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WO2015189143A1 - Aglycosyl anti-tweakr antibodies and uses thereof - Google Patents

Aglycosyl anti-tweakr antibodies and uses thereof Download PDF

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Publication number
WO2015189143A1
WO2015189143A1 PCT/EP2015/062693 EP2015062693W WO2015189143A1 WO 2015189143 A1 WO2015189143 A1 WO 2015189143A1 EP 2015062693 W EP2015062693 W EP 2015062693W WO 2015189143 A1 WO2015189143 A1 WO 2015189143A1
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Prior art keywords
antibody
antibodies
tpp
tweakr
seq
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PCT/EP2015/062693
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French (fr)
Inventor
Christian Votsmeier
Stefanie Hammer
Sandra Berndt
Uwe Gritzan
Dmitry Zubov
Ruprecht ZIERZ
Lars Linden
Sven Christian
Axel Harrenga
Jörg BIRKENFELD
Christoph Freiberg
Sven Golfier
Andrea Eicker
Simone Greven
Beatrix Stelte-Ludwig
Marian RASCHKE
Julian Marius GLÜCK
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Bayer Pharma AG
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Bayer Pharma AG
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Priority claimed from PCT/EP2014/062207 external-priority patent/WO2014198817A1/en
Application filed by Bayer Pharma AG filed Critical Bayer Pharma AG
Publication of WO2015189143A1 publication Critical patent/WO2015189143A1/en
Anticipated expiration legal-status Critical
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/72—Increased 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention provides recombinant antibodies that are specific for the TWEAKR (TNFRSF12A, FN 14) comprising a mutated Fc region lacking the glycans attached to the conserved N- linked site in the CH2 domains of the Fc region.
  • the antibodies accordingly, can be used to treat tumors and other disorders and conditions associated with expression of the TWEAKR.
  • the invention also provides nucleic acid sequences encoding the foregoing antibodies, vectors containing the same, pharmaceutical compositions and kits with instructions for use.
  • Antibody-based therapy is pr ving very effective in the treatment of various cancers, including solid tumors.
  • HERCEPTIN® has been used successfully to treat breast cancer and RITUXAN® is effective in B-celi related cancer types.
  • RITUXAN® is effective in B-celi related cancer types.
  • Central to the development of a novel successful antibody-based therapy is the isolation of antibodies against cell-surface proteins found to be preferentially expressed on tumor cells that are able to functionally modify the activity of the corresponding receptor.
  • Tumor necrosis factor like weak inducer of apoptosis (TWEAK) and the TWEAK receptor (TWEAKR, alias TNFRSF12A, FN 14, CD266; Swiss Pro! Acc. Q9NP84, NP_057723) are a TNF superfamily ligand-receptor pair involved in inflammation, proliferation, invasion, migration, differentiation, apoptosis and angiogenesis (Winkles J A, Nat Rev Drug Discov. 2008 May;7(5):411 -25; Michael son JS and Burkly LC, Results Probl Cell Differ. 2009;49: 145-60).
  • TWEAK binds to TWEAKR with an affinity of 0.8 - 2.4 nM and is the only member of the TNF family that binds this receptor (Wiley SR et al .. Immunity. 2001 Nov; 15(5):837-46).
  • the TWEAKR is expressed at relatively low levels in normal tissues, but is markedly increased locally in injured tissues, where it has a role in tissue remodeling (Winkles J A, Nat Rev Drug Discov. 2008 May;7(5):411 -25; Zhou et al., Mol Cancer Ther. 2011 Jul; 10(7): 1276-88 ; Burkly LC et al., Immunol Rev. 2011 Nov;244(l):99-114).
  • TWEAKR signaling is involved in processes as wound healing, chronic autoimmune disease and acute ischemic stroke (Burkly LC et al., Immunol Rev. 201 1 Nov;244(l):99-114).
  • the TWEAKR is highly expressed in various solid tumor types as for example pancreatic cancer, non-small-cell-lung-cancer (NSCLC), colorectal cancer (CRC), breast cancer, renal cancer, head and neck cancer, esophageal cancer, bladder cancer, hepatocellular carcinoma, ovarian cancer, melanoma as well as liver and bone metastasis (Culp P et al., Clin Cancer Res.
  • TWEAKR TWEAKR expression correlates with EGFR overexpression or activation in NSCLC (Whits ett TG et al., Am J Pathol. 2012 Jul; 181(l): l 1 1 -20) and HER2 expression in breast cancer (Wang J et al., Histol Histopathol. 2013 Jan 9 [Epub ahead of print]; Chao DT et al., J Cancer Res Clin Oncol. 2013 Feb; 139(2):315-25).
  • TWEAKR TNF -receptor associated factors
  • tumor cell lines Kym-1 , SKOV-3, OVCAR .
  • TNF tumor cell line
  • TRAF2cIAP complex apoptosis through TNF and the recruitment of TRAF2, followed by lysosomal degradation of the resulting TRAF2-cIAP complex.
  • HSC3, HT-2 cell lines
  • TWEAK induced apoptosis is reported to be TNF independent (Nakayama M et al., J Immunol. 2003 Jan l ; 170(l):341 -8; Wilson CA et al., Cell Death Differ. 2002 Dec;9(12): 1321-33).
  • I n a recent report induction of apoptosis by TWEAK was shown to be dependent on the stimulation of Stat-1 phosphorylation as treatment with a JAK- inhibitor abolished the ability of TWEAK to increase caspase3/7 activation in Wi Dr cells (Chapman MS et al., Cytokine. 2013 Jan;61 (l):210-7).
  • PDL-192 binds to the TWEAKR with a binding affinity of 5.5 nM (Culp PA et al., Clin Cancer Res. 2010 Jan 15; 16(2):497-508) and inhibits the growth of several TWEAKR expressing cancer cell lines. Yet, in comparison to TWEAK ligand PDL-192 was shown to be less potent in proliferation and apoptosis assays with respect to EC/IC50 and only reached reduced efficacy (V m ax) of caspase 3/7 activation (Culp PA et al., Clin Cancer Res. 2010 Jan 15; 16(2):497-508).
  • PDL-192 A further limitation of PDL-192 is the lack of species cross-reactivity, especially mouse and rat, not allowing e.g. assessment of common pre-clinical studies as toxicological studies.
  • First results of clinical trials with PD-L192 show relatively low dose limiting toxicity at doses of 1 -1.5 mg/kg (Lam et al., Abstract C 18, Mol. Cancer Ther. 2011 ; 10:C18).
  • the second agonistic anti-TWEAKR antibody described as drug candidate, ⁇ 036/ ⁇ 4 ⁇ 8 binds to TWEAKR with an affinity of 1.7 nM which is in a similar range as the endogenous ligand TWEAK (Michaeison JS et al., MAbs. 2011 Jul-Aug;3(4):362-75).
  • This antibody is shown to induce activation of NF-KB and cytokine release in cancer cells, albeit significantly less efficacious compared to Fc- TWEAK, a hlgGl Fc-fusion of soluble TWEAK (aa 106-249) with similar activity as recombinant soluble TWEAK (Michaeison JS et al. Oncogene.
  • the first anti-TWEAKR antibodies with anti-proliferative activity on cancer cells were antibodies Item 1 -4 described by Nakayama et al . (Nakayama M et al, Biochem Biophys Res Commun. 2003 Jul 1 1 ;306(4):819-25). These antibodies, however, harbor only relatively weak agonistic activity and were shown to act as partial agonists/antagonists with regard to TWEAK mediated TWEAKR activation.
  • Antibodies 136.1 and 18.3.3 show higher affinity binding compared to TWEAK ligand, which does not translate in more efficacious caspase activation.
  • Antibodies P3G5 and P2D3 induce cytokine release in cancer cells significantly less efficacious compared to Fc-TWEAK.
  • TWEAKR agonistic activity with regard to induction of apoptosis and inhibition of proliferation of the anti-TWEAKR antibodies described in the art is limited and does not reach or exceed the efficacy of the endogenous ligand TWEAK. This lack of agonistic activity is not due to a decreased affinity as these antibodies bind to the TWEAKR with affinities in a similar range as compared to the endogenous ligand TWEAK (Michaelson JS et al, MAbs.
  • This invention is related to antibodies comprising a mutated Fc region lacking the glycans attached to the conserved N-iinked site in the CH2 domains of the Fc region (called aglycosyl antibodies), and which lead to strong activation of the TWEAKR, thus leading to a strong induction of apoptosis in various cancer cells showing overexpression of the TWEAKR and having an improved safety and tolerateability profile.
  • Induction of cancer cell apoptosis by the antibodies described herein is more efficacious compared to all antibodies described in the art (e.g. PDL-192 or BIIB0036/P4A8; e.g. require the addition of a cross-linking agent).
  • the unique property of the antibodies of this invention is based on a novel binding epitope characterized by selective binding of the antibodies to amino acid at position 47 (D47) f
  • TWEAKR SEQ ID NO: 169; and see Figure 1).
  • the antibodies of the invention are thus suitable for the treatment of cancer as well as metastases thereof, in particular TWEAKR expressing tumors, such as colorectal cancer, non-small-cell lung cancer (NSCLC), head and neck cancer, esophageal cancer, melanoma, hepatocellular carcinoma, bladder cancer, gastric cancer, breast cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, ovarian cancer and cervical cancer in monotherapy as well as in combination with other targeted and non- targeted anti-tumor therapies.
  • the invention describes antibodies that are distinguished from existing anti-TWEAKR antibodies in that they induce strong activation of cancer cell apoptosis, at superior levels as compared to the endogenous ligand TWEAK in most cell lines.
  • the antibodies of the invention a) strongly activate the TWEAKR, b) induce apoptosis in cancer cells, c) induce cytokine secretion from cancer cells, d) all together resulting in anti-tumor activity of the antibodies in in vivo tumor experiments, e) additionally the antibodies lead to internalization of the TWEAKR and inhibition of cancer cell proliferation when incubated with saporine-conjugated secondary antibodies in experimental conditions where the antibody alone has no effect, f) are crossreactive to several species.
  • the antibodies of this invention comprise a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region. It was surprisingly observed that agonistic anti-TWEAKR antibodies comprising a mutation in the Fc region at N297A (using Kabat EU numbering) resulting in an aglycosyl antibody have less side effects and an improved toxicity profile compared to antibodies comprising the natural sequence of the Fc region. Oligomerization of TWEAKR by the anti-TWEAKR antibody via of FcyRIIB binding on cells, leading to inappropriate activation might play an important role in the observed side effects. Binding to other Fey receptors and complement could also potentiate this effect.
  • an antibody of the invention might be co-administered with known medicaments, and in some instances the antibody might itself be modified.
  • an antibody could be conjugated to a cytotoxic agent, immunotoxin, toxophore or radioisotope to potentially further increase efficacy.
  • the invention further provides antibodies which constitute a tool for diagnosis of malignant or dysplastic conditions in which TWEAKR expression is elevated compared to normal tissue.
  • anti-TWEAKR antibodies conjugated to a detectable marker.
  • Preferred markers are a radiolabel, an enzyme, a chromophore or a fiuorescer.
  • the invention is also related to polynucleotides encoding the antibodies of the invention, cells expressing the antibodies of the invention, methods for producing the antibodies of the invention, methods for inhibiting the growth of dysplastic cells using the antibodies of the invention, and methods for treating and detecting cancer using the antibodies of the invention.
  • the invention is also related to isolated nucleic acid sequences, each of which can encode an aforementioned antibody that is specific for an epitope f TWEAKR. Nucleic acids of the invention are suitable for recombinant production of antibodies. Thus, the invention also relates to vectors and host cells containing a nucleic acid sequence of the invention.
  • compositions of the invention may be used for therapeutic or prophylactic applications.
  • the invention therefore, includes a pharmaceutical composition comprising an inventive antibody and a pharmaceutically acceptable carrier or excipient therefore.
  • the invention provides a method for treating a disorder or condition associated with the undesired presence of TWEAKR expressing cells.
  • the aforementioned disorder is cancer.
  • Such method contains the steps of administering to a subject in need thereof an effective amount of the pharmaceutical composition that contains an inventive antibody as described or contemplated herein.
  • the invention also provides instructions for using an antibody library to isolate one or more members of such library that binds specifically to TWEAKR.
  • Fig. 1 Alignment of TWEAKR cysteine rich domain (aa 34-68) of different species. (Numbers indicate amino acid position in full length construct inclusive signal sequence; SEQ I D NO: 169)
  • Fig. 2 A - Schematic diagram of the structure of TWEAKR (SEQ ID NO: 169). The diagram shows the extracellular domain (aa 28-80) (SEQ I D NO: 168) including the cysteine rich domain (36-67), the transmembrane domain - TM (81-101), and the intracellular domain (102-129). TPP-2202 - the full ectodomain (28-80) fused to the Fc domain of hlgGl .
  • TPP-2203 Extracellular domain with N- and C- terminal truncation (34-68) fused to the Fc domain of hlgGl . Disulfide bridges Cys36-Cys49, Cys52- Cys67 and Cys55-Cys64 are indicated by black bars. N-terminally, TPP-2203 contains two amino acids and C -terminally, one amino acid more compared to the pure cysteine rich domain to ensure proper folding.
  • TPP-1984 Extracellular domain with C -terminal truncation (28-68) fused to HIS6 tag. All three constructs show comparable binding to the antibodies of this disclosure and PDL-192(TPP-1 104). P4A8(TPP-1324) does only bind to the full extracellular domain (TPP-2202).
  • Fig. 3 Interaction of TWEAK R ectodomain with antibodies of this disclosure and reference antibodies.
  • Y is "ELISA signal intensity [Rfu]”;
  • TWEAKR cysteine rich domain with antibodies of this disclosure and reference antibodies. Shown is the result of an ELI SA with TWEAKR(34-68)-Fc fusion protein (TPP-2203) coating (1 ⁇ g/ml) and 0.08 ⁇ g/ml (open bars) and 0.3 ⁇ g/ml (filled bars) of biotinylated IgG as soluble binding partner. Detection was done with Streptavidin-HRP and Amplex-Red substrate.
  • Y is "ELISA signal intensity [Rfu]”
  • X are "antibody constructs tested”: a is “TPP-2090”; b is “TPP-2084”; c is “PDL-192(TPP-1104)”; d is “P4A8(TPP-1324)”; e is “P3G5(TPP-2195)”; f is “136.1 (TPP-2194)”; h is “ITEM1”; i is “ITEM4"; j is a murine isotype control; k is a human isotype control.
  • the antibodies of the invention bind to the cysteine rich domain.
  • Fig. 5 Interaction of TWEAKR(28-68) with antibodies of this disclosure and reference antibodies. Shown is the result of an ELISA with TWEAKR(28-68)-HIS (TPP-1984) coating (1 ⁇ g/ml) and 0.08 ⁇ g/ml (open bars) and 0.3 ⁇ & ⁇ (filled bars) of biotinylated IgG as soluble binding partner. Detection was done with Streptavidin-HRP and Amplex-Red substrate.
  • Y is"ELISA signal intensity [Rfu]”;
  • X are "antibody constructs tested”: a is “TPP-2090”; b is “TPP-2084”; c is “PDL-192(TPP-1 104)”; d is “P4A8(TPP-1324)”; e is “P3G5(TPP-2195)”; f is “136.1 (TPP-2194)”; h is “ITEM ! “; i is "ITEM4"; j is a murine isotype control; k is a human isotype control.
  • the antibodies of the invention bind to the cysteine rich domain.
  • Antibodies P4A8(TPP-1324), P3G5(TPP-2195), ITEM- 1 and IT EM -4 show impaired binding.
  • Fig. 6 A - Alanine scan of cysteine rich domain. Muteins of TWEAKR(34-68)-Fc were analyzed for PDL-192(TPP-1104) (X) and TPP-2090 (Y) binding. S37A, R38A, S40A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A and 1.6 A muteins were expressed in HEK293 cells (black diamonds).
  • TPP-2090 PDL-192(TPP-1104) and TPP-2090 were coated (1 ⁇ g/ml) and an eight-fold diluted supernatant of the HEK293 fermentation broth was added for TWEAKR mutein binding.
  • X is "ELISA intensity of PDL-192(TPP-1 104) interaction [Rfu]”
  • Y is "ELISA intensity of TPP-2090 interaction [Rfu]”.
  • TPP-2090 (Y) shows impaired binding for the D47A TWEAKR mutein (closed box) and PDL-192(TPP-1 104) (X) shows impaired binding to R56A (dotted box).
  • B - Y is "% binding normalized by wt binding signal [%]", 1 is “TPP-2090”; 2 is “PDL-192(TPP- 1 104)"; 3 is “ITEM-1”.
  • Antibodies were coated (1 ⁇ g/ml), TWEAKR variant (D47A mutein of TWEAKR(34-68)-Fc and TWEAKR(34-68-Fc)were added at 250 ng/ml, detection via anti-HIS HRP.
  • TTP-2090 shows less than 5% binding to D47A mutein compared to the WT construct.
  • PDL-192 and ITEM- 1 bind with equal stringency to both constructs.
  • Y was calculated as follows: Signal intensity TWEAKR(34-68)Fc-D47A mutein / Signal intensity TWEAKR(34-68)-Fc) * 100.
  • C - Y is composition% binding to D47A mutein of TWEAKR(34-68)-Fc normalized by respective wt binding signal (TWEAKR(34-68-Fc) [%]", 1 is adjuvantTPP-2090"; 2 is "TPP-2149", 3 is “TPP-2093”; 4 is “TPP- 2148”; 5 is “TPP-2084”; 6 is “TPP-2077”; 7 is “TPP-1538”; 8 is “TPP-883”; 9 is “TPP-1854”; 10 is “TPP-1853”; 11 is “TPP-1857”; 12 is “TPP-1858”; 13 is “PDL-192(TPP-1 104)".
  • TWEAKR variant was added 250 ng ml, detection via anti-H I S HRP. All variants despite PDL-192 show less than 5% binding compared to the WT construct. Y was calculated as follows: Signal intensity TWEAKR(34-68)Fc-D47A mutein / Signal intensity TWEAKR(34-68)-Fc) * 100.
  • Fig. 7 NMR structure of TWEAKR ectodomain as published by Pellegrini et al (FEBS 280: 1818-1829). TWEAK binding depends on L46 (Pellegrini et al), TPP-2090 binding on D47 and PDL- 1 2 binding on R56. PDL- 1 2 binds opposite of the TWEAK ligand binding site, TPP-2090 binds directly to the TWEAK ligand site.
  • Fig. 8 To differentiate binding epitopes of antibodies of the disclosure and of reference antibodies competition experiments were performed. A lack of a second binding event after injection of the 2nd antibody indicates clear competition within a respective antibody pair. Non competing antibody pairs showed clear binding signal over background after 2nd antibody injection. In addition the investigation of self-competition (1 st & 2nd antibody identical) was monitored as an internal system control. (-) no 2 nd binding detected; (+) 2 nd binding. The antibodies of this disclosure compete with all tested antibodies.
  • Fig. 9 To differentiate binding epitopes of antibodies of the disclosure and of reference antibodies competition experiments were performed. In general all analyzed anti-TWEAKR antibodies could be clustered into three distinct "competition groups".
  • TPP-2084 and TPP- 2090 both showing competition to all other tested members. These other members could be split into two separate sets of antibodies, which do not show any competition between each other. Both antibodies of this disclosure bind to a new and unique epitope.
  • Fig. 10 Homology tree of all 29 known TNF receptor superfamily members. The closest homologs TNFRSF13C and TNFRSF17 have only about 30% sequence identity.
  • Fig. 11 Binding ELISA with all 29 TNF receptor superfamily members for selectivity assessment of TPP-2090. Shown is the result of an ELISA: Y is "ELISA signal intensity [Rfu]”; X are “TNF receptor superfamily proteins tested (Fc-fusion proteins)”: 1 is “TWEAKR”; 2 is “TWEAKR”; 3 is “Apo-3”; 4 is “Trail-Rl “; 5 is “Trail-R2”; 6 is “CD385"; 7 is “CD95”; 8 is “Rank”; 9 is “TNF-R1 “; 10 is “TNF-R2”; I I is “BAFF-R”; 12 is “DcR3”; 13 is “BCMA”; 14 is “TACI”; 15 is “OX40”; 16 is “CD30”; 17 is “CD27”; 18 is “CD40”; 19 is “Osteoprotegerin”; 20 is “EDAR”; 21 is “GITR”; 22 is “H
  • TPP-2090 In (1 ) 300 pM TPP-2090 were employed, in (2) 75 nM. TPP- 2090 binds at a very low concentration of 300 pM (1) and at a high concentration of 75 nM (2) in saturation to TWEAKR. For binding analysis to all other TNF receptor superfamily members (3 - 30) 75 nM TPP-2090 were used. TPP-2090 binds selectively to TWEAKR.
  • Fig. 12 FACS analysis for binding of anti-TWEAKR antibodies to HT-29 cells. Y is "background corrected Geo-Mean of FACS signal [au]".
  • Fig. 13 Caspase 3/7 activation by anti-TWEAKR antibodies in HT-29 cells.
  • X is "anti-TWEAKR antibodies tested [ ⁇ g/ml]”;
  • Y is "relative light units [RLU]”.
  • HT-29 cells were incubated with anti- TWEAKR antibodies at different concentrations as indicated (0.03-300 ⁇ g/mi) for 24h in the presence of IFNgamma.
  • Caspase 3/7 activity measured as luminescence by the Caspase 3/7 Gio reagent (Promega) was plotted against the antibody concentrations. Average values of 1 -3 representative experiments performed in triplicates are shown including standard deviations.
  • Filled symbols show antibodies of this disclosure, open symbols known antibodies [PDL-192(TPP-1 104); P4A8(TPP-1324), 136.1(TPP-2194) ].
  • the antibodies of this disclosure (TPP-1538, TPP-1854, TPP-2084, TPP-2090) dis lay a stronger efficacy to induce Cas ase 3/7 activation compared to the known antibodies [PDL- 192(TPP-1104); P4A8(TPP-1324) and 136.1(TPP-2194)].
  • Fig. 14 Antiproliferative activity of anti-TWEAKR antibodies in WiDr (A) and 786-0 (B) cells.
  • X is "anti-TWEAKR antibodies tested ⁇ g/ml]”;
  • Y is "C ell proliferation related to proliferation of untreated control cells [%]”. Ceils were incubated with anti-TWEAKR antibodies at different concentrations as indicated (0.03-300 ⁇ 3 ⁇ 4 ⁇ 1) for 96 h (WiDr cells absence, 786-0 cells in the presence of I FN gamma). Average values of a representative experiment performed in triplicates are shown and standard deviations are indicated by error bars. Filled symbols: antibodies of this disclosure, open symbols ltnown antibodies [PDL- !
  • the antibodies of this disclosure display a stronger efficacy to inhibit cellular proliferation compared to the known antibodies [PDL-192(TPP-1104) and P4A8(TPP-1324)].
  • Fig. 15 IL-8 secretion induced by anti-TWEAKR antibodies in A375 cells.
  • X is "anti-TWEAKR antibodies tested ⁇ g/ml]";
  • Y is "IL-8 levels [pg/ml]”.
  • A375 ceils were incubated with anti-TWEAKR antibodies at different concentrations as indicated (0.03-300 ⁇ g/ml).
  • Levels of IL-8 were determined in the supernatant of the cells after 24h treatment (and plotted against the used antibody concentrations. Average values of 1 -3 representative experiments performed in triplicates are shown including standard deviations.
  • Filled symbols show antibodies of this disclosure, open symbols ltnown antibodies [PDL- 192(TPP-1104); P4A8(TPP-1324), 136.1 (TPP-2194)], and treatment with an isotype control antibody is indicated (C).
  • the antibodies of this disclosure (TPP-1538, TPP-1854, TPP-2084, TPP-2090) display a stronger efficacy to induce IL-8 secretion from A375 cells compared to the known antibodies [PDL- 192(TPP-1104), P4A8(TPP-1324), 136.1 (TPP-2194)].
  • Fig. 15a IL-8 secretion induced by the anti-TWEAKR antibodies TPP-2090 and TPP-2658 in A375 cells in comparison to a non-binding isotype control (C).
  • X is "anti-TWEAKR antibodies tested ⁇ g/ml]";
  • Y is "IL-8 levels [pg ml]”.
  • A375 ceils were incubated with anti-TWEAKR antibodies at different concentrations as indicated (0.01 -100 ⁇ / ⁇ ). Levels of IL-8 were determined in the supernatant of the cells after 24h treatment (and plotted against the used antibody concentrations. Triplicate values are shown including standard deviations. TPP-2658 strongly induced IL-8 secretion comparable to TPP-2090.
  • Fig. 16 Human IL-8 secretion induced by anti-TWEAKR antibodies in xenografts in mice.
  • X is "hours after treatment [h]”; Y is "11-8 level [pg/ml]”. Results from 3 animals per group are indicated, error bars represent standard deviations.
  • Human IL-8 secretion is specifically induced after treatment with TPP-2090 in WiDr tumor bearing mice in a time dependent manner.
  • B A 75 tumor bearing (filled symbols) or non-tumor bearing (open symbols) mice were treated with a single dose of 10 mg/kg TPP-1538, vehicle or an isotpye control antibody.
  • CI is "vehicle control”
  • C2 is “isotype control antibody”
  • Y is "Level of human 11-8 [pg/ml]”.
  • Levels of human IL-8 were determined in the serum of 4 mice per group 7 h after treatment are shown. IL-8 secretion is specifically induced in A375 tumor bearing mice by TPP-1538 but not in equally treated tumor free animals.
  • Fig. 17 Microscopic evaluation of the time course of specific internalization of TWEAKR upon antibody binding to endogenous TWEAKR expressing cells (InCell Analyzer). Internalization of TPP- 1538 and TPP-2090 was investigated on renal cancer cell line 786-0. Granule count/cell after treatment with antibodies of this disclosure (at 1 ⁇ g/ml) or isotype control C- at 5 ⁇ g/ml) is plotted for different incubation times as indicated (X is "time [min]”; Y is "granule count/cell [quantity]”). Antibodies of this disclosure (TPP-1538, TPP-2090) show rapid and specific internalization in TWEAKR expressing cells.
  • Fig. 18 Inhibition of 786-0 cell proliferation by anti-TWEAKR antibodies after incubation with saporme-conjugated secondary antibodies (Hum-Zap Assay).
  • 786-0 cells were incubated with TWEAKR or isotype control antibodies in the presence or absence of saporine-conjugated secondary antibodies at 10 nM antibody concentration for 48h (in the absence of I FN gamma).
  • X is "antibody variant tested”
  • a is "vehicle control”
  • b is “isotype control antibody”
  • c is "TPP-2084”
  • d is "TPP-2090”
  • Y is "cell proliferation compared to untreated control cells [%]”.
  • Fig. 19 Efficacy of anti-TWEAKR antibodies in the human renal cell cancer xenograft 786-0 after treatment with 0.3, 1.0 and 3.0 mg/kg (i.v., q4dx3) started at day 7 after tumor cell inoculation. Shown are final tumor weights at day 40.
  • A is "Vehicle group, treated with PBS (i.v. q4dx3)".
  • B is "isotype, 3 mg/kg”
  • C is “TPP-2084, 0.3 mg/kg”
  • D is “TPP-2084, 1 mg kg”
  • E is “TPP-2084, 3 mg/kg”
  • F is "TPP- 2090, 0.3 mg/kg”
  • G is “TPP-2090, 1 mg/kg”
  • H is “TPP-2090, 3 mg/kg”.
  • B is "TPP-2090, 3 mg/kg”
  • C is “TPP-2090, 10 mg/kg”
  • D is “Irinotecan, 5 mg/kg”
  • E is “Combo TPP-2090 3 mg/kg + Irinotecan, 5 mg/kg”
  • F is "Regorafenib, 10 mg/kg”
  • G is "Combo TPP-2090, 3 mg/kg + Regorafenib 10 mg/kg”.
  • Fig. 21 Efficacy of 10 mg/kg TPP-2090 (i.v., q4dx8) in the human lung cancer xenograft NCI-H322 in monotherapy and combination therapy with Paciitaxei (16 mg kg, i.v., q7dx4).
  • Treatment started 14 d after inoculation with established tumors of about 45 mm 2 .
  • A is "Vehicle group, treated with PBS (i.v. q4dx8)".
  • B is "TPP-2090, 5 mg/kg”
  • C is "TPP-2090, 10 mg/kg”
  • D is "Paciitaxei, 16 mg kg”
  • E is "Combo TPP-2090 10 mg/kg + Paciitaxei 16 mg/kg”.
  • Fig. 22 Reduction of proliferative cells in xenografts after treatment with antibodies of this disclosure.
  • Cryo sections from Wi Dr xenograft tumors after treatment with PBS (i.v., q4dx7: A) or TPP-2090 (10 mg/kg, i.v. q4dx7:B) were stained for the proliferation marker Ki67 by immunohistochemistry.
  • Treatment started at day 7 after tumor cell inoculation and cryo sections were prepared from tumors taken at the end of the study (day 29). N 3 tumors per group were analyzed and representative images are shown.
  • Treatment with TPP-2090 leads to a strong reduction of Ki67 positive cells (cells with dark staining in image) in WiDr xenograft tumors in mice.
  • Fig. 23 Induction of Stat-1 and NF-kappaB2 signaling pathways by anti-TWEAKR antibodies in vivo.
  • Lysates of snap frozen WiDr xenograft tumors after treatment with PBS (i.v., q4dx7: lanes 1&2) or TPP-2090 (3 mg/kg, i.v., q4dx7: lanes 3&4) were subjected to Western Blot analysis detected with specific antibodies for P-Statl (a), Stat-1 (b), NF-kappa2 - p52 (c) and GAPDH (d).
  • Fig. 23A Induction of Stat-1 and NF-kappaB2 signaling pathways by anti-TWEAKR antibodies in Wi Dr cells in vitro.
  • Treatment with TPP-2090 and TPP-2658 leads to an equally strong induction of P-Stati & TWEAKR levels as well as NF-kappa
  • Fig. 24 Consensus sequences for an ti -TWEAKR antibodies.
