the human IgG1 heavy chain CH2 and CH3 domains on the knob. An AcTEV protease cleavage site was introduced between the antigen ectodomain and the Fc of human IgG1. An Avi tag was introduced at the C-terminus of the antigen-Fc knob for directed biotinylation. Combining an antigen Fc knob chain containing S354C / T366W mutations with an Fc hole chain containing Y349C / T366S / L368A / Y407V mutations allows the generation of heterodimers containing a single copy of the 4-1BB ectodomain-containing chain, creating a monomeric form of Fc-bound antigen. Table 5 shows the amino acid sequences of the antigen Fc fusion constructs.

[0259] TIFF0007675083000012.tif75170

[0260] All sequences encoding the 4-1BB-Fc fusion molecules were cloned into a plasmid vector that drives expression of the insert from the MPSV promoter and contains a synthetic polyA signal sequence located at the 3' end of the CDS. In addition, the vector contains the EBV OriP sequence for episomal maintenance of the plasmid.

[0261] To prepare biotinylated monomeric antigen / Fc fusion molecules, exponentially growing suspension HEK293 EBNA cells were cotransfected with three vectors encoding the two components of the fusion protein (knob and hole strands) and BirA, the enzyme required for the biotinylation reaction. The corresponding vectors were used in a ratio of 2:1:0.05 ("Antigen ECD-AcTEV-Fc knob":"Fc hole":"BirA").

[0262] For protein production in 500 ml shake flasks, 400 million HEK293 EBNA cells were seeded 24 hours prior to transfection. For transfection, cells were centrifuged at 210 g for 5 minutes and the supernatant was replaced with pre-warmed CD CHO medium. Expression vectors were resuspended in 20 mL of CD CHO medium containing 200 μg of vector DNA. After adding 540 μl of polyethyleneimine (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 and transferred to a 500 mL shake flask and incubated at 37°C for 3 hours in an incubator with a 5% CO2 atmosphere. After 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% Feed1 with supplements were added to the culture. After 7 days of culture, the cell supernatant was harvested by spinning down the cells at 210 g for 15 min. The solution was sterile filtered (0.22 μm filter), supplemented with sodium azide to a final concentration of 0.01% (w / v) and kept at 4°C.

[0263] Secreted proteins were purified from cell culture supernatants by affinity chromatography using Protein A followed by size exclusion chromatography. For affinity chromatography, the supernatant was loaded onto a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 40 mL of 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unbound proteins were removed by washing with at least 10 column volumes of a buffer containing 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride (pH 7.5). Bound proteins were eluted using a linear pH gradient of sodium chloride (0 to 500 mM) made against 20 column volumes of 20 mM sodium citrate, 0.01% (v / v) Tween-20, pH 3.0. The column was then washed with 10 column volumes of 20 mM sodium citrate, 500 mM sodium chloride, 0.01% (v / v) Tween-20, pH 3.0.

[0264] The pH of the collected fractions was adjusted by adding 1 / 40 (v / v) of 2 M Tris (pH 8.0). The protein was concentrated, filtered, and loaded onto a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 2 mM MOPS, 150 mM sodium chloride, 0.02% (w / v) sodium azide solution (pH 7.4).

[0265] Human PD-L1-Fc (recombinant human PD-L1 / B7-H1 Fc chimeric protein, 156-B7-100: R&D Systems) was commercially available and was used to measure binding to PD-L1.

[0266] Determining simultaneous binding The ability to simultaneously bind human 4-1BB Fc(kih) and human PD-L1 was assessed by surface plasmon resonance (SPR). All SPR experiments were performed at 25°C on a Biacore T200 using HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20, Biacore, Freiburg / Germany) as running buffer. Human 4-1BB-Fc(kih) protein was directly coupled to the flow cell of a CM5 chip by amine coupling. An immobilization level of approximately 900RU was used.

[0267] PD-L1 targeted trimeric split 4-1BBL construct was passed through the flow cell at a concentration range of 150 nM at a flow rate of 10 μL / min for 90 seconds and dissociation was set at 0 seconds. Human PD-L1-Fc (recombinant human PD-L1 / B7-H1 Fc chimeric protein, 156-B7-100: R&D Systems) as the second analyte was injected through the flow cell at a concentration of 200 nM at a flow rate of 30 μL / min for 90 seconds (Figure 3A). Dissociation was monitored for 240 seconds. Bulk refractive index differences were corrected by subtracting the response obtained with a reference flow cell where no protein was immobilized.

[0268] As seen in Figure 3B, PD-L1-targeted 4-1BBL can simultaneously bind to human PD-L1 and human 4-1BB.

