WO2018178074A1 - Molécules trimériques de liaison à l'antigène spécifiques pour récepteur de co-stimulation du tnf - Google Patents

Molécules trimériques de liaison à l'antigène spécifiques pour récepteur de co-stimulation du tnf Download PDF

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WO2018178074A1
WO2018178074A1 PCT/EP2018/057767 EP2018057767W WO2018178074A1 WO 2018178074 A1 WO2018178074 A1 WO 2018178074A1 EP 2018057767 W EP2018057767 W EP 2018057767W WO 2018178074 A1 WO2018178074 A1 WO 2018178074A1
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seq
amino acid
acid sequence
antigen binding
cdr
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Maria AMANN
Peter Bruenker
Christina CLAUS
Claudia Ferrara Koller
Sandra GRAU-RICHARDS
Christian Klein
Ekkehard Moessner
Pablo Umaña
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F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Publication of WO2018178074A1 publication Critical patent/WO2018178074A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • Trimeric antigen binding molecules specific for a costimulatory TNF receptor Trimeric antigen binding molecules specific for a costimulatory TNF receptor
  • the invention relates to novel trimeric antigen binding molecules comprising three fusion polypeptides, each of the three fusion polypeptides comprising (a) a moiety capable of specific binding to a costimulatory TNF receptor family member, (b) a trimerization domain, in particular a trimerization domain derived from human cartilage matrix protein (hCMP, SEQ ID NO: 1), and (c) a moiety capable of specific binding to a target cell antigen, and to methods of producing these molecules and to methods of using the same.
  • the invention further relates to methods of producing these molecules and to methods of using the same.
  • TNFR tumor necrosis factor receptor
  • T cell activation to sustain T cell responses and thus have pivotal roles in the organization and function of the immune system.
  • CD27, 4-1BB (CD137), OX40 (CD134), HVEM, CD30, and GITR can have costimulatory effects on T cells, meaning that they sustain T-cell responses after initial T cell activation (Watts T.H. (2005) Annu. Rev. Immunol. 23, 23-68).
  • the effects of these costimulatory TNFR family members can often be functionally, temporally, or spatially segregated from those of CD28 and from each other.
  • the sequential and transient regulation of T cell activation/survival signals by different costimulators may function to allow longevity of the response while maintaining tight control of T cell survival.
  • stimulation via costimulatory TNF family members can exacerbate or ameliorate disease.
  • TNFR family costimulators shows promise for several therapeutic applications, including cancer, infectious disease, transplantation, and autoimmunity.
  • the tumor necrosis factor (TNF) receptor family member OX40 (CD 134) plays a key role in the survival and homeostasis of effector and memory T cells (Croft M. et al. (2009), Immunological Reviews 229, 173-191).
  • OX40 (CD134) is expressed in several types of cells and regulates immune responses against infections, tumors and self-antigens and its expression has been demonstrated on the surface of T-cells, NKT-cells and NK-cells as well as neutrophils (Baumann R. et al. (2004), Eur. J. Immunol. 34, 2268-2275) and shown to be strictly inducible or strongly upregulated in response to various stimulatory signals.
  • This effect is likely to be responsible, at least in part, for the enhancing activity of OX40 on anti-tumor or anti-microbial immune responses.
  • OX40 engagement can expand T- cell populations, promote cytokine secretion, and support T-cell memory
  • agonists including antibodies and soluble forms of the ligand OX40L have been used successfully in a variety of preclinical tumor models (Weinberg et al. (2000), J. Immunol. 164, 2160-2169).
  • 4-1BB (CD137), a member of the TNF receptor superfamily, has been first identified as a molecule whose expression is induced by T-cell activation (Kwon Y.H. and Weissman S.M. (1989), Proc. Natl. Acad. Sci. USA 86, 1963-1967). Subsequent studies demonstrated expression of 4-1BB in T- and B-lymphocytes (Snell L.M. et al. (2011) Immunol. Rev. 244, 197-217 or Zhang X.et al. (2010), J. Immunol. 184, 787-795), NK-cells (Lin W. et al. (2008), Blood 112, 699-707, NKT-cells (Kim D.H.
  • TCR T-cell receptor
  • B-cell receptor triggering signaling induced through co-stimulatory molecules or receptors of pro-inflammatory cytokines
  • CD 137 signaling is known to stimulate IFNy secretion and proliferation of NK cells (Buechele C. et al. (2012), Eur. J. Immunol. 42, 737-748; Lin W. et al. (2008), Blood 112, 699- 707; Melero I. et al. (1998), Cell Immunol. 190, 167-172) as well as to promote DC activation as indicated by their increased survival and capacity to secret cytokines and upregulate co- stimulatory molecules (Choi B. K. et al. (2009), J. Immunol. 182, 4107-4115; Futagawa T. et al. (2002), Int. Immunol. 14, 275-286; Wilcox R. A. et al.
  • CD 137 is best characterized as a co-stimulatory molecule which modulates TCR- induced activation in both the CD4 + and CD8 + subsets of T-cells.
  • agonistic 4-lBB-specific antibodies enhance proliferation of T-cells, stimulate lymphokine secretion and decrease sensitivity of T-lymphocytes to activation- induced cells death (Snell L.M. et al. (2011) Immunol. Rev. 244, 197-217).
  • CD8 + T-cells play the most critical role in anti-tumoral effect of 4-lBB-specific antibodies.
  • contributions of other types of cells such as DCs, NK-cells or CD4 + T-cells have been reported (Murillo O. et al. (2009), Eur. J. Immunol. 39, 2424-2436; Stagg J. et al. (2011), Proc. Natl. Acad. Sci. USA 108, 7142-7147).
  • 4- IBB agonists can also induce infiltration and retention of activated T-cells in the tumor through 4-lBB-mediated upregulation of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) on tumor vascular endothelium (Palazon A. et al. (2011), Cancer Res. 71, 801-811).
  • IMM1 intercellular adhesion molecule 1
  • VCAM1 vascular cell adhesion molecule 1
  • 4- IBB triggering may also reverse the state of T-cell anergy induced by exposure to soluble antigen that may contribute to disruption of immunological tolerance in the tumor micro- environment or during chronic infections (Wilcox R.A. et al. (2004), Blood 103, 177-184).
  • fusion proteins usually has a large molecular weight and/or that the trimerization is rather inefficient.
  • WO 2007/014744, WO 2009/000538 and Wyzgol et al. (2009) disclose fusion proteins comprising a TNF cytokine and a trimerization domain from the chicken protein tenascin. Biological activity could be strongly enhanced.
  • huCMP Human cartilage matix protein
  • GITR ligands and OX40 ligands respectively, that are fused to a trimerization domain which is connected to a Fc domain.
  • these molecules lack the "tumor-targeting" through a moiety capable of binding to tumor-specific target and thus could lead to unspecific immune reactions.
  • the available pre-clinical and clinical data clearly demonstrate that there is a high clinical need for effective agonists of costimulatory TNFR family members such as OX40 and 4- IBB that are able to induce and enhance effective endogenous immune responses to cancer.
  • the antigen binding molecules of the invention combine a moiety capable of preferred binding to tumor-specific or tumor-associated targets with a moiety capable of agonistic binding to costimulatory TNF receptors.
  • the antigen binding molecules of this invention may be able to trigger TNF receptors not only effectively, but also very selectively at the desired site thereby reducing undesirable side effects.
  • the present invention provides a trimeric antigen binding molecule comprising three fusion polypeptides, each of the three fusion polypeptides comprising
  • novel trimeric antigen binding molecules of the present invention are stable, as the fusion polypeptides associate with each other through the huCMP trimerization domain.
  • the stable antigen binding molecule trimers are able to trigger TNF receptors highly effectively due to their trimeric structure, but also very selectively at the site where the target cell antigen is expressed, due to their binding capability towards a target cell antigen. Side effects may therefore be drastically reduced.
  • the trimeric antigen binding molecule comprises three fusion polypeptides, each of the three fusion polypeptides comprising a trimerization domain comprising an amino acid sequence having at least 95% identity to SEQ ID NO:2. More particularly, the trimerization domain comprises the amino acid sequence of SEQ ID NO:2.
  • the three fusion polypeptides are linked by disulphide bonds. In some embodiments, the three fusion polypeptides are linked by disulphide bonds formed between the trimerization domains of the fusion polypeptides.
  • the costimulatory TNF receptor family member is selected from OX40 and 4-lBB.
  • the moiety capable of specific binding to a costimulatory TNF receptor family member binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:3.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to OX40, wherein the moiety comprises a heavy chain variable domain (VH) comprising
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14,
  • VL light chain variable domain
  • a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO:19 and SEQ ID NO:20, and
  • a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to OX40, wherein the moiety capable of specific binding to OX40 comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:39 and a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 and SEQ ID NO:40.
  • VH heavy chain variable domain
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to OX40, wherein the moiety capable of specific binding to OX40 comprises (i) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:27 and light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO:28,
  • VH comprising the amino acid sequence of SEQ ID NO:37 and a VL comprising the amino acid sequence of SEQ ID NO:38, or
  • the moiety capable of specific binding to a costimulatory TNF receptor family member binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:41.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to 4- IBB, wherein the moiety comprises a VH comprising
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:43,
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:44 and SEQ ID NO:45
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50
  • VL comprising
  • a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51 and SEQ ID NO:52,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:53 and SEQ ID NO:54, and
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to 4-1BB, wherein the moiety capable of specific binding to 4-1BB comprises a VH comprising an amino acid sequence that is at least about 95%, 96%>, 97%, 98%, 99% or 100%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 and SEQ ID NO:68 and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 and SEQ ID
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to 4-1BB, wherein the moiety capable of specific binding to 4-1BB comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO:61,
  • VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO:67, or
  • a VH comprising the amino acid sequence of SEQ ID NO:68 and a VL comprising the amino acid sequence of SEQ ID NO:69.
  • the moiety capable of specific binding to a costimulatory TNF receptor family member is fused at the C-terminal amino acid to the N-terminal amino acid of the trimerization domain, optionally through a peptide linker.
  • the moiety capable of specific binding to a costimulatory TNF receptor family member is a Fab fragment or a scFv.
  • the target cell antigen is selected from the group consisting of Fibroblast Activation Protein (FAP), Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic Antigen (CEA), CD 19, CD20 and CD33.
  • FAP Fibroblast Activation Protein
  • MCSP Melanoma-associated Chondroitin Sulfate Proteoglycan
  • EGFR Epidermal Growth Factor Receptor
  • CEA Carcinoembryonic Antigen
  • CD 19, CD20 and CD33 CD19
  • the target cell antigen is FAP or CEA.
  • the target cell antigen is FAP.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to FAP, wherein the moiety capable of specific binding to FAP comprises a VH comprising
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:70 and SEQ ID NO:76,
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71 and SEQ ID NO:77, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72 and SEQ ID NO:78,
  • a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:73 and SEQ ID NO:79,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:74 and SEQ ID NO:80, and
  • a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:75 and SEQ ID NO:81.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to FAP, wherein the moiety capable of specific binding to FAP comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%o, 97%), 98%o, 99%> or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 82 and SEQ ID NO: 84 and a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%>, 96%>, 97%>, 98%>, 99%. or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 83 and SEQ ID NO:85.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to FAP, wherein the moiety capable of specific binding to FAP comprises
  • VH comprising the amino acid sequence of SEQ ID NO: 82 and a VL comprising the amino acid sequence of SEQ ID NO:83, or
  • a VH comprising the amino acid sequence of SEQ ID NO: 84 and a VL comprising the amino acid sequence of SEQ ID NO:85.
  • the moiety capable of specific binding to a target cell antigen is fused at the N-terminal amino acid to the C-terminal amino acid of the trimerization domain, optionally through a peptide linker.
  • three fusion polypeptides are identical.
  • the present invention also provides a fusion polypeptide comprising (a) a VH and/or VL of a moiety capable of specific binding to a costimulatory TNF receptor family member, (b) a trimerization domain derived from human cartilage matrix protein (huCMP) comprising the amino acid sequence of SEQ ID NO:2, wherein the trimerization domain is capable of mediating stable association of the fusion polypeptide with two further such fusion polypeptides and (c) a VH and/or VL of a moiety capable of specific binding to a target cell antigen.
  • huCMP human cartilage matrix protein
  • the fusion polypeptide comprises a moiety capable of specific binding to FAP, wherein the moiety capable of specific binding to FAP comprises a VH comprising
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:70 and SEQ ID NO:76,
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71 and SEQ ID NO:77, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72 and SEQ ID NO:78,
  • a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:73 and SEQ ID NO:79,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:74 and SEQ ID NO:80, and
  • a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:75 and SEQ ID NO:81.