  • CDR-H1 - X at position 5 M or I
  • CDR- H 2 X at position 8 S or
  • CDR-L1 X at position 8 G or S
  • CDR-L2 X at position 1 N, A or Q
  • CDR-L3 - X at position 5 T or S
  • X at position 6 S or T
  • X at position 8 F or G
  • Fig. 25 Continuous CDR sequence nomenclature.
  • A Positions in boxes were diversified for mutation gathering (maturation process).
  • B Single substitutions in boxes were recombined in one recombination library.
  • the present invention is based on the discovery of novel antibodies comprising a mutated Fc region lacking the giycans attached to the conserved N-linked site in the CH2 domains of the Fc region (called aglycosyl antibodies) that have a specific affinity for TWEAKR and can deliver a therapeutic benefit to a subject.
  • novel antibodies comprising a mutated Fc region lacking the giycans attached to the conserved N-linked site in the CH2 domains of the Fc region (called aglycosyl antibodies) that have a specific affinity for TWEAKR and can deliver a therapeutic benefit to a subject.
  • the antibodies of the invention which may be human, humanized or chimeric, can be used in many contexts, which are more fully described herein.
  • polypeptide and "protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • anti-TWEAKR antibody and "an antibody that binds to TWEAKR” refer to an antibody that is capable of binding TWEAKR with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting TWEAKR.
  • the extent of binding of an anti TWEAKR antibody to an unrelated, non-TWEAKR protein is less than about 10% of the binding of the antibody to TWEAKR as measured, e.g., by a surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • an antibody that binds to TWEAKR has a dissociation constant (KD) of ⁇ 1 ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 iiM, ⁇ 0.1 nM, ⁇ 0.01 n M . or ⁇ 0.001 n M (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).
  • KD dissociation constant
  • an anti-TWEAKR antibody binds to an epitope of TWEAKR that is conserved among TWEAKR from different species.
  • antibody is intended to refer to immunglobulin molecules, preferably comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains which are typically inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH ) and a heavy chain constant region.
  • the heavy chain constant region can comprise e.g. three domains CHI , CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain (CL).
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is typically composed of three CDRs and up to four FRs. arranged from amino terminus to carboxy-terminus e.g. in the following order: FR1 , CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • CDRs Complementarity Determining Regions
  • CDR 1 , CDR2, and CDR3 refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding.
  • Each variable domain typically has three CDR regions identified as CDR I .
  • Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat (e.g.
  • a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.
  • immunoglobulins Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, I G. and IgM, and several of these maybe further divided into "subclasses" (isotypes), e.g., I G I. I ( i2. IgG3, IgG4, IgAl , and I A 2.
  • a preferred class of immunoglobulins for use in the present invention is IgG.
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called [alpha], [delta], [epsilon], [gamma], and [mu], respectively.
  • the subunit structures and three- dimensional configurations of different classes of immunglobulins are well known.
  • antibodies are conventionally known antibodies and functional fragments thereof.
  • a “functional fragment” or "antigen-binding antibody fragment” of an antibody/immunoglobulin hereby is defined as a fragment of an antibody/immunoglobulin (e.g., a variable region of an IgG) that retains the antigen-binding region.
  • An "antigen-binding region" of an antibody typically is found in one or more hyper variable region(s) of an antibody, e.g., the CDR I , -2, and/or -3 regions; however, the variable "framework" regions can also play an important role in antigen binding, such as by providing a scaffold for the CDRs.
  • the "antigen-binding region” comprises at least amino acid residues 4 to 103 of the variable light (VL) chain and 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 of VL and 4 to 1 1 1 of VH, and particularly preferred are the complete VL and VH chains (amino acid positions 1 to 109 of VL and 1 to 1 13 f VH; numbering according to WO 97/08320).
  • “Functional fragments” or “antigen-binding antibody fragments” include Fab, Fab', F(ab r , and Fv fragments; diabodies; single domain antibodies (DAbs), linear antibodies; single-chain antibody molecules (scFv); and multispecific, such as bi- and tri-specific, antibodies formed from antibody fragments (C. A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering ( Springer Laboratory Manual), Springer Verlag).
  • An antibody other than a "multi-specific” or “multifunctional” antibody is understood to have each of its binding sites identical.
  • the F(ab' )2 or Fab may be engin cored to minimize or completely remove the intermolecular disulphide interactions that occur between the CHI and CL domains.
  • Fc region herein is used to define a ( ' -terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • 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.
  • an antibody herein is used to define an antibody or an antibody derivative which comprises a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region.
  • Aglycosyl antibodies can for example be prepared by mutation of the heavy chain glycosylation site of N297 (using Kabat EU numbering). Derivatives of such antibodies can be for example scFv-Fc comprising antibodies.
  • aglycosyl anti-TWEAKR antibody and "an aglycosyl antibody that binds to TWEAKR” refer to an aglycosyl antibody that is capable of binding TWEAKR with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting TWEA R .
  • aglycosyl agonistic anti-TWEAKR antibody or "agonistic aglycosyl anti-TWEAKR antibody” herein are used to define an aglycosyl anti-TWEAKR antibody having agonistic activity.
  • Variants of the antibodies or antigen-binding antibody fragments contemplated in the invention are molecules in which the binding activity of the antibody or antigen-binding antibody fragment is maintained.
  • Binding proteins contemplated in the invention are for example antibody mimetics, such as Affibodies, Adnectins, Anticalins, DA R ins. Avimers, Nanobodies (reviewed by Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S.D. et al., Curr. Opinion in Pharmacology 2008; 8:608-617).
  • a "human” antibody or antigen-binding fragment thereof is hereby defined as one that is not chimeric (e.g., not “humanized”) and not from (either in whole or in part) a non-human species.
  • a human antibody or antigen-binding fragment thereof can be derived from a human or can be a synthetic human antibody.
  • a "synthetic human antibody” is defined herein as an antibody having a sequence derived, in whole or in part, in silico from synthetic sequences that are based on the analysis of known luunan antibody sequences. In silico design of a human antibody sequence or fragment thereof can be achieved, for example, by analyzing a database of human antibody or antibody fragment sequences and devising a polypeptide sequence utilizing the data obtained there from.
  • human antibody or antigen-binding fragment thereof is one that is encoded by a nucleic acid isolated from a library of antibody sequences of human origin (e.g., such library being based on antibodies taken from a human natural source).
  • libraries of antibody sequences of human origin e.g., such library being based on antibodies taken from a human natural source.
  • human antibodies include antibodies as described in Soderlind et al., Nature Biotech. 2000, 18:853-856.
  • a “humanized antibody” or humanized antigen-binding fragment thereof is defined herein as one that is (i) derived from a non-human source (e.g., a transgenic mouse which bears a heterologous immune system), which antibody is based on a human germline sequence; (ii) where amino acids of the framework regions of a non-human antibody are partially exchanged to human amino acid sequences by genetic engineering or (iii) CDR-grafted, wherein the CDRs of the variable domain are from a non- human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.
  • a “chimeric antibody” or antigen-binding fragment thereof is defined herein as one, wherein the variable domains are derived from a non-human origin and some or all constant domains are derived from a human origin.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the term “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins . The term “monoclonal” is not to be construed as to require production of the antibody by any particular method. The term monoclonal antibody specifically includes chimeric, humanized and human antibodies.
  • an “isolated” antibody is one that has been identified and separated from a component of the cell that expressed it. Contaminant components of the cell are materials that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • An "isolated" nucleic acid is one that has been identified and separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • an antibody binds specifically to, is “specific to/for” or “specifically recognizes” an antigen of interest, e.g. a tumor-as so dated polypeptide antigen target, is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins or does not significantly cross-react with proteins other than orthologs and variants (e.g. mutant forms, splice variants, or proteolytically truncated forms) of the aforementioned antigen target.
  • an antigen of interest e.g. a tumor-as so dated polypeptide antigen target
  • the term “specifically recognizes” or “binds specifically to” or is “specific to/for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by an antibody, or antigen- binding fragment thereof, having a monovalent Kn for the antigen of less than about 10 ⁇ 4 M, alternatively less than about 10 "5 M, alternatively less than about 10 ⁇ 6 M, alternatively less than about 10 " 7 M, alternatively less than about 10 ⁇ 8 M, alternatively less than about 10 ⁇ 9 M, alternatively less than about 10 ⁇ 10 M, alternatively less than about 10 " " M, alternatively less than about 10 "12 M, or less.
  • an antibody “binds specifically to,” is “specific to/for” or “specifically recognizes” an antigen if such antibody is able to discriminate between such antigen and one or more reference antigen(s).
  • “specific binding”, “binds specifically to”, is “specific to/for” r “specifically recognizes” is referring to the ability of the antibody to discriminate between the antigen of interest and an unrelated antigen, as determined, for example, in accordance with one of the following methods.
  • Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.
  • a standard ELISA assay can be carried out.
  • the scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxidase and tetramethyl benzidine with hydrogen peroxide).
  • the reaction in certain wells is scored by the optical density, for example, at 450 nm.
  • determination of binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like.
  • Binding affinity refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule and its binding partner. 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. an antibody and an antigen).
  • the dissociation constant “KD” is commonly used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen) i.e. how tightly a ligand binds to a particular protein.
  • Ligand-protein affinities are influenced by non-covalent intermolecular interactions between the two molecules.
  • the "KD" or "KD value" according to this invention is measured by using surface plasmon resonance assays using a Biacore T200 instrument (GE Healthcare Biacore, Inc.).
  • a Biacore T100, a Biacore-2000, a Biacore-4000, a Biacore-3000, or a ProteOn XPR36 instrument Bio- Rad Laboratories, Inc.
  • epitopic determinants includes any protein determinant capable of specific binding to an immunoglobulin or T-celi receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids r sugar side chains, or combinations thereof and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • an “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
  • An exemplary competition assay is provided herein.
  • Antibody-dependent cell-mediated cytotoxicity or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc gamma receptors (FcyRs) present on certain cytotoxic cells (e.g.
  • NK cells, neutrophils, and macrophages enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell e.g. with cyto toxins.
  • an in vitro ADCC assay such as that described in US Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), may be performed.
  • Useful effector cells for such assays include PBMC anil N cells.
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass), which are bound to their cognate antigen.
  • Clq first component of the complement system
  • IX assay e.g., as described in Gazzano- Santoro et al., .1. Immunol. Methods 202: 163 (1996), may be performed.
  • Polypeptide variants with altered Fc region amino acid sequences polypeptides with a variant Fc region
  • increased or decreased Clq binding are described, e.g., in US Patent No. 6,194,551 Bl and WO 1999/51642.
  • naked antibody refers to an antibody that is not conjugated to a heterologous moiety (e.g. a cytotoxic moiety) or radiolabel. This naked antibody may be present in a pharmaceutical composition.
  • ADC antibody-drug conjugate
  • cytotoxic r cytostatic agents such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radio conjugate).
  • Immunoconjugates have been used for the local delivery of cytotoxic agents, i.e., drugs that kill or inhibit the growth or proliferation of cells, in the treatment of cancer (e.g. Liu et al., Proc Natl. Acad. Sci. (1996), 93, 8618-8623)). Immunoconjugates allow for the targeted delivery of a drug moiety to a tumor, and intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells and/or tissues.
  • Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin. The toxins may exert their cytotoxic effects by mechanisms including tubulin binding, ON A binding, or topoisomerase inhibition.
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence, respectively, is defined as the percentage of nucleic acid or amino acid residues, respectively, in a candidate sequence that are identical with the nucleic acid or amino acid residues, respectively, in the reference polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered as part of the sequence identity. Preferred are un -gapped alignments.
  • 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 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.
  • Sequence homology indicates the percentage of amino acids that either is identical or that represent conservative amino acid substitutions.
  • saturated antibodies or “maturated antigen-binding fragments” such as maturated Fab variants includes derivatives of an antibody or antibody fragment exhibiting stronger binding - i. e. binding with increased affinity - to a given antigen such as the extracellular domain of a target protein.
  • Maturation is the process of identifying a small number of mutations within the six CDRs of an antibody or antibody fragment leading to this affinity increase.
  • the maturation process is the combination of molecular biology methods for introduction of mutations into the antibody and screening for identifying the improved binders.
  • an “antagonistic” antibody or a “blocking” antibody is one which significantly inhibits (either partially or completely) a biological activity of the antigen it binds.
  • An “agonistic” antibody or an antibody with “agonistic activity” is one that binds to its target and induces the activation of the respective target (either partially or completely), that e.g. leads to activation of the signaling pathways or biological effects that are mediated by the respective target.
  • An ' ' agonist/agonistic antibody as used herein is an antibody which mimics at least one of the functional activities of a polypeptide of interest (here the TWEA R ligand TWEAK).
  • pharmaceutical formulation / "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.
  • Amino acids may be referred to herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • 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.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors.”
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “trans formants” 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 fully human antibody phage display library (Hoet RM et al., Nat Biotechnol 2005;23(3):344-8) was used to isolate TWEAKR-specific, human monoclonal antibodies of the present invention by protein panning (Hoogenboom I I .R.. Nat Biotechnol 2005;23(3): 1 105-16) with dimeric Fc-fused extracellular domains of human and murine TWEAKR as immobilized target.
  • the antibodies were purified by Protein A chromatography and further characterized by their binding affinity to soluble monomeric TWEAKR in EL ISA and BIAcore analysis as described in Example 2. To determine the cell binding characteristics of anti- TWEAKR antibodies, binding was tested by flow cytometry to a panel of cell lines (HT29, HS68, HS578).
  • NF-kappaB reporter gene assays were performed to assess the agonistic activity of all 1 1 identified antibodies (human IgGl). The antibody with the strongest in vitro efficacy (TPP-883) was selected for further potency and affinity maturation (see Example 1 for details). 1 single substitution variant was detected with improved agonistic activity: G102T of CDR-H3. Finally, 7 variants were selected based on enhanced affinity compared to the best single substitution variant, G102T. The corresponding ON A of these were re- cloned in a mammalian IgG expression vector and tested for functional activity in the afore mentioned NFkB reporter cell assay.
  • Antibodies of the invention can be further generated by methods known in the art like antibody phage display screening (for example see Hoet RM et al., Nat Biotechnol 2005;23(3):344-8), the well- established hybridoma technology (for example see Kohler and Milstein Nature. 1975 Aug 7;256(5517):495-7), or immunization of mice inter alia immunization of hMAb mice (e.g. Veloclmmune mouse ® ).
  • Antibodies of the invention is related to antibodies comprising a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region (aglycosyl antibodies) and which lead to strong activation of the TWEAKR (SEQ I D NO: 169 (protein); SEQ ID NO: 170 (DNA)), thus leading to a strong induction of apoptosis in various cancer cells showing overexpression of the TWEAKR (SEQ I D NO: 169 (protein); SEQ ID NO: 170 (DNA)), thus leading to a strong induction of apoptosis in various cancer cells showing overexpression of the TWEAKR (SEQ I D NO: 169 (protein); SEQ ID NO: 170 (DNA)), thus leading to a strong induction of apoptosis in various cancer cells showing overexpression of the TWEAKR (SEQ I D NO: 169 (protein); SEQ ID NO: 170 (DNA)), thus leading to a strong induction of
  • TWEAKR agonistic activity with regard to induction of apoptosis and inhibition of proliferation of the anti-TWEAKR antibodies described previously (e.g. PDL-192) is limited and does not reach the efficacy of the endogenous ligand TWEAK. This lack of agonistic activity is not due to a decreased affinity as these antibodies bind to the TWEAKR with affinities in a similar range as compared to the endogenous ligand TWEAK (Michaelson JS et al, MAbs. 201 1 Jul-Aug;3(4):362-75; G ulp PA et al.. Clin Cancer Res.
  • the invention provides antibodies which have such a strong agonistic activity with regard to induction of apoptosis and inhibition of proliferation that in vivo anti-tumor efficacy can be achieved without ADCC playing a significant role.
  • the human Fc gamma receptor (hFcyR) family consists of the activating receptors FcyRI, FcyRI IA, and FcyRIIIA, and the inhibitory receptor FcyRl i B. While FcyRI binds IgG with high affinity (nanomolar binding constants), FcyRI IA, FcyRl i B. and FcyR I I I A bind IgG with micromolar affinity, becoming activated only via avid multivalent interactions with opsonized antigen.
  • the binding of IgG to FcyR is highly sensitive to the presence of glycosylation at a single N-linked glycosylation site at asparagine 297 (N297) in its CH2 domain (Jefferis & Lund, 2002, Immunol Lett 82:57-65; Arnold et al., 2007, An nu Rev Immunol 25:21-50), with a loss of binding to the low-affinity FcyRs observed in N297 point mutants (Shields et al, 2001 , .1 Biol Chem 276:6591-6604; Tao & Morrison, 1989, J Immunol 143:2595-2601), enzymatic Fc deglycosylation (Mimura et al, 2001, .1 Biol Chem 276:45539-45547), recombinant IgG expression in the presence of the N-linked glycosylation-inhibitor tunicamycin
  • the present invention relates to aglycosyl anti-TWEAKR antibodies with a decreased effector function, which are characterized by a modification at the conserved N-linked site in the CI 12 domains of the Fc portion of said antibody. It is an embodiment of the invention to provide agonistic anti- TWEAKR antibodies which have a reduced Fc gamma receptor binding compared to antibodies comprising a wild type Fc region.
  • aglycosyl anti-TWEAKR antibodies having reduced Fc gamma receptor binding compared to antibodies comprising a wild type Fc region for receptors FcyRIIA, FcyRl i . and even more preferred for the FcyRIIB. Further preferred are the human Fc gamma receptors. Preferred are aglycosyl anti- TWEAKR antibodies having a binding affinity for FcyRIIB, preferentially human Fey RU B.
  • a binding affinity (KD) larger 200 ⁇ is highly preferred.
  • One way to modify an antibody is to mutate amino acids on the surface of the mAb that are involved in the effector binding interactions.
  • the other way relates to aglycosyl antibodies with decreased effector function, which are characterized by a modification at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody via for example by mutations of the residues at position 297 or 299 (using abat EU numbering).
  • the modification comprises a mutation at the heavy chain glycosylation site to prevent glycosylation at this site.
  • the aglycosyl anti-TWEAKR antibody was generated by mutation of the heavy chain glycosylation site, - i.e., mutation of N297 (using Kabat EU numbering) and was expressed in an appropriate host cell.
  • N297 using Kabat EU numbering
  • Preferred are mutations of S/T299 to Alanine or N297 to Alanine or Glutamine. Highly preferred is a mutation of N297 to Alanine.
  • the aglycosyl anti-TWEAKR antibody comprises a human I G Fc region and even more preferred a human I G Fc region selected for the group consisting of human I G I , I G 2. I G 3 and lgG4 Fc region. Hi hly preferred is a human I G I Fc region.
  • the modification comprises a mutation at the heavy chain glycosylation site to prevent glycosylation at this site in combination with one or more mutations influencing Fc receptor binding selected from the group constisting of E282V, M428I, S298G, T299A, N390D, E382V, and M428L (using Kabat EU numbering).
  • the combinations E282V together with M428I and S298G together with T299A, N390D, E382V, and M428L It is an embodiment of the invention to provide aglycosyl antibodies which have a strong induction of Caspase-3/7 in one or more TWEAKR expressing cell lines.
  • the one or more TWEAKR expressing cell line is comprised in the group consisting Wi Dr. A253, NCI-H322, HT-29 and 786-0 cells, preferably HT-29.
  • "Induction of Caspase 3/7" can be measured by common methods known in the art, including those described herein.
  • the "Induction of Caspase 3/7" according to this invention is measured by using activity determination with Caspase 3/7 Solution (Promega, #G8093) and reading of luminescence on a VICTOR V ( Perkin Elmer). At the end of the incubation time Caspase 3/7 activity was determined and the fold induction of Caspase 3/7 was calculated as compared to untreated cells.
  • An antibody is said to have "strong induction" of Caspase-3/7 if the fold of induction is greater than 1.2, preferably greater than 1.5, more preferably greater than 1.8, more preferably greater than 2.1 , more preferably grealer than 2.5.
  • anti-TWEAKR antibodies which lead to a stronger induction of Caspase 3/7 in HT-29 cells as compared to the agonistic antibodies previously described [e.g. PDL-192(TPP-1104), P4A8(TPP-1324), 136.1 (TPP-2194)] and also as compared to 300ng/ml recombinant human TWEAK.
  • This strong efficacy to induce Caspase 3/7 in cancer cells was also seen in WiDr, A253, NCI-H322 and 786-0 cells.
  • Some antibodies of this disclosure bind to the TWEAKR with only moderate affinity (> ⁇ ⁇ ) that is clearly lower compared to the affinity of the endogenous ligand TWEAK and lower compared to other known agonistic antibodies. This property provides further potential advantages as e.g. potentially improved tumor penetration.
  • aglycosyl antibodies that specifically bind to a TWEAKR at a novel epitope characterized by selective binding to aspartate (D) at position 47 (D47) of TWEAKR (SEQ ID NO: 169; and see Figure 1). It is a preferred embodiment of this invention to provide an antibody comprising a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region that specifically bind to a TWEAKR at a novel epitope characterized by selective binding to aspartate (D) at position 47 (D47) of TWEAKR (SEQ ID NO: 169; and see Figure 1).
  • N297 to Alanine or Glutamine using Kabat EU numbering
  • Highly preferred is a mutation of N297 to Alanine.
  • the identified dependencies on certain TWEAKR amino acids for antibody interaction correlate with the agonistic activity that has been determined for these antibodies.
  • the native ligand TWEAK shows efficient activation of TWEAKR and binds dependent on Leucin 46 in the cysteine rich domain of TWEAKR ( Pellegrini et al, FEBS 280: 1818-1829).
  • P4A8 shows very low agonistic activity and at least partially interacts with domains outside of the cysteine rich domain of TWEAKR.
  • PDL-192 shows moderate agonistic activity and binds dependent f R56 to the cysteine rich domain but opposite to the TWEAK ligand site.
  • Antibodies of this invention (exemplary TPP-2658) bind dependent on D47, and TWEAK binds dependent on 1.46. and binds to a similar but distinguishable binding site ( Figure 7). Therefore the antibodies of this invention which show a strong agonistic activity bind to a novel epitope ( 1)47 dependent) for antibodies which is connected to very strong agonistic activity.
  • Amino acid at position 47 (D47) of TWEAKR ( SEQ ID NO: 169) is regarded as critical for binding for the antibodies of the invention, which means the antibody specifically binds to the D at position 47 (D47) of TWE AKR ( SEQ ID NO: 169), when the antibody loses more than 20%, alternatively more than 30%, alternatively more than 40%, alternatively more than 50%, alternatively more than 60%, alternatively more than 70%o, alternatively more than 80%), alternatively more than 90%o, alternatively 100%, of its ELISA signal by changing this residue into an Alanine as described in Example 2 and Figure 6.
  • an antibody specifically binds to the D at position 47 (D47) of TWEAK R ( SEQ I D NO: 169), when the antibody loses more than 20%, alternatively more than 30%, alternatively more than 40%, alternatively more than 50%, alternatively more than 60%, alternatively more than 70%, alternatively more than 80%, alternatively more than 90%o, alternatively 100%o, of its ELISA signal on TPP-2614 compared to TPP-2203.
  • an antibody specifically binds to the D at position 47 (D47) of TWEAKR ( SEQ I D NO: 1 69), when the antibody loses more than 80% of its ELISA signal on TPP-2614 compared to TPP-2203.
  • a preferred embodiment of the invention is an aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR ( SEQ I D NO: 169).
  • a further preferred embodiment of the invention is an aglycosyl agonistic anti-TWEAK R antibody which specifically binds to aspartate 47 (D47) of TWEAKR ( SEQ I D NO: 169).
  • Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which has reduced ADCC activity or which lacks ADCC activity, and which specifically binds to aspartate 47 (D47) of TWEAKR ( SEQ I D NO: 169).
  • a further preferreii embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TW EAKR ( SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is selected from the group of agonistic activities consisting of induction of Caspase3/7, inhibition of proliferation of TWEAKR expressing cell lines, and induction of cytokine secretion.
  • Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti- TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR ( SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of Caspase3/7.
  • Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of Caspase3/7 in a TWEAKR expressing cancer cell line.
  • Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering).
  • Hi hly preferred is a mutation of N297 to Alanine.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of Caspase3/7 in a TWEAKR expressing cancer cell li ne comprised in t e group consisting Wi r. A253, NCT-H322, HT-2 and 786-0 cells.
  • Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • a further more preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is higher induction of Caspase3/7 in a HT-29 and/or 786-0 cell line compared to the induction by recombinant human TWEAK.
  • the concentration of anti-TWEAKR antibody used is 100 ⁇ g/ml and of recombinant human TWEAK is 300 ng/ml.
  • aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine. It is another embodiment of the invention to provide aglycosyl antibodies which bind specifically to the cysteine rich domain (aa 34-68 of SEQ I D: 169 ) of TWEAKR of different species ( Figure 1).
  • aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • the two species are human and mouse.
  • the antibodies of the current invention are more efficacious as compared to other known antibodies (PDL-192, P4A8) in inhibiting proliferation of various cancer cells.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is inhibition of proliferation of TWEAKR expressing cell lines.
  • the TWEAKR expressing cell line is comprised in the group consisting of 786-0, LOVO. NCI-H1975, SW480, WiDr, HT-29. A253, and SK-OV3.
  • the TWEAKR expressing cell line is Wi Dr.
  • a further more preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is stronger inhibition of proliferation of a786-0 and/or Wi Dr cell line compared to the inhibition by recombinant human TWEAK.
  • the concentration of anti-TWEAKR antibody used is 10( ⁇ g/ml and of recombinant human TWEAK is 300 ng ml.
  • Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • Cytokines induced include but are not limited to IL-8, I I.- 1 5.
  • a preferred cytokine which is induced is IL-8.
  • a preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion.
  • Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • a preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion in a TWEAKR expressing cancer cell line.
  • the TWEAKR expressing cancer cell line is a A375 or a Wi Dr cell line.
  • Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion wherein the cytokine is comprised in a group of cytokine consisting of IL-8, IL-15, IP- 10, IL-1RA and MCP-1.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion in a TWEAKR expressing cancer cell line secretion wherein the cytokine is comprised in a group of cytokine consisting of IL-8, IL-15, IP-10, I I .- ! RA and MCP-1.
  • Pre erred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Hi hly preferred is a mutation of N297 to Alanine.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion in a TWEAKR expressing cancer cell line secretion wherein the cytokine is comprised in a group of cytokine consisting of IL-8, IL-15, IP-10, 11.- 1 RA and MCP-1 and wherein the TWEAKR expressing cancer cell line is a A 75 or a WiDr cell line.
  • aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • the cytokine is IL-8, in an even more preferred embodiment the IL-8 is human IL-8.
  • a preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion in a mouse tumor xenograft model.
  • the secreted cytokine is a human cytokine derived from the tumor xenograft.
  • Preferred are aglycosyl antibodies comprising a mutation of N2 7 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of human IL-8 secretion in a mouse tumor xenograft model.
  • the mouse tumor xenograft model is a A375 or WiDr mouse xenograft model.
  • the induction of cytokine secretion is observed after injection of at 3 mg ' kg or higher or 10 mg kg or higher anti-TWEAKR antibody of the invention.
  • a further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of human IL-8 secretion in a mouse WiDr tumor xenograft model after injection of 3 mg kg of said antibody wherein no induction of the mouse IL-8 analogue KC is detected.
  • the induction of cytokine secretion is observed in the plasma of tumor bearing mice.
  • the examples in Table 21 include human and murine cell lines from many tumor origins (e.g. NSCLC, CRC, HNSCC, RCC, PancCA, OvC ' a. BreastCA, Melanoma, GastricCA, Esophageal CA, Bladder CA, HCC, Prostate CA, Neuroblastoma).
  • Tumor models include but are not limited to 786-0, A375, A253, SK-OV-3, WiDr, SW480, Co5682, NCI- H1975, NCI-H322, Lu7343, Co5676, Co 5841 SCaBER and SCC4.
  • efficacy of TPP-2658 is shown in Example 13 for WiDr and SCaBER.
  • Aglycosyl antibodies of this invention have less side effects and an improved toxicity pr fil compared to antibodies comprising the natural sequence of the Fc region.
  • said other species is a rodent, such as for example mouse or rat.
  • the antibodies bind to human TWEAKR and are cross - reactive to murine TWEAKR.
  • aglycosyl anti-TWEAKR antibodies conjugated to a detectable marker are a radiolabel, an enzyme, a chromophore or a fluorescer.
  • I n the following in addition to such aglycosyl anti-TWEAKR antibodies (like TPP-2658) antibodies are described (like TPP-2090) which can be transformed via mutations into the corresponding agclycosyl antibodies.
  • the antibody TPP-2090 is provided which has been transformed into the aglycosyl antibody TPP-2658 by mutation of N297 into Alanine.
  • I n order to transform such glycosylated antibodies into aglycosyl antibodies mutation of N297 to Alanine or Glutamine in the Fc region (using Rabat EU numbering) is preferred. Highly preferred is a mutation of N297 to Alanine.
  • Table 31 reference is made to the following antibodies as depicted in Table 31 :
  • TPP-2658 is a highly preferred antibody of this invention.
  • TPP-2658 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO: 213 and a light chain region corresponding to SEQ ID NO: 1.
  • TPP-2090 represents an antibody comprising a heavy chain region corresponding to SEQ I NO: 2 and a light chain region corresponding to SEQ I D NO: 1.
  • TPP-2149 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 12 and a light chain region corresponding to SEQ I D NO: 1 1.
  • TPP-2093 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO:
  • TPP-2148 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 32 and a light chain region corresponding to SEQ I D NO: 31.
  • TPP-2084 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 42 and a light chain region corresponding to SEQ ID NO: 41.
  • TPP-2077 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO: 52 and a light chain region corresponding to SEQ ID NO: 51.
  • TPP-1538 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 62 and a light chain region corresponding to SEQ I D NO: 61.
  • TPP-883 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 72 and a light chain region corresponding to SEQ I D NO: 71.