[0269] Example 3 3. Functional characterization of PD-L1-targeting 4-1BB ligand trimer-containing antigen-binding molecules by in vitro assays. 3.1. Binding to human PD-L1-expressing cell lines First, we generated a cell line expressing human PD-L1. The full-length cDNA encoding human PD-L1 was subcloned into a mammalian expression vector. The plasmid was transfected into MKN45 (DSMZ 409) cells using Lipofectamine LTX Reagent (Invitrogen, #15338100) according to the manufacturer's protocol. Stably transfected PD-L1 positive PD-L1 cells were maintained in RPMI1640 medium (GIBCO from Life Technologies, Catalog No. 42401-042) supplemented with 10% fetal bovine serum (FBS, GIBCO from Life Technologies, Catalog No. 16000-044, Lot 941273, gamma irradiated mycoplasma free, heat inactivated) and 2 mM L-alanyl-L-glutamine dipeptide (Gluta-MAX-I, GIBCO from Life Technologies, Catalog No. 35050-038) and either 200 μg / mL hygromycin B (Roche, Catalog No. 10843555001) or 1.5 μg / mL puromycin (Gibco from Life Technologies, Catalog No. A11138-02). For the binding assay, MKN45 cells and MKN45-huPD-L1 were harvested, washed with DPBS (GIBCO from life technologies, #14190-136) and stained with fixable viability dye eF450 (eBioscience #65-0863-18) in DPBS for 30 min at 4°C. Cells were washed and plated in 384-well plates (Corning #3830) at 3 × 10 4Cells were seeded at 1000 x g / well. Cells were centrifuged (350 x g, 5 min), the supernatant removed, and resuspended in 10 μL / well of FACS buffer (DPBS supplemented with 2% FBS, 5 nM EDTA, 7.5 mM sodium azide) containing titrated concentrations of PD-L1-4-1BBL or control (starting concentration 300 nM). Cells were incubated for 30 min at 4°C and then washed twice with 80 μL / well of DPBS. Cells were resuspended in 10 μL / well of FACS buffer containing 2.5 μg / mL of PE-conjugated AffiniPure anti-human IgG Fcγ fragment specific goat F(ab')2 fragment (Jackson ImmunoResearch, Cat. No. 109-116-098) for 30 min at 4°C. Cells were washed twice with 80 μL / well DPBS and then fixed in 30 μL / well DPBS containing 1% formaldehyde for at least 15 min. On the same or following day, cells were resuspended in 50 μL / well FACS buffer and acquired using a MACSQuant Analyzer X (Miltenyi Biotec).

[0270] As shown in Figures 4A and 4B, the PD-L1-4-1BBL construct (black triangles and lines), but not the non-PD-L1 control, efficiently bound to human PD-L1-expressing MKN45-huPD-L1 cells, but not to the parental cell line MKN45. 50 The values ​​and area under the curve values ​​are shown in Table 5.

[0271] Shown is the binding of PD-L1-4-1BBL to the parental cell line MKN45 and the PD-L1 expressing cell line MKN45-PD-L1. Concentrations of PD-L1-4-1BBL or control molecules are blotted against the geometric mean of the fluorescence intensity of the PE-conjugated secondary detection antibody. All values ​​are baseline corrected by subtracting the baseline value of the blank control (e.g., secondary only detection antibody, no primary). PD-L1-4-1BBL binds efficiently to human PD-L1 expressing MKN45-huPD-L1 cells (Figure 4B), but not to the parental cell line MKN45 (Figure 4A). The bispecific 4-1BBxPDL1 antibody showed even stronger binding to MKN45-huPD-L1 cells expressing human PD-L1 as PD-L1-4-1BBL.

[0272] TIFF0007675083000013.tif43170

[0273] 3.2 Activation of NF-κB in the reporter cell line Jurkat-hu4-1BB-NFκB-luc2 expressing human 4-1BB and NFκB-luciferase reporter genes Agonistic binding of the 4-1BB (CD137) receptor to its ligand (4-1BBL) induces 4-1BB downstream signaling through activation of nuclear factor kappa B (NFkB), promoting survival and activity of CD8 T cells (Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS. 4-1BB promotes the survival of CD8(+)T lymphocytes by increasing expression of Bcl-x(L) and Bfl-1. J Immunol 2002;169:4882-4888). To monitor this NFkB-activation mediated by 2+1H2H anti-4-1BB and anti-PD-L1 huIgG1 PGLALA bispecific antibody, Jurkat-hu4-1BB-NFkB-luc2 reporter cell line was purchased from Promega (Germany). Cells were cultured as previously described. For the assay, cells were harvested and resuspended in assay medium, RPMI 1640 medium supplemented with 10% (v / v) FBS and 1% (v / v) GlutaMAX-I. 3 10μl containing 1x10 Jurkat-hu4-1BB-NFκB-luc2 reporter cells were transferred to each well of a sterile white 384-well flat-bottom tissue culture plate with lid (Corning, Cat#: 3826). 10μL of assay medium containing titrated concentrations of PD-L1-4-1BBL antibodies or control molecules were added. Finally, 10μL of assay medium alone or 1x10 4 Cells were fed with assay medium containing parental MKN45 or MKN45 cells transfected with human PD-L1 and plates were incubated for 6 hours at 37°C and 5% CO2 in a cell incubator. 6μl of freshly thawed One-Glo Luciferase Assay Detection Solution (Promega, Cat#: E6110) was added to each well and luminescence emission was immediately measured using a Tecan microplate reader (500ms integration time, no filter collection at all wavelengths).