  • the fusion polypeptide comprises a VH of a moiety capable of specific binding to OX40, wherein the VH comprises
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7, and
  • the fusion polypeptide comprises a VH of a moiety capable of specific binding to 4- IBB, wherein the VH comprises
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:43,
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50.
  • the present invention also provides a polynucleotide encoding the trimeric antigen binding molecule of the invention, or the fusion polypeptide of the invention.
  • the present invention also provides a vector, e.g. an expression vector, comprising the polynucleotide of the invention.
  • the present invention also provides a host cell comprising the polynucleotide of the invention or the expression vector of the invention.
  • the host cell is a eukaryotic cell, particularly a mammalian cell.
  • the present invention also provides a method of producing a trimeric antigen binding molecule, comprising culturing the host cell of the invention under conditions suitable for the expression of the trimeric antigen binding molecule, and isolating the trimeric antigen binding molecule.
  • the invention also encompasses a trimeric antigen binding molecule produced by the method of the invention.
  • the present invention also provides a pharmaceutical composition comprising the trimeric antigen binding molecule of the invention and at least one pharmaceutically acceptable excipient.
  • the present invention also provides the trimeric antigen binding molecule of the invention, or the pharmaceutical composition of the invention, for use as a medicament.
  • the present invention also provides the trimeric antigen binding molecule as described herein before, or the pharmaceutical composition of the invention, for use
  • the present invention also provides the trimeric antigen binding molecule of the invention, or the pharmaceutical composition of the invention, for use in the treatment of a disease in an individual in need thereof. Also provided is the trimeric antigen binding molecule of the invention, or the pharmaceutical composition of the invention, for use in the treatment of cancer.
  • the present invention also provides the use of the trimeric antigen binding molecule of the invention, or the pharmaceutical composition of the invention, in the manufacture of a disease in an individual in need thereof, in particular for the manufacture of a medicament for the treatment of cancer.
  • the present invention also provides a method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising the trimeric antigen binding molecule of the invention in a pharmaceutically acceptable form. Also provided is a method of treating an individual having cancer, said method comprising administering to the individual an effective amount of the trimeric antigen binding molecule of the invention, or the pharmaceutical composition of the invention.
  • the present invention also provides the trimeric antigen binding molecule of the invention, or the pharmaceutical composition of the invention, for use in up-regulating or prolonging cytotoxic T cell activity. Also provided is the use of the trimeric antigen binding molecule of the invention, or the pharmaceutical composition of the invention, in the manufacture of a medicament for up-regulating or prolonging cytotoxic T cell activity. Also provided is a method of up-regulating or prolonging cytotoxic T cell activity in an individual having cancer, comprising administering to the individual an effective amount of the trimeric antigen binding molecule of the invention, or the pharmaceutical composition of the invention.
  • the individual is a mammal, particularly a human.
  • FIG. 1 shows the monomeric form of Fc-linked TNF receptor antigen that was used for the preparation of TNF receptor antibodies.
  • Figure 2A shows a schematic representation of the trimeric, bispecific, antigen binding molecules comprising three anti-OX40 Fab, and three scFv capable of specific binding to FAP.
  • Figure 2B shows the trimeric, bispecific antigen binding molecules comprising three anti-4-lBB Fab, and three scFv capable of specific binding to FAP.
  • Figure 3A shows a schematic representation of the set up of the surface plasmon resonance assays for simultaneous binding of human OX40 and human FAP by the trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules.
  • Figure 3B shows a schematic representation of the set up of the surface plasmon resonance assays for simultaneous binding of human 4- IBB and human FAP by the trimeric, bispecific anti-4-lBB, anti-FAP antigen binding molecules.
  • Figures 4A to 4E show the binding of the trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules to recombinant OX40 Fc (kih) receptor and human FAP protein, as assessed by surface plasmon resonance.
  • Figure 4A shows binding of molecules comprising anti-OX40 clone 8H9
  • Figure 4B shows binding of molecules comprising anti-OX40 clone 1G
  • Figure 4C shows binding of molecules comprising anti-OX40 clone 49B
  • Figure 4D shows binding of molecules comprising anti-OX40 clone 21H
  • Figure 4E shows binding of molecules comprising anti-OX40 clone CLC-563.
  • Figures 5A to 5C show the binding of the trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules to resting and activated human CD4+ and CD 8+ T cells.
  • Figure 5 A shows binding to resting CD4+ T cells
  • Figure 5B shows binding to activated CD4+ T cells
  • Figure 5C shows binding to resting CD8+ T cells
  • Figure 5D shows binding to activated CD8+ T cells. Binding is shown as the median of fluorescence intensity (MFI) of FITC conjugated anti- human IgG F(ab') 2 -fragment-specific goat IgG F(ab")2 fragment, which is used as secondary detection antibody.
  • MFI was measured by flow cytometry and baseline corrected by subtracting the MFI of the blank control.
  • the x-axis shows the concentration of the antigen binding molecules. All OX40 clones bind to activated, OX40 expressing human CD4+ T cells, and to a lower extent to activated human CD8+ T cells. OX40 is not expressed on resting human PBMCs ( Figure 5A and 5C). After activation, OX40 is up-regulated on CD4+ and CD8+ T cells ( Figure 5B and 5D). OX40 expression on human CD8+ T cells is lower than on CD4+ T cells. The clones vary in the strength of binding (EC50 values as well as signal strength) to OX40 positive cells.
  • Figures 6A and 6B show the binding of the trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules to tumor cells which do not express FAP (FAP-), or which express FAP (FAP+).
  • Figure 6 A shows binding to CHO cells, which are negative for FAP.
  • Figure 6B shows binding to WM266-4 cells, which express high levels of human fibroblast activation protein (huFAP). Binding is shown as the median of fluorescence intensity (MFI) of FITC conjugated anti-human IgG F(ab') 2 -fragment-specific goat IgG F(ab " )2 fragment, which is used as secondary detection antibody. MFI was measured by flow cytometry. The x-axis shows the concentration of antibody constructs. The molecules are shown to bind to FAP-expressing WM266-4 cells ( Figure 6B), but not to FAP-negative CHO cells ( Figure 6A).
  • MFI median of fluorescence intensity
  • Figures 7A to 7E show activation of NFKB by the trimeric, bispecific anti-OX40, anti- FAP antigen binding molecules, in the presence or absence of crosslinking by FAP -positive cells.
  • Figure 7 A shows NFKB activation by molecules comprising anti-OX40 clone 49B4
  • Figure 7B shows NFKB activation by molecules comprising anti-OX40 clone 1G4
  • Figure 7C shows NFKB activation by molecules comprising anti-OX40 clone CLC-563
  • Figure 7D shows NFKB activation by molecules comprising anti-OX40 clone 8H9
  • Figure 7E shows NFKB activation by molecules comprising anti-OX40 clone 21H4.
  • NF-KB-mediated luciferase activity was characterized by plotting the units of released light (URL), measured during 0.5 s, versus the concentration of the antigen binding molecule (in nM). URLs are emitted due to luciferase- mediated oxidation of luciferin to oxyluciferin. The values were baseline-corrected by
  • Figures 8A to 8D show activation of NFKB by the trimeric, bispecific anti-OX40, anti- FAP antigen binding molecules, in the presence or absence of crosslinking by FAP -positive cells.
  • Figure 8A shows activation of NFKB by molecules comprising anti-OX40 clones 49B4, 1G4, CLC-356, 21H4 and 8H9, or DP47 hulgGI P329GLALA (isotype control), following hyper- crosslinking by culture in the presence of FAP-expressing NIH/3T3-huFAP clone 39 cells (2: 1 ratio of FAP + tumor cells to reporter cells),
  • Figure 8B shows activation of NFKB by molecules comprising anti-OX40 clones 49B4, 1G4, CLC-356, 21H4 and 8H9 in the absence of hyper- crosslinking.
  • NF-KB-mediated luciferase activity was characterized by plotting the units of released light (URL), measured during 0.5 s, versus the concentration of the antigen binding molecule (in nM). URLs are emitted due to luciferase-mediated oxidation of luciferin to oxyluciferin. The values are baseline-corrected by subtracting the URLs for a 'blank control' condition. Limited, dose-dependent NFkB activation observed in the absence of FAP+ cells, in line with the observation that the trimeric OX40L naturally engages three OX40 receptors on the cell surface to form the basic signaling unit.
  • a drop in bioactivity was observed after an initial increase; it may be that at high concentration, efficient hyper- crosslinking is limited by insufficient FAP molecules at the cell surface, or by steric hindrance.
  • Figure 8C shows NFKB activation in the presence of hyper-crosslinking (Figure 8A) minus NFKB activation in the absence of hyper-crosslinking (Figure 8B).
  • Figure 8D shows the data of Figure 8C represented as area under the curve (AUC).
  • Figures 9A to 9D show rescue of suboptimal TCR restimulation of preactivated CD4 T cells with plate-immobilized trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules.
  • Suboptimally PHA-L pre-activated CFSE-labeled human CD4 T cells were cultured for four days on plates pre-coated with mouse IgG Fey-specific antibodies (2 ug/mL), human IgG Fab specific antibodies (2 ug/mL), mouse anti- human CD3 antibodies (clone OKT3, [3 ng/mL]) and titrated trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules, or DP47 hulgGI P329GLALA (isotype control).
  • FIG. 9A shows the number of events
  • Figure 9B shows the percentage of proliferating (i.e. CFSElow) cells
  • Figure 9C shows the percentage of effector T cells (i.e. CD1271ow)
  • Figure 9D shows the percentage of cells with an activated phenotype (Tim-3 positive cells). All of the trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules were able to rescue suboptimal TCR stimulation of preactivated, OX40+ CD4 T cells when coated to plate.
  • Figure 10 shows the EC50 values of the plate-immobilized trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules for rescuing suboptimal TCR stimulation, as calculated from the data of Figure 9A to 9D.
  • Event count, the percentage of proliferating (CFSElow) cells, the percentage of CD1271ow and Tim-3 positive cells at day 4 were plotted against the antigen binding molecule concentration concentration, and EC50 values as measure for agonistic strength were calculated using the inbuilt sigmoidal dose response quotation in Prism4
  • Figures 11A to 11D show rescue of suboptimal TCR restimulation of preactivated CD4 T cells with plate-immobilized trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules, in the presence or absence of crosslinking by FAP-positive cells.
  • CFSE-labeled human PBMCs were activated with anti- human CD3 antibodies (clone V9, human IgGl; 2 nM) and trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules or non-targeted trivalent OX40 antigen binding molecules, at the indicated concentrations, for four days.
  • Cells were activated with anti- human CD3 antibodies (clone V9, human IgGl; 2 nM) and trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules or non-targeted trivalent OX40 antigen binding molecules, at the indicated concentrations, for four days. Cells were
  • Figures 11A and llC show the MFI of CD25 (as a marker of activation) on vital CD4+ T cells.
  • Figures 11B and 11D show the percentage of CD4+ T cells which were CD4+ CD25+ T cells. Only constructs containing FAP binding moiety were able to rescue suboptimal TCR stimulation of preactivated, OX40 + CD4 T cells when crosslinking was provided by FAP positive cells.
  • the high affinity clone 8H9 ( Figures 11A and 11B), showed peak activity at concentration of -0.1-1 nM, and a reduced activity at higher concentration.
  • Figures 12A and 12B show the correlation between the strength of binding to cells, and the strength of NFKB activation (as determined as AUC for NFKB activation; Figure 12A) and the strength of T cell activation (Figure 12B) shown for trimeric, bispecific anti-OX40, anti-FAP antigen binding molecules.
  • the binding strength is the MFI of at the highest concentration (see Figure 5) for each clone.
  • the AUC of bioactivity is the AUC calculated for NFkB activation as in Figure 8D.
  • the AUC of T cell bioactivity was calculated from dose response curves shown in Figure 11. A negative correlation between binding strength to OX40 and agonism of bioactivity was observed.
  • Figures 13 A to 13C show the binding of the trimeric, bispecific anti-4-lBB, anti-FAP antigen binding molecules to recombinant 4- IBB Fc (kih) receptor and human FAP protein, as assessed by surface plasmon resonance.
  • Figure 13A shows binding of molecules comprising anti-4-lBB clone 25G7
  • Figure 13B shows binding of molecules comprising anti-4-lBB clone 12B3
  • Figure 13C shows binding of molecules comprising anti-4-lBB clone 9B11.
  • Figures 14A to 14D show the binding of the trimeric, bispecific anti-4-lBB, anti-FAP antigen binding molecules; trimeric, bispecific isotype control DP47, anti-FAP antigen binding molecules; trimeric, monospecific anti-4-lBB antigen binding molecules; and isotype control DP47 hulgG P329G LALA molecules, to resting (naive) and activated human CD4+ and CD8+ T cells.