  • TPP-1854 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 82 and a light chain region corresponding to SEQ I D NO: 81.
  • TPP-1853 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 92 and a light chain region corresponding to SEQ I D NO: 91.
  • TPP-1857 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 102 and a light chain region corresponding to SEQ I D NO: 101.
  • TPP-1858 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO:
  • TPP-2658 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 10 and a variable light chain region corresponding to SEQ I D NO: 9.
  • TPP-2090 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 10 and a variable light chain region corresponding to SEQ ID NO: 9.
  • TPP-2149 represents an antibody comprising a variable heavy chain region corresponding to SEQ
  • TPP-2093 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 30 and a variable light chain region corresponding to SEQ I D NO: 29.
  • TPP-2148 represents an antibody comprising a variable heavy chain region corresponding to SEQ
  • TPP-2084 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 50 and a variable light chain region corresponding to SEQ I D NO: 49.
  • TPP-2077 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 60 and a variable light chain region corresponding to SEQ ID NO: 59.
  • TPP-1538 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 70 and a variable light chain region corresponding to SEQ I D NO: 69.
  • TPP-883 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 80 and a variable light chain region corresponding to SEQ I D NO: 79.
  • TPP-1854 represents an antibody comprising a variable heavy chain region corresponding to SEQ
  • TPP-1853 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 100 and a variable light chain region corresponding to SEQ I D NO: 99.
  • TPP-1857 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 110 and a variable light chain region corresponding to SEQ ID NO: 109.
  • TPP-1858 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 120 and a variable light chain region corresponding to SEQ ID NO: 119.
  • the aglycosyl antibodies comprise heavy or light chain CDR sequences which are at least 50%, 55%, 60% 70%, 80%, 90, or 95% identical to at least one, preferably corresponding, CDR sequence of the antibodies "TPP-2658", “TPP-2090", “TPP-2149", “TPP-2093”, “TPP-2148", “TPP-2084”, “TPP-2077”, “TPP-1538", “TPP-883", “TPP-1854", “TPP-1853", “TPP- 1857” or “TPP-1858” or at least 50%, 60%, 70%, 80%, 90%, 92% or 95% identical to the VH or VI.
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:6 ( H-CDRl ).
  • SEQ ID NO:7 (H-CDR2) and SEQ ID NO:8 ( II-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:3 (L- CDR1), SEQ ID NO:4 (L-CDR2) and SEQ ID NO:5 (L-CDR3).
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO: 16 (H-CDRl ), SEQ ID NO: 17 (H-CDR2) and SEQ ID NO: 18 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:13 (L-CDRl ).
  • SEQ ID NO: 14 I.-CDR2
  • SEQ ID NO: 15 L-CDR3
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:26 (H-CDRl ).
  • SEQ ID NO:27 (II -CDR 2) and SEQ ID NO:28 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:23 (L-CDRl ).
  • SEQ ID NO:24 (L-CDR2) and SEQ ID NO:25 (L-CDR3).
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:36 (H-CDRl ).
  • SEQ ID NO:34 (L-CDR2) and SEQ ID NO:35 I.-CDR3
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:46 (H-CDRl), SEQ ID NO:47 (H-CDR2) and SEQ ID NO:48 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:43 (L-CDR1), SEQ ID NO:44 (L-CDR2) and SEQ ID NO:45 (L-CDR3).
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:56 (H-CDRl ).
  • SEQ ID NO:54 (L-CDR2) and SEQ ID NO:55 I.-CDR3
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:66 (H-CDRI), SEQ ID NO:67 (H-CDR2) and SEQ ID NO:68 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:63 (L-CDRI ).
  • SEQ ID NO:64 (L-CDR2) and SEQ ID NO:65 (I.-CDR3) comprises a heavy chain antigen-binding region that comprises SEQ ID NO:66 (H-CDRI), SEQ ID NO:67 (H-CDR2) and SEQ ID NO:68 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:63 (L-CDRI ).
  • SEQ ID NO:64 (L-CDR2) and SEQ ID NO:65 (I.-CDR3) H-CDRI
  • SEQ ID NO:65 I.-CDR3
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO: 76 (H-CDRl ).
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO: 86 (H-CDRl ).
  • SEQ ID NO:87 H-CDR2 and SEQ ID NO:88 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:83 (L-CDRI), SEQ ID NO:84 (L-CDR2) and SEQ ID NO:85 (L-CDR3).
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:96 (H-CDR! ).
  • SEQ ID NO:97 (H-CDR2) and SEQ ID NO:98 (II-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:93 (L-CDRI), SEQ ID NO:94 (I.-CDR2) and SEQ ID NO:95 (I.-CDR3).
  • the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO: 106 (H-CDR! ).
  • SEQ ID NO: 104 (L-CDR2) and SEQ ID NO: 105 (L-CDR3) comprises a heavy chain antigen-binding region that comprises SEQ ID NO: 106 (H-CDR! ).
  • SEQ ID NO: 107 H-CDR2
  • SEQ ID NO: 108 (H-CDR3) comprises a light chain antigen-binding region that comprises SEQ ID NO:103 (L-CDRI).
  • the aglycosyl antibody of the invention comprises SEQ ID NO:116 (H-CDRl ).
  • Sequence alignment of the CDRs of the antibodies of this invention reveals a consensus sequence (see Figure 24).
  • the antibodies of the invention or antigen-binding fragment thereof comprise:
  • variable heavy chain comprising
  • a heavy chain CDR2 encoded by an amino acid sequence comprising the formula YISPSGGXTHYADSVKG (SEQ I D NO: 172), wherein X is S or K;
  • variable light chain comprising
  • a light chain CDR3 encoded by an amino acid sequence comprising the formula QQSYXXPXIT (SEQ I D NO: 176), wherein X at position 5 is T or S, and X at position 6 is T or S, and X at position 8 is G, or F.
  • Antibodies differ in sequence, not only within their complementarity determining regions (CDRs), but also in the framework (FR). These sequence differences are encoded in the different V-genes.
  • the human antibody germline repertoire has been completely sequenced. There are about 50 functional VH germline genes which can be grouped into six subfamilies according to sequence homology VH 1 , VH2, VH3, VH4. VH5 and VI I 6 (Tomlinson et al, 1992, J. Mol. Biol. 227, 776-798; Matsuda & Honjo, 1996, Advan. Immunol. 62, 1 -29). About 40 functional VL kappa genes comprising seven subfamilies are known (Cox et al, 1994, Eur. J.
  • Vkappa l Vkappa2.
  • Vkappa4, Vkappa5, Vkappa6 and Vkappa7 Disclosed herein are heavy chains of antibodies of this invention that belong to the human VH3 subfamily and the light chains of antibodies of this invention that belong to the human Vkappa 1 subfamily, respectively. It is known that framework sequences of antibodies belonging to the same subfamily are closely related, e.g. antibodies comprising a human VI 13 subfamily member all share comparable stability (Honegger et al., 2009, Protein Eng Des Sei. 22(3): 121-134).
  • the antibody or antigen-binding fragment of the invention comprises at least one CDR sequence of an antibody of the invention as depicted in Table 31 and a human variable chain framework sequence.
  • the aglycosyl antibody of the invention comprises a variable light chain or light chain antigen-binding region comprising the L-CDRl, L-CDR2 and L-CDR3 sequence of the variable light chain and a variable heavy chain or heavy chain antigen-binding region comprising the H- CDR1, H-CDR2 and H-CDR3 sequence of the variable heavy chain antibody of the invention as depicted in Table 31 and a human variable light and human variable heavy chain framework sequence.
  • the aglycosyl antibody of the invention comprises a variable light chain or light chain antigen-binding region comprising the L-CDRl, L-CDR2 and L-CDR3 sequence of the variable light chain and a variable heavy chain or heavy chain antigen-binding region comprising the H-CDR1, H-CDR2 and H-CDR3 sequence of the variable heavy chain antibody of the invention as depicted in Table 31 and a human VH3 subfamily framework sequence for the variable heavy chain and a human Vkappa 1 subfamily framework sequence for the variable light chain.
  • the human VH3 subfamily framework sequence for the variable heavy chain is comprised in the group f VH3 subfamily framework sequence consisting of VH3-07, VH3-09, VI 13- 1 1. VH3- 13. VH3-15, VH3-20, VH3-21. VI I3-23. VH3-30, VH3-30.3, VH3-30.5, VI I3-33. VH3-43. VH3-48, VH3- 49, VH3-53, VH3-64. VH3-66, VH3-72, VH3-73, VH3-74 and VH3-d. in an even more preferred embodiment the human VH3 framework sequence has less than 16 or less than 15 amino acid exchanges compared to a human VH3-23 framework sequence.
  • the human Vkappal subfamily framework sequence for the variable light chain is comprised in the group of Vkappa I subfamily framework sequence consisting of Vkappa 1-5, Vkappa 1-6, Vkappa 1-8, Vkappa 1D-8, Vkappa 1-9, Vkappa 1-12, Vkappa 1D-12, Vkappa 1-13, Vkappa I D- 13.
  • Vkappa 1 D-33 Vkappa 1 D-33.
  • the human Vkappa 1 framework sequence has less than 15 or less than 13 amino acid exchanges compared to a human Vkappa 1-39 framework sequence.
  • the aglycosyl antibody of the invention comprises a variable light chain or light chain antigen-binding region comprising the L- DR I . L-CDR2 and I .-CDR3 sequence of the variable light chain and a variable heavy chain or heavy chain antigen-binding region comprising the H-CDR1, H-CDR2 and H-CDR3 sequence of the variable heavy chain antibody of the invention as depicted in Table 31 and a human VI I 3 subfamily framework sequence for the variable heavy chain and a human Vkappa 1-39 framework sequence for the variable light chain.
  • the aglycosyl antibody of the invention comprises a variable light chain or light chain antigen-binding region comprising the L-CDRl, L-CDR2 and L-CDR3 sequence of tfae variable light chain and a variable heavy chain or heavy chain antigen-binding region comprising the H-CDR1 , H-CDR2 and H-CDR3 sequence of the variable heavy chain antibody of the invention as depicted in Table 31 and a human VH3-3 framework sequence for the variable heavy chain and a human
  • variable light chain framework sequence belongs to the human
  • Vkappal subfamily and the variable heavy chain framework sequence belongs to the human VH3 subfamily.
  • a VH3 subfamily or Vkappa I subfamily variable chain framework sequence may comprises sequence variations compared to the respective WT framework sequence to adopt the framework for insertion of the respective CDR sequence.
  • a VH3 subfamily or Vkappa I subfamily variable chain framework sequence comprising a sequence variation compared to the WT framework sequence is a VII 3 subfamily member or Vkappa ! subfamily member, respectively.
  • a variant framework sequence has up to 15 sequence variations, more preferably up to 10 sequence variations, more preferably up to 5 sequence variations, most preferably up to 3 sequence variations.
  • An antibody of the invention may be an lg( i (e.g. IgGl Ig( i2. IgG3, IgG4).
  • the agiycosyl antibodies of the invention are monoclonal. In a further preferred embodiment the agiycosyl antibodies of the invention are human, humanized or chimeric.
  • the invention provides agiycosyl antibodies having an antigen-binding region that binds specifically to and/or has a high affinity for TWEAKR.
  • An antibody is said to have a "high affinity" for an antigen if the affinity measurement is less than 250 nM (monovalent affinity of the antibody or aniigen-binding fragment).
  • An inventive agiycosyl antibody can bind to human TWEAKR with an affinity of less than 250 nM, preferably less than 150 nM, more preferably less than 100 nM, more preferably less than 50 nM, more preferably less than 30 nM, more preferably less than 20 nM, determined as monovalent affinity to human TWEAKR.
  • the invention provides antibodies having an antigen-binding region that binds specifically to TWEAKR and does not bind to other members of the TNF receptor superfamily (see Table 20).
  • An antibody of the invention might be co- administered with known medicaments, and in some instances the antibody might itself be modified.
  • an antibody could be conjugated to a cytotoxic agent, immunotoxin, toxophore or radioisotope to potentially further increase efficacy.
  • An antibody internalizes "efficiently" when its time of half maximal internalization (t 1 ⁇ 2) as measured by granule count/cell into TWEAKR expressing tumor cells is shorter than 400 min or more preferably shorter than 300 min and still more preferably shorter than 200 min.
  • Further preferred are antibodies or antigen-binding fragments with half maximal internalization times (t 1 ⁇ 2) of 100 minutes or less as determined by the protocol described in Example 7 and Figure 17.
  • Internalizable antibodies are suitable as targeting moiety of an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • An antibody or antigen-binding fragment is suitable in an in vitro or in vivo method to deliver a compound, preferably a cytotoxic agent, into a TWEAKR expressing cell.
  • the efficient internalization is shown with fluorescently labeled antibodies (Example 7).
  • the efficient use as an antibody drug conjugate is exemplified with a Saporin-conjugated antibody (Example 7).
  • antibodies of the invention or nucleic acids encoding the same are isolated.
  • An isolated biological component (such as a nucleic acid molecule or protein such as an antibody) is one that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, e.g., other chromosomal and extra-chromosomal ON A and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods as described for example in Sambrook et al., 1989 (Sambrook, J., Frits ch, E. F. and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  • An antibody of the invention may be derived from a recombinant antibody library that is based on amino acid sequences that have been isolated from the antibodies of a large number of healthy volunteers e.g. using the n-CoDeR® technology the fully human CDRs are recombined into new antibody molecules.
  • antibody libraries as the fully human antibody phage display library described in Hoet RM et a!.. Nat Biotechnol 2005;23(3):344-8) can be used to isolate TWEAKR- specific antibodies.
  • Antibodies or antigen-binding fragments of the invention are not limited to the specific peptide sequences provided herein. Rather, the invention also embodies variants of these polypeptides. With reference to the instant disclosure and conventionally available technologies and references, the skilled worker will be able to prepare, test and utilize functional variants of the antibodies disclosed herein, while appreciating these variants having the ability to bind to TWEAKR fall within the scope of the present invention.
  • a variant can include, for example, an antibody that has at least one altered complementary determining region (CDR) (hyper-variable) and/or framework (FR) (variable) domain/position, vis-a-vis a peptide sequence disclosed herein. To better illustrate this concept, a brief description of antibody structure follows.
  • An antibody is composed of two peptide chains, each containing one (light chain) or three (heavy chain) constant domains and a variable region (VL, VH), the latter of which is in each case made up of four FR regions and three interspaced CDRs.
  • the antigen-binding site is formed by one or more CDRs, yet the F R regions provide the structural framework for the CDRs and, hence, play an important role in antigen binding.
  • the skilled worker routinely can generate mutated or diversified antibody sequences, which can be screened against the antigen, for new or improved properties, for example.
  • a further preferred embodiment of the invention is an antibody or antigen-binding fragment in which the VH and VL sequences are selected as shown in Table 31.
  • the skilled worker can use the data in Table 31 to design peptide variants that are within the scope of the present invention. It is preferred that variants are constructed by changing amino acids within one or more CDR regions; a variant might also have one or more altered framework regions. Alterations also may be made in the framework regions. For example, a peptide FR domain might be altered where there is a deviation in a residue compared to a germiine sequence.
  • variants may be obtained by using one antibody as starting point for further optimization by diversifying one or more amino acid residues in the antibody, preferably amino acid residues in one or more CDRs, and by screening the resulting collection of antibody variants for variants with improved properties. Particularly preferred is diversification of one or more amino acid residues in CDR3 of VL and/or VH. Diversification can be done e.g. by synthesizing a collection of DMA molecules using trinucleotide mutagenesis (TRIM) technology (Virnekas B. et al., Nucl. Acids Res. 1994, 22 : 5600.).
  • Antibodies or antigen-binding fragments thereof include molecules with modifications/variations including but not limited to e.g. modifications leading to altered hal - life (e.g. modification of the Fc part or attachment of further molecules such as PEG), altered binding affinity or altered A IX or CDC activity. Conservative Amino Acid Variants
  • Polypeptide variants may be made that conserve the overall molecular structure of an antibody peptide sequence described herein. Given the properties of the individual amino acids, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • n n polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophane, and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;
  • positively charged (basic) amino acids include arginine, lysine, and histidine; and
  • (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions typically may be made within groups (a)-(d).
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • certain amino acids such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in a-helices
  • valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in ⁇ -pleated sheets.
  • Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns.
  • substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I .
  • sequence identity indicates the percentage of amino acids that are identical between the sequences.
  • sequence homology indicates the percentage of amino acids that either is identical or that represent conservative amino acid substitutions.
  • the present invention also relates to the DNA molecules that encode an antibody of the invention or antigen-binding fragment thereof.
  • the DNA sequences used for the antibodies expressed are given in Table 32. These sequences are optimized for mammalian expression.
  • DNA molecules of the invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants within the invention may be described by reference to their physical properties in hybridization. The skilled worker will recognize that DNA can be used to identify its complement and, since DNA is double stranded, its equivalent or homoiog, using nucleic acid hybridization techniques. It also will be recognized that hybridization can occur with less than 100% complementarity. However, given appropriate choice of conditions, hybridization techniques can be used to differentiate among DNA sequences based on their structural relatedness to a particular probe.
  • Structural similarity between two polynucleotide sequences can be expressed as a function of
  • stringency of the conditions under which the two sequences will hybridize with one another.
  • T m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatched base pairs.
  • ⁇ and ⁇ 2 are the ionic strengths of two solutions.
  • Hybridization stringency is a function of many factors, including overall DNA concentration, ionic strength, temperature, probe size and the presence of agents which disrupt hydrogen bonding. Factors promoting hybridization include high DNA concentrations, high ionic strengths, low temperatures, longer probe size and the absence of agents that disrupt hydrogen bonding. Hybridization typically is performed in two phases: the "binding" phase and the “washing” phase.
  • variants of DNA molecules provided herein can be constructed in several different ways. For example, they may be constructed as completely synthetic DNAs. Methods of efficiently synthesizing oligonucleotides in the range of 20 to about 150 nucleotides are widely available. See Ausubel et ah, section 2.11, Supplement 21 (1993). Overlapping oligonucleotides may be synthesized and assembled in a fashion first reported by Khorana et ah, J. Mo I. Biol. 72:209-217 (1971); see also Ausubel et ah, supra, Section 8.2. Synthetic DNAs preferably are designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.
  • a method of generating variants is to start with one of the DNAs disclosed herein and then to conduct site-directed mutagenesis. See Ausubel et al., supra, chapter 8, Supplement 37 (1997).
  • a target DNA is cloned into a single-stranded DNA bacteriophage vehicle.
  • Single- stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide alteration(s).
  • the complementary strand is synthesized and the double stranded phage is introduced into a host.
  • Some of the resulting progeny will contain the desired mutant, which can be confirmed using DNA sequencing.
  • various methods are available that increase the probability that the progeny phage will be the desired mutant. These methods are well known to those in the field and kits are commercially available for generating such mutants.
  • ADC Antibody-Drug Conjugates
  • the invention also provides antibody-drug conjugates (ADC, immunoconjugates) comprising an aglycosyl anti-TWEAKR antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • toxins e
  • an auri statin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof; an anthracycline such as daunomycin or doxorubicin; methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.
  • an immunoconjugate comprises an aglycosyl antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alphas arcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (P API, P A I I, and PAP-S ), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • an aglycosyl antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain
  • an immunoconjugate comprises an aglycosyl antibody as described herein conjugated to a radioactive atom to form a radioconjugate.
  • radioactive isotopes are available for the production of radioconjugates. Examples include Th 227 , Ac 225 , At 2 ", I !3 ! , I 125 , Y 90 , Re 186 , Re 188 , Sin 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • the radioconjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example Tc99m, or a spin label for nuclear magnetic resonance (NMR) imaging, such as iodine- 123 again, iodine-131 , indium- 1 1 1, fluorine- 19, carbon- 13, nitrogen- 1 . oxygen- 1 7, gadolinium, manganese or iron.
  • a radioactive atom for scintigraphic studies for example Tc99m
  • NMR nuclear magnetic resonance
  • the present invention further provides recombinant DNA constructs comprising one or more of the nucleotide sequences of the present invention (see Table 32).
  • the recombinant constructs of the present invention are used in connection with a vector, such as a plasmid, phagemid, phage or viral vector, into which a DNA molecule encoding an antibody of the invention or antigen-binding fragment thereof or variant thereof is inserted.
  • An antibody provided herein can be prepared by recombinant expression of nucleic acid sequences encoding light and heavy chains or portions thereof in a host cell.
  • a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the light and/or heavy chains or portions thereof such that the light and heavy chains are expressed in the host cell.
  • Standard recombinant DNA methodologies are used to prepare and/or obtain nucleic acids encoding the heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor.
  • nucleic acid sequences encoding variable regions of the heavy and/or light chains can be converted, for example, to nucleic acid sequences encoding full-length antibody chains, Fab fragments, or to scFv.
  • the VL- or VH-encoding DNA fragment can be operatively linked, (such that the amino acid sequences encoded by the two DNA fragments are in-frame) to another DNA fragment encoding, for example, an antibody constant region or a flexible linker.
  • sequences of human heavy chain and light chain constant regions are known in the art (see e.g., Kabat, E. A., el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • an expression of the antibodies of this invention as murine IgG is preferred, e.g. immunohistochemistry with human samples can be analyzed more easily by using murine antibodies.
  • the VH- and VL-encoding nucleic acids can be operatively linked to another fragment encoding a flexible linker such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al, Nature (1990) 348:552-554).
  • DNA encoding the desired polypeptide can be inserted into an expression vector which is then transfected into a suitable host cell.
  • suitable host cells are prokaryotic and eukaryotic cells. Examples for prokaryotic host cells are e.g. bacteria, examples for eukaryotic host cells are yeast, insect or mammalian cells.
  • the DNAs encoding the heavy and light chains are inserted into separate vectors. In other embodiments, the DNA encoding the heavy and light chains is inserted into the same vector. It is understood that the design of the expression vector, including the selection of regulatory sequences is affected by factors such as the choice of the host cell, the level of expression of protein desired and whether expression is constitutive or inducible.
  • an embodiment of the present invention are also host cells comprising the vector or a nucleic acid molecule, whereby the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.
  • the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.
  • Another embodiment of the present invention is a method of using the host cell to produce an antibody and antigen binding fragments, comprising culturing the host cell under suitable conditions and recovering said antibody. Therefore another embodiment of the present invention is the production of the antibodies according to this invention with the host cells of the present invention and purification of these antibodies to at least 95% homogeneity by weight.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include but are not limited to E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • Bacterial vectors may be, for example, bacteriophage-, piasmid- or phagemid-based. These vectors can contain a selectable marker and a bacterial ori in of replication derived from commercially available plasmids typically containing elements of the well-known cloning vector pBR322 (ATCC 37017). Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • appropriate means e.g., temperature shift or chemical induction
  • a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • an embodiment of the present invention is an expression vector comprising a nucleic acid sequence encoding for the novel antibodies of the present invention. See Example 1 for an exemplary description.
  • Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic host, including, for example, E. coli, Bacillus suhtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably, from E. coli cells.
  • Mammalian Expression Preferred regulatory sequences for mammalian host cell expression include viral elements that direct hi h levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the recombinant expression vectors can also include origins of replication and selectable markers (see e.g., U.S. 4.399.2 ! 6. 4.634.665 and U.S. 5,179,017).
  • Suitable selectable markers include genes that confer resistance to drugs such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • drugs such as G418, hygromycin or methotrexate
  • the dihydrofolate reductase I DI I FR confers resistance to methotrexate
  • the neo gene confers resistance to G418.
  • Trans fection of the expression vector into a host cell can be carried out using standard techniques such as electroporation, calcium-phosphate precipitation, and DEAE-dextran transfection.
  • Suitable mammalian host cells for expressing the antibodies, antigen binding fragments thereof or variants thereof provided herein include Chinese Hamster Ovary (CHO cells) [including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601 -621], NSO myeloma cells, COS cells and SP2 cells.
  • HEK Human Embryonic Kidney
  • Purification Antibodies of the invention can be recovered and purified from recombinant cell cultures by well- known methods including, but not limited to ammonium sulfate or ethanol precipitation, acid extraction, Protein A chromatography, Protein G chromatography, anion or cation exchange chromatography, phospho-celiulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydro xylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification.
  • HPLC high performance liquid chromatography
  • Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from an eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.
  • the antibody is purified (1) to greater than 95% by weight of antibody as determined e.g. by the Lowry method, UV-Vis spectroscopy or by by SDS-C apillary Gel electrophoresis (for example on a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer device), and in further preferred embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated naturally occurring antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • Therapeutic methods involve administering to a subject in need of treatment a therapeutically effective amount of an antibody contemplated by the invention.
  • a "therapeutically effective" amount hereby is defined as the amount of an antibody that is of sufficient quantity to reduce proliferation of TWEAK R positive cell or to reduce size of a TWEAK expressing tumor in a treated area of a subject - either as a single dose or according to a multiple dose regimen, alone r in combination with other agents, which leads to the alleviation of an adverse condition, yet which amount is toxicologically tolerable.
  • the subject may be a human or non-human animal (e.g. , rabbit, rat, mouse, dog, monkey or other lower-order primate).
  • the cancer is a solid tumor.
  • inventive antibodies can be used as a therapeutic or a diagnostic tool in a variety of situations with aberrant TWEAKR-signaling, e.g. cell proliferative disorders such as cancer or fibrotic diseases.
  • disorders and conditions particularly suitable for treatment with an antibody of the inventions are solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, and their distant metastases.
  • Those disorders also include lymphomas, sarcomas and leukemias.
  • Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers.
  • esophageal cancer examples include, but are not limited to esophageal cell carcinomas and Adeno carcinomas , as well as squamous cell carcinomas, Leiomyosarcoma, malignant melanoma, rhabdomyosarcoma and lymphoma.
  • gastric cancer examples include, but are not limited to intestinal type and diffuse type gastric adeno carcinoma .
  • pancreatic cancer examples include, but are not limited to ductal adenocarcinoma, adenosquamous carcinomas and pancreatic endocrine tumors.
  • breast cancer examples include, but are not limited to triple negative breast cancer, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
  • cancers of the respiratory tract include, but are not limited to small-cell and non-small- cell lung carcinoma, as well as bronchial adenoma and pleuropuimonary blastoma.
  • brain cancers include, but are not limited to brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, glioblastoma, medul loblastoma, ependymoma, as well as neuroectodermal and pineal tumor.
  • Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer.
  • Tumors of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal and vulvar cancer, as well as sarcoma of the uterus.
  • ovarian cancer examples include, but are not limited to serous tumour, endometrioid tumor, mucinous cystadenocarcinoma, granulosa cell tumor, Sertoii-Leydig cell tumor and arrhenoblastoma
  • cervical cancer examples include, but are not limited to squamous cell carcinoma, adenocarcinoma, adeno squamous carcinoma, small cell carcinoma, neuroendocrine tumour, glassy cell carcinoma and villoglandular adeno carcinoma .
  • Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, urethral, and hereditary and sporadic papillary renal cancers.
  • kidney cancer examples include, but are not limited to renal cell carcinoma, urothelial cell carcinoma, juxtaglomerular cell tumor (reninoma), angiomyolipoma, renal oncocytoma, Bellini duct carcinoma, clear-cell sarcoma of the kidney, mesoblastic nephroma and Wilms' tumor.
  • reninoma juxtaglomerular cell tumor
  • angiomyolipoma renal oncocytoma
  • Bellini duct carcinoma clear-cell sarcoma of the kidney
  • mesoblastic nephroma and Wilms' tumor examples include, but are not limited to renal cell carcinoma, urothelial cell carcinoma, juxtaglomerular cell tumor (reninoma), angiomyolipoma, renal oncocytoma, Bellini duct carcinoma, clear-cell sarcoma of the kidney, mesoblastic nephroma and Wilms' tumor.
  • bladder cancer examples include, but are not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma and small cell carcinoma.
  • Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.
  • liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibroiamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.
  • Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
  • Head-and-neck cancers include, but are not limited to squamous cell cancer of the head and neck, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, salivary gland cancer, lip and oral cavity cancer, and squamous cell cancer.
  • Lymphomas include, but are not limited to AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.
  • Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lympho sarcoma, and rhabdomyosarcoma.
  • Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
  • the antibodies are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease.
  • the antibodies of the invention are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease wherein the cancer is a solid cancer.
  • the antibodies of the invention are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease comprised in a group consisting of gastric cancer, breast cancer, pancreatic cancer, colorectal cancer, kidney cancer, prostate cancer, ovarian cancer, cervical cancers, lung cancer, endometrial cancer, esophageal cancer, head and neck cancer, hepatocellular carcinoma, melanoma and bladder cancer.
  • the antibodies are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease comprised in a group consisting of colorectal cancer, non small cell lung cancer, kidney cancer, melanoma, ovarian cancer, head and neck cancer and pancreatic cancer.
  • inventive antibodies can also be used as a therapeutic or a diagnostic tool in a variety of other disorders wherein TWEAKR is involved such as, but not limited to fibrotic diseases such as intraalveolar fibrosis, silica-induced pulmonary fibrosis, experimental lung fibrosis, idiopathic lung fibrosis, renal fibrosis, as well as lymphangioleiomyomatosis, polycystic ovary syndrome, acne, psoriasis, cholesteatoma, cholesteatomatous chronic otitis media, periodontitis, solar lentigines, bowel disease, atherosclerosis or endometriosis.
  • fibrotic diseases such as intraalveolar fibrosis, silica-induced pulmonary fibrosis, experimental lung fibrosis, idiopathic lung fibrosis, renal fibrosis, as well as lymphangioleiomyomatosis, polycystic ovary syndrome, acne, psoriasis,
  • an antibody of the invention might be co-administered with known medicaments, and in some instances the antibody might itself be modified.
  • an antibody could be conjugated to a cytotoxic agent or radioisotope to potentially further increase efficacy.
  • Antibodies of the present invention may be administered as the sole pharmaceutical agent or in combination with one or more additional therapeutic agents where the combination causes no nacceptable adverse effects.
  • This combination therapy includes administration of a single pharmaceutical dosage formulation which contains an antibody of the invention and one or more additional therapeutic agents, as well as administration of an antibody of the invention and each additional therapeutic agent in its own separate pharmaceutical dosage formulation.