[0274] As shown in Figures 5A to 5D, in the absence of PD-L1 expressing cells, PD-L1-4-1BBL failed to induce strong human 4-1BB receptor activation in the Jurkat-hu4-1BB-NFkB-luc2 reporter cell line, resulting in activation of NFkB and therefore luciferase expression in two independent experiments. In the presence of human PD-L1 expressing MKN45 cells, crosslinking of PD-L1-4-1BBL led to a strong increase in NFkB-activated luciferase activity in the Jurkat-hu4-1BB-NFkB-luc2 reporter cell line, which exceeded the activation mediated by the non-targeting control DP47-4-1BBL. The bispecific 4-1BBxPDL1 antibody produced similar, but still slightly lower, activity. Furthermore, the anti-human 4-1BB clone 20H4.9 induced some baseline activity indicative of the superagonistic activity recently reported for this clone as huIgG1 P329G LALA (Sun K Ho et al. Mol Cancer Ther. 2020, 19(4), 1040-1051). EC 50 The values ​​and the areas under the curves (AUC) of the activation curves are shown in Table 6.

[0275] TIFF0007675083000014.tif39170

Claims

1. A 4-1BBL trimer-containing antigen binding molecule, (a) a heavy chain variable region (V Ｈ PD-L1) and a light chain variable region (V Ｌ PD-L1), and a Fab molecule capable of specifically binding to PD-L1; (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule comprises a first polypeptide comprising a CH1 or CL domain and a second polypeptide comprising a CL or CH1 domain, respectively, and the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, wherein the first polypeptide comprises two ectodomains of 4-1BBL comprising the amino acid sequence of SEQ ID NO: 5 or a 4-1BB-binding fragment thereof, connected to each other and to the CH1 or CL domain by a peptide linker, and the second polypeptide comprises one ectodomain of 4-1BBL comprising the amino acid sequence of SEQ ID NO: 5 or a 4-1BB-binding fragment thereof, connected to the CL or CH1 domain of the polypeptide via a peptide linker; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, wherein the Fc domain is an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and P329G (numbering according to the Kabat EU index) and comprises a knob-into-hole modification that promotes association of the first and second subunits of the Fc domain; Including, A 4-1BBL trimer-containing antigen-binding molecule, comprising: a first heavy chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:29; a first light chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:30; a second heavy chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:21; and a second light chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

22.

2. A first peptide comprising two ectodomains of 4-1BBL or their 4-1BB-binding fragments connected to each other by a first peptide linker is fused at its C-terminus to a CL domain that is a part of a heavy chain by a second peptide linker, and a second peptide comprising one ectodomain of 4-1BBL or its 4-1BB-binding fragment is fused at its C-terminus to a CH1 domain that is a part of a light chain by a third peptide linker. The 4-1BBL trimer-containing antigen binding molecule of claim 1.

3. The 4-1BBL trimer-containing antigen-binding molecule of claim 1 or 2, comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29, a first light chain comprising the amino acid sequence of SEQ ID NO: 30, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a second light chain comprising the amino acid sequence of SEQ ID NO:

22.

4. An isolated nucleic acid molecule encoding the 4-1BBL trimer-containing antigen-binding molecule of any one of claims 1 to 3.

5. A vector comprising the isolated nucleic acid molecule of claim 4.

6. The vector of claim 5 which is an expression vector.

7. 7. A host cell comprising the isolated nucleic acid molecule of claim 4 or the vector of claim 5 or 6.

8. A method for producing a 4-1BBL trimer-containing antigen-binding molecule according to any one of claims 1 to 3, comprising culturing a host cell according to claim 7 under conditions suitable for expression of the 4-1BBL trimer-containing antigen-binding molecule.

9. The method of claim 8, further comprising recovering the antibody from the host cell.

10. A pharmaceutical composition comprising the 4-1BBL trimer-containing antigen binding molecule of any one of claims 1 to 3 and at least one pharma- ceutically acceptable excipient.

11. 11. The pharmaceutical composition of claim 10, further comprising an additional therapeutic agent.

12. A 4-1BBL trimer-containing antigen-binding molecule according to any one of claims 1 to 3, or a pharmaceutical composition according to claim 10 or 11, for use as a medicament.

13. A 4-1BBL trimer-containing antigen binding molecule according to any one of claims 1 to 3, or a pharmaceutical composition according to claim 10 or 11, for use in the treatment of cancer.

14. The 4-1BBL trimer-containing antigen binding molecule according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 10 or 11, for use according to claim 13, wherein the 4-1BBL trimer-containing antigen binding molecule is used in combination with another therapeutic agent.

15. Use of the 4-1BBL trimer-containing antigen binding molecule according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of cancer.

16. Use of the 4-1BBL trimer-containing antigen binding molecule according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of cancer, in which the 4-1BBL trimer-containing antigen binding molecule is used in combination with another therapeutic agent.

17. A pharmaceutical for treating an individual having cancer, comprising an effective amount of a 4-1BBL trimer-containing antigen binding molecule according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 10 or 11.

18. A pharmaceutical for upregulating or prolonging cytotoxic T cell activity in an individual with cancer, comprising an effective amount of a 4-1BBL trimer-containing antigen binding molecule according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 10.

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