  • Figure 14A shows binding to naive CD8+ T cells
  • Figure 14B shows binding to naive CD4+ T cells
  • Figure 14C shows binding to activated CD8+ T cells
  • Figure 14D shows binding to activated CD4+ T cells.
  • Binding is shown as the median of fluorescence intensity (MFI) of PE conjugated anti- human IgG F(ab') 2 -fragment-specific goat IgG F(ab " )2 fragment, which is used as secondary detection antibody. MFI was measured by flow cytometry and baseline corrected by subtracting the MFI of the blank control. The x-axis shows the
  • Figure 15 shows the binding of the trimeric, bispecific anti-4-lBB, anti-FAP antigen binding molecules; trimeric, bispecific anti-4-lBB, isotype control DP47 antigen binding molecules; and monospecific, anti-4-lBB hulgGI P329G LALA antigen binding molecules to FAP-expressing NIH/3T3-huFAP clone 39 cells.
  • the trimeric, bispecific anti-4-lBB, anti-FAP antigen binding molecules are efficiently targeted to FAP, whereas the molecules lacking anti- FAP scFv did not bind to FAP-expressing NHI/3T3-huFAP clone 39 cells.
  • Figures 16A and 16B show activation of NFKB by the trimeric, bispecific anti-4-lBB, anti-FAP antigen binding molecules, in the presence of crosslinking by FAP -positive cells.
  • NF- ⁇ -mediated luciferase activity was characterized by plotting the units of released light (URL), measured during 0.5 s, versus the concentration of the antigen binding molecule (in nM). URLs are emitted due to luciferase-mediated oxidation of luciferin to oxyluciferin ( Figure 16A). The values were baseline-corrected by subtracting the URLs for a 'blank control' condition.
  • Figure 16B shows that the trimeric, bispecific anti-4-lBB, anti-FAP antigen binding molecules (clone 25G7, filled diamonds and clone 12B3, filled squares) induced NFkB activation in human 4- lBB-positive HeLa reporter cells, but the control monospecific anti-4-lBB hulgGI P329G LALA and trimeric monospecific anti-4-lBB antigen binding molecules lacking anti-FAP scFv did not trigger activation of NFKB.
  • antigen binding molecule refers in its broadest sense to a molecule that specifically binds an antigenic determinant.
  • antigen binding molecules are antibodies, antibody fragments and scaffold antigen binding proteins.
  • moiety capable of specific binding to a target cell antigen or
  • antigen binding domain capable of specific binding refers to a polypeptide molecule that specifically binds to an antigenic determinant.
  • the antigen binding moiety is able to activate signaling through its target cell antigen.
  • the antigen binding moiety is able to direct the entity to which it is attached to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant.
  • Moieties capable of specific binding to a target cell antigen include antibodies and fragments thereof as further defined herein.
  • a "moiety capable of specific binding to a target cell antigen” is an antigen binding domain comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) capable of specific binding to a target cell antigen.
  • the "moiety capable of specific binding to a costimulatory TNF receptor family member” may be a Fab fragment, a cross-Fab fragment or a scFv.
  • moieties capable of specific binding to a target cell antigen include scaffold antigen binding proteins as further defined herein, e.g. binding domains which are based on designed repeat proteins or designed repeat domains (see e.g. WO 2002/020565).
  • the term "moiety capable of specific binding to a target cell antigen” refers to the part of the molecule that comprises the area which 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 called antibody variable regions).
  • a moiety capable of specific antigen binding comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
  • VL antibody light chain variable domain
  • VH antibody heavy chain variable domain
  • the "moiety capable of specific binding to a target cell antigen” may be a scFv, a Fab fragment or a cross-Fab fragment.
  • moiety capable of specific binding to a costimulatory TNF receptor family member or "antigen binding domain capable of specific binding to a costimulatory TNF receptor family member” refers to a polypeptide molecule that specifically binds to a costimulatory TNF receptor family member.
  • the antigen binding moiety is able to activate signaling through a costimulatory TNF receptor family member.
  • Moieties capable of specific binding to a target cell antigen include antibodies and fragments thereof as further defined herein.
  • moieties capable of specific binding to a costimulatory TNF receptor family member include scaffold antigen binding proteins as further defined herein, e.g. binding domains which are based on designed repeat proteins or designed repeat domains (see e.g.
  • a moiety capable of specific binding to a costimulatory TNF receptor family member comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
  • VL antibody light chain variable domain
  • VH antibody heavy chain variable domain
  • the "moiety capable of specific binding to a costimulatory TNF receptor family member” may be a Fab fragment, a cross-Fab fragment or a scFv.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g. containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations 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.
  • the term "monospecific" antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.
  • bispecific means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants.
  • a bispecific antigen binding molecule comprises at least two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.
  • the trimeric antigen binding molecules of the present invention are bispecific, comprising a moiety capable of specific binding to a costimulatory TNF receptor family member and a moiety capable of specific binding to a target cell antigen.
  • valent denotes the presence of a specified number of binding sites in an antigen binding molecule.
  • bivalent tetravalent
  • hexavalent denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen binding molecule.
  • Valency of an antigen binding molecule may also be expressed in relation to the number of binding sites for a given antigenic determinant.
  • the trimeric antigen binding molecules of the present invention are trivalent with respect to the target target cell antigen, and trivalent with respect to the
  • the trimeric antigen binding molecules comprise six binding sites (3+3).
  • full length antibody “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.
  • Native antibodies refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded.
  • each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region.
  • VH variable heavy domain
  • CH2 heavy chain variable domain
  • each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region.
  • the heavy chain of an antibody may be assigned to one of five types, called a (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG), or ⁇ (IgM), some of which may be further divided into subtypes, e.g.
  • the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • An "antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • 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.
  • scFv single-chain antibody molecules
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson et al, Nat Med 9, 129-134 (2003); and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al, Nat Med 9, 129-134 (2003).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S. Patent No. 6,248,516 Bl).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab fragment refers to an antibody fragment comprising a light chain fragment comprising a light chain variable domain (VL) and a constant domain of a light chain (CL), and a heavy chain variable domain (VH) and a first constant domain (CHI) of a heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteins from the antibody hinge region.
  • Fab'-SH are Fab' fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.
  • cross-Fab fragment or "xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged.
  • Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are 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 (CHI), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL).
  • This crossover Fab molecule is also referred to as CrossFab (VLVH).
  • the crossover Fab molecule comprises 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 (CHI).
  • This crossover Fab molecule is also referred to as CrossFab (CLCHI).
  • a “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, b) VL-CL-linker-VH-CHl, c) VH-CL-linker-VL-CHl or d) VL-CH1 -linker- VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.
  • Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CHI domain.
  • these single chain Fab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g. position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).
  • a “crossover single chain Fab fragment” or “x-scFab” is a is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N- terminal to C-terminal direction: a) VH-CL-linker-VL-CHl and b) VL-CH1 -linker- VH-CL; wherein VH and VL form together an antigen-binding site which binds specifically to an antigen and wherein said linker is a polypeptide of at least 30 amino acids.
  • these x-scFab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g. position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).
  • a "single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy (V H ) and light chains (V L ) of an antibody, connected with a short linker peptide of ten to about 25 amino acids.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V H with the C-terminus of the V L , or vice versa.
  • This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.
  • scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96).
  • antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.
  • fibronectin and designed ankyrin repeat proteins have been used as alternative scaffolds for antigen- binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008).
  • a scaffold antigen binding protein is selected from the group consisting of CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derived molecule such as Z-domain of Protein A (Affibody), an A-domain (Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrin repeat protein (DARPin), a variable domain of antibody light chain or heavy chain (single-domain antibody, sdAb), a variable domain of antibody heavy chain (nanobody, aVH), V NAR fragments, a fibronectin (AdNectin), a C-type lectin domain (Tetranectin); a variable domain of a new antigen receptor beta-lactamase (V NAR fragments), a human gamma- crystallin or ubiquitin (Affilin molecules); a kunitz type domain of human protease inhibitors, microbodies such as the proteins from the knot
  • CTLA-4 Cytotoxic T Lymphocyte-associated Antigen 4
  • CTLA-4 is a CD28-family receptor expressed on mainly CD4+ T-cells. Its extracellular domain has a variable domain- like Ig fold. Loops corresponding to CDRs of antibodies can be substituted with heterologous sequence to confer different binding properties.
  • CTLA-4 molecules engineered to have different binding specificities are also known as Evibodies (e.g. US7166697B1). Evibodies are around the same size as the isolated variable region of an antibody (e.g. a domain antibody). For further details see Journal of Immunological Methods 248 (1-2), 31-45 (2001).
  • Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid beta-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B1 and US20070224633.
  • An affibody is a scaffold derived from Protein A of Staphylococcus aureus which can be engineered to bind to antigen.
  • the domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP 1641818A1.
  • Avimers are multidomain proteins derived from the A-domain scaffold family.
  • the native domains of approximately 35 amino acids adopt a defined disulfide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature Biotechnology 23(12), 1556 - 1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).
  • a transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop. Examples of engineered transferrin scaffolds include the Trans-body. For further details see J. Biol. Chem 274, 24066-24073 (1999). Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33 residue motif consisting of two alpha- helices and a beta-turn.
  • DARPins Designed Ankyrin Repeat Proteins
  • a single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain.
  • the first single domains were derived from the variable domain of the antibody heavy chain from came lids (nanobodies or V H H fragments).
  • the term single-domain antibody includes an autonomous human heavy chain variable domain (aVH) or VN AR fragments derived from sharks.
  • Fibronectin is a scaffold which can be engineered to bind to antigen.
  • Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the .beta. -sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest.
  • Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site. For further details see Expert Opin. Biol. Ther. 5, 783-797 (2005).
  • Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges - examples of microproteins include KalataBI and conotoxin and knottins.
  • the microproteins have a loop which can beengineered to include upto 25 amino acids without affecting the overall fold of the microprotein.
  • engineered knottin domains see WO2008098796.
  • an "antigen binding molecule that binds to the same epitope" as a reference molecule refers to an antigen binding molecule that blocks binding of the reference molecule to its antigen in a competition assay by 50% or more, and conversely, the reference molecule blocks binding of the antigen binding molecule to its antigen in a competition assay by 50% or more.
  • an antigen binding domain refers to the part of an antigen binding molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen binding molecule may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions).
  • an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • antigenic determinant is synonymous with “antigen” and “epitope,” and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety- antigen complex.
  • Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • the proteins useful as antigens herein can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the antigen is a human protein.
  • the term encompasses the "full-length", unprocessed protein as well as any form of the protein that results from processing in the cell.
  • the term also encompasses naturally occurring variants of the protein, e.g. splice variants or allelic variants.
  • ELISA enzyme-linked immunosorbent assay
  • SPR Surface Plasmon Resonance
  • the extent of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the binding of the antigen binding molecule to the antigen as measured, e.g. by SPR.
  • an molecule that binds to the antigen has a dissociation constant (Kd) of ⁇ 1 ⁇ , ⁇ 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. from 10 ⁇ 8 M to 10 "13 M, e.g. from 10 "9 M to 10 "13 M).
  • Binding affinity refers to the strength of the sum total 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 indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g.
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (koff and kon, respectively).
  • Kd dissociation constant
  • equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same.
  • Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).
  • target cell antigen refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma.
  • the target cell antigen is an antigen on the surface of a tumor cell.
  • target cell antigen is selected from the group consisting of Fibroblast Activation Protein (FAP), Carcinoembryonic Antigen (CEA), Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), CD 19, CD20 and CD33.
  • FAP Fibroblast Activation Protein
  • CEA Carcinoembryonic Antigen
  • MCSP Melanoma-associated Chondroitin Sulfate Proteoglycan
  • EGFR Epidermal Growth Factor Receptor
  • CD 19, CD20 and CD33 CD19
  • the target cell antigen is Fibroblast Activation Protein (FAP).
  • FAP Fibroblast activation protein
  • Prolyl endopeptidase FAP or Seprase EC 3.4.21
  • FAP Fibroblast activation protein
  • mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the term encompasses "full-length,” unprocessed FAP as well as any form of FAP that results from processing in the cell.
  • the term also encompasses naturally occurring variants of FAP, e.g., splice variants or allelic variants.
  • the antigen binding molecule of the invention is capable of specific binding to human, mouse and/or cynomolgus FAP.
  • the amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession no. Q12884 (version 149, SEQ ID NO:86), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP 004451.2.
  • the extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760.
  • the amino acid and nucleotide sequences of a His- tagged human FAP ECD is shown in SEQ ID NOs 87 and 88, respectively.
  • mouse FAP The amino acid sequence of mouse FAP is shown in UniProt accession no. P97321 (version 126, SEQ ID NO:89), or NCBI RefSeq NP 032012.1.
  • the extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761.