  • an antibody of the invention and a therapeutic agent may be administered to the patient together in a single liquid composition, or each agent may be administered in separate dosage formulation.
  • an antibody of the invention and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially).
  • antibodies of the present invention may be used in fixed or separate combination with other anti-tumor agents such as alkylating agents, anti-metabolites, plant-derived anti-tumor agents, hormonal therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and/or biological response modifiers, anti-angiogenic compounds, and other anti-tumor drugs.
  • anti-tumor agents such as alkylating agents, anti-metabolites, plant-derived anti-tumor agents, hormonal therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and/or biological response modifiers, anti-angiogenic compounds, and other anti-tumor drugs.
  • secondary agents that may be used in
  • Alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, thiotepa, ranimustine, nimustine, temozolomide, altretamine, apaziquone, brostallicin, bendamustine, carmustine, estramustine, fotemustine, glufosfamide, inafosfamide, bendamustin, and mitolactol; platinum-coordinated alkylating compounds include, but are not limited to, cisplatin, carboplatin, eptaplatin, lobaplatin, nedaplatin, oxaliplatin, and satraplatin;
  • Anti-metabolites include, but are not limited to, methotrexate, 6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil alone or in combination with leucovorin, tegafur, doxifluridine, carmofur, cytarabine, cytarabine ocfosfate, enocitabine, gemcitabine, fludarabin, 5-azacitidine, capecitabine, cladribine, clofarabine, decitabine, eflornithine, ethynylcytidine, cytosine arabinoside, hydroxyurea, melphalan, nelarabine, nolatrexed, ocfosfite, disodium premetrexed, pentostatin, pelitrexol, raltitrexed, triapine, trimetrexate, vidarabine, vincristine, and vinorelbine;
  • Hormonal therapy agents include, but are not limited to, exemestane, Lupron, anastrozole, doxercalciferol, fadrozole, formestane, 11 -beta hydro xysteroid dehydrogenase 1 inhibitors, 1 7 -alpha hydroxylase/ 17,20 lyase inhibitors such as abiraterone acetate, 5-alpha reductase inhibitors such as finasteride and epristeride, anti-estrogens such as tamoxifen citrate and fulvestrant, Trelstar, toremifene, raloxifene, lasofoxifene, letrozole, anti-androgens such as bicalutamide, flutamide, mifepristone, nilutamide, Casodex, and anti-progesterones and combinations thereof; Plant-derived anti-tumor substances include, e.g., tho e selected from mitotic
  • Cytotoxic topoisomerase inhibiting agents include, but are not limited to, aclarubicin, doxorubicin, amonafide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, Irinotecan, topotecan, edotecarin, epimbicin, etoposide, exatecan, gimatecan, lurtotecan, mitoxantrone, pirambicin, pixantrone, rubitecan, sobuzoxane, tafluposide, and combinations thereof;
  • Immunologicals include interferons such as interferon alpha, interferon alpha-2a, interferon alpha- lb. interferon beta, interferon gamma- la and interferon gamma-nl, and other immune enhancing agents such as L I -11.2 and other 11.2 derivatives, filgrastim, lentinan, sizofilan, TheraCys, ubenimex, aldesleukin, alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab, ozogamicin, ibritumomab, imiquimod, lenograstim, lentinan, melanoma vaccine (Corixa), molgramostim, sargramostim, tasonermin, tecleukin, thymalasin, tositumomab, Vimlizin, epratuzumab, mitum
  • Biological response modifiers are agents that modify defense mechanisms of living organisms or biological responses such as survival, growth or differentiation of tissue cells to direct them to have antitumor activity; such agents include, e.g., krestin, lentinan, sizofiran, picibanil, ProMune, and ubenimex;
  • Anti-angiogenic compounds include, but are not limited to, acitretin, aflibercept, angiostatin, aplidine, asentar, axitinib, bevacizumab, brivanib alaninat, cilengtide, combreta statin, endostatin, fenretinide, halofuginone, pa/opanib. ranibizumab, rebimastat, recentin, regorafenib, removab, revlimid, sorafenib, squalamine, sunitinib, telatinib, thalidomide, ukrain, vatalanib, and vitaxin;
  • Antibodies include, but are not limited to, trastuzumab, cetuximab, bevacizumab, rituximab, ticilimumab, ipilimumab, lumiliximab, catumaxomab, atacicept, oregovomab, panitumumab and alemtu- vonab;
  • VEGF inhibitors such as, e.g., sorafenib, regorafenib, bevacizumab, sunitinib, recentin, axitinib, aflibercept, telatinib, brivanib alaninate, vatalanib, pa/opanib. and ranibizumab;
  • EGFR (HER1) inhibitors such as, e.g., cetuximab, panitumumab, vectibix, gefitinib, erlotinib, and Zactima
  • H ER2 inhibitors such as, e.g., lapatinib, tratuzumab, and pertuzumab
  • inTOR inhibitors such as, e.g., temsirolimus, sirolimus/Rapamycin, and everolimus
  • PI3K inhibitors such as PI3K inhibitor 1 (2-3 ⁇ - ⁇ -[7- ⁇ 6 ⁇ -8-(3- ⁇ ⁇ -4- ⁇ 1 ⁇ )- 2,3 -dihydroimidazo [ 1 ,2-c]quinazolin-5-yl]pyrimidine-5 -carboxamide dihydro chloride (see compound of Examples 1 and 2 WO 2012/136553, (which is incorporated herein by reference in its entirety) and AKT inhibitors;
  • CDK inhibitors such as roscovitine and flavopiridol
  • Spindle assembly checkpoints inhibitors and targeted anti-mitotic agents such as PLK inhibitors, Aurora inhibitors (e.g. Hesperadin), checkpoint kinase inhibitors, and KSP inhibitors;
  • HDAC inhibitors such as, e.g., panobinostat, vorinostat, MS275, belinostat, and LBH589;
  • Proteasome inhibitors such as bortezomib and carfilzomib;
  • S erine/threonine kinase inhibitors including MEK inhibitors and Raf inhibitors such as sorafenib; Farnesyl transferase inhibitors such as, e.g., tipifarnib;
  • Tyrosine kinase inhibitors including, e.g., dasatinib, nilotibib, regorafenib, bosutinib, sorafenib, bevacizumab, sunitinib, cediranib, axitinib, aflibercept, telatinib, imatinib mesylate, brivanib alaninate, pazopanib, ranibizumab, vatalanib, cetuximab, panitumumab, vectibix, gefitinib, erlotinib, lapatinib, tratuzumab, pertuzumab, and c-Kit inhibitors; Vitamin D receptor agonists;
  • Bcl-2 protein inhibitors such as obatoclax, oblimersen sodium, and gossypol;
  • Cluster of differentiation 20 receptor antagonists such as, e.g., rituximab;
  • Ribonucleotide reductase inhibitors such as, e.g., gemcitabine;
  • Tumor necrosis factor related apoptosis inducing ligand receptor 1 agonists such as, e.g., mapatumumab
  • Tumor necrosis factor related apoptosis inducing ligand receptor 2 agonists such as e.g.. lexatumumab, conatumumab, CS-1008, PRO95780;
  • 5 -Hydro xytryptamine receptor antagonists such as, e.g., rEV598, xaliprode, palonosetron hydrochloride, granisetron, Zindol, and AB-1001 ;
  • Integrin inhibitors including alpha5-betal integrin inhibitors such as, e.g., E7820, JSM 6425, volociximab, and endo statin;
  • Androgen receptor antagonists including, e.g., nandrolone decanoate, fluoxymesterone, Android, Prost-aid, andromustine, bicalutamide, flutamide, apo-cyproterone, apo-flutamide, chlormadinone acetate, Androcur, Tabi, cyproterone acetate, and nilutamide;
  • Aromatase inhibitors such as, e.g., anastrozole, letrozole, testolactone, exemestane, amino- glutethimide, and formestane;
  • anti-cancer agents including, e.g., alitretinoin, ampligen, atrasentan bexarotene, bortezomib, bosentan, calcitriol, exisulind, fotemustine, ibandronic acid, miltefosine, mitoxantrone, i -asparaginase. procarbazine, dacarbazine, hydroxy carbamide, pegaspargase, pentostatin, tazaroten, velcade, gallium nitrate, canfosfamide, compactsin, and tretinoin.
  • the antibodies of the present invention may be used in combination with chemotherapy (i.e. cytotoxic agents), anti-hormones and/or targeted therapies such as other kinase inhibitors (for example, EGFR inhibitors), mTOR inhibitors and angiogenesis inhibitors.
  • chemotherapy i.e. cytotoxic agents
  • anti-hormones and/or targeted therapies such as other kinase inhibitors (for example, EGFR inhibitors), mTOR inhibitors and angiogenesis inhibitors.
  • the antibodies of the present invention may be used in combination with check-point inhibitors and antibodies to immunotherapy receptors which empower any ongoing anticancer immune response that might have been too weak or exhausted.
  • mAb monoclonal antibodies interfering with CTLA4, PD-1 (CD279, PDCD1), PD-L 1 (B7-H1 , CD274, PDCDILGI), PD-L2 (PDCD1LG2, B7-DC, CD273), CD276 (B7-H3), TNFRSF4 (OX-40, CD 1 34).
  • CD27 TNFRSF7
  • CD70 CD27 Ligand
  • TNFRSF9 CD137, CDwl37, 4- 1 B, CD 1 37.
  • ILA TNFRSF18
  • KIR3 KIR, NKAT3, NKB1 , CD158e
  • LAG3 CD223 .
  • Such antibodies include, but are not limited to, ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, RG-7446.
  • the antibodies of the present invention are used in combination with anti-CTLA4, anti-PD-1 , and/ or anti- PD-L1 antibodies including but not limited to ipilimumab, nivolumab, pembrolizumab, BMS-936559, durvalumab, RG-7446.
  • the compounds of the present invention may also be employed in cancer treatment in conjunction with radiation therapy and/or surgical intervention.
  • the antibodies of the invention may be utilized, as such or in compositions, in research and diagnostics, or as analytical reference standards, and the like, which are well known in the art.
  • An ti -TWEAKR antibodies can be used for detecting the presence of TWEAKR-expressing tumors.
  • the presence of TWEAKR-containing cells or shed TWEAKR within various biological samples, including serum, and tissue biopsy specimens, may be detected with anti-TWEAKR antibodies.
  • anti-TWEAKR antibodies may be used in various imaging methodologies such as immuno s cintigraphy with a "Tc (or other isotope) conjugated antibody.
  • an imaging protocol similar to the one described using a ' "in conjugated anti-PS MA antibody may be used to detect pancreatic or ovarian carcinomas (Sodee et al., Clin. Nuc. Med. 21 : 759-766, 1997).
  • Another method of detection that can be used is positron emitting tomography by conjugating the antibodies of the invention with a suitable isotope (see Herzog et al., J. Nucl. Med. 34:2222-2226, 1993).
  • compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
  • An antibody of the invention can be administered by any suitable means, which can vary, depending on the type of disorder being treated. Possible administration routes include parenteral (e.g., intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous), intrapulmonary and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration.
  • an antibody of the invention might be administered by pulse infusion, with, e.g., declining doses of the antibody.
  • the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • the amount to be administered will depend on a variety of factors such as the clinical symptoms, weight of the individual, whether other drugs are administered. The skilled artisan will recognize that the route of administration will vary depending on the disorder or condition to be treated.
  • An embodiment of the present invention are pharmaceutical compositions which comprise anti-
  • TWEAKR antibodies alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • a stabilizing compound which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • a further embodiment are pharmaceutical compositions comprising a TWEAKR binding antibody and a further pharmaceutically active compound that is suitable to treat TWEAKR related diseases such as cancer. Any of these molecules can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier is pharmaceutically inert.
  • the present invention also relates to the administration of pharmaceutical compositions. Such administration is accomplished orally or parenterally.
  • Methods of parenteral delivery include topical, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl-cellulose, hydro xypropylmethylcellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, earbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e. dosage.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • compositions for parenteral administration include aqueous solutions of active compounds.
  • the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances that increase viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • 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 oleate or triglycerides, or liposomes.
  • 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.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1 %-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
  • compositions comprising a compound of the invention formulated in an acceptable carrier
  • they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency and method of administration. Kits
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Associated with such containers can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose, i.e. treatment of a particular disease state characterized by TWEAKR expression.
  • Determining a therapeutically effective amount of the novel antibody of this invention largely will depend on particular patient characteristics, route of administration, and the nature of the disorder being treated. General guidance can be found, for example, in the publications of the International Conference on Harmonization and in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528 (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. C o.. 1990). More specifically, determining a therapeutically effective amount will depend on such factors as toxicity and efficacy of the medicament. Toxicity may be determined using methods well known in the art and found in the foregoing references. Efficacy may be determined utilizing the same guidance in conjunction with the methods described below in the Examples.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., neoplastic ceils, or in animal models, usually mice, rabbits, dogs, pigs or monkeys. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of antibody or antigen-binding fragment thereof, that ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, ED50 LD50.
  • Pharmaceutical compositions that exhibit large therapeutic indices are pre erred.
  • the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease state, e.g., tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered for example every 3 to 4 days, every week, once every two weeks, or once every three weeks, depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 2 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature. See U.S. Pat. No. 4,657,760; 5,206,344; or 5,225.212.
  • Those skilled in the art will employ different formulations for polynucleotides than for proteins or their inhibitors.
  • delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • Preferred specific activities for a radiolabelled antibody may range from 0.1 to 10 mCi/mg of protein (Riva et al, Clin. Cancer Res. 5:3275-3280, 1999; Ulaner et al, 2008 Radiology 246(3):895-902)
  • a further preferred embodiment of the Invention is: 1.
  • An isolated anti-TWEAKR antibody comprising a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region, which specifically binds to the D at position 47 (D47) of TWEAK R (SEQ ID NO: 169).
  • variable heavy chain comprising:
  • GGDTYFDYFDY (SEQ ID NO: 173); and a variable light chain comprising:
  • variable heavy chain comprising the variable heavy chain CDR1 sequence as presented by SEQ I D NO: 6, the variable heavy chain CDR2 sequence as presented by SEQ I D NO: 7, and the variable heavy chain CDR3 sequence as presented by SEQ I D NO: 8, and a variable light chain comprising the variable light chain CDR1 sequence presented by SEQ I D NO: 3, the variable light chain CDR2 sequence presented by SEQ I D NO: 4, and the variable light chain CDR3 sequence presented by SEQ I D NO: 5, or a variable heavy chain comprising the variable heavy chain CDR1 sequence as presented by SEQ ID NO: 16, the variable heavy chain CDR2 sequence as presented by SEQ I NO: 17, the variable heavy chain CDR3 sequence as presented by SEQ ID NO: 18, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 13, the variable light chain CDR2 sequence presented by SEQ ID NO: 14, and the variable light chain CDR3 sequence presented by SEQ ID NO: 15,
  • variable heavy chain comprising the variable heavy chain CDRl sequence as presented by SEQ ID NO: 36, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 37, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:38, and a variable light chain comprising the variable light chain CDR l sequence presented by SEQ ID NO: 33, the variable light chain CDR2 sequence presented by SEQ ID NO: 34.
  • variable heavy chain comprising the variable heavy chain CDRl sequence as presented by SEQ ID NO: 46, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 47, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:48, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 43, the variable light chain CDR2 sequence presented by SEQ ID NO: 44, and the variable light chain CDR3 sequence presented by SEQ ID NO:45, or a variable heavy chain comprising the variable heavy chain CDRl sequence as presented by SEQ ID NO: 56, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 57, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:58, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 53, the variable light chain CDR2 sequence presented by SEQ ID NO: 54, and the variable light chain CDR3 sequence presented by SEQ ID NO:35, or a variable heavy chain comprising the variable heavy chain
  • variable heavy chain comprising the variable heavy chain CDR1 sequence as presented by SEQ ID NO: 1 16, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 1 1 7. the variable heavy chain CDR3 sequence as presented by SEQ I D NO: l 18, and a variable light chain comprising the variable light chain CDR1 sequence presented by SEQ I D NO: 1 13, the variable light chain CDR2 sequence presented by SEQ I D NO: 114, and the variable light chain CDR3 sequence presented by SEQ I D NO: l 15,.
  • the antibody according to anyone of the preceding embodiments comprising: a. a variable heavy chain sequence as presented by SEQ I D NO: 10 and a variable light chain sequences as presented by SEQ I D NO:9, or b.
  • the antibody according to any one of the preceding embodiments which is an IgG antibody, preferably a human IgGl .
  • the antibody according to anyone of the preceding embodiments comprising a heavy chain sequence as presented by SEQ ID NO: 2 1 3 and a light chain sequences as presented by SEQ ID NO: l .
  • the antibody according to any one of the preceding embodiments which is a human, humanized or chimeric antibody.
  • An antibody-drug conjugate comprising an antibody according to any one of the embodiments 1 to 14.
  • An isolated nucleic acid sequence that encodes the antibody according to any one of the embodiments 1 to 14.
  • a vector comprising a nucleic acid sequence according to embodiment 16.
  • An isolated cell expressing an antibody according to any one of the embodiments 1 to 14 and /or comprising a nucleic acid according to embodiment 16 or a vector according to embodiment 17.
  • An isolated cell according to embodiment 18, wherein said cell is a prokaryotic or a eukaryotic cell.
  • a method of producing an antibody according to any one of the embodiments 1 - 14 comprising culturing of a cell according to embodiment 19 and purification of the antibody.
  • An antibody according to any one of the embodiments 1 - 14 or an antibody-drug conjugate according to embodiment 15 for use as a medicament.
  • An antibody according to any one of the embodiments 1 - 14 for use as a diagnostic agent.
  • An antibody according to any one of the embodiments 1 - 14 or an antibody-drug conjugate according to embodiment 1 5 for use as a medicament in the treatment of cancer.
  • a pharmaceutical composition comprising an antibody according to any one of the embodiments 1 14 or an antibody-drug conjugate according to embodiment 1 5. 25.
  • a combination according to claim 25, wherein a therapeutically active compound is an anti- CTLA4, an anti-PD-1, or an anti-PD-Ll antibody 27.
  • a method for treating a disorder or condition associated with the undesired presence of TWEAK R. comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to embodiment 24 or a combination according to embodiment 25 or embodiment 26.
  • antibodies in addition to aglycosyl anti-TWEAKR antibodies (like TPP-2658) antibodies are described (like TPP-2090) which can be transformed via mutations into the corresponding aglycosyl antibodies.
  • the antibody TPP-2090 is provided which has been transformed into the aglycosyl antibody TPP-2658 by mutation of N297 into Alanine.
  • mutation of N297 to Alanine or Glutamine in the Fc region is preferred.
  • Highly preferred is a mutation of N297 to Alanine.
  • a fully human antibody phage display library (Hoet RM et al., Nat Biotechnol 2005;23(3):344-8) was used to isolate TWEAKR-specific, human monoclonal antibodies of the present invention by protein panning (Hoogenboom H.R., Nat Biotechnol 2005;23(3): 1 105-16) with dimeric Fc-fused extracellular domains of human and murine TWEAK as immobilized target.
  • Table 1 List of recombinant antigens used for antibody selection
  • the antigens were biotinylated using an approximately 2-fold molar excess of biotin-LC-NHS (Pierce; Cat. No. 21347) according to manufacturer ' s instructions and desalted using Zeba desalting columns (Pierce; Cat. No. 89889). Washed Magnetic beads (Dynabeads) were incubated o/n with 200 nM of biotinylated human antigen at 4 °C and blocked for lh at 4 °C with blocking buffer (PBS with 3% BSA, 0.05% Tween-20).
  • Fab-phages were identified and the corresponding antibodies were re-cloned into a mammalian IgG expression vector which provides the missing CH2-CH3 domains not present in the soluble Fab.
  • the resulting IgGs were transiently expressed in mammalian cells as described in Tom et al., Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves D 11 roc her. Scion Publishing Ltd, 2007. Briefly, a CMV-Promoter based expression plasmid was transfected into HEK293-6E cells and incubated in Fembach -Flasks or Wave-Bags.
  • Table 2 List of recombinant antigen used for affinity measurement
  • NF-kappaB reporter gene assays were performed to assess the agonistic activity of all 1 1 identified antibodies (human IgGl).
  • HEK293 cells were transiently transfected with a NF-kappaB reporter construct (BioCat, cat. No. LR-00 1 -PA ) using 293fectin according to manufacturer's instruction.
  • White poly-lysine coated 384well plates (BD) were seeded with transfected cells in F 17 media (serum- free; Invitrogen) at 37 °C, 5% C02. On the next day cells were stimulated with purified antibodies at different concentrations for 6h and subsequently a luciferase assay was carried out following standard procedures. Internalization is followed by fluorescence labeling of anti-TWEAKR antibodies (CypHer 5E mono
  • TPP-883 The antibody with the strongest in vitro efficacy (TPP-883) was selected for further potency and affinity maturation.
  • the DNA of all single NNK saturation mutagenesis libraries were re-cloned in a mammalian IgG expression vector for potency maturation and in a phagemid vector for affinity maturation, respectively.
  • Affinity maturation was done by phage panning. Washed magnetic beads (Dynabeads) were incubated o/n with 10 nM, 1 nM, 100 pM and 10 pM of biotinylated human antigen at 4°C and blocked for lh at 4°C with blocking buffer (PBS with 3% BSA, 0.05% Tween-20).
  • the blocked Fab-phage library was added with 10000-fold, 1000-fold and 100-fold excess compared to the theoretical library complexity to the blocked TWEAKR-Dynabeads and incubated for 30min at room temperature. Thus in total, 12 strategies were followed (4 antigen concentrations x 3 Fab-phage titers).
  • the phagemid vector was digested with the restriction endonuclease Mlul to remove the gene!II membrane anchor sequence required for Fab display on phage and religated.
  • 96 variants of each of the 12 selection pools were expressed as soluble Fabs and tested in an ELISA format. Therefore, 2.5 nM biotinylated TWEAKR-Fc antigen were coated and binding of soluble Fabs was detected by Anti-c-Myc antibody (Abeam ab62928). 7 single substitutions variants (continuous amino acid nomenclature, compare Figure 25) were detected with improved binding to TWEAKR-Fc (Seq I D No 138): S36G of CDR-L 1.
  • HEK293 cells were rransfected with an NF-kappaB reporter (BioCat, cat. No. LR-0051 -PA).
  • White poly-lysine coated 384weli plates (BD) were seeded with rransfected cells in F17 media (serum-free; Invitrogen) and individual variants of the NNK- diversified positional antibody (human IgGl) libraries were transiently expressed in mammalian cells.
  • NF-kappaB reporter cells were stimulated with the expressed single NNK mutagenesis antibody variants for 6h and subsequently a luciferase assay was carried out following standard procedures.
  • 1 single substitution variant was detected with improved agonistic activity: G102T of C DRIB. This variant was also obtained from affinity maturation and showed also there the greatest affinity enhancement.
  • affinity and potency screening all 7 beneficial single substitutions were recombined (library complexity: 128 variants) in one recombination library.
  • oligonucleotides were synthesized to introduce selected mutations or the corresponding wild type amino acid at each selected position. Library construction was performed using sequential rounds of overlap extension PCR.
  • the final PCR product was ligated into a bacterial soluble Fab expression vector and 528 variants were randomly selected ( ⁇ 4fold oversampling) for equilibrium ELISA screening with soluble Fabs as described before. Finally, 7 variants were selected based on enhanced affinity compared to the best single substitution variant, G102T. The corresponding DNA of these were re-cloned in a mammalian IgG expression vector and tested for functional activity in the afore mentioned NF-kappaB reporter ceil assay. Finally, the obtained sequences were compared with human germline sequences and deviations without significant impact on affinity and potency were adjusted. Antibodies depicted in Table 31 were obtained by antibody library screening and by affinity and/or potency maturation.
  • QQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ ID NO. I) and heavy (SEQ I D NO.2) chains of TPP-2090; CDRs of both the heavy and light chain are underlined.
  • QQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ I D NO.81) and heavy (SEQ I D NO.82) chains of TPP-1854; CDRs of both the heavy and light chain are underlined.
  • TPP-1858 Amino acid sequences of the light (SEQ ID NO.101) and heavy (SEQ ID NO.102) chains of TPP-1857; CDRs of both the heavy and light chain are underlined. TPP-1858
  • Binding affinities of anti-TWEAKR antibodies were determined by surface plasmon resonance analysis on a Biacore T100 instrument (GE Healthcare Biacore, Inc.). Antibodies were immobilized onto a CM 5 sensor chip through an indirect capturing reagent, anti-human IgG(Fc). Reagents from the "Human Antibody Capture Kit” (BR-1008-39, GE Healthcare Biacore, Inc.) were used as described by the manufacturer. Anti-TWEAKR antibodies were injected at a concentration of 10 ⁇ g/ml at 10 ⁇ /min for 10 sec. Table 3: List of recombinant antigen (TWEAKR) used for affinity measurement
  • the dissociation equilibrium constant (KD) was calculated based on the ratio of association ( ) and dissociation rate (koff) constants, obtained by fitting sensorgrams with a first order 1 : 1 binding model.
  • Table 6 Monovalent KD values of anti-TWEAKR antibodies measured by Biacore with TWEAKR kon (1/ s) k 0 ff (1/s) K D (nM)
  • the antibodies of the disclosure were determined to bind TWEAK R with moderate affinity (KD valus of 10 200 nM) whereas some antibodies used for comparison (e.g. PDL-192(TPP-1 104),
  • TPP-2093, TPP-2149 and TPP-2090 bind TWEAKR with moderate affinity (K D 10 - 200 nM) .
  • human, rat, murine, dog, pig and macaca fascicuiaris TWEAKR were expressed and purified as human Fc fragment fusion proteins and immobilized onto a CM5 sensor chip using amine coupling via a standard EDC/NHS-mediated chemistry (BR- 1006-33, GE
  • Table 7 List of recombinant proteins used in ELISA for profiling interspecies binders
  • concentrations 200 nM, 100 nM, 50 nM, 25 nM, I 2.5 nM, 6.25 nM, 3.12 nM, 1.56 nM
  • HEPES-EP buffer GE Healthcare Biacore, Inc.
  • Sensorgrams were generated after in-line reference cell correction followed by buffer sample subtraction.
  • the dissociation equilibrium constant (KD) was calculated based on the ratio of association (koni) and dissociation rated (kofn) constants, obtained by fitting sensorgrams with a bivalent analyte model using Biavaluation Software.
  • the species cross reactivity of anti-TWEAKR antibodies has been determined in "avidity mode" with immobilized bivalent antigen which does not provide "absolute” K D values, but gives good comparative data.
  • Table 8 KD values (nM) of anti-TWEAKR antibodies to different species measured by Biacore
  • TPP-1538, TPP-2077, TPP-2084 and TPP-2090 show affinity to ail tested species (human, rat, murine, dog, pig and macaca fascicularis TWEAKR).
  • TWEAKR cysteine rich domain (aa 34-68) of different species ( Figure 1) shows that it is well conserved throughout all 6 analyzed species.
  • PDL-192 binds dependent of R56 (WO2009/020933 : Figure 2B) and therefore does not bind to rat, pig and mouse TWEAKR.
  • TPP-2090 binds dependent of the conserved amino acid 047 and therefore binds to all depicted species.
  • a - and C- terminal truncation mutant of the TWEAKR ectodomain was generated and tested for its ability to bind to the different anti-TWEAKR antibodies.
  • the antibodies of the present disclosure show saturated binding to both constructs, whereas antibodies P4A8(TPP-1324), P3G5(TPP-2195) and ITEM-4 show saturated binding only to the full length ectodomain and impaired binding to the N- and C -terminally truncated construct ( Figure 3 & Figure 4). This indicates that the binding epitope of the antibodies of the present invention is located within the cysteine rich domain between amino acid 34-68.
  • Table 10 List of antibodies used in ELISA anal sis for e ito e rofilin
  • the binding epitope of TPP-2090, TPP-2084, PDL-192(TPP-1 104) and 136.1 is located within the cysteine rich domain and the binding epitope of P4A8 (TPP- 1324) and P3G5(TPP- 2195) is located at least partially outside of the cysteine rich domain.
  • Table 11 List of recombinant proteins used in ELISA analysis for mutein binding
  • the different TWEAKR-Fc muteins were coated with a low concentration (62 ng ml) in a 384-well Maxisorb ELI SA plate and a serial 2fold dilution of biotinylated lg( i starting with a concentration of 100 nM was used as soluble binding partner. Detection was done with Streptavidin-HRP and Amplex Red.
  • the tested igGs were TPP-2090 and TPP-2084, PDL-192, 136.1 and 18.3.3 from WO2009/020933, P4A8 and P3G5 from WO2009/140177, and ITEM - ! and ITEM-4 from Nakayama et al [Biochem Biophys Res Com 306: 819-825].
  • Table 13 List of commercially available antibodies used in ELI SA for mutein binding
  • IgGs were biotinylated using an approximately 2-fold molar excess of biotin-LC-NHS (Pierce; Cat.
  • IT EM -4 shows impaired binding to the H60K mutein [WO2009/140177: Figure 23 F] and PDL-192 to the R56P mutein [WO2009/020933 : Figure 22B].
  • ITEM- ! shows impaired binding to R56P and all antibodies to W42A [WO2009/140177: Figure 23E, Figure 23F]. This difference can be explained by the method chosen; the extreme low coating concentration favors the discrimination of off-rate impairments since it minimizes avidity effects. As none of the analyzed antibodies shows unimpaired binding to the W42A mutein, this substitution seems to cause rather structural changes and not a direct alteration of the binding epitope.
  • TWEAKR(34-68)-Fc construct S37A, R38A, S40A, S41 A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A and L65A.
  • Table 15 List of TWEAKR mutein constructs for alanine scan of cysteine rich domain
  • TPP-2624 TweakR-ECD-34-68-hIgGFe-His-F63A 150
  • TPP-2623 TweakR-ECD-34-68-hIgGFc-His-D62A 151
  • TPP-2622 TweakR-ECD-34-68-hIgGFc-His-S61A 152
  • TPP-2621 TweakR-ECD-34-68-hIgGFc-His-H60A 1 53
  • TPP-2620 TweakR-ECD-34-68-hIgGFc-His-P59A 154
  • TPP-2618 TweakR-ECD-34-68-hIgGFc-His-R56A 156
  • TPP-2617 TweakR-ECD-34-68-hIgGFc-His-S54A 157
  • TPP-2616 TweakR-ECD-34-68-hIgGFc-His-D51A 158
  • TPP-2615 TweakR-ECD-34-68-hIgGFc-His-K48A 159
  • TPP-2614 TweakR-ECD-34-68-hIgGFc-His-D47A 160
  • TPP-2613 TweakR-ECD-34-68-hIgGFc-His-D45A 161
  • TPP-261 1 TweakR-ECD-34-68-hIgGFc-His-W42A 163
  • TPP-2610 TweakR-ECD-34-68-hIgGFc-His-S41A 164
  • TPP-2609 TweakR-ECD-34-68-hIgGFc-His-S40A 165
  • TPP-2608 TweakR-ECD-34-68-hIgGFc-His-R38A 166
  • TPP-2607 TweakR-ECD-34-68-hIgGFc-His-S37A 167 Tfaese TWEAKR(34-68)-Fc muteins were expressed in HEK293 cells.
  • the tested IgGs were TPP-2090, PDL-192 from WO2009/020933 and ITEM- 1 (purchased from Abeam).
  • Table 16 List of antibodies used for alanine scan of c steine rich domain
  • TWEAK shows efficient activation of TWEAKR and binds dependent of Leucin 46 in the cysteine rich domain of TWEAKR (Pellegrini et al., FEBS 280: 1818-1829).
  • P4A8 shows very low agonistic activity and at least partially interacts with domains outside of the cysteine rich domain of TWEAKR ( Figure 4).
  • PDL-192 shows moderate agonistic activity and binds dependent of R56 to the cysteine rich domain but opposite to the TWEAK ligand site.
  • T - 2090 and TWEAK bind dependent on D47 and L46, respectively, and therefore bind to a similar binding site ( Figure 7).
  • TPP-2090 TPP-2149, TPP-2093, TPP-2148, TPP- 2084, TPP-2077, TPP- 1 538.
  • TPP- 1 858 were tested.
  • the antibodies of this disclosure bind to TWEAKR dependent on
  • TWEAK The identified dependencies on certain TWEAKR amino acids for antibody interaction correlate with the agonistic activity that has been determined for these antibodies.
  • the native ligand TWEAK shows efficient activation of TWEAKR and binds dependent on Leucin 46 in the cysteine rich domain of TWEAKR (Pellegrini et al.. FEBS 280: 1818-1829).
  • P4A8 shows very low agonistic activity and at least partially interacts with domains outside of the cysteine rich domain of TWEAKR.
  • PDL-192 shows moderate agonistic activity and binds dependent of R56 to the cysteine rich domain but opposite to the TWEAK ligand site.
  • Antibodies of this disclosure (see Figure 6C) bind dependent on D47, and TWEAK binds dependent on L46, and binds to a similar but distinguishable binding site ( Figure 7). Therefore the antibodies of this disclosure which show a strong agonistic activity bind to a novel epitope (D47 dependent) for antibodies which is connected to very strong agonistic activity.
  • Michaelson et al (see page 369, left column in Michaelson JS et al., MAbs. 2011 Jul-Aug;3(4):362-75) gave an explanation why all agonistic antibodies examined by them have weaker agonistic activity compared to the natural ligand TWEAK.
  • the decreased efficacy might be a function of the dimeric binding interaction of an antibody with TWEAKR wherein TWEAK presumably engages in a trimeric interaction. Therefore, it is a surprising finding that an antibody of the invention, though in a dimeric interaction with TWEAKR has even higher agonistic activity. This surprising effect is coupled to the specific binding property of the antibodies of this disclosure, hence specific binding to D47 of TWEAKR.
  • Table 17 List of antibodies used for com etition ex eriments
  • Table 18 List of commerciall available antibodies used for com etition ex eriments
  • Table 1 List of recombinant antigen used for competition experiments
  • the antibody TPP-2090 was also tested for binding to other members of the TNF receptor superfamily to assess its selectivity.
  • the TNF receptor superfamily shows very high sequence divergence as depicted in Figure 10. Most similar to TWEAKR are TNFRSF13C and TNFRSF 17 with only about 30% sequence identity.
  • the ectodomains of all 29 known TNF receptor superfamily members were purchased as Fc fusion proteins (Table 20) and 1 ⁇ g/ml were coated in a Maxisorp EL IS A plate.
  • TPP-2090 binds already at a very low concentration of 300 pM in saturation to TWEAKR whereas also at a very high concentration of 75 nM it does not bind to all other 28 TNF receptor superfamily members.
  • TPP-2090 binds selectively to TWEAKR.
  • Table 20 List of recombinant proteins used in ELI SA for selectivity profiling
  • TWEAKR (TNFRSF 12) 1 Human 1610-TW-050
  • Trail-R2 (TNFRSF10B) 5 Human 631-T2-100/CF
  • CD95 (TNFRSF6) 7 Human 326-FS-050/CF
  • TNF-R1 (TNFRSF1A) 9 Human 636-R1-025/CF
  • TNF-R2 (TNFRSFI B) 10 Human 1089-R2-025/CF
  • BCMA (TNFRSF17) 13 Human 193-BC-050
  • TACI TNFRSF13B 14 Human 174-TC-050
  • CD30 (TNFRSF8) 16 Human 6126-CD-l OO
  • CD27 (TNFRSF7) 17 Human 382-CD-lOO
  • CD40 (TNFRSF5) 18 Human 1493-CD-050
  • GITR TNFRSF18 21 Human 689-GR-100
  • HVEM (TNFRSF 14) 22 Human 356-HV-lOO/CF
  • NGF R (TNFRSF16) 23 Human 367-NR-050/CF
  • Trail R3 (TNFRSF10C) 24 Human 630-TR-lOO/CF
  • TROY (TNFRSF19) 28 Human 1548-TR-100
  • binding was tested by flow cytometry to a panel of cell lines.
  • Adherent cells were washed twice with PBS without ( a and Mg (Biochrom #L1825: aqueous solution containing 8000 mg/l NaCl, 200 mg/l KCl, 1 150 mg/l Na 2 HP0 4 , and 200 mg/l KH 2 P0 4 ) and detached by enzyme-free PBS based cell dissociation buffer (Invitrogen). Cells were suspended at approximately 10 5 cells/well in FACS buffer (PBS without Ca/Mg, containing 3% FCS, Biochrom).
  • Cells were centrifuged (250 g, 5 min, 4 °C) and supernatant discarded. Cells were resuspended in dilutions of the antibodies of interest (10 ⁇ g/ml in 80 ⁇ if not indicated otherwise) in FACS buffer, and incubated on ice for 1 h. In the following cells were washed once with 100 ⁇ cold FACS buffer and 80 ⁇ secondary antibody diluted at 1 : 150 (PE goat anti-human IgG, Dianova #109-1 15-098, or PE Goat Anti-Mouse IgG, Jackson Immuno Research #1 15-1 15-164) was added.
  • 150 PE goat anti-human IgG, Dianova #109-1 15-098, or PE Goat Anti-Mouse IgG, Jackson Immuno Research #1 15-1 15-164 was added.
  • Geo Mean - Geo Mean of secondary antibody alone >10: +, >100: ++, >1000: +++, 10000: ++++, close to category border in ().
  • the sources of the cell lines are given in Table 21.
  • ail anti-TWEAKR antibodies of this disclosure used at a concentration of 10 ⁇ g/ml bind a broad range of tumor cells expressing TWEAKR of murine (4T1 , Lewis Lung) and human (all other cell lines included in the table) origin representing a variety of tumor entities.
  • Table 21 Binding of anti-TWEAKR antibodies (10 ⁇ g/ml) to different cell lines by scoring of FACS analysis: TPP-1538 and TPP-2090 bind to a broad panel of murine and human tumor cell lines representing a variety of tumor indications.
  • HNSCC A253 ATCC HTB-41 11 +(+) n.d.
  • Murine C A cell lines Lewis Lung ATCC CRL-1642 3 + n.d.
  • Table 23 Binding of different anti-TWEAKR antibodies 10 ⁇ g/ml to a panel of cell lines by scoring of
  • HT-29 cells were plated at a density of 4000 cells/75 ⁇ /well in 96 well plates in assay medium (DMEM/Ham'sF12, Biochrom #FG4815 + 10% FCS + 100 ng/ml I F gamma (R&D Systems #285-IF)). 24h later cells were incubated with antibodies to the TWEAK R (see Table 24), recombinant human TWEAK ( R& D. #1090-TW- 025/CF, E.
  • the capacity to induce Caspase 3/7 by anti-TWEAKR antibodies as compared to recombinant human TWEAK was evaluated in a panel of cell lines.
  • WiDr cells were plated at 3000 cells/well and incubated for 48h in the presence of TWEAK or the described antibodies, A 253 cells were plated at 2500 cells/well and incubated for 24h, NCI-H322 cells were plated at 5000 cells/well and incubated for 48h and 786-0 cells were plated at 2500 cells/well and incubated for 48h.
  • the cells were plated in the media as described in Table 21 , for A253, NCI-H322 and 786-0 cells lOOng/ml I FN gamma (R&D Systems #285-IF) was added. 24 h after plating antibodies at 100 ⁇ g/ml or TWEAK at 300 ng/ml (100 ng/ml TWEAK for NCI-H322 cells) were added and the cells were further incubated for the time periods indicated above. At the end of the incubation time Caspase 3/7 activity was determined as described for HT-29 cells. The fold induction of Caspase 3/7 was calculated as compared to untreated cells. As shown in Table 25, all tested antibodies including TPP-2658, the aglycosylated variant of TPP-
  • Table 25 Fold induction of Caspase 3/7 in different cancer cells after incubation with 100 ⁇ g/ml anti- TWEAKR antibodies or recombinant human TWEAK (300 ng/ml or *100 ng/ml). Results from 1 -3 representative experiment carried out in triplicates are shown, including standard deviations. Tested antibodies show enhanced induction of Caspase 3/7 in different cell types as compared to known antibodies or recombinant TWEAK.
  • EXAMPLE 5 Inhibition of proliferation by agonistic anti-TWEAKR antibodies in cancer ceil lines
  • cells were plated in 96 well plates in 75 ⁇ assay medium (growth media from Table 21 , plus 100 ng/ml I FN gamma for 786-0 cells) at the following cell numbers: WiDr cells 3000 cells/well, 786-0 cells 2500 cells/well. 24h later cells were incubated with anti-TWEAKR antibodies (see Table 26), recombinant human TWEAK or isotype control IgG (not shown) at the indicated concentrations (antibodies from 0.03-300 ⁇ g/ml, TWEAK 100 or 300 ng/ml).
  • NCI-H1975, SW-480 (all 3000 cells/well), HT-29 (4000 cells/well), A253 and SK-OV3 (both 2500 cells/well) cells were incubated with 100 ⁇ anti-TWEAKR antibodies or TWEAK ligand for the time periods as indicated in Table 27. All cells were seeded in the growth media indicated in Table 21 and for all cells except for WiDr cells 100 ng/ml IFNgamma was added to the assay medium when seeding the cells. The percentage of growth inhibition for treated cells as compared to proliferation in untreated control cells was measured and calculated as described above.
  • the antibodies of the current disclosure are more efficacious as compared to other known antibodies in inhibiting proliferation of various cancer cell lines at 100 ⁇ g/ml. In most experiments the antibodies of the current disclosure also show equal or stronger efficacy as compared to 100-300 ng/ml TWEAK ligand. Thus, the antibodies described in this invention are unique in their activity to induce apoptosis and proliferation inhibition in a broad panel of cancer cell lines.
  • Table 27a shows the results of a separate experiment in WiDr cells with or without of 100 ng/ml IFNgamma added to the medium under the same conditions as described above.
  • TPP-2658 the aglycosylated variant of TPP-2090, shows anti-proliferative activity comparable to TPP-2090 and human recombinant TWEAKR ligand after 96h incubation. No activity was found with the isotype control antibody.
  • Table 26 List of antibodies used for roliferation assa
  • Table 27 % Inhibition of proliferation induced by incubation with 100 ⁇ g/m ⁇ anti-TWEAKR antibodies or TWEAK ligand (*100 ng/ml, **300 ng/ml). Incubation time in the presence of the agents is indicated as time of assay in [h]. Results from 1 -3 representative experiments carried out in triplicates are shown.
  • Antibodies show stronger inhibition of cancer cell proliferation as compared to known antibodies (PDL- 192(TPP-1104), P4A8(TPP-1324)) and equal or stronger activity as compared to recombinant TWEAK.
  • Table 27a Inhibition of proliferation (IC50) of WiDr cells induced by incubation with anti-TWEAKR antibodies or TWEAK ligand. Incubation time was 96h. Results from a representative experiment carried out in triplicates are shown. TPP-2658 demonstrates comparable potency to TPP-2090 and recombinant human TWEAK in the same nanomolar range.
  • EXAMPLE 6 Cytokine secretion induced by anti-TWEAKR antibodies from cancer ceils and xenograft tumors
  • A375 cells were plated at 2500 cells/well in 96 well plates in growth medium DM EM (Biochrom;# FG 0435. with stable Glutamin), 10% PC ' S. 24h later cells were incubated with anti- TWEAKR antibodies, recombinant human TWEAK at various concentrations as indicated or corresponding isotype control IgG. 24h after start of the incubation with the antibodies, cell supernatant or dilutions thereof were added to the Capture Elisa Plate of the human CXCL8/IL-8 ELISA Kit (R&D Systems DY208) and incubated over night at 4 °C by shaking 300 rpm.
  • the antibodies including TPP-2658, the aglycosylated variant of TPP-2090 showed increased induction of IL-8 release as compared to other antibodies previously known.
  • TPP-1538/-1854/-2084/-2090 reached 134/129/113/103% of the activation as compared to 300 ng/ml TWEAK ligand respectively.
  • the antibodies used for comparison PDL-192(TPP-1 104)/P4A8(TPP- 1324)/! 36.1 (TPP-2194), reached only 66/29/93%o, respectively.
  • the antibodies show the strongest activity with regard to induction of IL-8 secretion as compared to previously known antibodies and 300 ng/ml TWEAK ligand.
  • mice human and murine cytokines in serum/plasma from tumor bearing (A375, Wi Dr) as well as tumor free mice were investigated.
  • mice were treated by a single intravenous injection of TPP- 1538 (10 mg/kg) or TPP-2090 (3 mg/kg) both diluted in PBS into the tail vein. Mice were then sacrificed at given time-points (0, 6h, 24h for A375-bearing mice and 0, 7h, 24h, 72h, 168h, 240h for WiDr-b earing mice) to harvest serum/plasma samples. Blood was collected after decapitation and serum was prepared by 30 minutes clotting with subsequent centrifugation at lOOOxg.
  • Cytokines were quantified using Luminex® bead immunoassays. Human cytokines were determined with Human Cytokine Magnetic 2 -pi ex panel (Invitrogen®, Cat-No. LHC0009M), comprising IL- ⁇ ⁇ , I L- I RA. I I .-2. I L-2 R. IL-4, I L-5. IL-6, 11. -7, IL-8, IL-10, IL-12 (p40), IL-13, I L- 1 5. IL-17, TNF-a, IFN-a, IFN- ⁇ , GM-CSF, MIP- l a, ⁇ - ⁇ , I - 1 0. MIG, Eotaxin, RANTES, and MCP-1).
  • Murine cytokines were determined with Mouse Cytokine Magnetic 20-piex panel (Invitrogen®, Cat-No. LHC0006M) comprising FGF basic, GM-CSF, IFN- ⁇ , IL-l a, IL- ⁇ , 1 1.-2. IL-4, IL-5, IL-6, IL-10, I L- 1 . I L- 1 2 (p40/p70), IL-17, I P- 1 0. KC, MCP-1 , M IG. MIP-l a, TNF-a und VEGF).
  • the assays were conducted according to the manufacturer's instructions and measured by the Luminex reader io- PI ex 200 (Bio-Rad GmbH). Cytokine concentrations were interpolated from standard curves, as part of the assay procedure, by the operating software Bio-Plex Manger (Bio-Rad GmbH).
  • human IL-8 is released from WiDr xenograft by a single treatment with TPP-2090 3 mg/kg in a time dependent manner.
  • induction of secretion of human MCP-1 , I P- 1 0 and I L- 1 5 was observed after treatment with TPP-2090 (not shown).
  • human IL-8 is released from A375 xenografts in tumor bearing mice 6 h after treatment with TPP-1538 at 10 mg/kg.
  • increased levels of human MCP- 1 . IP-10 and IL-IRA were observed (not shown).
  • in the plasma of treated tumor free mice no increased secretion of human cytokines was detected ( Figure 16B and not shown).
  • no increase of murine cytokines including the murine IL-8 analogue KC was detected in the plasma of neither tumor bearing nor tumor free mice after treatment with TPP-1538 (data not shown).
  • the antibodies of the present disclosure potently induce secretion of cytokines from cancer cells and xenografts in vivo in a tumor specific manner.
  • EX AM PLE 7 Internalization of anti-TWEAKR antibodies and usability for drug conjugate approaches
  • TWEAK R specific antibodies TPP-1538 and TPP-2090 and an isotype control antibody were selected.
  • the antibodies were conjugated in the presence of a twofold molar excess of CypHer 5E mono NHS ester (batch 357392, GE Healthcare) at pH 8.3. After the conjugation the reaction mixture was dialyzed (slide- A-Lyser Dialysis Cassettes MWCD 1 OkD, Fa. Pierce) overnight at 4 °C to eliminate excess dye and to adjust the pH-value. Afterwards the protein solution was concentrated (VIVASPIN 500, Fa Sartorius Stedim Biotec). In addition to the pH-dependent fluorescent dye CypHer5E the pH-independent dye Alexa 488 was used.
  • the dye load of the antibody was determined with a spectrophotometer (Fa. NanoDrop).
  • the dye load of TPP-1538, TPP-2090 and an isotype control antibody were in a similar range.
  • the affinity of the labeled antibodies was tested in a cell binding-assay to ensure that labeling did not alter the binding to TWEAK R. These labeled antibodies were used in the following internalization assays. Prior to treatment cells (2xl0 4 /well) were seeded in 100 ⁇ medium in a 96-MTP (fat, black, clear bottom No 4308776, Fa. Applied Biosy stems).
  • TPP-1538 and TPP-2090 were assessed for TPP-1538 and TPP-2090 in endogenous TWEAKR expressing cancer ceil lines 786-0 (renal cancer) and HT-29 (colon cancer).
  • anti-TWEAKR antibodies of the present disclosure show rapid internalization and targeted delivery of conjugated payloads and are thus well suitable for the generation and use as ADC ' s.
  • EXAMPLE 8 Anti-Tumor efficacy of anti-TWEAKR antibodies in xenograft models in vivo
  • anti-TWEAKR antibodies show anti-tumor activity in vivo xenograft tumors derived from different cancer cell lines or patient derived tumor models were tested for their sensitivity against tumor growth inhibition by agonistic anti-TWEAKR antibodies in mono- or combination therapy.
  • expression of TWEAKR in the selected xenograft models was evaluated by immunohistochemistry. Therefore, frozen sections (5 ⁇ ) of the corresponding xenografts were fixed with acetone for 5 min at 4 °C and blocked against unspecific protein binding and peroxidase activity.
  • Tissue sections were incubated with rabbit anti-TWEAKR antibody (Fnl4, Epitomics, 3488-1) at room temperature for 60 min, followed by peroxidase labeled anti rabbit polymer (DAKO, K401 1) incubation for 30 min. Sections were developed with diaminobenzidine and finally counters tained with hematoxylin. Only models that were positive for expression of the TWEAKR were used for in vivo experiments
  • nude mice bearing xenografts from different human tumor cell lines or patient-derived tumors were treated by repeated intravenous injections.
  • Tumor cell lines were cultivated as described in the parts above and 100 ⁇ containing cell line specific numbers of tumor cells inoculated subcutaneously (s.c.) into female athymic nude mice (NMRI nu/nu, 6-8 weeks, 20-25 g, Taconic). Mice were housed under standardized pathogen free conditions and treated according to the animal welfare guidelines.
  • Regorafenib 10 mg kg daily, per os
  • PI3K- inhibitor 1 10 mg kg.
  • BID, 2 d on, 5 d off (applications twice daily on two consecutive days, followed by five days without treatment), i.v.) were diluted in their respective formulations whereas the standard of care therapies Irinotecan (5 mg/kg, 4 d on, 3 d off (applications once daily on four consecutive days followed by three days without treatment) i.v.) and Paclitaxel (16 mg/kg, q7dx4 (applications once per week, four applications in total), i.v.) were diluted in 0.9% NaCi. Animals injected with PBS served as the control (vehicle) group. The applied volume of the compounds was 5 ml/kg body weight per mouse.
  • Tumor growth was monitored 2-3 times per week by caliper measurement (length x width in mm) as well as body weight (in g). At the end of study tumors were dissected, weighted and used for the calculation of tumor-to-control (T/C) ratios (mean tumor weight of treated animals divided by mean tumor weight of control/vehicle animals).
  • T/C tumor-to-control
  • Tumor-to-control ratios listed in Table 28 demonstrate good efficacy in the 786-0 model and in further tumor models (A375, A253, SK-OV-3, Bx-PC3, treated with 3-10 mg/kg anti-TWEAKR antibodies TPP-1538, TPP-2094 or TPP-2090 in a q7dx3 (applications once per week, three applications in total) or q4dx3 (applications twice per week, three applications in total) schedule with the exception of MDA-MB-231 where more intense dosing schedules of anti-TWEAKR antibodies might be required to reach monotherapy efficacy.
  • Table 28 Final Tumor-to-Control (T/C) ratios in 786-0 and further tumor models after treatment with TPP-1538, TPP-2084 or TPP-2090.
  • Anti-TWEAKR antibodies show strong anti-tumor activity in a variety of xenograft models from different solid tumor indications.
  • T/C tumor-to- control ratio based on final tumor weight after dissection or based on measurement of tumor area ( * ).
  • Figure 20 shows the efficacy of the anti-TWEAKR antibody TPP-2090 in the human colon cancer xenograft WiDr (which represents a subclone of the HT-29 tumor cell line) in monotherapy and combination therapy with Irinotecan and Regorafenib.
  • WiDr which represents a subclone of the HT-29 tumor cell line
  • 5xl0 6 WiDr cells in Matrigel/Medium (1 : 1) were s.c. inoculated in immunodeficient NMRI nude mice. Treatment started 7d after inoculation with established tumors of about 40 mm 2 . Even 3 mg ' kg of TPP-2090 (i.v., q4dx7: applications twice per week, seven applications in total) in monotherapy was strongly effective to control tumor growth.
  • TPP-2090 was also investigated in other colorectal tumor models such as SW480 and the patient- derived tumor model Co5682 with similar good results (Table 29).
  • a dose of 10 mg/kg TPP-2090 was effective in monotherapy in SW480 to control tumor growth (T/C 0.49) and to lead to tumor regression in combination with 5 mg kg Irinotecan (T/C 0.22) or 10 mg kg Regorafenib (T/C 0.37).
  • 3 mg/kg of TPP-2090 showed synergistic efficacy with tumor regression in combination with Irinotecan (T/C 0.23) and tumor stasis in combination with Regorafenib (T/C 0.27).
  • Table 29 Final Tiimor-to-Control (T/C) ratios of two colon cancer cell lines WiDr and SW480 and one patient-derived colon cancer xenograft Co5682 after treatment with TPP-2090 and combination partners based on tumor weights at study end.
  • T/C tumor-to-control ratio based on final tumor weight after dissection
  • Tx therapy
  • combo combination therapy
  • Figure 21 shows the efficacy of the anti-TWEAKR antibody TPP-2090 in the human non-small-cell lung cancer xenograft NCI-H322 in monotherapy and combination therapy with Paclitaxel.
  • 5x10 6 NCI- H322 cells in Matrigei were s.c. inoculated in immunodeficient NMRI nude mice. Treatment started 14d after inoculation with established tumors of about 45 mm 2 .
  • At a dose of 5 mg kg TPP-2090 i.v., q4dx8) was strongly effective in monotherapy demonstrating tumor regression.
  • TPP-2090 was also investigated in other lung cancer models such as NCI-H 1 975 and the patient-derived tumor models Lu7343 and Lu7433 with comparable results (Table 30).
  • a dose of 3 mg/kg TPP-2090 showed additive effects in NCI-H 1 975 in combination with 16mg/kg Paclitaxel resulting in tumor regression (T/C 0.08).
  • Additive effects were also achieved with the same dose (3 mg/kg) of the aglycosylated variant TPP-2658 in combination with paclitaxel (16 mg/kg) in NCI- H 1975 xenografts.
  • Lu7343 and Lu7433 combination of 3 mg/kg TPP-2090 with 10 mg/kg of the PI3K-inhibitor 1 led to tumor control or regression (T/C 0.18- 0.36) in an additive efficacious manner.
  • PI3K-inhibitor 1 is (2-amino-N-[7-methoxy-8-(3-mo holin-4-ylpropoxy)-2,3-dihydroimidazo[l ,2- c]quinazolin-5-yl]pyiimidine-5-carboxamide dihydrochloride.
  • T/C Tumor-to-Control
  • T/C tumor-to-control ratio based on final tumor weight after dissection
  • Tx therapy
  • combo combination therapy
  • tumors from WiDr xenografts were taken at study end as described in Example 8 and investigated by immunohistochemistry and Western Blot Analysis.
  • Sections were developed with diaminobenzidine and finally counters tained with hematoxylin.
  • entire tumor sections were scanned and analyzed using AR IOL automated microscopy version 3.2 (Applied Imaging, San Jose CA, USA).
  • Representative images of PBS (i.v., q4dx7) and TPP-2090 (10 mg/kg, i.v., q4dx7) treated xenografts stained for Ki67 are shown in Figure 22 A and B respectively.
  • the quantification using the ARIOL image system revealed 355+A59 Ki67 positive cells / mm 2 in the group treated with 10 mg/kg TPP-2090 (i.v.
  • Samples were diluted to a final concentration of 4mg/ml and 10 ⁇ of sample were mixed with 3.08 ⁇ of (10*) Sample Reducing agend, 10 ⁇ H 2 0 and 7,68 ⁇ (4*) NuPAGE Sample Buffer (Invitrogen). Samples corresponding to 40 ⁇ g of protein were applied to NuPage 4-12% SOS page gels from Invitrogen and run for 2 h 45 min at 120 V. Blotting was carried out by an iBlot system (Invitrogen) according to the manufacturer's recommendations. Membranes were blocked for 2h at room temperature in 5% BLOT QuickBlocker in PBST (Invitrogen), followed by incubation with primary antibodies over night at 4 °C.
  • Primary antibodies were as follows: Phospho-Statl #9167S, Stat-1 #9172, both Cell Signaling Technology, dilution 1 : 1000; TWEAKR Fn l 4 #3488-1 Epitomics, dilution 1 : 10000; NF-kappaB2 pl 00/p52 #4882S, Ceil Signaling Technology, dilution 1 : 1000 in in 3% BLOT QuickBlocker in PBST. On the next day membranes were washed three times in PBST, followed by incubation with secondary antibodies (Peroxidase-conjugated donkey anti-rabbit IgG # NA934, GE Healthcare 1 : 10000 in 3% BLOT QuickBlocker/PBST) for 2h at room temperature.
  • secondary antibodies Peroxidase-conjugated donkey anti-rabbit IgG # NA934, GE Healthcare 1 : 10000 in 3% BLOT QuickBlocker/PBST
  • membranes were washed four times for 10 min with PBST and signals were detected by chemoluminescence after incubation with ECL reagent.
  • FIG. 23 Representative Blots from tumors of 2 animals per group treated with TPP-2090 3 mg kg side by side with tumors from vehicle treated animals are shown in Figure 23.
  • Treatment with TPP-2090 leads to strong increase of total and phosphorylated Stat-1 levels as well as a strong activation of NF-kappaB2 as indicated by the appearance of the p52 fragment.
  • the NF-kappaB2 as wells as Stat-1 pathways are activated by agonistic anti-TWEAKR antibodies in xenograft tumors and this activation is potentially involved in the anti-tumor activity of the corresponding antibodies.
  • Table 31 Protein se uences of the antibodies:
  • EXAMPLE 10 Anti-tumor efficacy of anti-TWEAKR antibody TPP-2090 in further human colorectal cancer models in vivo
  • Standard dosing schedule was 10 mg/kg of TPP-2090 twice weekly for 4 weeks in monotherapy or in combination with regorafenib or the standard of cares (SoCs) irinotecan (5-15 mg/kg i.p., 4 d on, 3 d off), oxaliplatin (3-8 mg/kg i.p., twice weekly), 5-fhioroiiracil (50-100 mg/kg i.p., once weekly) and cetuximab (15 mg/kg i.p., twice weekly) .
  • TPP-2090 and cetuximab were formulated in PBS, which was also used as the vehicle in the control group, and the SoCs were formulated in 0.9% NaCl.
  • the formulation of regorafenib is described in example 8.
  • TPP-2090 in these human colorectal cancer patient-derived and cell line based models was moderate with final Tumor-to-Control (T/C) ratios in the range of 0.48-1.07.
  • T/C Tumor-to-Control
  • SoCs in particular 5-FU and irinotecan
  • Table 33-35 the monotherap eutic efficacy of T - 2090 in these models were limited more intense dosing schedules of anti-TWEAKR antibodies might be required to reach higher monotherapy efficacy, as has been shown in example 8 for colorectal cancer.
  • Table 33 Final Tumor-to-Control (T/C) ratios of colorectal cancer models treated with TPP-2090
  • T/C tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
  • T/C tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
  • T/C Tumor-to-Control
  • T/C tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
  • TPP-2090 was formulated in PBS, which was also used as the vehicle in the control group, and the standard of cares (SoCs) were formulated in 0.9% NaCl.