  • SEQ ID NOs 90 and 91 show the amino acid and nucleotide sequences, respectively, of a His-tagged mouse FAP ECD.
  • SEQ ID NOs 92 and 93 show the amino acid and nucleotide sequences, respectively, of a His-tagged cynomolgus FAP ECD.
  • an anti-FAP binding molecule of the invention binds to the extracellular domain of FAP. Exemplary anti-FAP binding molecules are described in International Patent Application No. WO 2012/020006 A2.
  • CEA Carcinoembryonic antigen-related cell adhesion molecule 5
  • CEACAM5 Carcinoembryonic antigen- related cell adhesion molecule 5
  • CEA cynomolgus monkeys
  • rodents e.g. mice and rats
  • the amino acid sequence of human CEA is shown in UniProt accession no. P06731 (version 151, SEQ ID NO: 94).
  • CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med., 121 :439-462, 1965; Berinstein N. L., J Clin Oncol, 20:2197-2207, 2002). Originally classified as a protein expressed only in fetal tissue, CEA has now been identified in several normal adult tissues.
  • tumors of epithelial origin contain CEA as a tumor associated antigen. While the presence of CEA itself does not indicate transformation to a cancerous cell, the distribution of CEA is indicative. In normal tissue, CEA is generally expressed on the apical surface of the cell (Hammarstrom S., Semin Cancer Biol.
  • CEA tends to be expressed over the entire surface of cancerous cells (Hammarstrom S., Semin Cancer Biol. 9(2):67-81 (1999)). This change of expression pattern makes CEA accessible to antibody binding in cancerous cells.
  • CEA expression increases in cancerous cells.
  • increased CEA expression promotes increased intercellular adhesions, which may lead to metastasis (Marshall J., Semin Oncol, 30(a Suppl. 8):30-6, 2003).
  • the prevalence of CEA expression in various tumor entities is generally very high.
  • CEA colorectal carcinoma
  • NSCLC non-small cell lung cancer
  • HER3 non-small cell lung cancer
  • HER3 non-small cell lung cancer
  • breast cancer low expression was found in small cell lung cancer and glioblastoma.
  • CEA is readily cleaved from the cell surface and shed into the blood stream from tumors, either directly or via the lymphatics. Because of this property, the level of serum CEA has been used as a clinical marker for diagnosis of cancers and screening for recurrence of cancers, particularly colorectal cancer (Goldenberg D M., The International Journal of Biological
  • MCSP Chondroitin Sulfate Proteoglycan
  • CSPG4 Chondroitin Sulfate Proteoglycan 4
  • the amino acid sequence of human MCSP is shown in UniProt accession no. Q6UVK1 (version 103, SEQ ID NO:95).
  • Epidermal Growth Factor Receptor also named Proto- oncogene c-ErbB-1 or Receptor tyro sine-protein kinase erbB-1, refers to any native EGFR 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), unless otherwise indicated.
  • the amino acid sequence of human EGFR is shown in UniProt accession no. P00533 (version 211, SEQ ID NO:96).
  • CD19 refers to B-lymphocyte antigen CD19, also known as B-lymphocyte surface antigen B4 or T-cell surface antigen Leu- 12 and includes any native CD 19 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), unless otherwise indicated.
  • the amino acid sequence of human CD19 is shown in Uniprot accession no. P15391 (version 160, SEQ ID NO:97). The term encompasses "full-length" unprocessed human CD19 as well as any form of human CD 19 that results from processing in the cell as long as the antibody as reported herein binds thereto.
  • CD 19 is a structurally distinct cell surface receptor expressed on the surface of human B cells, including, but not limited to, pre-B cells, B cells in early development ⁇ i.e., immature B cells), mature B cells through terminal differentiation into plasma cells, and malignant B cells.
  • CD 19 is expressed by most pre-B acute lymphoblastic leukemias (ALL), non- Hodgkin's lymphomas, B cell chronic lymphocytic leukemias (CLL), pro-lymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias, and some Null-acute lymphoblastic leukemias.
  • ALL pre-B acute lymphoblastic leukemias
  • CLL B cell chronic lymphocytic leukemias
  • pro-lymphocytic leukemias pro-lymphocytic leukemias
  • hairy cell leukemias common acute lymphocytic leukemias
  • CD 19 on plasma cells further suggests it may be expressed on differentiated B cell tumors such as multiple myeloma. Therefore, the CD 19 antigen is a target for immunotherapy in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia.
  • CD20 refers to B-lymphocyte antigen CD20, also known as membrane-spanning 4- domains subfamily A member 1 (MS4A1), B-lymphocyte surface antigen Bl or Leukocyte surface antigen Leu- 16, and includes any native CD20 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), unless otherwise indicated.
  • the amino acid sequence of human CD20 is shown in Uniprot accession no. PI 1836 (version 149, SEQ ID NO:98).
  • CD33 refers to Myeloid cell surface antigen CD33, also known as SIGLEC3 or gp67, and includes any native CD33 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), unless otherwise indicated.
  • the amino acid sequence of human CD33 is shown in Uniprot accession no. P20138 (version 157, SEQ ID NO:99).
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL may be sufficient to confer antigen-binding specificity.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops").
  • native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
  • HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition.
  • CDRs complementarity determining regions
  • Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50- 52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3).
  • Exemplary CDRs CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of HI, 50-65 of H2, and 95-102 of H3.
  • Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • Hypervariable regions are also referred to as complementarity determining regions (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions. This particular region has been described by Kabat et al, U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al, J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other.
  • Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody.
  • Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody.
  • One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering” to any variable region sequence, without reliance on any experimental data beyond the sequence itself.
  • Kabat numbering refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system.
  • CDRs generally comprise the amino acid residues that form the hypervariable loops.
  • CDRs also comprise "specificity determining residues,” or "SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.
  • Exemplary a-CDRs (a-CDR-Ll, a-CDR-L2, a-CDR-L3, a- CDR-H1 , a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI , 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3.
  • HVR residues and other residues in the variable domain are numbered herein according to Kabat et al., supra.
  • affinity matured in the context of antigen binding molecules (e.g., antibodies) 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 binds to the same epitope, as the reference antibody; and has a higher affinity for the antigen than that of 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.
  • the affinity matured antigen binding molecule binds to the same epitope as the initial reference antigen binding mo lecule .
  • FR Framework
  • HVR hypervariable region residues
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • An "acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
  • an acceptor human framework "derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 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 human consensus framework sequence.
  • the term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ respectively.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non- human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, 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 region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
  • a "human” antibody is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non- human antigen-binding residues.
  • Fc domain or "Fc region” herein is used to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain.
  • the "CH2 domain” of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340.
  • a carbohydrate chain is attached to the CH2 domain.
  • the CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain.
  • the "CH3 domain” comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e.
  • the CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with an introduced "protuberance” ("knob”) in one chain thereof and a corresponding introduced “cavity” ("hole”) in the other chain thereof; see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • 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 "knob-into-hole" technology is described e.g. in US 5,731,168; US 7,695,936;
  • the method involves introducing a protuberance ("knob”) at the interface of a first polypeptide and a corresponding cavity ("hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain
  • the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain.
  • the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C
  • the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in 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)).
  • a "region equivalent 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 alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to mediate effector functions (such as antibody- dependent cellular cytotoxicity).
  • one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function.
  • Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J. U. et al, Science 247: 1306-10 (1990)).
  • effector functions refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • antibody effector functions include: Clq 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, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.
  • Fc receptor binding dependent effector functions can be mediated by the interaction of the Fc-region of an antibody with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells.
  • Fc receptors belong to the immunoglobulin superfamily, and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC) (see e.g. Van de Winkel, J.G. anderson, C.L., J. Leukoc. Biol. 49 (1991) 511-524).
  • ADCC antibody dependent cell mediated cytotoxicity
  • FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcyR. Fc receptor binding is described e.g. in Ravetch, J.V.
  • FcyR Fc-region of IgG antibodies
  • FcyRI binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils.
  • Modification in the Fc-region IgG at least at one of the amino acid residues E233-G236, P238, D265, N297, A327 and P329 (numbering according to EU index of Kabat) reduce binding to FcyRI.
  • FcyRII A is found on many cells involved in killing (e.g. macrophages, monocytes, neutrophils) and seems able to activate the killing process.
  • FcyRIIB seems to play a role in inhibitory processes and is found on B cells, macrophages and on mast cells and eosinophils. On B-cells it seems to function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class.
  • FcyRIIB acts to inhibit phagocytosis as mediated through FcyRIIA.
  • the B-form may help to suppress activation of these cells through IgE binding to its separate receptor.
  • Reduced binding for FcyRIIA is found e.g. for antibodies comprising an IgG Fc-region with mutations at least at one of the amino acid residues E233- G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to EU index of Kabat).
  • FcyRIII (CD 16) binds IgG with medium to low affinity and exists as two types.
  • FcyRIIIA is found on NK cells, macrophages, eosinophils and some monocytes and T cells and mediates ADCC.
  • Fc ⁇ RIIIB is highly expressed on neutrophils. Reduced binding to FcyRIIIA is found e.g.
  • antibodies comprising an IgG Fc-region with mutation at least at one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 (numbering according to EU index of Kabat).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCC is investigated by measuring their binding to Fey receptors expressing cells, such as cells, recombinantly expressing FcyRI and/or FcyRIIA or NK cells (expressing essentially FcyRIIIA). In particular, binding to FcyR on NK cells is measured.
  • an “activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions.
  • Activating Fc receptors include FcyRIIIa (CD 16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).
  • a particular activating Fc receptor is human FcyRIIIa (see UniProt accession no. P08637, version 141).
  • TNF receptor superfamily or "TNF receptor superfamily” currently consists of 27 receptors. It is a group of cytokine receptors characterized by the ability to bind tumor necrosis factors (TNFs) via an extracellular cysteine-rich domain (CRD). These pseudorepeats are defined by intrachain disulphides generated by highly conserved cysteine residues within the receptor chains. With the exception of nerve growth factor (NGF), all TNFs are homologous to the archetypal TNF-alpha. In their active form, the majority of TNF receptors form trimeric complexes in the plasma membrame. Accordingly, most TNF receptors contain transmembrane domains (TMDs).
  • TNFs transmembrane domains
  • TNF superfamily receptors that recruit caspase-interacting proteins following ligand binding to initiate the extrinsic pathway of caspase activation.
  • Other TNF superfamily receptors that lack death domains bind TNF receptor-associated factors and activate intracellular signaling pathways that can lead to proliferation or differentiation. These receptors can also initiate apoptosis, but they do so via indirect mechanisms.
  • TNF superfamily receptors are involved in regulating immune cell functions such as B cell homeostasis and activation, natural killer cell activation, and T cell co-stimulation.
  • TNF superfamily receptors are involved in regulating immune cell functions such as B cell homeostasis and activation, natural killer cell activation, and T cell co-stimulation.
  • TNF type-specific responses such as hair follicle development and osteoclast development.
  • TNF receptor superfamily include the following: Tumor necrosis factor receptor 1 (1 A) (TNFRSF1A, CD120a), Tumor necrosis factor receptor 2 (IB) (TNFRSF1B, CD120b), Lymphotoxin beta receptor (LTBR, CD 18), OX40 (TNFRSF4, CD 134), CD40 (Bp50), Fas receptor (Apo-1, CD95, FAS), Decoy receptor 3 (TR6, M68, TNFRSF6B), CD27 (S152, Tp55), CD30 (Ki-1, TNFRSF8), 4- IBB (CD137, TNFRSF9), DR4 (TRAILR1, Apo-2, CD261, TNFRSF10A), DR5 (TRAILR2, CD262, TNFRSF10B), Decoy Receptor 1 (TRAILR3, CD263, TNFRSF10C), Decoy Receptor 2 (TRAILR4, CD264, TNFRSF10D), RANK (CD265,
  • TNFRSF11A Osteoprotegerin
  • OCIF Osteoprotegerin
  • TR1, TNFRSF1 IB TWEAK receptor
  • Fnl4 CD266, TNFRSF12A
  • TACI CD267, TNFRSF13B
  • BAFF receptor CD268, TNFRSF13C
  • HVEM Herpesvirus entry mediator
  • p75NTR Nerve growth factor receptor
  • p75NTR B-cell maturation antigen
  • B-cell maturation antigen CD269, TNFRSF17
  • Glucocorticoid- induced TNFR-related GITR, AITR, CD357, TNFRSF18
  • TROY TNFRSF19
  • DR6 CD358, TNFRSF21
  • DR3 Apo-3, TRAMP, WS-1, TNFRSF25
  • Ectodysplasin A2 receptor XEDAR, EDA2R
  • costimulatory TNF receptor family member or “costimulatory TNF family receptor” refers to a subgroup of TNF receptor family members, which are able to costimulate proliferation and cytokine production of T-cells.
  • the term refers to any native TNF family receptor 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), unless otherwise indicated.
  • costimulatory TNF receptor family members are selected from the group consisting of OX40 (CD134), 4-1BB (CD137), CD27, HVEM (CD270), CD30, and GITR, all of which can have costimulatory effects on T cells. More particularly, the costimulatory TNF receptor family member is selected from the group consisting of OX40 and 4- IBB.
  • sequences, of the TNF receptor family members may be obtained from publically accessible databases such as Uniprot (www.uniprot.org).
  • the human costimulatory TNF receptors have the following amino acid sequences: human OX40 (UniProt accession no. P43489, SEQ ID NO: 100), human 4-1BB (UniProt accession no.
  • OX40 refers to any native OX40 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses "full-length,” unprocessed OX40 as well as any form of OX40 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of OX40, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human OX40 is shown in SEQ ID NO: 100 (Uniprot P43489, version 112) and the amino acid sequence of an exemplary murine OX40 is shown in SEQ ID NO: 106 (Uniprot P47741, version 101).
  • an antibody that specifically binds to OX40 refers to an antibody that is capable of binding OX40 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting OX40.
  • the extent of binding of an anti-OX40 antibody to an unrelated, non- OX40 protein is less than about 10% of the binding of the antibody to OX40 as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry (FACS).
  • an antibody that binds to OX40 has a dissociation constant ( K D ) of ⁇ ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM,
  • nM ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 "6 M or less, e.g. from 10 ⁇ 68 M to 10 "13 M, e.g., from 10 ⁇ 8 M to 10 "10 M).
  • 4-lBB refers to any native 4-lBB from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses "full-length,” unprocessed 4- IBB as well as any form of 4- IBB that results from processing in the cell.
  • the term also encompasses naturally occurring variants of 4-lBB, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human 4- IBB is shown in SEQ ID NO: 101 (Uniprot accession no.
  • amino acid sequence of an exemplary murine 4- IBB is shown in SEQ ID NO: 107 (Uniprot accession no. P20334) and the amino acid sequence of an exemplary cynomolgous 4-lBB (from Macaca mulatta) is shown in SEQ ID NO: 108 (Uniprot accession no. F6W5G6).
  • anti-4-lBB antibody refers to an antibody that is capable of binding 4- IBB with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting 4- IBB.
  • the extent of binding of an anti-4-lBB antibody to an unrelated, non- 4-1BB protein is less than about 10% of the binding of the antibody to 4-lBB as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry (FACS).
  • an antibody that binds to 4- IBB has a dissociation constant ( K D ) of ⁇ ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM,
  • nM ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 "6 M or less, e.g. from 10 ⁇ 68 M to 10 "13 M, e.g., from 10 ⁇ 8 M to 10 ⁇ 10 M).
  • peptide linker refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids.
  • Peptide linkers are known in the art or are described herein.
  • Suitable, non-immunogenic linker peptides are, for example, (G 4 S) n , (SG 4 ) n or G 4 (SG 4 ) n peptide linkers, wherein "n” is generally a number between 1 and 10, typically between 1 and 4, in particular 2, i.e.
  • GGGGS GGGGGGS
  • GGGGSGGGGS SEQ ID NO: 110
  • SGGGGSGGGG SEQ ID NO: 111
  • G 4 S 3 or GGGGSGGGGSGGGGS
  • GGGGGGGGGG or G4(SG4) 2 SEQ ID NO: 113
  • G 4 S 4 or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 114)
  • GSPGSSSSGS SEQ ID NO: 115
  • GSGSGS SEQ ID NO: 116
  • GSGSGNGS SEQ ID NO: 117
  • GGSGSGSG SEQ ID NO: 118
  • GGSGSG SEQ ID NO: 119
  • GGSG SEQ ID NO: 120
  • GGSGNGSG SEQ ID NO: 121
  • GGNGSGSGSG SEQ ID NO:122
  • GGNGSG SEQ ID NO:
  • amino acid denotes the group of naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, 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).
  • a “fusion polypeptide” as used herein refers to a single chain polypeptide comprising a heavy chain variable domain (VH) and/or light chain variable domain (VL) of a moiety capable of specific binding to a costimulatory TNF receptor family member, fused to a trimerization domain.
  • the fusion polypeptide further comprises a VH and/or VL of a moiety capable of specific binding to a target cell antigen.
  • the fusion may occur by direc linking of the N- or C-terminal amino acids of the respective domains via a peptide linker.
  • fused or “connected” it is meant that the components are linked by peptide bonds, either directly or via one or more peptide linkers.
  • the fusion polypeptide comprises a scFv fragment capable of specific binding to a costimulatory TNF receptor family member (i.e. the VH and VL domains of a scFv fragment). In some embodiments, the fusion polypeptide comprises the VH and CHI domains of a Fab fragment capable of binding to a costimulatory TNF receptor family member. In some embodiments, the fusion polypeptide comprises the VL and CL domains of a Fab fragment capable of binding to a costimulatory TNF receptor family member. In some embodiments, the fusion polypeptide comprises a scFv fragment capable of specific binding to a target cell antigen (i.e. the VH and VL domains of a scFv fragment). In some embodiments, the fusion polypeptide comprises the VH and CHI domains of a Fab fragment capable of binding to a target cell antigen. In some embodiments, the fusion polypeptide comprises the fusion polypeptide comprises the VH and CHI domain
  • polypeptide comprises the VL and CL domains of a Fab fragment capable of binding to a target cell antigen.
  • trimerization domain refers to an amino acid sequence within a polypeptide that promotes self-assembly by associating with two other trimerization domains to form a trimer. The term is also use to refer to the polynucleotide encoding said amino acid sequence.
  • the trimerization domain comprises an amino acid sequence able to form an alpha-helicial coiled-coil domain or an isoleucine zipper domain. Suitable trimerization domains include
  • TRAF2 (UniProt accession no. Q12933, SEQ ID NO: 124), in particular amino acids 299 to 348 or amino acids 310 to 349), Thrombospondin 1 (UniProt accession no. P07996, SEQ ID NO: 124
  • Matrilin-4 (UniProt accession no. 095460, SEQ ID NO: 126), in particular amino acids 594 to 618; CMP (matrilin-1) (Uniprot accession No. P21941, SEQ ID NO: l), in particular amino acids 454 to 496, and Cubilin (UniProt accession no. 060494, SEQ ID NO: 127), in particular amino acids 104 to 138.
  • An exemplary isoleucine zipper domain is the engineered yeast GCN4 isoleucine variant described by Harbury et al. (1993) Science 262, 1401- 1407 comprising the amino acid sequence of SEQ ID NO: 128.
  • a particular trimerization domain is "huCMP” or “CMP” or "human cartilage matrix protein", a protein that is also known as matrilin-1 or MATN1 or CRTM (UniProt accession no. P21941, SEQ ID NO: l).
  • the term “huCMP trimerization domain” or “trimerization domain derived from human cartilage matrix protein (CMP)” refers to a polypeptide structure capable of associating with two similar or identical polypeptides to form a stable trimer. The trimerisation is mediated through ionic bonds and other non-covalent bonds formed between adjacent charged amino acids of the polypeptide chains.
  • the huCMP trimerization domain has been described e.g. in Beck et al (1996), J.
  • a huCMP trimerization domain of particular context comprises a sequence having at least 95% identity and most preferably at least 98% identity to SEQ ID NO 2.
  • said trimerization domain comprises the sequence of SEQ ID NO. 2. "Percent (%) amino acid sequence identity" with respect to a reference polypeptide
  • protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN. SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN- 2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 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 the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • amino acid sequence variants of the trimeric antigen binding molecules are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the trimeric antigen binding molecules.
  • Amino acid sequence variants of the trimeric antigen binding molecules may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecules, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
  • Sites of interest for substitutional mutagenesis include the HVRs and Framework (FRs). Conservative substitutions are provided in Table B under the heading "Preferred Substitutions" and further described below in reference to amino acid side chain classes (1) to (6). Amino acid substitutions may be introduced into the molecule of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Table B.
  • Amino acids may be grouped according to common side-chain properties:
  • amino acid sequence variants includes substantial variants wherein 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).
  • a parent antigen binding molecule e.g. a humanized or human antibody.
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antigen binding molecule and/or will have substantially retained certain biological properties of the parent antigen binding molecule.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antigen binding molecules displayed on phage and screened for a particular biological activity (e.g. binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antigen binding molecule to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antigen binding molecule complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include the fusion to the N- or C-terminus to a polypeptide which increases the serum half-life of the trimeric antigen binding molecules.
  • the trimeric antigen binding molecules provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation variants of the molecules may be conveniently obtained by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the trimeric antigen binding molecules provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antigen binding molecule 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), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane,
  • polyethylene/maleic anhydride copolymer ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • polynucleotide refers to an isolated nucleic acid molecule or construct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA).
  • mRNA messenger RNA
  • pDNA virally-derived RNA
  • a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g. an amide bond, such as found in peptide nucleic acids (PNA).
  • nucleic acid molecule refers to any one or more nucleic acid segments, e.g. DNA or RNA fragments, present in a polynucleotide.
  • isolated nucleic acid molecule or polynucleotide is intended a nucleic acid molecule,
  • DNA or RNA which has been removed from its native environment.
  • DNA or RNA which has been removed from its native environment.
  • recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention.
  • Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.
  • a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
  • nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g.
  • expression cassette refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell.
  • the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
  • the expression cassette of the invention comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
  • vector or "expression vector” is synonymous with "expression construct” and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • the expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery.
  • the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • a host cell is any type of cellular system that can be used to generate the bispecific antigen binding molecules of the present invention.
  • Host cells include cultured cells, e.g.
  • mammalian cultured cells such as CHO cells, HEK cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • An "effective amount" of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.
  • a “therapeutically effective amount” of an agent 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 for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
  • an “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non- human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human. In some embodiments, an individual or subject may be a patient.
  • pharmaceutical composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable excipient” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable excipient includes, but is not limited to, a buffer, a stabilizer, or a preservative.
  • treatment and grammatical variations thereof such as “treat” or
  • treating refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect
  • the molecules of the invention are used to delay development of a disease or to slow the progression of a disease.
  • cancer refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, 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
  • the invention provides novel trimeric antigen binding molecules with particularly advantageous properties such as producibility, stability, binding affinity, biological activity, targeting efficiency and reduced toxicity.
  • the present invention provides a trimeric antigen binding molecule comprising three fusion polypeptides, each of the three fusion polypeptides comprising
  • a trimeric antigen binding molecule comprising three fusion polypeptides, each of the three fusion polypeptides comprising
  • a trimerization domain can be derived from a polypeptide derived from a protein selected from the group consisting of human TRAF2, human thrombospondin 1, human matrilin-4, human cartilage matrix protein (huCMP) and human cubilin. It can also be an isoleucine zipper domain, for example the peptide with the amino acid sequence of SEQ ID NO: 128.
  • a trimeric antigen binding molecule of the invention comprises a trimerization domain derived from human cartilage matrix protein (huCMP) of amino acid sequence of SEQ ID NO: 1.
  • the trimerization domain derived from human cartilage matrix protein (huCMP) comprises at least a part of SEQ ID NO.: 1.
  • the trimerization domain comprises an amino acid sequence having at least 95% identity and most preferably at least 98% identity to SEQ ID NO:2. More particularly, the trimerization domain comprises the amino acid sequence of SEQ ID NO:2.
  • the trimerization domain derived from human cartilage matrix protein (CMP) is herein further referred to as "huCMP trimerization domain”.
  • the trimeric antigen binding molecule according to the present invention may associate to form trimers through disulphide bonds formed between the trimerisation domains of the fusion polypeptides.
  • the trimeric antigen binding molecule comprises three fusion polypeptides, each of the three fusion polypeptides comprising a trimerization domain comprising an amino acid sequence having at least 95% identity and in particular at least 98% identity to SEQ ID NO:2. More particularly, each of the three fusion polypeptides comprises one trimerization domain comprising the amino acid sequence of SEQ ID NO:2.
  • the moiety capable of specific binding to a costimulatory TNF receptor family member is selected from the group consisting of is selected from the group consisting of an antibody, an antibody fragment and a scaffold antigen binding protein.
  • the moiety capable of specific binding to a costimulatory TNF receptor family member is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, an aVH and a scaffold antigen binding protein.
  • the invention provides a trimeric antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to a costimulatory TNF receptor family member is an antibody fragment.
  • the antibody fragment is selected from the group consisting of a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, and aVH.
  • the moiety capable of specific binding to a target cell antigen is a single chain Fab molecule.
  • the moiety capable of specific binding to a target cell antigen is a single domain antibody or an aVH.