  • TPP-2090 was found in SCaBER xenograft model.
  • the combination of TPP-2090 in the human bladder cancer patient-derived bladder cancer models BXF1352 and BXF1228 with SoCs (Cisplatin and Gemcitabine) resulted in significant synergistic effects (see
  • Table 36 Tumor-to-Control (T/C) ratios of human bladder cancer models treated with TPP-2090 in monotherapy or combination with cisplatin or gemcitabine
  • T/C tumor-to-control ratio based on final tumor weight
  • EXAMPLE 12 Anti-tumor efficacy of anti-TWEAKR antibody TPP-2090 in further human cancer models in vivo
  • TPP-2090 was formulated in PBS, which was also used as the vehicle in the control group. Strong monotherapeutic efficacy of TPP-2090 was found in SCC4 (head & neck cancer) and A375
  • T/C Tumor-to-control
  • T/C tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
  • EXAMPLE 13 Further mode of action of anti-TWEAKR antibodies in xenograft models
  • TPP-2090 is dependent on antibody-dependent cellular cytotoxicity (ADCC) or agonistic activity alone is already sufficient
  • ADCC antibody-dependent cellular cytotoxicity
  • SCID beige mice Janvier
  • the aglycosyl variant of TPP-2090, namely TPP-2658 was investigated in NMRi nude mice.
  • Binding of TPP-2090 and TPP-2658 to human FcgR2B/C (CD32b/c, R&D Systems, Inc., catalog number 1875-CD), cynomolgus (Macaca fascicularis) FcgR2B (CD32b, Sino Biological Inc., catalog number 90014-C08H) and murine FcgR2B (CD32b, R&D Systems, Inc., catalog number 1460-CD) were measured by using surface plasmon resonance (SPR) assays.
  • SPR surface plasmon resonance
  • Binding assays were carried out at 25 °C with assay buffer HBS-EP+ (10 mM HEPES pH 7.4, 150 nM Nad. 0.05% SP20; GE Healthcare Biacore, Inc.). Polyhistidine-tagged FcyR proteins were captured with an anti-His capture antibody covalently immobilized to the chip surface via amine coupling chemistry. Reagents for amine coupling ( 1 -ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanolamine-HCi pH 8.5) were used from the Amine Coupling Kit (GE Healthcare, product code BR-1000-50).
  • EDC -ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • ethanolamine-HCi pH 8.5 were used from the Amine Coupling Kit
  • anti-His capture antibody was obtained from (Molecular Probes, Inc., catalog number P21215) and immobilization buffer (10 mM sodium acetate pH 4.0) was used from the Human Antibody Capture Kit (GE Healthcare, BR- 1008-39).
  • the sensor chip surface was activated with a freshly prepared solution of 0.2 M EDC and 0.05 M NHS passed over the chip surface for 420 s at a flow rate of 10 ⁇ /min, followed by an injection of anti-His capture antibody (dissolved to 10 ⁇ g/m ⁇ in immobilization buffer) for 180 s at a flow rate of 5 ⁇ /min. Excess of activated groups were blocked with a 1 molar solution of ethanolamine injected at a flow rate of 10 ⁇ /min for 420 s.
  • TPP-2090 and TPP-2658 were used as analyte.
  • FcyR variants were captured at a flow rate of 10 ⁇ /min to a final response of - 26 RU prior to each analyte injection.
  • various concentrations between 0.64 and 25 ⁇ of human IgG in assay buffer were injected over the captured FcyR variants at a flow rate of 30 ⁇ /min for 3 minutes and the dissociation was monitored for 5 minutes.
  • Obtained sensorgrams were double-referenced, i.e. in-line reference cell correction followed by buffer sample subtraction. Data evaluation was performed with the Biacore T200 Evaluation Software Package.
  • TPP-2090 and TPP-2658 were captured with an anti-hlgG capture antibody (Human Antibody Capture Kit, GE Healthcare, BR-1008-39) covalently immobilized to the chip surface via amine coupling chemistry as described above.
  • Purified recombinant human TWEAKR-ECD protein (Fitzgerald Industries International, MA, USA, catalog number 30R-AT080) was used as analyte. After capture of TPP-2090 and TPP-2658 at 5 ⁇ g/ml in assay buffer at a flow rate of 10 ⁇ /min for 60 seconds to a final response of -220 RU.
  • TWEAKR-ECD protein was injected at concentrations ranging from
  • Tabie 43 SPR analysis of TWEAK interactions: deglycosylation does not influence binding affinity to soluble human TWEAKR-ECD
  • TPP-2658 13 Animal studies were conducted as described in example 8 in WiDr (human colorectal cancer) and SCaBER (human bladder cancer) xenografts in SCID beige mice with tumor growth of control groups comparable to those in NMRI nude mice of previous studies. Standard dosing schedule was 10 mg/kg of TPP-2090 i.v. (formulated in PBS) twice weekly for 2 weeks in monotherapy.
  • TPP-2090 A similar strong monotherapeutic efficacy of TPP-2090, including its aglycosyl variant TPP-2658, in NK-cell lacking SCID beige mice xenograft models (WiDr and SCaBER) was found as seen in NMRI nudes mice. This indicates an in vivo mode of action independent from ADCC (see Table 38).
  • T/C Tumor-to-Control
  • T/C tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
  • TPP-2090 namely TPP- 2658
  • TPP-2658 was used in either a Wi Dr- or A375 -xenograft model (see Table 39).
  • the variant TPP-2658 showed equally strong monotherapeutic efficacy as the TPP-2090 in both models indicating an ADCC- independent mode of action.
  • TPP-2658 was combined with anti-CLLA4, anti PD-1 , anti-PD-Ll antibodies binding to the murine proteins (mouse surrogates).
  • Tfae anti-CLLA4 (BE0131 -R005mg) and anti PD-1 antibody (BE0146-R005mg) were purchased from BioXcell. The endotoxin concentration was confirmed to be below 2 EU/mg by standard methology and antibodies were used directly for in vivo experiemnts.
  • variable domain of anti-PD-Ll antibody RG7446 from patent US8217149 was fused with mlgGl CH1-CH3 sequences to optain a full length chimeric IgG with the following sequence: anti-PD-L1 -mlgGl Kappa_RG7 46 light chain (SEQ ID NO:214):
  • the anti-PD-Ll antibody was expressed as described above in the chapter antibody generation.
  • T/C Tumor-to-Control
  • Tabie 39a Combination (Combo) effects in syngeneic MC38 and CT26 tumors treated with TPP- 2658 and immune-checkpoint inhibitors (anti-PD-Ll, -CTLA4- und -PD-1 [mouse surrogates])
  • T/C Tumor-to-Controi ratios
  • Table 40 In vitro ADCC assay with HT-29 target cells and NK92V effector cells for testing antibody TPP-2090 (MgGl) and TPP-2658 (aglycosylated counterpart of TPP-2090 - gGl N297A):
  • the liver-specific enzymes alanin-aminotransferase (ALAT) and glutamatedehydrogenase (GLDH) as well as the pancreatic enzymes lipase and amylase and the kidney markers creatinine (CREA) and urea were determined in blood samples of the monkeys as laboratory parameters. Increase in these liver and pancreas enzymes in the blood indicate cellular damages of these particular organs.
  • Tweak R is expressed in liver, pancreas and kidneys only at a very low level
  • T - 2090 induced toxicities due to its agonistic mode of action strongly potentiated by FcyR-mediated cross- linking by immune cells.
  • TPP-2658 lacking FcyR binding ability drastically reduced these toxicities to minor grades.

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Abstract

The present invention provides recombinant antibodies that are specific for the TWEAKR (TNFRSF12A, FN14) comprising a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region. Agonistic anti-TWEAKR antibodies comprising a mutation in the Fc region at N297 (using Kabat EU numbering) resulting in an aglycosyl antibody have less side effects and an improved toxicity profile compared to antibodies comprising the natural sequence of the Fc region. The antibodies, accordingly, can be used to treat tumors and other disorders and conditions associated with expression of the TWEAKR. The invention also provides nucleic acid sequences encoding the foregoing antibodies, vectors containing the same, pharmaceutical compositions and kits with instructions for use.

Description

. i -
Ag!ycosyl Anti-TWEAKR Antibodies and Uses Thereof
The present invention provides recombinant antibodies that are specific for the TWEAKR (TNFRSF12A, FN 14) comprising a mutated Fc region lacking the glycans attached to the conserved N- linked site in the CH2 domains of the Fc region. The antibodies, accordingly, can be used to treat tumors and other disorders and conditions associated with expression of the TWEAKR. The invention also provides nucleic acid sequences encoding the foregoing antibodies, vectors containing the same, pharmaceutical compositions and kits with instructions for use.
BACKGROUND OF THE INVENTION Antibody-based therapy is pr ving very effective in the treatment of various cancers, including solid tumors. For example, HERCEPTIN® has been used successfully to treat breast cancer and RITUXAN® is effective in B-celi related cancer types. Central to the development of a novel successful antibody-based therapy is the isolation of antibodies against cell-surface proteins found to be preferentially expressed on tumor cells that are able to functionally modify the activity of the corresponding receptor.
Tumor necrosis factor (TNF) like weak inducer of apoptosis (TWEAK) and the TWEAK receptor (TWEAKR, alias TNFRSF12A, FN 14, CD266; Swiss Pro! Acc. Q9NP84, NP_057723) are a TNF superfamily ligand-receptor pair involved in inflammation, proliferation, invasion, migration, differentiation, apoptosis and angiogenesis (Winkles J A, Nat Rev Drug Discov. 2008 May;7(5):411 -25; Michael son JS and Burkly LC, Results Probl Cell Differ. 2009;49: 145-60). TWEAK binds to TWEAKR with an affinity of 0.8 - 2.4 nM and is the only member of the TNF family that binds this receptor (Wiley SR et al .. Immunity. 2001 Nov; 15(5):837-46). The TWEAKR is expressed at relatively low levels in normal tissues, but is markedly increased locally in injured tissues, where it has a role in tissue remodeling (Winkles J A, Nat Rev Drug Discov. 2008 May;7(5):411 -25; Zhou et al., Mol Cancer Ther. 2011 Jul; 10(7): 1276-88; Burkly LC et al., Immunol Rev. 2011 Nov;244(l):99-114). TWEAKR signaling is involved in processes as wound healing, chronic autoimmune disease and acute ischemic stroke (Burkly LC et al., Immunol Rev. 201 1 Nov;244(l):99-114). In addition, the TWEAKR is highly expressed in various solid tumor types as for example pancreatic cancer, non-small-cell-lung-cancer (NSCLC), colorectal cancer (CRC), breast cancer, renal cancer, head and neck cancer, esophageal cancer, bladder cancer, hepatocellular carcinoma, ovarian cancer, melanoma as well as liver and bone metastasis (Culp P et al., Clin Cancer Res. 2010 Jan 15; 16(2):497-508; Zhou H et al., J Invest Dermatol. 2013 Apr; 133(4): 1052-62). Association of increased TWEAKR expression and higher tumor grade and/or poor prognosis has been described in brain (Tran NL et al., Cancer Res. 2006 Oct 1 ;66(19):9535- 42), breast (Willis AL et al., Mol Cancer Res. 2008 May;6(5):725-34; Wang J et al., Histol Histopathol. 2013 Jan 9 [Epub ahead of print]), esophageal (Watts GS et al., Int J Cancer. 2007 Nov 15; 121(10):2132-9 2007), prostate (Huang M et al, Carcinogenesis. 201 1 Nov;32(l l): 1589-96), gastric (Kwon OH et al, Cancer Lett. 2012 Jan 1 ;314(1):73-81), neuroblastoma (Pettersen I et al., Int J Oncol. 2013 Apr;42(4): 1239-48) and bladder cancer (Shimada K et al, Clin Cancer Res. 2012 Oct l ; 18(19):5247-55).
Expression of TWEAKR is induced by growth factors as FGF, PDGF and VEGF (Winkles J A, Nat Rev Drug Discov. 2008 May;7(5):41 1 -25). In line with this observation, it has been shown that TWEAKR expression correlates with EGFR overexpression or activation in NSCLC (Whits ett TG et al., Am J Pathol. 2012 Jul; 181(l): l 1 1 -20) and HER2 expression in breast cancer (Wang J et al., Histol Histopathol. 2013 Jan 9 [Epub ahead of print]; Chao DT et al., J Cancer Res Clin Oncol. 2013 Feb; 139(2):315-25).
Activation of the TWEAKR by TWEAK leads to recruitment of TNF -receptor associated factors (TRAF) to the intracellular binding domain resulting in prolonged NF-κΒ activation via the canonical and non-canonical NF-κΒ pathway and induction of cytokine secretion as I I. -8 and MCP-1 (reviewed in Michaelson .IS and Buddy LC, Results Probl Cell Differ. 2009;49: 145-60). This is well in accordance with the described pro-inflammatory role of the TWEAK/TWEAKR pathway. However, the signaling pathways responsible for cell killing via TWEAKR are less clear, as the TWEAKR lacks a characteristic "death domain". In some tumor cell lines (Kym-1 , SKOV-3, OVCAR ) it induces apoptosis through TNF and the recruitment of TRAF2, followed by lysosomal degradation of the resulting TRAF2-cIAP complex (Nakayama M. et al., J Immunol. 2002 Jan 15; 168(2):734-43; Schneider P et al., Eur .1 Immunol. 1999 Jun;29(6): 1785-92; Vince JE et al, J Cell Biol. 2008 Jul 14; 182(l): 171-84). In other cell lines (HSC3, HT-2 . KATO-I I I ) TWEAK induced apoptosis is reported to be TNF independent (Nakayama M et al., J Immunol. 2003 Jan l ; 170(l):341 -8; Wilson CA et al., Cell Death Differ. 2002 Dec;9(12): 1321-33). I n a recent report induction of apoptosis by TWEAK was shown to be dependent on the stimulation of Stat-1 phosphorylation as treatment with a JAK- inhibitor abolished the ability of TWEAK to increase caspase3/7 activation in Wi Dr cells (Chapman MS et al., Cytokine. 2013 Jan;61 (l):210-7).
Several studies validated TWEAKR as an oncologic target. Michaelson et al. have shown that the administration of TWEAK reduces tumor growth in murine xenograft models (Michaelson JS et al., MAbs. 201 1 Jul-Aug;3(4):362-75). This anti-tumor effect has been imitated by several groups with agonistic anti-TWEAKR antibodies. Potential drug candidates, namely BIIB0036/P4A8 (Michaelson JS et al, MAbs. 201 1 Jul-Aug;3(4):362-75) and PDL-192, (Culp PA et al, Clin Cancer Res. 2010 Jan 15; 16(2):497-508) have been generated by immunization of mice and subsequent clonal selection and humanization. PDL-192 binds to the TWEAKR with a binding affinity of 5.5 nM (Culp PA et al., Clin Cancer Res. 2010 Jan 15; 16(2):497-508) and inhibits the growth of several TWEAKR expressing cancer cell lines. Yet, in comparison to TWEAK ligand PDL-192 was shown to be less potent in proliferation and apoptosis assays with respect to EC/IC50 and only reached reduced efficacy (Vmax) of caspase 3/7 activation (Culp PA et al., Clin Cancer Res. 2010 Jan 15; 16(2):497-508). Profiling in a larger panel of breast cancer cell lines confirmed the only modest anti-proliferative activity of monomeric PDL-192 (Culp PA et al, Clin Cancer Res. 2010 Jan 15; 16(2):497-508; Chao DT et al, J Cancer Res Clin Oncol. 2013 Feb; 139(2):315-25) with only 5 of 27 cell lines responding with >20% of proliferation inhibition. Anti -proli ferative activity of the antibody is slightly enhanced by cross-linking or immobilization of the antibody. In addition, PDL-192 exhibits ADCC and the anti-tumor activity described in xenograft models is thought to be a mixture of ADCC and tumor cell growth inhibition effects (Culp PA et al., Clin Cancer Res. 2010 Jan 15; 16(2):497-508). A further limitation of PDL-192 is the lack of species cross-reactivity, especially mouse and rat, not allowing e.g. assessment of common pre-clinical studies as toxicological studies. First results of clinical trials with PD-L192 show relatively low dose limiting toxicity at doses of 1 -1.5 mg/kg (Lam et al., Abstract C 18, Mol. Cancer Ther. 2011 ; 10:C18).
The second agonistic anti-TWEAKR antibody described as drug candidate, ΒΠΒ036/Ρ4Α8 binds to TWEAKR with an affinity of 1.7 nM which is in a similar range as the endogenous ligand TWEAK (Michaeison JS et al., MAbs. 2011 Jul-Aug;3(4):362-75). This antibody is shown to induce activation of NF-KB and cytokine release in cancer cells, albeit significantly less efficacious compared to Fc- TWEAK, a hlgGl Fc-fusion of soluble TWEAK (aa 106-249) with similar activity as recombinant soluble TWEAK (Michaeison JS et al. Oncogene. 2005 Apr 14;24(16):2613-24). The same holds true in cell proliferation assays as well as for induction of apoptosis as shown in a TUNEL staining after treatment of cells with antibodies, where potency of ΒΠΒ036/Ρ4Α8 is also significantly decreased compared to Fc-TWEAK, Anti-proliferative activity increases after multimerization of the antibody, but also the multimerized form is still less efficacious as compared to recombinant Fc-TWEAK. In contrast, BIIB036/P4A8 is a potent inducer of ADCC and anti-tumor activity in xenograft models was shown to be largely dependent on Fc effector function.
Besides both drug candidates several murine antibodies have been described that would need antibody engineering for humanization to be useful for a human therapy. The first anti-TWEAKR antibodies with anti-proliferative activity on cancer cells were antibodies Item 1 -4 described by Nakayama et al . (Nakayama M et al, Biochem Biophys Res Commun. 2003 Jul 1 1 ;306(4):819-25). These antibodies, however, harbor only relatively weak agonistic activity and were shown to act as partial agonists/antagonists with regard to TWEAK mediated TWEAKR activation. Antibodies 136.1 and 18.3.3 (WO2009/020933) show higher affinity binding compared to TWEAK ligand, which does not translate in more efficacious caspase activation. Antibodies P3G5 and P2D3 (WO2009/140177) induce cytokine release in cancer cells significantly less efficacious compared to Fc-TWEAK. To summarize, TWEAKR agonistic activity with regard to induction of apoptosis and inhibition of proliferation of the anti-TWEAKR antibodies described in the art is limited and does not reach or exceed the efficacy of the endogenous ligand TWEAK. This lack of agonistic activity is not due to a decreased affinity as these antibodies bind to the TWEAKR with affinities in a similar range as compared to the endogenous ligand TWEAK (Michaelson JS et al, MAbs. 201 1 Jul-Aug;3(4):362-75; G ulp PA et al, Clin Cancer Res. 2010 Jan 15; 16(2):497-508) and also antibodies with higher binding affinity do not necessarily exhibit more potent signaling activity (Culp PA et al., Clin Cancer Res. 2010 Jan 15;16(2):497-508). Anti-tumor activity of the antibodies described previously is shown to be dependent on Fc effector function and ADCC is shown to play a significant role for the in vivo efficacy in mouse models. Additionally, patients carrying low-affinity alleles of FcyRIIIA would exhibit a reduced benefit from the treatment due to lower Fc-FcR interaction capacity (Varchetta S et al., Cancer Res. 2007 Dec 15;67(24): 1 1991-9).
Thus, developable human antibodies with strong intrinsic capacity to induce cancer ceil apoptosis and growth inhibition by hyper-activation of the TWEAKR to the same or even higher extend as compared to the endogenous ligand TWEAK are highly demanded. Currently established is the opinion that in vivo cross linking via Fc-Fc receptor (FcR) interactions is a mandatory feature for agonistic anti
TWEAKR antibodies.
In addition in the light of the first results of clinical trials with PDL-192 showing relatively low dose limiting toxicity at doses of 1 - 1 .5 mg/kg (Lam et al., Abstract C18, Mol. Cancer Ther. 201 1 ; 10:C18) antibodies with an improved safety and tolerateability profile are highly demanded.
As induction of apoptosis and inhibition of proliferation is since many years a valid concept in inducing anti-tumor response in patients (Hanahan D and Weinberg RA, Cell. 2000 Jan 7; 100(1):57-70; Kim R et al, Cancer Chemother Pharmacol. 2002 Nov;50(5):343-52; Fesik SW, Nat Rev Cancer. 2005 Nov;5(l l):876-855) these antibodies are expected to show increased anti-tumor activity in solid tumors in human and are therefore promising drug candidates for the treatment of cancer.
SUMMARY OF THE INVENTION
This invention is related to antibodies comprising a mutated Fc region lacking the glycans attached to the conserved N-iinked site in the CH2 domains of the Fc region (called aglycosyl antibodies), and which lead to strong activation of the TWEAKR, thus leading to a strong induction of apoptosis in various cancer cells showing overexpression of the TWEAKR and having an improved safety and tolerateability profile.
Induction of cancer cell apoptosis by the antibodies described herein is more efficacious compared to all antibodies described in the art (e.g. PDL-192 or BIIB0036/P4A8; e.g. require the addition of a cross-linking agent). The unique property of the antibodies of this invention is based on a novel binding epitope characterized by selective binding of the antibodies to amino acid at position 47 (D47) f
TWEAKR ( SEQ ID NO: 169; and see Figure 1).
The antibodies of the invention are thus suitable for the treatment of cancer as well as metastases thereof, in particular TWEAKR expressing tumors, such as colorectal cancer, non-small-cell lung cancer (NSCLC), head and neck cancer, esophageal cancer, melanoma, hepatocellular carcinoma, bladder cancer, gastric cancer, breast cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, ovarian cancer and cervical cancer in monotherapy as well as in combination with other targeted and non- targeted anti-tumor therapies. The invention describes antibodies that are distinguished from existing anti-TWEAKR antibodies in that they induce strong activation of cancer cell apoptosis, at superior levels as compared to the endogenous ligand TWEAK in most cell lines. The antibodies of the invention a) strongly activate the TWEAKR, b) induce apoptosis in cancer cells, c) induce cytokine secretion from cancer cells, d) all together resulting in anti-tumor activity of the antibodies in in vivo tumor experiments, e) additionally the antibodies lead to internalization of the TWEAKR and inhibition of cancer cell proliferation when incubated with saporine-conjugated secondary antibodies in experimental conditions where the antibody alone has no effect, f) are crossreactive to several species. These and other objects of the invention are more fully described herein.
In addition the antibodies of this invention comprise a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region. It was surprisingly observed that agonistic anti-TWEAKR antibodies comprising a mutation in the Fc region at N297A (using Kabat EU numbering) resulting in an aglycosyl antibody have less side effects and an improved toxicity profile compared to antibodies comprising the natural sequence of the Fc region. Oligomerization of TWEAKR by the anti-TWEAKR antibody via of FcyRIIB binding on cells, leading to inappropriate activation might play an important role in the observed side effects. Binding to other Fey receptors and complement could also potentiate this effect.
These antibodies comprising mutations in the Fc region resulting in aglycosyl antibodies having a reduced affinity for Fc gamma receptors compared to antibodies comprising a wild type Fc region do not depend any more on in vivo cross linking via Fc-Fc receptor (FcR) interactions which is thought to be mandatory for agonistic anti TWEAKR antibodies. In contradiction to the current opinion these antibodies are still almost as functional as the glycosylated antibody in their anti-cancer activity.
While removal of the glycans in the CH2 domains appears to have a significant effect on effector function and in the case of agoniostic anti-TWEAKR antibodies on the side effects and toxicity profile, other functional and physical properties of the antibody (binding properties etc.) remain unaltered. Specifical!y, it is well known that removal of the glycans has little to no effect on serum half-life (Leabman et al., MAbs. 2013 Nov-Dec;5(6):896-903) and binding to antigen (see examples).
An antibody of the invention might be co-administered with known medicaments, and in some instances the antibody might itself be modified. For example, an antibody could be conjugated to a cytotoxic agent, immunotoxin, toxophore or radioisotope to potentially further increase efficacy.
The invention further provides antibodies which constitute a tool for diagnosis of malignant or dysplastic conditions in which TWEAKR expression is elevated compared to normal tissue. Provided are anti-TWEAKR antibodies conjugated to a detectable marker. Preferred markers are a radiolabel, an enzyme, a chromophore or a fiuorescer. The invention is also related to polynucleotides encoding the antibodies of the invention, cells expressing the antibodies of the invention, methods for producing the antibodies of the invention, methods for inhibiting the growth of dysplastic cells using the antibodies of the invention, and methods for treating and detecting cancer using the antibodies of the invention.
The invention is also related to isolated nucleic acid sequences, each of which can encode an aforementioned antibody that is specific for an epitope f TWEAKR. Nucleic acids of the invention are suitable for recombinant production of antibodies. Thus, the invention also relates to vectors and host cells containing a nucleic acid sequence of the invention.
Compositions of the invention may be used for therapeutic or prophylactic applications. The invention, therefore, includes a pharmaceutical composition comprising an inventive antibody and a pharmaceutically acceptable carrier or excipient therefore. In a related aspect, the invention provides a method for treating a disorder or condition associated with the undesired presence of TWEAKR expressing cells. In a preferred embodiment the aforementioned disorder is cancer. Such method contains the steps of administering to a subject in need thereof an effective amount of the pharmaceutical composition that contains an inventive antibody as described or contemplated herein. The invention also provides instructions for using an antibody library to isolate one or more members of such library that binds specifically to TWEAKR.
DESCRIPTION OF THE FIGURES
Fig. 1 : Alignment of TWEAKR cysteine rich domain (aa 34-68) of different species. (Numbers indicate amino acid position in full length construct inclusive signal sequence; SEQ I D NO: 169) Fig. 2: A - Schematic diagram of the structure of TWEAKR (SEQ ID NO: 169). The diagram shows the extracellular domain (aa 28-80) (SEQ I D NO: 168) including the cysteine rich domain (36-67), the transmembrane domain - TM (81-101), and the intracellular domain (102-129). TPP-2202 - the full ectodomain (28-80) fused to the Fc domain of hlgGl . TPP-2203 - Extracellular domain with N- and C- terminal truncation (34-68) fused to the Fc domain of hlgGl . Disulfide bridges Cys36-Cys49, Cys52- Cys67 and Cys55-Cys64 are indicated by black bars. N-terminally, TPP-2203 contains two amino acids and C -terminally, one amino acid more compared to the pure cysteine rich domain to ensure proper folding. TPP-1984 - Extracellular domain with C -terminal truncation (28-68) fused to HIS6 tag. All three constructs show comparable binding to the antibodies of this disclosure and PDL-192(TPP-1 104). P4A8(TPP-1324) does only bind to the full extracellular domain (TPP-2202).
B Amino acid sequence of extracellular domain: aa46 has been published to be essential for TWEAK ligand binding, aa47 has been characterized to be essential for binding of the antibodies of this invention.
Fig. 3: Interaction of TWEAK R ectodomain with antibodies of this disclosure and reference antibodies.
Shown is the result of an ELiSA with TWEAKR-Fc fusion protein (TPP-2202) coating (1 μg/mί) and 0.08 μg/ml (open bars) and 0.3 μg/mί (filled bars) of biotinylated IgG as soluble binding partner. Detection was done with Streptavidin-HRP and Amplex-Red substrate. Y is "ELISA signal intensity [Rfu]"; X are "antibody constructs tested": a is "TPP-2090"; b is "TPP-2084"; c is "PDL-192(TPP- 1 104)"; d is "P4A8(TPP-1324)"; e is "P3G5(TPP-2195)"; f is "136.1 (TPP-2194)"; h is "ITEM ! "; i is "ITEM4"; j is a murine isotype control; k is a human isotype control. All tested antibodies show saturated binding with a concentration of 80 ng/ml. Fig. 4: Interaction of TWEAKR cysteine rich domain with antibodies of this disclosure and reference antibodies. Shown is the result of an ELI SA with TWEAKR(34-68)-Fc fusion protein (TPP-2203) coating (1 μg/ml) and 0.08 μg/ml (open bars) and 0.3 μg/ml (filled bars) of biotinylated IgG as soluble binding partner. Detection was done with Streptavidin-HRP and Amplex-Red substrate. Y is "ELISA signal intensity [Rfu]"; X are "antibody constructs tested": a is "TPP-2090"; b is "TPP-2084"; c is "PDL-192(TPP-1104)"; d is "P4A8(TPP-1324)"; e is "P3G5(TPP-2195)"; f is "136.1 (TPP-2194)"; h is "ITEM1"; i is "ITEM4"; j is a murine isotype control; k is a human isotype control. The antibodies of the invention bind to the cysteine rich domain.
Fig. 5: Interaction of TWEAKR(28-68) with antibodies of this disclosure and reference antibodies. Shown is the result of an ELISA with TWEAKR(28-68)-HIS (TPP-1984) coating (1 μg/ml) and 0.08 μg/ml (open bars) and 0.3 μ&<ϊηΙ (filled bars) of biotinylated IgG as soluble binding partner. Detection was done with Streptavidin-HRP and Amplex-Red substrate. Y is"ELISA signal intensity [Rfu]"; X are "antibody constructs tested": a is "TPP-2090"; b is "TPP-2084"; c is "PDL-192(TPP-1 104)"; d is "P4A8(TPP-1324)"; e is "P3G5(TPP-2195)"; f is "136.1 (TPP-2194)"; h is "ITEM ! "; i is "ITEM4"; j is a murine isotype control; k is a human isotype control. The antibodies of the invention bind to the cysteine rich domain. Antibodies P4A8(TPP-1324), P3G5(TPP-2195), ITEM- 1 and IT EM -4 show impaired binding.
Fig. 6: A - Alanine scan of cysteine rich domain. Muteins of TWEAKR(34-68)-Fc were analyzed for PDL-192(TPP-1104) (X) and TPP-2090 (Y) binding. S37A, R38A, S40A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A and 1.6 A muteins were expressed in HEK293 cells (black diamonds). PDL-192(TPP-1104) and TPP-2090 were coated (1 μg/ml) and an eight-fold diluted supernatant of the HEK293 fermentation broth was added for TWEAKR mutein binding. X is "ELISA intensity of PDL-192(TPP-1 104) interaction [Rfu]", Y is "ELISA intensity of TPP-2090 interaction [Rfu]". TPP-2090 (Y) shows impaired binding for the D47A TWEAKR mutein (closed box) and PDL-192(TPP-1 104) (X) shows impaired binding to R56A (dotted box).