  • the invention provides a trimeric antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to a costimulatory TNF receptor family member is a scaffold antigen binding protein.
  • the moiety capable of specific binding to a costimulatory TNF receptor family member can be a specifically designed ankyrin repeat protein.
  • the invention is concerned with a trimeric antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to a costimulatory TNF receptor family member is a Fab molecule capable of specific binding to a costimulatory TNF receptor family member.
  • the invention provides an antigen binding molecule comprising (a) a Fab molecule capable of specific binding to a costimulatory TNF receptor family member, (b) a trimerization domain derived from human cartilage matrix protein (huCMP) of amino acid sequence of SEQ ID NO: 1 , and (c) a moiety capable of specific binding to a target cell antigen.
  • said Fab molecule is fused at the C-terminal amino acid of the CHI domain to the N-terminal amino acid of the trimerization domain, optionally through a peptide linker as defined herein.
  • the costimulatory TNF receptor family member is selected from the group consisting of 4- IBB, OX40 and GITR. More particularly the costimulatory TNF receptor family member is selected from OX40 and 4- IBB.
  • the moiety capable of specific binding to a costimulatory TNF receptor family member binds to OX40. In some embodiments, the moiety capable of specific binding to a costimulatory TNF receptor family member binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:3.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to OX40, wherein the moiety comprises a VH comprising
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12, SEQ ID NO:
  • a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20, and
  • a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to OX40, wherein the moiety capable of specific binding to OX40 comprises a VH comprising an amino acid sequence that is at least about 95%, 96%>, 97%, 98%>, 99% or 100%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO :37 and SEQ ID NO:39 and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%) or 100%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 and SEQ ID NO:40.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to OX40, wherein the mo
  • VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, CDR-H2 comprising the amino acid sequence of SEQ ID NO:6, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 8 and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO : 15 , CDR-L2 comprising the amino acid sequence of SEQ ID NO : 18 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:21,
  • VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, CDR-H2 comprising the amino acid sequence of SEQ ID NO:6, CDR-H3 comprising the amino acid sequence of SEQ ID NO:9 and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO : 15 , CDR-L2 comprising the amino acid sequence of SEQ ID NO : 18 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:22,
  • VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, CDR-H2 comprising the amino acid sequence of SEQ ID NO:6, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 18 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:23,
  • VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:5, CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13 and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:25, or
  • VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:5, CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14 and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 18 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:26.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to OX40, wherein the moiety capable of specific binding to OX40 comprises
  • VH comprising the amino acid sequence of SEQ ID NO:37 and a VL comprising the amino acid sequence of SEQ ID NO:38, or
  • the moiety capable of specific binding to a costimulatory TNF receptor family member binds to 4- IBB. In some embodiments, the moiety capable of specific binding to a costimulatory TNF receptor family member binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:41. In some embodiments, the trimeric antigen binding molecule comprises a moiety capable of specific binding to 4- IBB, wherein the moiety comprises a VH comprising
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:43,
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:44 and SEQ ID NO:45, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, and a VL comprising
  • a CDR-Ll comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51 and SEQ ID NO:52,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:53 and SEQ ID NO:54, and
  • a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to 4-1BB, wherein the moiety capable of specific binding to 4-1BB comprises a VH comprising an amino acid sequence that is at least about 95%, 96%>, 97%, 98%, 99% or 100%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 and SEQ ID NO:68 and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:61 , SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 and SEQ ID NO:69.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to 4-1BB, wherein the moiety capable of specific binding to 4-1BB comprises
  • VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:42, CDR-H2 comprising the amino acid sequence of SEQ ID NO:44, CDR-H3 comprising the amino acid sequence of SEQ ID NO:46 and a VL comprising CDR-Ll comprising the amino acid sequence of SEQ ID NO: 51, CDR-L2 comprising the amino acid sequence of SEQ ID NO:53 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:55,
  • VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:42, CDR-H2 comprising the amino acid sequence of SEQ ID NO:44, CDR-H3 comprising the amino acid sequence of SEQ ID NO:49 and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 51, CDR-L2 comprising the amino acid sequence of SEQ ID NO:53 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:58, or
  • VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:42, CDR-H2 comprising the amino acid sequence of SEQ ID NO:44, CDR-H3 comprising the amino acid sequence of SEQ ID NO:50 and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:51, CDR-L2 comprising the amino acid sequence of SEQ ID NO:53 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:59.
  • the trimeric antigen binding molecule comprises a moiety capable of specific binding to 4-1BB, wherein the moiety capable of specific binding to 4-1BB comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO:61,
  • VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO:67, or
  • a trimeric antigen binding molecule comprising three fusion polypeptides as described before, wherein the moiety capable of specific binding to a
  • costimulatory TNF receptor family member is fused at the C-terminal amino acid to the N- terminal amino acid of the trimerization domain, optionally through a peptide linker.
  • the fusion can be a direct bond between the moiety capable of specific binding to a costimulatory TNF receptor family member and the trimerization domain, or the moiety capable of specific binding to a costimulatory TNF receptor family member and the trimerization domain may be connected through a peptide linker.
  • the moiety capable of specific binding to a costimulatory TNF receptor family member and the trimerization domain are connected through a peptide linker.
  • the peptide linker is a peptide comprising 2 to 20 amino acids.
  • the peptide linker is a peptide selected from the group consisting of GGGGS (SEQ ID NO: 109), GGGGSGGGGS (SEQ ID NO: 110), SGGGGSGGGG (SEQ ID NO: 111), (G 4 S) 3 or
  • GGGGSGGGGSGGGGS SEQ ID NO: l 12
  • GGGGSGGGGSGGGG or G4(SG4) 2 SEQ ID NO: 113
  • (G 4 S) 4 or GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 114
  • the sequences GSPGSSSSGS SEQ ID NO: 115
  • GSGSGSGS SEQ ID NO: 116
  • GSGSGNGS SEQ ID NO: 117
  • GGSGSGSG SEQ ID NO: 118
  • GGSGSG SEQ ID NO: 119
  • GGSG SEQ ID NO: 120
  • GGSGNGSG SEQ ID NO: 121
  • GGNGSGSGSG SEQ ID NO: 122
  • GGNGSG SEQ ID NO: 123
  • the peptide linker is selected from (G4S)i or GGGGS (SEQ ID NO : 109), (G 4 S) 2 or GGGGSGGGGS (SEQ ID NO : 110), (G 4
  • GGGGSGGGGSGGGGS SEQ ID NO: 112
  • G 4 S 4 or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: l 14).
  • the moiety capable of specific binding to a costimulatory TNF receptor family member and the huCMP trimerization domain are connected by a peptide linker of SEQ ID NO: 114.
  • a trimeric antigen binding molecule comprising three fusion polypeptides as described before, wherein the moiety capable of specific binding to a target cell antigen is fused at the N-terminal amino acid to the C-terminal amino acid of the trimerization domain, optionally through a peptide linker.
  • the trimerization domain and the moiety capable of specific binding to a target cell antigen are connected through a peptide linker.
  • the peptide linker is a peptide selected from the group consisting of GGGGS (SEQ ID NO: 109), GGGGSGGGGS (SEQ ID NO: 110), SGGGGSGGGG (SEQ ID NO: 111), (G 4 S) 3 or GGGGSGGGGSGGGGS (SEQ ID NO : 112), GGGGSGGGGSGGGG or G4(SG4) 2 (SEQ ID NO: l 13), and (G 4 S) 4 or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: l 14), but also include the sequences GSPGSSSSGS (SEQ ID NO: l 15), GSGSGS (SEQ ID NO: l 16), GSGSGNGS (SEQ ID NO: 117), GGSGSGSG (SEQ ID NO: 118), GGSGSG (SEQ ID NO: 119), GGSG (SEQ ID NO: 120), GGSGNGSG (SEQ ID NO: 121), GGNGSGSG (SEQ ID NO: 111
  • the peptide linker is selected from (G4S)i or GGGGS (SEQ ID NO: 109), (G 4 S) 2 or GGGGSGGGGS (SEQ ID NO: l 10), (G 4 S) 3 or GGGGSGGGGSGGGGS (SEQ ID NO: 112) and (G 4 S) 4 or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: l 14).
  • the huCMP trimerization domain and the moiety capable of specific binding to a target cell antigen are connected by a peptide linker of SEQ ID NO: 110.
  • the invention provides a trimeric antigen binding molecule comprising three fusion polypeptides as described before, wherein the moiety capable of specific binding to a target cell antigen is selected from the group consisting of is selected from the group consisting of an antibody, an antibody fragment and a scaffold antigen binding protein.
  • the moiety capable of specific binding to a target cell antigen is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, an aVH and a scaffold antigen binding protein.
  • the invention provides a trimeric antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to a target cell antigen is an antibody fragment.
  • the antibody fragment is selected from the group consisting of a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, and aVH.
  • the moiety capable of specific binding to a target cell antigen is a single chain Fab molecule.
  • the moiety capable of specific binding to a target cell antigen is a single domain antibody or an aVH.
  • the invention provides a trimeric antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to a target cell antigen is a scaffold antigen binding protein.
  • the moiety capable of specific binding to a target cell antigen can be a specifically designed ankyrin repeat protein.
  • the invention is concerned with a trimeric antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to a target cell antigen is a Fab molecule capable of specific binding to a target cell antigen.
  • the invention provides an antigen binding molecule comprising (a) a moiety capable of specific binding to a
  • TNF receptor family member (b) a trimerization domain derived from human cartilage matrix protein (huCMP) of amino acid sequence of SEQ ID NO: 1 , and (c) a Fab molecule capable of specific binding to a target cell antigen.
  • huCMP human cartilage matrix protein
  • said Fab molecule is fused at the C-terminal amino acid of the CHI domain to the N-terminal amino acid of the trimerization domain, optionally through a peptide linker as defined above.
  • a trimeric antigen binding molecule comprising three fusion polypeptides as described before, wherein the target cell antigen is selected from the group consisting of Fibroblast Activation Protein (FAP), Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic Antigen (CEA), CD 19, CD20 and CD33.
  • FAP Fibroblast Activation Protein
  • MCSP Melanoma-associated Chondroitin Sulfate Proteoglycan
  • EGFR Epidermal Growth Factor Receptor
  • CEA Carcinoembryonic Antigen
  • the target cell antigen is Fibroblast Activation Protein (FAP).
  • FAP Fibroblast Activation Protein
  • the moiety capable of specific binding to a target cell antigen binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:86.
  • a trimeric antigen binding molecule comprising three fusion polypeptides as described before, wherein the moiety capable of specific binding to FAP comprises
  • VH comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:71 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72, and a VL comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:73, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75 or
  • VH comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:76, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 77 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:78, and a VL comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:79, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO : 80 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO : 81.
  • the moiety capable of specific binding to FAP comprises a VH 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: 82 and a VL 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: 83.
  • the moiety capable of specific binding to FAP comprises a VH 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: 84 and a VL 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: 85.
  • the moiety capable of specific binding to FAP comprises
  • a VH comprising the amino acid sequence of SEQ ID NO: 82 and a VL comprising the amino acid sequence of SEQ ID NO:83
  • a VH comprising the amino acid sequence of SEQ ID NO: 84 and a VL comprising the amino acid sequence of SEQ ID NO: 85.
  • a trimeric antigen binding molecule comprising three fusion polypeptides as described before, wherein said three fusion polypeptides are identical.
  • the trimeric antigen binding molecules of the present invention are characterized by agonistic binding to a costimulatory TNF receptor family member.
  • the costimulatory TNF receptor family member is selected from the group consisting of OX40 and 4- IBB.
  • the invention provides a fusion polypeptide comprising (a) a VH and/or VL of a moiety capable of specific binding to a costimulatory TNF receptor family member, (b) a trimerization domain derived from human cartilage matrix protein (huCMP) comprising the amino acid sequence of SEQ ID NO:2, wherein the trimerization domain is capable of mediating stable association of the fusion polypeptide with two further such fusion polypeptides and (c) a VH and/or VL of a moiety capable of specific binding to a target cell antigen.
  • a fusion polypeptide comprising (a) a VH and/or VL of a moiety capable of specific binding to a costimulatory TNF receptor family member, (b) a trimerization domain derived from human cartilage matrix protein (huCMP) comprising the amino acid sequence of SEQ ID NO:2, wherein the trimerization domain is capable of mediating stable association of the fusion polypeptide with two further such fusion polypeptide
  • the fusion polypeptide comprises a VH of a moiety capable of specific binding to FAP, wherein the VH comprises
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:70 and SEQ ID NO:76,
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71 and SEQ ID NO:77, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72 and SEQ ID NO:78.
  • the fusion polypeptide comprises a VH of a moiety capable of specific binding to FAP, wherein the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:82 and SEQ ID NO:84.
  • the fusion polypeptide comprises a VL of a moiety capable of specific binding to FAP, wherein the VL comprises
  • a CDR-Ll comprising an amino acid sequence selected from the group consisting of SEQ ID NO:73 and SEQ ID NO:79,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:74 and SEQ ID NO:80, and
  • a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:75 and SEQ ID NO:81.
  • the fusion polypeptide comprises a VL of a moiety capable of specific binding to FAP, wherein the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:83 and SEQ ID NO:85.
  • the fusion polypeptide comprises a moiety capable of specific binding to FAP, wherein the moiety capable of specific binding to FAP comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 82 and a VL comprising the amino acid sequence of SEQ ID NO:83, or
  • the fusion polypeptide comprises a VH of a moiety capable of specific binding to OX40, wherein the moiety comprises a VH comprising
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
  • the fusion polypeptide comprises a VH of a moiety capable of specific binding to OX40, wherein the VH comprises an amino acid sequence that is at least about 95%, 96%), 97%, 98%>, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:39.
  • the fusion polypeptide comprises a VL of a moiety capable of specific binding to OX40, wherein the moiety comprises a VL comprising
  • a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20, and
  • SEQ ID NO:21 SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.
  • the fusion polypeptide comprises a VL of a moiety capable of specific binding to OX40, wherein the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 and SEQ ID NO:40.
  • the fusion polypeptide comprises comprises a moiety capable of specific binding to OX40, wherein the moiety capable of specific binding to OX40 comprises (i) a VH comprising the amino acid sequence of SEQ ID NO:27 and a VL comprising the amino acid sequence of SEQ ID NO:28,
  • VH comprising the amino acid sequence of SEQ ID NO:37 and a VL comprising the amino acid sequence of SEQ ID NO:38, or
  • the present invention provides a fusion polypeptide comprising, or consisting of, the amino acid selected from the group consisting of SEQ ID NO:227, 229, 231, 233, 235 and 237.
  • the fusion polypeptide comprises a VH of a moiety capable of specific binding to 4-1BB, wherein the moiety comprises a VH comprising
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:43,
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:44 and SEQ ID NO:45, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50.
  • the fusion polypeptide comprises a VH of a moiety capable of specific binding to 4-1BB, wherein the VH comprises an amino acid sequence that is at least about 95%, 96%o, 97%, 98%>, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66 and SEQ ID NO:68.
  • the fusion polypeptide comprises a VL of a moiety capable of specific binding to 4-1BB, wherein the moiety comprises a VL comprising
  • a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51 and SEQ ID NO:52
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:53 and SEQ ID NO:54
  • the fusion polypeptide comprises a VL of a moiety capable of specific binding to 4-1BB, wherein the VL comprises an amino acid sequence that is at least about 95%, 96%o, 97%, 98%>, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 and SEQ ID NO:69.
  • the fusion polypeptide comprises comprises a moiety capable of specific binding to 4- IBB, wherein the moiety capable of specific binding to 4- IBB comprises
  • VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO:67, or
  • the present invention provides a fusion polypeptide comprising, or consisting of, the amino acid selected from the group consisting of SEQ ID NO:246, 248, 250 and 252.
  • the invention further provides isolated polynucleotides encoding a trimeric antigen binding molecule of the invention as described herein, or a fragment thereof.
  • the isolated polynucleotides encoding trimeric antigen binding molecules of the invention may be expressed as a single polynucleotide that encodes the entire antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed.
  • Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional trimeric antigen binding molecule.
  • the light chain portion of a moiety capable of specific binding to a target cell antigen may be encoded by a separate polynucleotide from the heavy chain portion of the capable of specific binding to a target cell antigen. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the moiety capable of specific binding to a target cell antigen.
  • the light chain portion of a moiety capable of specific binding to a costimulatory TNF receptor family member may be encoded by a separate polynucleotide from the heavy chain portion of the capable of specific binding to a costimulatory TNF receptor family member.
  • the heavy chain polypeptides When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the moiety capable of specific binding to a costimulatory TNF receptor family member.
  • the invention relates to an isolated polynucleotide encoding a fusion polypeptide of the present invention as described herein.
  • the present invention provides a polynucleotide encoding a fusion polypeptide comprising, or consisting of, the amino acid selected from the group consisting of SEQ ID NO:227, 229, 231, 233, 235 and 237. More particularly, provided is a polynucleotide comprising, or consisting of, the sequence selected from the group consisting of SEQ ID NO:215, 217, 219, 221 , 223 and 225.
  • the present invention provides a polynucleotide encoding a fusion polypeptide comprising, or consisting of, the amino acid selected from the group consisting of SEQ ID NO: 246, 248, 250 and 252. More particularly, provided is a
  • RNA for example, in the form of messenger R A (mR A).
  • RNA of the present invention may be single stranded or double stranded.
  • an isolated polynucleotide encoding a trimeric antigen binding molecule as defined herein before or a fusion polypeptide as described herein before.
  • the invention further provides a vector, particularly an expression vector, comprising the isolated polynucleotide of the invention and a host cell comprising the isolated polynucleotide or the vector of the invention.
  • the host cell is a eukaryotic cell, particularly a mammalian cell.
  • a method for producing the trimeric antigen binding molecule of the invention comprising the steps of (i) culturing the host cell of the invention under conditions suitable for expression of said antigen binding molecule, and (ii) isolating said trimeric antigen binding molecule.
  • the invention also encompasses a trimeric antigen binding molecule produced by the method of the invention.
  • Trimeric antigen binding molecules of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production.
  • solid-state peptide synthesis e.g. Merrifield solid phase synthesis
  • polynucleotide encoding the antigen binding molecule or polypeptide fragments thereof, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • Such polynucleotide may be readily isolated and sequenced using conventional procedures.
  • a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided.
  • the expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment.
  • the expression vector includes an expression cassette into which the polynucleotide encoding the trimeric antigen binding molecule or polypeptide fragments thereof (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements.
  • a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids.
  • a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, and the like, are not part of a coding region.
  • Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors.
  • any vector may contain a single coding region, or may comprise two or more coding regions, e.g.
  • a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage.
  • a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the trimeric antigen binding molecule of the invention or polypeptide fragments thereof, or variants or derivatives thereof.
  • Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g.
  • a polypeptide is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
  • Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated” if induction of promoter function results in the transcription of 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 regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was 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 predetermined cells.
  • Other transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
  • transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus).
  • transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit a-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells.
  • tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins).
  • inducible promoters e.g. promoters inducible tetracyclins
  • translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
  • the expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).
  • LTRs retroviral long terminal repeats
  • AAV adeno-associated viral inverted terminal repeats
  • Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding a trimeric antigen binding molecule of the invention or polypeptide fragments thereof.
  • proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide.
  • the native signal peptide e.g.
  • an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
  • a heterologous mammalian signal peptide, or a functional derivative thereof may be used.
  • the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse ⁇ - glucuronidase.
  • TP A tissue plasminogen activator
  • fusion protein e.g. a histidine tag
  • assist in labeling the fusion protein may be included within or at the ends of the polynucleotide encoding a trimeric antigen binding molecule of the invention or polypeptide fragments thereof.
  • a host cell comprising one or more polynucleotides of the invention.
  • a host cell comprising one or more vectors of the invention.
  • the polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively.
  • a host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) a trimeric antigen binding molecule of the invention.
  • the term "host cell" refers to any kind of cellular system which can be engineered to generate the fusion proteins of the invention or fragments thereof.
  • Host cells suitable for replicating and for supporting expression of antigen binding molecules are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the antigen binding molecule for clinical applications.
  • Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), human embryonic kidney (HEK) cells, insect cells, or the like.
  • prokaryotic microorganisms such as E. coli
  • various eukaryotic cells such as Chinese hamster ovary cells (CHO), human embryonic kidney (HEK) cells, insect cells, or the like.
  • polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with a partially or fully human glycosylation pattern.
  • fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with a partially or fully human glycosylation pattern.
  • Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells.
  • baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. US Patent Nos. 5,959,177, 6,040,498,
  • Vertebrate cells may also be used as hosts.
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • TM4 cells as described, e.g., 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 BBL 3A
  • human lung cells W138
  • human liver cells Hep G2
  • mouse mammary tumor cells MMT 060562
  • TRI cells as described, e.g., in Mather et al, Annals N.Y.
  • MRC 5 cells MRC 5 cells
  • FS4 cells Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells (Urlaub et al, Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.
  • CHO Chinese hamster ovary
  • 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.
  • Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
  • 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 lymphoid cell (e.g., Y0, NS0, Sp20 cell). Standard technologies are known in the art to express foreign genes in these systems.
  • Cells expressing a polypeptide comprising either the heavy or the light chain of an antigen binding domain may be engineered so as to also express the other of the
  • immunoglobulin chains such that the expressed product is an antigen binding domain that has both a heavy and a light chain.
  • a method for producing the trimeric antigen binding molecule of the invention comprising the steps of (i) culturing the host cell of the invention under conditions suitable for expression of said antigen binding molecule, and (ii) isolating said trimeric antigen binding molecule form the host cell or host cell culture medium.
  • the components of the trimeric antigen binding molecule are genetically fused to each other.
  • Trimeric antigen binding molecules can be designed such that its components are fused directly to each other or indirectly through a linker sequence.
  • the composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Examples of linker sequences between different components of trimeric antigen binding molecules are found in the sequences provided herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence.
  • the moieties capable of specific binding to a target cell antigen (e.g. Fab fragments or scFv) forming part of the antigen binding molecule comprise at least an immunoglobulin variable region capable of binding to a target cell antigen.
  • the moieties capable of specific binding to a costimulatory TNF receptor family member (e.g. Fab fragments or scFv) forming part of the antigen binding molecule comprise at least an immunoglobulin variable region capable of binding to a costimulatory TNF receptor family member.
  • Variable regions can form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof.
  • Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. patent No. 4,186,567) or can be obtained, for example, by screening
  • any animal species of immunoglobulin can be used in the 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, a chimeric form of immunoglobulin may be used wherein the constant regions of the immunoglobulin are from a human.
  • a humanized or fully human form of the immunoglobulin can also be prepared in accordance with methods well known in the art (see e. g. U.S. Patent No. 5,565,332 to Winter).
  • Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues.
  • a grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions)
  • SDRs or a-CDRs the residues critical for the antibody-antigen interaction
  • Particular immunoglobulins according to the invention are human immunoglobulins.
  • Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form part of and be derived from human monoclonal antibodies made by the hybridoma method (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 a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variable regions may also be generated by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see e.g., Hoogenboom et al.
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • the moieties capable of specific binding to the relevant target e.g. Fab fragments or scFv
  • the moieties capable of specific binding to the relevant target are engineered to have enhanced binding affinity according to, for example, the methods disclosed in PCT publication WO 2012/020006 (see Examples relating to affinity maturation) or U.S. Pat. Appl. Publ. No. 2004/0132066.
  • the ability of the antigen binding molecules of the invention to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g.
  • ELISA enzyme-linked immunosorbent assay
  • Competition assays may be used to identify an antigen binding molecule that competes with a reference antibody for binding to a particular antigen.
  • a competing antigen binding molecule binds to the same epitope (e.g. a linear or a conformational epitope) that is bound by the reference antigen binding molecule.
  • epitope e.g. a linear or a conformational epitope
  • immobilized antigen is incubated in a solution comprising 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 a hybridoma supernatant.
  • immobilized antigen is incubated in a solution comprising the first labeled antigen binding molecule but not the second unlabeled antigen binding molecule.
  • Trimeric antigen binding molecules of the invention prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • 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 those having skill in the art.
  • affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the trimeric antigen binding molecule binds.
  • a matrix with protein A or protein G may be used.
  • Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate an antigen binding molecule essentially as described in the Examples.
  • the purity of the trimeric antigen binding molecule or fragments thereof can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.
  • the trimeric antigen binding molecule expressed as described in the Examples were shown to be intact and properly assembled as demonstrated by reducing and non-reducing SDS-PAGE.
  • the invention also encompasses a trimeric antigen binding molecule produced by the methods of the invention.
  • the trimeric antigen binding molecules provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art. 1. Affinity assays
  • the affinity of the trimeric antigen binding molecule provided herein for the corresponding TNF receptor can be determined in accordance with the methods set forth in the Examples by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare), and receptors or target proteins such as may be obtained by recombinant expression.
  • the affinity of the trimeric antigen binding molecule for the target cell antigen can also be determined by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare), and receptors or target proteins such as may be obtained by recombinant expression.
  • SPR surface plasmon resonance
  • a specific illustrative and exemplary embodiment for measuring binding affinity is described in Example 4.1.
  • K D is measured by surface plasmon resonance using a BIACORE® T200 machine (GE Healthcare) at 25 °C.