B - Y is "% binding normalized by wt binding signal [%]", 1 is "TPP-2090"; 2 is "PDL-192(TPP- 1 104)"; 3 is "ITEM-1". Antibodies were coated (1 μg/ml), TWEAKR variant (D47A mutein of TWEAKR(34-68)-Fc and TWEAKR(34-68-Fc)were added at 250 ng/ml, detection via anti-HIS HRP. TTP-2090 shows less than 5% binding to D47A mutein compared to the WT construct. PDL-192 and ITEM- 1 bind with equal stringency to both constructs. Y was calculated as follows: Signal intensity TWEAKR(34-68)Fc-D47A mutein / Signal intensity TWEAKR(34-68)-Fc) * 100.
C - Y is„% binding to D47A mutein of TWEAKR(34-68)-Fc normalized by respective wt binding signal (TWEAKR(34-68-Fc) [%]", 1 is„TPP-2090"; 2 is "TPP-2149", 3 is "TPP-2093"; 4 is "TPP- 2148"; 5 is "TPP-2084"; 6 is "TPP-2077"; 7 is "TPP-1538"; 8 is "TPP-883"; 9 is "TPP-1854"; 10 is "TPP-1853"; 11 is "TPP-1857"; 12 is "TPP-1858"; 13 is "PDL-192(TPP-1 104)". Antibodies were coated (1 μg/ml), TWEAKR variant was added 250 ng ml, detection via anti-H I S HRP. All variants despite PDL-192 show less than 5% binding compared to the WT construct. Y was calculated as follows: Signal intensity TWEAKR(34-68)Fc-D47A mutein / Signal intensity TWEAKR(34-68)-Fc) * 100. Fig. 7: NMR structure of TWEAKR ectodomain as published by Pellegrini et al (FEBS 280: 1818-1829). TWEAK binding depends on L46 (Pellegrini et al), TPP-2090 binding on D47 and PDL- 1 2 binding on R56. PDL- 1 2 binds opposite of the TWEAK ligand binding site, TPP-2090 binds directly to the TWEAK ligand site.
Fig. 8: To differentiate binding epitopes of antibodies of the disclosure and of reference antibodies competition experiments were performed. A lack of a second binding event after injection of the 2nd antibody indicates clear competition within a respective antibody pair. Non competing antibody pairs showed clear binding signal over background after 2nd antibody injection. In addition the investigation of self-competition (1 st & 2nd antibody identical) was monitored as an internal system control. (-) no 2nd binding detected; (+) 2nd binding. The antibodies of this disclosure compete with all tested antibodies. Fig. 9: To differentiate binding epitopes of antibodies of the disclosure and of reference antibodies competition experiments were performed. In general all analyzed anti-TWEAKR antibodies could be clustered into three distinct "competition groups". One group contains exclusively TPP-2084 and TPP- 2090, both showing competition to all other tested members. These other members could be split into two separate sets of antibodies, which do not show any competition between each other. Both antibodies of this disclosure bind to a new and unique epitope.
Fig. 10: Homology tree of all 29 known TNF receptor superfamily members. The closest homologs TNFRSF13C and TNFRSF17 have only about 30% sequence identity.
Fig. 11: Binding ELISA with all 29 TNF receptor superfamily members for selectivity assessment of TPP-2090. Shown is the result of an ELISA: Y is "ELISA signal intensity [Rfu]"; X are "TNF receptor superfamily proteins tested (Fc-fusion proteins)": 1 is "TWEAKR"; 2 is "TWEAKR"; 3 is "Apo-3"; 4 is "Trail-Rl "; 5 is "Trail-R2"; 6 is "CD385"; 7 is "CD95"; 8 is "Rank"; 9 is "TNF-R1 "; 10 is "TNF-R2"; I I is "BAFF-R"; 12 is "DcR3"; 13 is "BCMA"; 14 is "TACI"; 15 is "OX40"; 16 is "CD30"; 17 is "CD27"; 18 is "CD40"; 19 is "Osteoprotegerin"; 20 is "EDAR"; 21 is "GITR"; 22 is "HVEM"; 23 is "NGF R"; 24 is "Trail R3"; 25 is "Lymphotioxin β R"; 26 is "Trail R4"; 27 is "EDA2R"; 28 is "TROY"; 29 is "REIT"; 30 is "4-1BB". In (1 ) 300 pM TPP-2090 were employed, in (2) 75 nM. TPP- 2090 binds at a very low concentration of 300 pM (1) and at a high concentration of 75 nM (2) in saturation to TWEAKR. For binding analysis to all other TNF receptor superfamily members (3 - 30) 75 nM TPP-2090 were used. TPP-2090 binds selectively to TWEAKR. Fig. 12: FACS analysis for binding of anti-TWEAKR antibodies to HT-29 cells. Y is "background corrected Geo-Mean of FACS signal [au]". Shown is the fluorescence after FACS analysis of HT-29 cells incubated with the antibodies as indicated at 10μ¾/ιη1 subtracted by the Geo-Mean of fluorescence of HT-29 cells incubated with the secondary antibody alone. Antibodies of this disclosure (TPP-1538, TPP-2084, TPP-2090) show lower cellular binding at this concentration as compared to known antibodies [PDL-192(TPP-1 104) and P4A8(TPP-1324)].
Fig. 13: Caspase 3/7 activation by anti-TWEAKR antibodies in HT-29 cells. X is "anti-TWEAKR antibodies tested [^g/ml]"; Y is "relative light units [RLU]". HT-29 cells were incubated with anti- TWEAKR antibodies at different concentrations as indicated (0.03-300 μg/mi) for 24h in the presence of IFNgamma. Caspase 3/7 activity measured as luminescence by the Caspase 3/7 Gio reagent (Promega) was plotted against the antibody concentrations. Average values of 1 -3 representative experiments performed in triplicates are shown including standard deviations. Filled symbols show antibodies of this disclosure, open symbols known antibodies [PDL-192(TPP-1 104); P4A8(TPP-1324), 136.1(TPP-2194) ]. The antibodies of this disclosure (TPP-1538, TPP-1854, TPP-2084, TPP-2090) dis lay a stronger efficacy to induce Cas ase 3/7 activation compared to the known antibodies [PDL- 192(TPP-1104); P4A8(TPP-1324) and 136.1(TPP-2194)].
Fig. 14: Antiproliferative activity of anti-TWEAKR antibodies in WiDr (A) and 786-0 (B) cells. X is "anti-TWEAKR antibodies tested ^g/ml]"; Y is "C ell proliferation related to proliferation of untreated control cells [%]". Ceils were incubated with anti-TWEAKR antibodies at different concentrations as indicated (0.03-300 μ¾ ιη1) for 96 h (WiDr cells absence, 786-0 cells in the presence of I FN gamma). Average values of a representative experiment performed in triplicates are shown and standard deviations are indicated by error bars. Filled symbols: antibodies of this disclosure, open symbols ltnown antibodies [PDL- ! 92(TPP- I 1 04 ) and P4A8(TPP-1324)]. The antibodies of this disclosure (TPP-1538, TPP-1854, TPP-2084, TPP-2090) display a stronger efficacy to inhibit cellular proliferation compared to the known antibodies [PDL-192(TPP-1104) and P4A8(TPP-1324)].
Fig. 15: IL-8 secretion induced by anti-TWEAKR antibodies in A375 cells. X is "anti-TWEAKR antibodies tested ^g/ml]"; Y is "IL-8 levels [pg/ml]". A375 ceils were incubated with anti-TWEAKR antibodies at different concentrations as indicated (0.03-300 μg/ml). Levels of IL-8 were determined in the supernatant of the cells after 24h treatment (and plotted against the used antibody concentrations. Average values of 1 -3 representative experiments performed in triplicates are shown including standard deviations. Filled symbols show antibodies of this disclosure, open symbols ltnown antibodies [PDL- 192(TPP-1104); P4A8(TPP-1324), 136.1 (TPP-2194)], and treatment with an isotype control antibody is indicated (C). The antibodies of this disclosure (TPP-1538, TPP-1854, TPP-2084, TPP-2090) display a stronger efficacy to induce IL-8 secretion from A375 cells compared to the known antibodies [PDL- 192(TPP-1104), P4A8(TPP-1324), 136.1 (TPP-2194)].
Fig. 15a: IL-8 secretion induced by the anti-TWEAKR antibodies TPP-2090 and TPP-2658 in A375 cells in comparison to a non-binding isotype control (C). X is "anti-TWEAKR antibodies tested ^g/ml]"; Y is "IL-8 levels [pg ml]". A375 ceils were incubated with anti-TWEAKR antibodies at different concentrations as indicated (0.01 -100 μ§/τηϊ). Levels of IL-8 were determined in the supernatant of the cells after 24h treatment (and plotted against the used antibody concentrations. Triplicate values are shown including standard deviations. TPP-2658 strongly induced IL-8 secretion comparable to TPP-2090.
Fig. 16: Human IL-8 secretion induced by anti-TWEAKR antibodies in xenografts in mice.
A: Wi r xenograft tumor bearing mice were treated with a single dose of 3 mg/kg TPP-2090 (open symbols) or vehicle (C - filled symbols) and levels of human IL-8 (IL-8 pg/ml) determined at different time points after treatment in the plasma of tumor bearing mice. X is "hours after treatment [h]"; Y is "11-8 level [pg/ml]". Results from 3 animals per group are indicated, error bars represent standard deviations. Human IL-8 secretion is specifically induced after treatment with TPP-2090 in WiDr tumor bearing mice in a time dependent manner.
B: A 75 tumor bearing (filled symbols) or non-tumor bearing (open symbols) mice were treated with a single dose of 10 mg/kg TPP-1538, vehicle or an isotpye control antibody. CI is "vehicle control"; C2 is "isotype control antibody"; Y is "Level of human 11-8 [pg/ml]". Levels of human IL-8 were determined in the serum of 4 mice per group 7 h after treatment are shown. IL-8 secretion is specifically induced in A375 tumor bearing mice by TPP-1538 but not in equally treated tumor free animals.
Fig. 17: Microscopic evaluation of the time course of specific internalization of TWEAKR upon antibody binding to endogenous TWEAKR expressing cells (InCell Analyzer). Internalization of TPP- 1538 and TPP-2090 was investigated on renal cancer cell line 786-0. Granule count/cell after treatment with antibodies of this disclosure (at 1 μg/ml) or isotype control C- at 5 μg/ml) is plotted for different incubation times as indicated (X is "time [min]"; Y is "granule count/cell [quantity]"). Antibodies of this disclosure (TPP-1538, TPP-2090) show rapid and specific internalization in TWEAKR expressing cells.
Fig. 18: Inhibition of 786-0 cell proliferation by anti-TWEAKR antibodies after incubation with saporme-conjugated secondary antibodies (Hum-Zap Assay). 786-0 cells were incubated with TWEAKR or isotype control antibodies in the presence or absence of saporine-conjugated secondary antibodies at 10 nM antibody concentration for 48h (in the absence of I FN gamma). X is "antibody variant tested", a is "vehicle control", b is "isotype control antibody", c is "TPP-2084", d is "TPP-2090"; Y is "cell proliferation compared to untreated control cells [%]". Cell proliferation compared to untreated control cells was plotted for 786-0 cells treated with different antibodies in the presence (open bars) or absence (filled bars) of saporine-conjugated secondary antibodies. Results from one representative experiment in triplicates are shown and standard deviations indicated by error bars. At the experimental conditions used only antibodies of this disclosure (TPP-2084, TPP-2090) in the presence of saporine-coupled secondary antibodies inhibit proliferation of 786-0 cells almost completely. Thus, the anti-pro liferative effect observed from the anti-TWEAKR antibodies in the presence of saporine- conjugated secondary antibodies is a result of specific internalization of the saporine after binding of the antibody-complexes to TWEAKR expressing cells.
Fig. 19: Efficacy of anti-TWEAKR antibodies in the human renal cell cancer xenograft 786-0 after treatment with 0.3, 1.0 and 3.0 mg/kg (i.v., q4dx3) started at day 7 after tumor cell inoculation. Shown are final tumor weights at day 40. A is "Vehicle group, treated with PBS (i.v. q4dx3)". B is "isotype, 3 mg/kg", C is "TPP-2084, 0.3 mg/kg", D is "TPP-2084, 1 mg kg", E is "TPP-2084, 3 mg/kg", F is "TPP- 2090, 0.3 mg/kg", G is "TPP-2090, 1 mg/kg", H is "TPP-2090, 3 mg/kg". (Y is "Tumor weights means of n=8; SD [g]"). Fig. 20: Efficacy of 3 mg kg TPP-2090 (i.v., q4dx7) in the human colon cancer xenograft WiDr in monotherapy and combination therapy with Irinotecan (5 mg/kg, i.v., 4 d on, 3 d off) and Regorafenib (10 mg/kg, p.o., daily). Treatment started 7 d after inoculation with established tumors of about 40 mm2. A is "Vehicle group, treated with PBS (i.v. q4dx7)". B is "TPP-2090, 3 mg/kg", C is "TPP-2090, 10 mg/kg", D is "Irinotecan, 5 mg/kg", E is "Combo TPP-2090 3 mg/kg + Irinotecan, 5 mg/kg", F is "Regorafenib, 10 mg/kg", G is "Combo TPP-2090, 3 mg/kg + Regorafenib 10 mg/kg". (X is "Time after inoculation [days]", Y is "Tumor area, means of n=10; SD [mm2])
Fig. 21: Efficacy of 10 mg/kg TPP-2090 (i.v., q4dx8) in the human lung cancer xenograft NCI-H322 in monotherapy and combination therapy with Paciitaxei (16 mg kg, i.v., q7dx4). Treatment started 14 d after inoculation with established tumors of about 45 mm2. A is "Vehicle group, treated with PBS (i.v. q4dx8)". B is "TPP-2090, 5 mg/kg", C is "TPP-2090, 10 mg/kg", D is "Paciitaxei, 16 mg kg", E is "Combo TPP-2090 10 mg/kg + Paciitaxei 16 mg/kg". (X is "Time after inoculation [days]"; Y is "Tumor area , means of n=10; SD [mm2]")
Fig. 22: Reduction of proliferative cells in xenografts after treatment with antibodies of this disclosure. Cryo sections from Wi Dr xenograft tumors after treatment with PBS (i.v., q4dx7: A) or TPP-2090 (10 mg/kg, i.v. q4dx7:B) were stained for the proliferation marker Ki67 by immunohistochemistry. Treatment started at day 7 after tumor cell inoculation and cryo sections were prepared from tumors taken at the end of the study (day 29). N=3 tumors per group were analyzed and representative images are shown. Treatment with TPP-2090 leads to a strong reduction of Ki67 positive cells (cells with dark staining in image) in WiDr xenograft tumors in mice.
Fig. 23: Induction of Stat-1 and NF-kappaB2 signaling pathways by anti-TWEAKR antibodies in vivo. Lysates of snap frozen WiDr xenograft tumors after treatment with PBS (i.v., q4dx7: lanes 1&2) or TPP-2090 (3 mg/kg, i.v., q4dx7: lanes 3&4) were subjected to Western Blot analysis detected with specific antibodies for P-Statl (a), Stat-1 (b), NF-kappa2 - p52 (c) and GAPDH (d). Treatment of mice started at day 7 after tumor cell inoculation and lysates were prepared from snap frozen tumors taken at the end of the study (day 29). Blots from 2 representative animals per group are shown. Treatment with TPP-2090 leads to a strong induction of P-Statl & Total Statl levels as well as NF-kappaB2 activation (shown by the appearance of the p52 band) in Wi Dr xenograft tumors.
Fig. 23A: Induction of Stat-1 and NF-kappaB2 signaling pathways by anti-TWEAKR antibodies in Wi Dr cells in vitro. Cell lysates of Wi Dr cells after 24 h treatment with PBS (lane 1), with 100 μg/ml of an IgGl -isotype control antibody (lane 2), with 0.1/1/10/100 μg/ml of TPP-2090 (lanes 3-6), with 0.1/1/10/100 μ^πιΐ of TPP-2658 (lanes 9-12) or with 10/300 ng/ml recombinant human TWEAK (lanes 7&8) were subjected to Western Blot analysis detected with specific antibodies for P-Statl (a), Stat-1 (b), NF-kappa2 - p52 (c), TWEAK R (d) and GAPDH (e). Treatment with TPP-2090 and TPP-2658 leads to an equally strong induction of P-Stati & TWEAKR levels as well as NF-kappaB2 activation (shown by the appearance of the p52 band) in WiDr cells.
Fig. 24: Consensus sequences for an ti -TWEAKR antibodies. CDR-H1 - X at position 5: M or I; CDR- H 2 X at position 8: S or ; CDR-L1 X at position 8: G or S; CDR-L2 X at position 1 : N, A or Q; CDR-L3 - X at position 5: T or S; X at position 6: S or T; X at position 8: F or G
Fig. 25: Continuous CDR sequence nomenclature. (A) Positions in boxes were diversified for mutation gathering (maturation process). (B) Single substitutions in boxes were recombined in one recombination library.
Fig. 26: Sequences of this disclosure
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the discovery of novel antibodies comprising a mutated Fc region lacking the giycans attached to the conserved N-linked site in the CH2 domains of the Fc region (called aglycosyl antibodies) that have a specific affinity for TWEAKR and can deliver a therapeutic benefit to a subject. The antibodies of the invention, which may be human, humanized or chimeric, can be used in many contexts, which are more fully described herein.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references, however, can provide one of skill in the art to which this invention pertains with a general definition of many of the terms used in this invention, and can be referenced and used so long as such definitions are consistent with the meaning commonly understood in the art. Such references include, but are not limited to, Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al.. The Dictionary of Cell & Molecular Biology (3rd ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, W.B. Saunders Company. Any additional technical resource available to the person of ordinary skill in the art providing definitions of terms used herein having the meaning commonly understood in the art can be consulted. For the purposes of the present invention, the following terms are further defined. Additional terms are defined elsewhere in the description. As used herein and in the appended claims, the singular forms "a," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth.
The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
The terms "anti-TWEAKR antibody" and "an antibody that binds to TWEAKR" refer to an antibody that is capable of binding TWEAKR with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting TWEAKR. In one embodiment, the extent of binding of an anti TWEAKR antibody to an unrelated, non-TWEAKR protein is less than about 10% of the binding of the antibody to TWEAKR as measured, e.g., by a surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to TWEAKR has a dissociation constant (KD) of < 1 μΜ, < 100 nM, < 10 nM, < 1 iiM, < 0.1 nM, < 0.01 n M . or < 0.001 n M (e.g. 10"8 M or less, e.g. from 10"8 M to 10"13 M, e.g., from 10~9M to 10~13 M). In certain embodiments, an anti-TWEAKR antibody binds to an epitope of TWEAKR that is conserved among TWEAKR from different species.
The term "antibody", as used herein, is intended to refer to immunglobulin molecules, preferably comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains which are typically inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH ) and a heavy chain constant region. The heavy chain constant region can comprise e.g. three domains CHI , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is typically composed of three CDRs and up to four FRs. arranged from amino terminus to carboxy-terminus e.g. in the following order: FR1 , CDR1, FR2, CDR2, FR3, CDR3, FR4.
As used herein, the term "Complementarity Determining Regions (CDRs; e.g., CDR 1 , CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR I . CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat (e.g. about residues 24-34 (LI), 50- 56 (L2) and 89-97 (L3) in the light chain variable domain and 3 1 -35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g. about residues 26-32 (LI), 50-52 (L2) and 91 -96 (L3) in the light chain variable domain and 26- 32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk; J Mo I Biol 196: 901 -917 (1987)). In some instances, a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.
Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, I G. and IgM, and several of these maybe further divided into "subclasses" (isotypes), e.g., I G I. I ( i2. IgG3, IgG4, IgAl , and I A 2. A preferred class of immunoglobulins for use in the present invention is IgG.
The heavy-chain constant domains that correspond to the different classes of antibodies are called [alpha], [delta], [epsilon], [gamma], and [mu], respectively. The subunit structures and three- dimensional configurations of different classes of immunglobulins are well known. As used herein antibodies are conventionally known antibodies and functional fragments thereof.
A "functional fragment" or "antigen-binding antibody fragment" of an antibody/immunoglobulin hereby is defined as a fragment of an antibody/immunoglobulin (e.g., a variable region of an IgG) that retains the antigen-binding region. An "antigen-binding region" of an antibody typically is found in one or more hyper variable region(s) of an antibody, e.g., the CDR I , -2, and/or -3 regions; however, the variable "framework" regions can also play an important role in antigen binding, such as by providing a scaffold for the CDRs. Preferably, the "antigen-binding region" comprises at least amino acid residues 4 to 103 of the variable light (VL) chain and 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 of VL and 4 to 1 1 1 of VH, and particularly preferred are the complete VL and VH chains (amino acid positions 1 to 109 of VL and 1 to 1 13 f VH; numbering according to WO 97/08320).
"Functional fragments" or "antigen-binding antibody fragments" include Fab, Fab', F(abr , and Fv fragments; diabodies; single domain antibodies (DAbs), linear antibodies; single-chain antibody molecules (scFv); and multispecific, such as bi- and tri-specific, antibodies formed from antibody fragments (C. A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering ( Springer Laboratory Manual), Springer Verlag). An antibody other than a "multi-specific" or "multifunctional" antibody is understood to have each of its binding sites identical. The F(ab' )2 or Fab may be engin cored to minimize or completely remove the intermolecular disulphide interactions that occur between the CHI and CL domains.
The term "Fc region" herein is used to define a ('-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
The term "aglycosyl antibody" herein is used to define an antibody or an antibody derivative which comprises a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region. Aglycosyl antibodies can for example be prepared by mutation of the heavy chain glycosylation site of N297 (using Kabat EU numbering). Derivatives of such antibodies can be for example scFv-Fc comprising antibodies.
The terms "aglycosyl anti-TWEAKR antibody" and "an aglycosyl antibody that binds to TWEAKR" refer to an aglycosyl antibody that is capable of binding TWEAKR with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting TWEA R .
The terms "aglycosyl agonistic anti-TWEAKR antibody" or "agonistic aglycosyl anti-TWEAKR antibody" herein are used to define an aglycosyl anti-TWEAKR antibody having agonistic activity.
Variants of the antibodies or antigen-binding antibody fragments contemplated in the invention are molecules in which the binding activity of the antibody or antigen-binding antibody fragment is maintained.
"Binding proteins" contemplated in the invention are for example antibody mimetics, such as Affibodies, Adnectins, Anticalins, DA R ins. Avimers, Nanobodies (reviewed by Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S.D. et al., Curr. Opinion in Pharmacology 2008; 8:608-617).
A "human" antibody or antigen-binding fragment thereof is hereby defined as one that is not chimeric (e.g., not "humanized") and not from (either in whole or in part) a non-human species. A human antibody or antigen-binding fragment thereof can be derived from a human or can be a synthetic human antibody. A "synthetic human antibody" is defined herein as an antibody having a sequence derived, in whole or in part, in silico from synthetic sequences that are based on the analysis of known luunan antibody sequences. In silico design of a human antibody sequence or fragment thereof can be achieved, for example, by analyzing a database of human antibody or antibody fragment sequences and devising a polypeptide sequence utilizing the data obtained there from. Another example of a human antibody or antigen-binding fragment thereof is one that is encoded by a nucleic acid isolated from a library of antibody sequences of human origin (e.g., such library being based on antibodies taken from a human natural source). Examples of human antibodies include antibodies as described in Soderlind et al., Nature Biotech. 2000, 18:853-856.
A "humanized antibody" or humanized antigen-binding fragment thereof is defined herein as one that is (i) derived from a non-human source (e.g., a transgenic mouse which bears a heterologous immune system), which antibody is based on a human germline sequence; (ii) where amino acids of the framework regions of a non-human antibody are partially exchanged to human amino acid sequences by genetic engineering or (iii) CDR-grafted, wherein the CDRs of the variable domain are from a non- human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin. A "chimeric antibody" or antigen-binding fragment thereof is defined herein as one, wherein the variable domains are derived from a non-human origin and some or all constant domains are derived from a human origin.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the term "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins . The term "monoclonal" is not to be construed as to require production of the antibody by any particular method. The term monoclonal antibody specifically includes chimeric, humanized and human antibodies.
An "isolated" antibody is one that has been identified and separated from a component of the cell that expressed it. Contaminant components of the cell are materials that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
An "isolated" nucleic acid is one that has been identified and separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
As used herein, an antibody "binds specifically to", is "specific to/for" or "specifically recognizes" an antigen of interest, e.g. a tumor-as so dated polypeptide antigen target, is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins or does not significantly cross-react with proteins other than orthologs and variants (e.g. mutant forms, splice variants, or proteolytically truncated forms) of the aforementioned antigen target. The term "specifically recognizes" or "binds specifically to" or is "specific to/for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by an antibody, or antigen- binding fragment thereof, having a monovalent Kn for the antigen of less than about 10~4 M, alternatively less than about 10"5 M, alternatively less than about 10~6 M, alternatively less than about 10" 7 M, alternatively less than about 10~8 M, alternatively less than about 10~9 M, alternatively less than about 10~10 M, alternatively less than about 10"" M, alternatively less than about 10"12 M, or less. An antibody "binds specifically to," is "specific to/for" or "specifically recognizes" an antigen if such antibody is able to discriminate between such antigen and one or more reference antigen(s). in its most general form, "specific binding", "binds specifically to", is "specific to/for" r "specifically recognizes" is referring to the ability of the antibody to discriminate between the antigen of interest and an unrelated antigen, as determined, for example, in accordance with one of the following methods. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. For example, a standard ELISA assay can be carried out. The scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxidase and tetramethyl benzidine with hydrogen peroxide). The reaction in certain wells is scored by the optical density, for example, at 450 nm. Typical background (=negative reaction) may be 0.1 OD; typical positive reaction may be 1 OD. This means the difference positive/negative is more than 5 -fold, 10-fold, 50-fold, and preferably more than 100-fold. Typically, determination of binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like. "Binding affinity" or "affinity" refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule and its binding partner. 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. an antibody and an antigen). The dissociation constant "KD" is commonly used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen) i.e. how tightly a ligand binds to a particular protein. Ligand-protein affinities are influenced by non-covalent intermolecular interactions between the two molecules. Affinity can be measured by common methods known in the art, including those described herein. In one embodiment, the "KD" or "KD value" according to this invention is measured by using surface plasmon resonance assays using a Biacore T200 instrument (GE Healthcare Biacore, Inc.). Other suitable devices are e.g. a Biacore T100, a Biacore-2000, a Biacore-4000, a Biacore-3000, or a ProteOn XPR36 instrument (Bio- Rad Laboratories, Inc.).
As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-celi receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids r sugar side chains, or combinations thereof and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein. "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc gamma receptors (FcyRs) present on certain cytotoxic cells (e.g. NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell e.g. with cyto toxins. To assess ADCC activity of an antibody of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), may be performed. Useful effector cells for such assays include PBMC anil N cells.
"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass), which are bound to their cognate antigen. To assess complement activation, a IX assay, e.g., as described in Gazzano- Santoro et al., .1. Immunol. Methods 202: 163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased Clq binding are described, e.g., in US Patent No. 6,194,551 Bl and WO 1999/51642.
As used herein, a "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g. a cytotoxic moiety) or radiolabel. This naked antibody may be present in a pharmaceutical composition.
The term ' 'immuno conj ugate" (interchangeably referred to as "antibody-drug conjugate," or "ADC") refers to an antibody conjugated to one or more cytotoxic r cytostatic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radio conjugate). Immunoconjugates have been used for the local delivery of cytotoxic agents, i.e., drugs that kill or inhibit the growth or proliferation of cells, in the treatment of cancer (e.g. Liu et al., Proc Natl. Acad. Sci. (1996), 93, 8618-8623)). Immunoconjugates allow for the targeted delivery of a drug moiety to a tumor, and intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells and/or tissues. Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin. The toxins may exert their cytotoxic effects by mechanisms including tubulin binding, ON A binding, or topoisomerase inhibition.
"Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence, respectively, is defined as the percentage of nucleic acid or amino acid residues, respectively, in a candidate sequence that are identical with the nucleic acid or amino acid residues, respectively, in the reference polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered as part of the sequence identity. Preferred are un -gapped alignments. 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 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.
"Sequence homology" indicates the percentage of amino acids that either is identical or that represent conservative amino acid substitutions.
The term "maturated antibodies" or "maturated antigen-binding fragments" such as maturated Fab variants includes derivatives of an antibody or antibody fragment exhibiting stronger binding - i. e. binding with increased affinity - to a given antigen such as the extracellular domain of a target protein. Maturation is the process of identifying a small number of mutations within the six CDRs of an antibody or antibody fragment leading to this affinity increase. The maturation process is the combination of molecular biology methods for introduction of mutations into the antibody and screening for identifying the improved binders.
An "antagonistic" antibody or a "blocking" antibody is one which significantly inhibits (either partially or completely) a biological activity of the antigen it binds.
An "agonistic" antibody or an antibody with "agonistic activity" is one that binds to its target and induces the activation of the respective target (either partially or completely), that e.g. leads to activation of the signaling pathways or biological effects that are mediated by the respective target. An ' ' agonist/agonistic antibody" as used herein is an antibody which mimics at least one of the functional activities of a polypeptide of interest (here the TWEA R ligand TWEAK).
The term "pharmaceutical formulation" / "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.
Amino acids may be referred to herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. 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. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."
The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "trans formants" 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.
Antibody Generation
A fully human antibody phage display library (Hoet RM et al., Nat Biotechnol 2005;23(3):344-8) was used to isolate TWEAKR-specific, human monoclonal antibodies of the present invention by protein panning (Hoogenboom I I .R.. Nat Biotechnol 2005;23(3): 1 105-16) with dimeric Fc-fused extracellular domains of human and murine TWEAKR as immobilized target.