  • Binding of the trimeric antigen binding molecule provided herein to the corresponding receptor expressing cells may be evaluated using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS).
  • FACS flow cytometry
  • PBMCs peripheral blood mononuclear cells
  • activated mouse splenocytes expressing the TNF receptor molecule
  • cancer cell lines expressing the target cell antigen were used to demonstrate the binding of the antigen binding molecules to the target cell antigen.
  • competition assays may be used to identify an antigen binding molecule that competes with a specific antibody or antigen binding molecule for binding to the target or TNF receptor, respectively.
  • a competing antigen binding molecule binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by a specific anti-target antibody or a specific anti-TNF receptor antibody.
  • epitope e.g., a linear or a conformational epitope
  • Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
  • assays are provided for identifying trimeric antigen binding molecules that bind to a specific target cell antigen and to a specific TNF receptor having biological activity.
  • Biological activity may include, e.g., agonistic signalling through the TNF receptor on cells expressing the TNF receptor.
  • Trimeric antigen binding molecules identified by the assays as having such biological activity in vitro are also provided.
  • a reporter cell assay detecting NF- ⁇ activation in Hela cells expressing human 4- IBB or human OX40 and co- cultured with FAP-expressing tumor cells is provided (see e.g. Example 5.1).
  • trimeric antigen binding molecules of the invention are tested for such biological activity.
  • Assays for detecting the biological activity of the molecules of the invention are those described in Example 5 or Example 8.
  • the biological activity of such complexes can be assessed by evaluating their effects on survival, proliferation and lymphokine secretion of various lymphocyte subsets such as NK cells, NKT-cells or ⁇ T-cells or assessing their capacity to modulate phenotype and function of antigen presenting cells such as dendritic cells, monocytes/macrophages or B-cells.
  • the invention provides pharmaceutical compositions comprising any of the trimeric antigen binding molecules provided herein, e.g., for use in any of the below therapeutic methods.
  • a pharmaceutical composition comprises a trimeric antigen binding molecule and at least one pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprises any of the trimeric antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.
  • pharmaceutically acceptable excipient refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • pharmaceutical composition that contains at least one trimeric antigen binding molecule and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's
  • compositions are lyophilized formulations or aqueous solutions.
  • pharmaceutically acceptable excipient includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers and combinations thereof, as would be known to one of ordinary skill in the art.
  • Parenteral compositions include those designed for administration by injection, e.g.
  • the trimeric antigen binding molecules of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the fusion proteins may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Sterile injectable solutions are prepared by incorporating the fusion proteins or trimeric antigen binding molecules of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required.
  • Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients.
  • the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered liquid medium thereof.
  • the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
  • composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium 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; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monos
  • Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in 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, e.g. films, or microcapsules.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g.
  • prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
  • Exemplary pharmaceutically acceptable excipients herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rHuPH20 HYLENEX®, Baxter International, Inc.
  • Certain exemplary sHASEGPs and methods of use, including rHuPH20 are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and
  • the trimeric antigen binding molecules may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by
  • the trimeric antigen binding molecules may be formulated with suitable polymeric or hydrophobic materials (for example as emulsion in a pharmaceutically acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example as emulsion in a pharmaceutically acceptable oil
  • ion exchange resins for example, as a sparingly soluble salt.
  • Pharmaceutical compositions comprising the trimeric antigen binding molecules of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the trimeric antigen binding molecules may be formulated into a composition in a free acid or base, neutral or salt form.
  • Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g. those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
  • the pharmaceutical compositions may also contain more than one active ingredients 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.
  • the pharmaceutical composition comprises a trimeric antigen binding molecule and another active anti-cancer agent.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • any of the trimeric antigen binding molecules provided herein may be used in therapeutic methods.
  • the antigen binding molecules of the invention can be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context 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 agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the trimeric antigen binding molecules of the invention are provided for use as a medicament. In further aspects, the trimeric antigen binding molecules of the invention are provided for use in treating a disease, in particular for use in the treatment of cancer. In certain embodiments, the trimeric costimulatory antigen binding molecules of the invention are provided for use in a method of treatment. In one embodiment, the invention provides a trimeric antigen binding molecule as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the invention provides a trimeric antigen binding molecule for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the trimeric antigen binding molecule. In certain embodiments the disease to be treated is cancer.
  • the disease to be treated is a proliferative disorder, particularly cancer.
  • cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.
  • neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases.
  • the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer.
  • the subject, patient, or "individual” in need of treatment is typically a mammal, more specifically a human.
  • the trimeric antigen binding molecule of the invention or the pharmaceutical composition of the invention, for use in up-regulating or prolonging cytotoxic T cell activity.
  • the invention provides for the use of a trimeric antigen binding molecule of the invention in the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof.
  • the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament.
  • the disease to be treated is a proliferative disorder, particularly cancer.
  • cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.
  • neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases.
  • the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer.
  • the trimeric antigen binding molecule may not provide a cure but may only provide partial benefit.
  • a physiological change having some benefit is also considered therapeutically beneficial.
  • an amount of trimeric antigen binding molecule that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount”.
  • the individual is preferably a mammal, particularly a human.
  • the invention provides a method for treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a trimeric antigen binding molecule of the invention.
  • a composition is administered to said individual, comprising a fusion protein of the invention in a pharmaceutically acceptable form.
  • the disease to be treated is a proliferative disorder.
  • the disease is cancer.
  • 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 if the disease to be treated is cancer.
  • An "individual" according to any of the above embodiments may be a mammal, preferably a human.
  • the appropriate dosage of a trimeric antigen binding molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the type of fusion protein, the severity and course of the disease, whether the fusion protein is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the fusion protein, and the discretion of the attending physician.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • the trimeric antigen binding molecule is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1 mg/kg - 10 mg/kg) of the antigen binding molecule can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • One exemplary dosage of the fusion protein would be in the range from about 0.005 mg/kg to about 10 mg/kg.
  • a dose may also comprise from about 1 ⁇ g/kg body weight, about 5 ⁇ g/kg body weight, about 10 ⁇ g/kg body weight, about 50 ⁇ g/kg body weight, about 100 ⁇ g/kg body weight, about 200 ⁇ g/kg body weight, about 350 ⁇ g/kg body weight, about 500 ⁇ g/kg body weight, about 1 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg body weight, to about 1000 mg/kg body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 ⁇ g/kg body weight to about 500 mg/kg body weight etc. can be administered, based on the numbers described above.
  • 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) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the trimeric antigen binding molecule).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the trimeric antigen binding molecule of the invention will generally be used in an amount effective to achieve the intended purpose.
  • the trimeric antigen binding molecules of the 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.
  • a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays.
  • a dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the trimeric antigen binding molecules which are sufficient to maintain therapeutic effect.
  • Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from 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.
  • the effective local concentration of the trimeric antigen binding molecules may not be related to plasma concentration.
  • One skilled in the art will be able to optimize therapeutically effective local dosages without undue
  • a therapeutically effective dose of the trimeric antigen binding molecules described herein will generally provide therapeutic benefit without causing substantial toxicity.
  • Toxicity and therapeutic efficacy of a fusion protein can be determined by standard pharmaceutical
  • Cell culture assays and animal studies can be used to determine the LD 50 (the dose lethal to 50% of a population) and the ED 50 (the dose therapeutically effective in 50% of a population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 /ED 50 .
  • Trimeric antigen binding molecules that exhibit large therapeutic indices are preferred.
  • the trimeric antigen binding molecule according to the present invention exhibits a high therapeutic index.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et al, 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).
  • the attending physician for patients treated with the trimeric antigen binding molecules of the invention will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.
  • the trimeric antigen binding molecules of the invention may be administered in any order.
  • a fusion protein of the invention may be co-administered with at least one additional therapeutic agent.
  • therapeutic agent encompasses any agent that can be administered for treating a symptom or disease in an individual in need of such treatment.
  • additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • an additional therapeutic agent is another anti-cancer agent.
  • Such other agents are suitably present in combination in amounts that are effective for the purpose intended.
  • the effective amount of such other agents depends on the amount of fusion protein used, the type of disorder or treatment, and other factors discussed above.
  • the trimeric antigen binding molecules are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the trimeric antigen binding molecule of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is a trimeric antigen binding molecule of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a trimeric antigen binding molecule of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • Ringer's solution such as phosphate
  • a trimeric antigen binding molecule comprising three fusion polypeptides, each of the three fusion polypeptides comprising (a) a moiety capable of specific binding to a costimulatory TNF receptor family member,
  • trimerization domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:2.
  • trimerization domain comprises the amino acid sequence of SEQ ID NO:2.
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, and a VL comprising
  • a CDR-Ll comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20, and
  • a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.
  • the trimeric antigen binding molecule as defined in any one of the preceding points comprising a moiety capable of specific binding to OX40, wherein the moiety capable of specific binding to OX40 comprises a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:39 and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 and SEQ ID NO:40.
  • VH comprising the amino acid sequence of SEQ ID NO:37 and a VL comprising the amino acid sequence of SEQ ID NO:38, or
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:43,
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:44 and SEQ ID NO:45, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, and a VL comprising
  • a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51 and SEQ ID NO:52,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:53 and SEQ ID NO:54, and
  • SEQ ID NO:55 SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.
  • the trimeric antigen binding molecule as defined in any one of points (1) to (6), or points (11) to (13) above, comprising a moiety capable of specific binding to 4-1BB, wherein the moiety capable of specific binding to 4-1BB comprises
  • VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO:61
  • VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 63
  • VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 67, or
  • the moiety capable of specific binding to a costimulatory TNF receptor family member is a Fab fragment or a scFv.
  • FAP Melanoma-associated Chondroitin Sulfate Proteoglycan
  • MCSP Melanoma-associated Chondroitin Sulfate Proteoglycan
  • EGFR Epidermal Growth Factor Receptor
  • CEA Carcinoembryonic Antigen
  • CD19 CD20 and CD33.
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:70 and SEQ ID NO:76,
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72 and SEQ ID NO:78,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:74 and SEQ ID NO:80
  • a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:75 and SEQ ID NO:81.
  • the trimeric antigen binding molecule as defined in any one of the preceding points comprising a moiety capable of specific binding to FAP, wherein the moiety capable of specific binding to FAP comprises a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 82 and SEQ ID NO: 84, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 82 and SEQ ID NO: 84, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence selected from the group consisting of SEQ
  • VH comprising the amino acid sequence of SEQ ID NO: 82 and a VL comprising the amino acid sequence of SEQ ID NO:83, or
  • a fusion polypeptide comprising (a) a VH and/or VL of a moiety capable of specific binding to a costimulatory TNF receptor family member, (b) a trimerization domain derived from human cartilage matrix protein (huCMP) comprising the amino acid sequence of SEQ ID NO:2, wherein the trimerization domain is capable of mediating stable association of the fusion polypeptide with two further such fusion polypeptides and
  • the moiety capable of specific binding to FAP comprises a VH comprising
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:70 and SEQ ID NO:76
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71 and SEQ ID NO:77
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72 and SEQ ID NO:78,
  • a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:73 and SEQ ID NO:79,
  • a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:74 and SEQ ID NO:80, and
  • polypeptide comprises a VH of a moiety capable of specific binding to OX40, wherein the VH comprises
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7, and
  • SEQ ID NO:8 SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
  • polypeptide comprises a VH of a moiety capable of specific binding to 4- IBB, wherein the VH comprises
  • a CDR-H1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:43,
  • a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:44 and SEQ ID NO:45, and
  • a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50.
  • An expression vector comprising the polynucleotide of point (26) above.
  • a host cell comprising the polynucleotide point (26) above or the expression vector of point (27) above.
  • a method of producing a trimeric antigen binding molecule comprising culturing the host cell of point (28) above under conditions suitable for the expression of the trimeric antigen binding molecule, and isolating the trimeric antigen binding molecule.
  • a pharmaceutical composition comprising the trimeric antigen binding molecule of any one of points (1) to (21) above and at least one pharmaceutically acceptable excipient.

Abstract

L'invention concerne de nouvelles molécules trimériques de liaison à l'antigène comprenant trois polypeptides de fusion, chacun des trois polypeptides de fusion comprenant (a) une fraction capable de se lier de manière spécifique à un membre de la famille des récepteurs de co-stimulation du TNF, (b) un domaine de trimérisation dérivé de la protéine de matrice cartilagineuse humaine (huCMP) d'une séquence d'acides aminés de SEQ ID NO : l, et (c) une fraction capable de se lier de manière spécifique à un antigène de cellule cible. L'invention concerne également des procédés de production et d'utilisation de ces molécules.
PCT/EP2018/057767 2017-03-29 2018-03-27 Molécules trimériques de liaison à l'antigène spécifiques pour récepteur de co-stimulation du tnf WO2018178074A1 (fr)

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