1 1 different Fab-phages were identified and the corresponding antibodies were re-cloned into a mammalian IgG expression vector which provides the missing CH2-CH3 domains not present in the soluble Fab. After identification of preferred antibodies these were expressed as full length IgGs. Theses constructs were for example transiently expressed in mammalian cells as described in Tom et al.,
Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007 (see Example 1 ). The antibodies were purified by Protein A chromatography and further characterized by their binding affinity to soluble monomeric TWEAKR in EL ISA and BIAcore analysis as described in Example 2. To determine the cell binding characteristics of anti- TWEAKR antibodies, binding was tested by flow cytometry to a panel of cell lines (HT29, HS68, HS578).
NF-kappaB reporter gene assays were performed to assess the agonistic activity of all 1 1 identified antibodies (human IgGl). The antibody with the strongest in vitro efficacy (TPP-883) was selected for further potency and affinity maturation (see Example 1 for details). 1 single substitution variant was detected with improved agonistic activity: G102T of CDR-H3. Finally, 7 variants were selected based on enhanced affinity compared to the best single substitution variant, G102T. The corresponding ON A of these were re- cloned in a mammalian IgG expression vector and tested for functional activity in the afore mentioned NFkB reporter cell assay. Finally, the obtained sequences were compared with human germline sequences and deviations without significant impact on affinity and potency were adjusted. The following antibodies were obtained by antibody library screening and by affinity and/or potency maturation: "TPP-2658", "TPP-2090", "TPP-2149", "TPP-2093", "TPP-2148", "TPP-2084", "TPP- 2077", "TPP-1538", "TPP-883", "TPP-1854", "TPP-1853", "TPP-1857", and "TPP-1858". Some of these antibodies have a glycosylated Fc region. These can easily be converted into aglycosyl antibodies by standard molecular biology methods.
Antibodies of the invention can be further generated by methods known in the art like antibody phage display screening (for example see Hoet RM et al., Nat Biotechnol 2005;23(3):344-8), the well- established hybridoma technology (for example see Kohler and Milstein Nature. 1975 Aug 7;256(5517):495-7), or immunization of mice inter alia immunization of hMAb mice (e.g. Veloclmmune mouse ® ).
Antibodies of the invention The invention is related to antibodies comprising a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region (aglycosyl antibodies) and which lead to strong activation of the TWEAKR (SEQ I D NO: 169 (protein); SEQ ID NO: 170 (DNA)), thus leading to a strong induction of apoptosis in various cancer cells showing overexpression of the
TWEAKR. TWEAKR agonistic activity with regard to induction of apoptosis and inhibition of proliferation of the anti-TWEAKR antibodies described previously (e.g. PDL-192) is limited and does not reach the efficacy of the endogenous ligand TWEAK. This lack of agonistic activity is not due to a decreased affinity as these antibodies bind to the TWEAKR with affinities in a similar range as compared to the endogenous ligand TWEAK (Michaelson JS et al, MAbs. 201 1 Jul-Aug;3(4):362-75; G ulp PA et al.. Clin Cancer Res. 2010 Jan 15; 16(2):497-508) and also antibodies with higher binding affinity do not necessarily exhibit more efficacious signaling activity (Gulp PA, et al., Clin Cancer Res. 2010 Jan 15;16(2):497-508). In addition, anti-tumor activity of the antibodies described previously is shown to be dependent on Fc effector function and ADC C is shown to play a significant role for the in vivo efficacy in mouse models.
The invention provides antibodies which have such a strong agonistic activity with regard to induction of apoptosis and inhibition of proliferation that in vivo anti-tumor efficacy can be achieved without ADCC playing a significant role.
The human Fc gamma receptor (hFcyR) family consists of the activating receptors FcyRI, FcyRI IA, and FcyRIIIA, and the inhibitory receptor FcyRl i B. While FcyRI binds IgG with high affinity (nanomolar binding constants), FcyRI IA, FcyRl i B. and FcyR I I I A bind IgG with micromolar affinity, becoming activated only via avid multivalent interactions with opsonized antigen. The binding of IgG to FcyR is highly sensitive to the presence of glycosylation at a single N-linked glycosylation site at asparagine 297 (N297) in its CH2 domain (Jefferis & Lund, 2002, Immunol Lett 82:57-65; Arnold et al., 2007, An nu Rev Immunol 25:21-50), with a loss of binding to the low-affinity FcyRs observed in N297 point mutants (Shields et al, 2001 , .1 Biol Chem 276:6591-6604; Tao & Morrison, 1989, J Immunol 143:2595-2601), enzymatic Fc deglycosylation (Mimura et al, 2001, .1 Biol Chem 276:45539-45547), recombinant IgG expression in the presence of the N-linked glycosylation-inhibitor tunicamycin
( Walker et al., 1989, Biochem J 259:347 353 ). or expression in bacteria (Mazor et al, 2007, Nat Biotechnoi 25 :563 565: Simmons et al.. 2002, .1 Immunol Methods 263 : 133-147).
The present invention relates to aglycosyl anti-TWEAKR antibodies with a decreased effector function, which are characterized by a modification at the conserved N-linked site in the CI 12 domains of the Fc portion of said antibody. It is an embodiment of the invention to provide agonistic anti- TWEAKR antibodies which have a reduced Fc gamma receptor binding compared to antibodies comprising a wild type Fc region. Preferred are antibodies which bind an Fc gamma receptor (the activating receptors FcyRI, FcyRIIA, or FcyRIIIA, or the inhibitory receptor FcyRliB) with a KD value which is more than 2 fold, alternatively more 5 fold, alternatively more than 10 fold higher than the parent antibody comprising an unmodified Fc region. Highly preferred is an antibody which binds to the FcyRliB with a KD value which is more than 10 fold higher than the parent antibody comprising an unmodified Fc region.
These antibodies of this invention do not depend any more on in vivo cross linking via Fc-Fc receptor (FcyR) interactions which is thought to be mandatory for agonistic anti TWEAKR antibodies. Preferred are aglycosyl anti-TWEAKR antibodies having reduced Fc gamma receptor binding compared to antibodies comprising a wild type Fc region for receptors FcyRIIA, FcyRl i . and even more preferred for the FcyRIIB. Further preferred are the human Fc gamma receptors. Preferred are aglycosyl anti- TWEAKR antibodies having a binding affinity for FcyRIIB, preferentially human Fey RU B. which is larger 50 μΜ, alternatively larger 100 μΜ, and alternatively larger 200 μΜ. A binding affinity (KD) larger 200 μΜ is highly preferred. Currently there are two ways to reduce the effector function of a mAb while retaining the other valuable attributes of the Fc portion thereof. One way to modify an antibody is to mutate amino acids on the surface of the mAb that are involved in the effector binding interactions. The other way relates to aglycosyl antibodies with decreased effector function, which are characterized by a modification at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody via for example by mutations of the residues at position 297 or 299 (using abat EU numbering).
In one embodiment of the present invention, the modification comprises a mutation at the heavy chain glycosylation site to prevent glycosylation at this site. Thus, in one preferred embodiment of this invention, the aglycosyl anti-TWEAKR antibody was generated by mutation of the heavy chain glycosylation site, - i.e., mutation of N297 (using Kabat EU numbering) and was expressed in an appropriate host cell. Preferred are mutations of S/T299 to Alanine or N297 to Alanine or Glutamine. Highly preferred is a mutation of N297 to Alanine.
In a preferred embodiment of the invention the aglycosyl anti-TWEAKR antibody comprises a human I G Fc region and even more preferred a human I G Fc region selected for the group consisting of human I G I , I G 2. I G 3 and lgG4 Fc region. Hi hly preferred is a human I G I Fc region. In one embodiment of present invention, the modification comprises a mutation at the heavy chain glycosylation site to prevent glycosylation at this site in combination with one or more mutations influencing Fc receptor binding selected from the group constisting of E282V, M428I, S298G, T299A, N390D, E382V, and M428L (using Kabat EU numbering). Preferred are the combinations E282V together with M428I and S298G together with T299A, N390D, E382V, and M428L It is an embodiment of the invention to provide aglycosyl antibodies which have a strong induction of Caspase-3/7 in one or more TWEAKR expressing cell lines. In a preferred embodiment the one or more TWEAKR expressing cell line is comprised in the group consisting Wi Dr. A253, NCI-H322, HT-29 and 786-0 cells, preferably HT-29. "Induction of Caspase 3/7" can be measured by common methods known in the art, including those described herein. In one embodiment, the "Induction of Caspase 3/7" according to this invention is measured by using activity determination with Caspase 3/7 Solution (Promega, #G8093) and reading of luminescence on a VICTOR V ( Perkin Elmer). At the end of the incubation time Caspase 3/7 activity was determined and the fold induction of Caspase 3/7 was calculated as compared to untreated cells. An antibody is said to have "strong induction" of Caspase-3/7 if the fold of induction is greater than 1.2, preferably greater than 1.5, more preferably greater than 1.8, more preferably greater than 2.1 , more preferably grealer than 2.5. Provided are anti-TWEAKR antibodies which lead to a stronger induction of Caspase 3/7 in HT-29 cells as compared to the agonistic antibodies previously described [e.g. PDL-192(TPP-1104), P4A8(TPP-1324), 136.1 (TPP-2194)] and also as compared to 300ng/ml recombinant human TWEAK. This strong efficacy to induce Caspase 3/7 in cancer cells was also seen in WiDr, A253, NCI-H322 and 786-0 cells. Some antibodies of this disclosure bind to the TWEAKR with only moderate affinity (> Ι ΟηΜ) that is clearly lower compared to the affinity of the endogenous ligand TWEAK and lower compared to other known agonistic antibodies. This property provides further potential advantages as e.g. potentially improved tumor penetration.
Toward these ends, it is an embodiment of the invention to provide aglycosyl antibodies that specifically bind to a TWEAKR at a novel epitope characterized by selective binding to aspartate (D) at position 47 (D47) of TWEAKR (SEQ ID NO: 169; and see Figure 1). It is a preferred embodiment of this invention to provide an antibody comprising a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region that specifically bind to a TWEAKR at a novel epitope characterized by selective binding to aspartate (D) at position 47 (D47) of TWEAKR (SEQ ID NO: 169; and see Figure 1). It is a preferred embodiment of this invention to provide an antibody comprising a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region that bind an Fc gamma receptor with worse affinity than the parent antibody comprising an unmodified Fc region and specifically bind to a TWEAKR at a novel epitope characterized by selective binding to aspartate (D) at position 47 (D47) of TWEAKR ( SEQ ID NO: 169; and see Figure 1). Preferred are antibodies which bind an Fc gamma receptor (the activating receptors FcyRI, FcyRIIA, or FcyRI I I A, or the inhibitory receptor FcyRIIB) with a KD value which is more than 2 fold, alternatively more 5 fold, alternatively more than 10 fold higher than the parent antibody comprising an unmodified Fc region. Highly preferred is an antibody which binds to the FcyRIIB with a KD value which is more than 10 fold higher than the parent antibody comprising an unmodified Fc region. Preferred are such aglycosyl anti-TWEAKR antibodies having a binding affinity for Fey RU B. preferentially human FcyRI I B. which is larger 50 μΜ, alternatively larger 100 μΜ, and alternatively larger 200 μΜ. A binding affinity (KD) larger 200 μΜ is highly preferred. Preferred are mutations of N297 to Alanine or Glutamine (using Kabat EU numbering) in the Fc region. Highly preferred is a mutation of N297 to Alanine. The identified dependencies on certain TWEAKR amino acids for antibody interaction correlate with the agonistic activity that has been determined for these antibodies. The native ligand TWEAK shows efficient activation of TWEAKR and binds dependent on Leucin 46 in the cysteine rich domain of TWEAKR ( Pellegrini et al, FEBS 280: 1818-1829). P4A8 shows very low agonistic activity and at least partially interacts with domains outside of the cysteine rich domain of TWEAKR. PDL-192 shows moderate agonistic activity and binds dependent f R56 to the cysteine rich domain but opposite to the TWEAK ligand site. Antibodies of this invention (exemplary TPP-2658) bind dependent on D47, and TWEAK binds dependent on 1.46. and binds to a similar but distinguishable binding site (Figure 7). Therefore the antibodies of this invention which show a strong agonistic activity bind to a novel epitope ( 1)47 dependent) for antibodies which is connected to very strong agonistic activity.
Amino acid at position 47 (D47) of TWEAKR ( SEQ ID NO: 169) is regarded as critical for binding for the antibodies of the invention, which means the antibody specifically binds to the D at position 47 (D47) of TWE AKR ( SEQ ID NO: 169), when the antibody loses more than 20%, alternatively more than 30%, alternatively more than 40%, alternatively more than 50%, alternatively more than 60%, alternatively more than 70%o, alternatively more than 80%), alternatively more than 90%o, alternatively 100%, of its ELISA signal by changing this residue into an Alanine as described in Example 2 and Figure 6. Alternatively, an antibody specifically binds to the D at position 47 (D47) of TWEAK R ( SEQ I D NO: 169), when the antibody loses more than 20%, alternatively more than 30%, alternatively more than 40%, alternatively more than 50%, alternatively more than 60%, alternatively more than 70%, alternatively more than 80%, alternatively more than 90%o, alternatively 100%o, of its ELISA signal on TPP-2614 compared to TPP-2203. Preferably, an antibody specifically binds to the D at position 47 (D47) of TWEAKR ( SEQ I D NO: 1 69), when the antibody loses more than 80% of its ELISA signal on TPP-2614 compared to TPP-2203.
A preferred embodiment of the invention is an aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR ( SEQ I D NO: 169).
A further preferred embodiment of the invention is an aglycosyl agonistic anti-TWEAK R antibody which specifically binds to aspartate 47 (D47) of TWEAKR ( SEQ I D NO: 169). Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine. A further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which has reduced ADCC activity or which lacks ADCC activity, and which specifically binds to aspartate 47 (D47) of TWEAKR ( SEQ I D NO: 169). A further preferreii embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TW EAKR ( SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is selected from the group of agonistic activities consisting of induction of Caspase3/7, inhibition of proliferation of TWEAKR expressing cell lines, and induction of cytokine secretion. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine. A further preferred embodiment of the invention is an agonistic aglycosyl anti- TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR ( SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of Caspase3/7. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine. A further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of Caspase3/7 in a TWEAKR expressing cancer cell line. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Hi hly preferred is a mutation of N297 to Alanine.
A further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of Caspase3/7 in a TWEAKR expressing cancer cell li ne comprised in t e group consisting Wi r. A253, NCT-H322, HT-2 and 786-0 cells. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
A further more preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is higher induction of Caspase3/7 in a HT-29 and/or 786-0 cell line compared to the induction by recombinant human TWEAK. In a further preferred embodiment the concentration of anti-TWEAKR antibody used is 100 μg/ml and of recombinant human TWEAK is 300 ng/ml. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine. It is another embodiment of the invention to provide aglycosyl antibodies which bind specifically to the cysteine rich domain (aa 34-68 of SEQ I D: 169 ) of TWEAKR of different species (Figure 1).
It is another preferred embodiment of the invention to provide aglycosyl antibodies which bind specifically to the cysteine rich domain (aa 34-68 of SEQ ID: 169) of TWEAKR and which bind specifically to the D at position 47 (D47) of TWEAKR. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
It is another preferred embodiment of the invention to provide aglycosyl antibodies which bind specifically to the cysteine rich domain (aa 34-68 of SEQ ID: 169) of TWEAKR of at least two species comprised in the group TWEAKR species consisting of human, mouse, dog, pig, rat, and macaca fascicularis and which bind specifically to the D at position 47 (D47) of TWEAKR. In a preferred embodiment the two species are human and mouse.
It is another embodiment of the invention to provide aglycosyl antibodies which inhibit the proliferation of different TWEAKR expressing cell lines. In line with the strong induction of Caspase 3/7 an efficacious inhibition of proliferation of different cancer cell lines is observed. The antibodies of the current invention are more efficacious as compared to other known antibodies (PDL-192, P4A8) in inhibiting proliferation of various cancer cells.
A further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is inhibition of proliferation of TWEAKR expressing cell lines. In a preferred embodiment of the invention the TWEAKR expressing cell line is comprised in the group consisting of 786-0, LOVO. NCI-H1975, SW480, WiDr, HT-29. A253, and SK-OV3. In a more preferred embodiment of the invention the TWEAKR expressing cell line is Wi Dr. A further more preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is stronger inhibition of proliferation of a786-0 and/or Wi Dr cell line compared to the inhibition by recombinant human TWEAK. In a further preferred embodiment the concentration of anti-TWEAKR antibody used is 10(^g/ml and of recombinant human TWEAK is 300 ng ml. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
It is another embodiment of the invention to provide aglycosyl antibodies which strongly induce cytokine secretion from various cancer cells including but not limited to A 75, Wi Dr cells and xenografts. Cytokines induced include but are not limited to IL-8, I I.- 1 5. IP- 10, I L- 1 RA and MCP-1. A preferred cytokine which is induced is IL-8.
A preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
A preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion in a TWEAKR expressing cancer cell line. In a more preferred embodiment the TWEAKR expressing cancer cell line is a A375 or a Wi Dr cell line. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine. A further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion wherein the cytokine is comprised in a group of cytokine consisting of IL-8, IL-15, IP- 10, IL-1RA and MCP-1. A further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion in a TWEAKR expressing cancer cell line secretion wherein the cytokine is comprised in a group of cytokine consisting of IL-8, IL-15, IP-10, I I .- ! RA and MCP-1. Pre erred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Hi hly preferred is a mutation of N297 to Alanine.
A further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion in a TWEAKR expressing cancer cell line secretion wherein the cytokine is comprised in a group of cytokine consisting of IL-8, IL-15, IP-10, 11.- 1 RA and MCP-1 and wherein the TWEAKR expressing cancer cell line is a A 75 or a WiDr cell line. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine. In a further preferred embodiment the cytokine is IL-8, in an even more preferred embodiment the IL-8 is human IL-8.
A preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of cytokine secretion in a mouse tumor xenograft model. In a further preferred embodiment the secreted cytokine is a human cytokine derived from the tumor xenograft. Preferred are aglycosyl antibodies comprising a mutation of N2 7 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
A further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ I D NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of human IL-8 secretion in a mouse tumor xenograft model. In a further preferred embodiment the mouse tumor xenograft model is a A375 or WiDr mouse xenograft model. In a further preferred embodiment the induction of cytokine secretion is observed after injection of at 3 mg 'kg or higher or 10 mg kg or higher anti-TWEAKR antibody of the invention.
A further preferred embodiment of the invention is an agonistic aglycosyl anti-TWEAKR antibody which specifically binds to aspartate 47 (D47) of TWEAKR (SEQ ID NO: 169) wherein the agonistic activity of the anti-TWEAKR antibody is induction of human IL-8 secretion in a mouse WiDr tumor xenograft model after injection of 3 mg kg of said antibody wherein no induction of the mouse IL-8 analogue KC is detected.
In a further preferred embodiment the induction of cytokine secretion is observed in the plasma of tumor bearing mice.
It is another embodiment of the invention to provide aglycosyl antibodies which bind to a broad range of different TWEAKR expressing cell lines including, but not limited to the ones shown in Table 21. The examples in Table 21 include human and murine cell lines from many tumor origins (e.g. NSCLC, CRC, HNSCC, RCC, PancCA, OvC'a. BreastCA, Melanoma, GastricCA, Esophageal CA, Bladder CA, HCC, Prostate CA, Neuroblastoma).
It is an embodiment of the invention to provide aglycosyl antibodies which have a strong antitumor efficacy in a broad panel of cell line-derived and patient-derived human tumor models. Tumor models include but are not limited to 786-0, A375, A253, SK-OV-3, WiDr, SW480, Co5682, NCI- H1975, NCI-H322, Lu7343, Co5676, Co 5841 SCaBER and SCC4. For example efficacy of TPP-2658 is shown in Example 13 for WiDr and SCaBER.
It is another embodiment of the invention to provide agonistic aglycosyl anti-TWEAKR antibodies that are safe for human administration. Aglycosyl antibodies of this invention have less side effects and an improved toxicity pr fil compared to antibodies comprising the natural sequence of the Fc region. Preferred are aglycosyl antibodies comprising a mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering). Highly preferred is a mutation of N297 to Alanine.
It is another embodiment of the invention to provide aglycosyl antibodies which bind to human TWEAKR and are cross-reactive to TWEAKR of another species including, but not limited to murine, rat, pig, dog, macaca fascicularis with similar affinity. Preferably, said other species is a rodent, such as for example mouse or rat. Most preferably, the antibodies bind to human TWEAKR and are cross - reactive to murine TWEAKR.
It is another embodiment of the invention to provide aglycosyl antibodies which constitute a tool for diagnosis of malignant or dysplastic conditions in which TWEAKR expression is elevated compared to normal tissue or where TWEAKR is shed from the cell surface and becoming detectable in serum. Provided are aglycosyl anti-TWEAKR antibodies conjugated to a detectable marker. Preferred markers are a radiolabel, an enzyme, a chromophore or a fluorescer.
I n the following in addition to such aglycosyl anti-TWEAKR antibodies (like TPP-2658) antibodies are described (like TPP-2090) which can be transformed via mutations into the corresponding agclycosyl antibodies. As an example the antibody TPP-2090 is provided which has been transformed into the aglycosyl antibody TPP-2658 by mutation of N297 into Alanine. I n order to transform such glycosylated antibodies into aglycosyl antibodies mutation of N297 to Alanine or Glutamine in the Fc region (using Rabat EU numbering) is preferred. Highly preferred is a mutation of N297 to Alanine. Throughout this document, reference is made to the following antibodies as depicted in Table 31 :
"TPP-2090", "TPP-2I49", "TPP-2093", "TPP-2148", "TPP-2084", "TPP-2077", "TPP-1538", "TPP- 883", "TPP-1854", "TPP-1853", "TPP-1857", "TPP-1858", and "TPP-2658".
TPP-2658 is a highly preferred antibody of this invention.
TPP-2658 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO: 213 and a light chain region corresponding to SEQ ID NO: 1.
TPP-2090 represents an antibody comprising a heavy chain region corresponding to SEQ I NO: 2 and a light chain region corresponding to SEQ I D NO: 1.
TPP-2149 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 12 and a light chain region corresponding to SEQ I D NO: 1 1. TPP-2093 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO:
22 and a light chain region corresponding to SEQ I D NO: 21.
TPP-2148 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 32 and a light chain region corresponding to SEQ I D NO: 31.
TPP-2084 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 42 and a light chain region corresponding to SEQ ID NO: 41.
TPP-2077 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO: 52 and a light chain region corresponding to SEQ ID NO: 51.
TPP-1538 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 62 and a light chain region corresponding to SEQ I D NO: 61. TPP-883 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 72 and a light chain region corresponding to SEQ I D NO: 71.
TPP-1854 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 82 and a light chain region corresponding to SEQ I D NO: 81. TPP-1853 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 92 and a light chain region corresponding to SEQ I D NO: 91.
TPP-1857 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO: 102 and a light chain region corresponding to SEQ I D NO: 101. TPP-1858 represents an antibody comprising a heavy chain region corresponding to SEQ I D NO:
1 12 and a light chain region corresponding to SEQ ID NO: 1 11.
TPP-2658 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 10 and a variable light chain region corresponding to SEQ I D NO: 9.
TPP-2090 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 10 and a variable light chain region corresponding to SEQ ID NO: 9.
TPP-2149 represents an antibody comprising a variable heavy chain region corresponding to SEQ
ID NO: 20 and a variable light chain region corresponding to SEQ ID NO: 19.
TPP-2093 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 30 and a variable light chain region corresponding to SEQ I D NO: 29. TPP-2148 represents an antibody comprising a variable heavy chain region corresponding to SEQ
I D NO: 40 and a variable light chain region corresponding to SEQ I D NO: 39.
TPP-2084 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 50 and a variable light chain region corresponding to SEQ I D NO: 49.
TPP-2077 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 60 and a variable light chain region corresponding to SEQ ID NO: 59.
TPP-1538 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 70 and a variable light chain region corresponding to SEQ I D NO: 69.
TPP-883 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 80 and a variable light chain region corresponding to SEQ I D NO: 79. TPP-1854 represents an antibody comprising a variable heavy chain region corresponding to SEQ
I D NO: 90 and a variable light chain region corresponding to SEQ I D NO: 89.
TPP-1853 represents an antibody comprising a variable heavy chain region corresponding to SEQ I D NO: 100 and a variable light chain region corresponding to SEQ I D NO: 99. TPP-1857 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 110 and a variable light chain region corresponding to SEQ ID NO: 109.
TPP-1858 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 120 and a variable light chain region corresponding to SEQ ID NO: 119. In a further preferred embodiment the aglycosyl antibodies comprise heavy or light chain CDR sequences which are at least 50%, 55%, 60% 70%, 80%, 90, or 95% identical to at least one, preferably corresponding, CDR sequence of the antibodies "TPP-2658", "TPP-2090", "TPP-2149", "TPP-2093", "TPP-2148", "TPP-2084", "TPP-2077", "TPP-1538", "TPP-883", "TPP-1854", "TPP-1853", "TPP- 1857" or "TPP-1858" or at least 50%, 60%, 70%, 80%, 90%, 92% or 95% identical to the VH or VI. sequence of "TPP-2658", "TPP-2090", "TPP-2149", "TPP-2093", "TPP-2148", "TPP-2084", "TPP- 2077", "TPP-1538", "TPP-883", "TPP-1854", "TPP-1853", "TPP-1857" or "TPP-1858", respectively.
In a further preferred embodiment the aglycosyl antibody of the invention comprises at least one
CDR sequence or at least one variable heavy chain or variable light chain sequence as depicted in Table 31. In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:6 ( H-CDRl ). SEQ ID NO:7 (H-CDR2) and SEQ ID NO:8 ( II-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:3 (L- CDR1), SEQ ID NO:4 (L-CDR2) and SEQ ID NO:5 (L-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO: 16 (H-CDRl ), SEQ ID NO: 17 (H-CDR2) and SEQ ID NO: 18 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:13 (L-CDRl ). SEQ ID NO: 14 (I.-CDR2) and SEQ ID NO: 15 (L-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:26 (H-CDRl ). SEQ ID NO:27 (II -CDR 2) and SEQ ID NO:28 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:23 (L-CDRl ). SEQ ID NO:24 (L-CDR2) and SEQ ID NO:25 (L-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:36 (H-CDRl ). SEQ ID NO:37 (H-CDR2) and SEQ ID NO:38 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:33 (L-CDRl ). SEQ ID NO:34 (L-CDR2) and SEQ ID NO:35 (I.-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:46 (H-CDRl), SEQ ID NO:47 (H-CDR2) and SEQ ID NO:48 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:43 (L-CDR1), SEQ ID NO:44 (L-CDR2) and SEQ ID NO:45 (L-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:56 (H-CDRl ). SEQ ID NO:57 (H-CDR2) and SEQ ID NO:58 (II-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:53 IL-CDR! ). SEQ ID NO:54 (L-CDR2) and SEQ ID NO:55 (I.-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:66 (H-CDRI), SEQ ID NO:67 (H-CDR2) and SEQ ID NO:68 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:63 (L-CDRI ). SEQ ID NO:64 (L-CDR2) and SEQ ID NO:65 (I.-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO: 76 (H-CDRl ). SEQ ID NO:77 (H-CDR2) and SEQ ID NO:78 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO: 73 (L-CDRI), SEQ ID NO:74 (L-CDR2) and SEQ ID NO:75 (L-CDR3). In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO: 86 (H-CDRl ). SEQ ID NO:87 (H-CDR2) and SEQ ID NO:88 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:83 (L-CDRI), SEQ ID NO:84 (L-CDR2) and SEQ ID NO:85 (L-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO:96 (H-CDR! ). SEQ ID NO:97 (H-CDR2) and SEQ ID NO:98 (II-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:93 (L-CDRI), SEQ ID NO:94 (I.-CDR2) and SEQ ID NO:95 (I.-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises a heavy chain antigen-binding region that comprises SEQ ID NO: 106 (H-CDR! ). SEQ ID NO: 107 (H-CDR2) and SEQ ID NO: 108 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:103 (L-CDRI). SEQ ID NO: 104 (L-CDR2) and SEQ ID NO: 105 (L-CDR3).
In a more preferred embodiment the aglycosyl antibody of the invention comprises SEQ ID NO:116 (H-CDRl ). SEQ ID NO:117 (H-CDR2) and SEQ ID NO:118 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:113 (L-CDRI ). SEQ ID NO: 114 (L-CDR2) and SEQ ID NO:l 15 (L-CDR3). Sequence alignment of the CDRs of the antibodies of this invention reveals a consensus sequence (see Figure 24). In a more preferred embodiment the antibodies of the invention or antigen-binding fragment thereof comprise:
a variable heavy chain comprising
· a heavy chain CDR1 encoded by an amino acid sequence comprising the formula PYPMX (SEQ
I D NO: 171), wherein X is I or M;
• a heavy chain CDR2 encoded by an amino acid sequence comprising the formula YISPSGGXTHYADSVKG (SEQ I D NO: 172), wherein X is S or K; and
• a heavy chain CDR3 encoded by an amino acid sequence comprising the formula GGDTYFDYFDY (SEQ I D NO: 1 73);
and a variable light chain comprising
• a light chain CDR1 encoded by an amino acid sequence comprising the formula RASQSISXYL (SEQ I D NO: 174), wherein X is G or S;
• a light chain CDR2 encoded by an amino acid sequence comprising the formula XASSLQS (SEQ I D NO: 175), wherein X is Q, A, or N; and
• a light chain CDR3 encoded by an amino acid sequence comprising the formula QQSYXXPXIT (SEQ I D NO: 176), wherein X at position 5 is T or S, and X at position 6 is T or S, and X at position 8 is G, or F.
Antibodies differ in sequence, not only within their complementarity determining regions (CDRs), but also in the framework (FR). These sequence differences are encoded in the different V-genes. The human antibody germline repertoire has been completely sequenced. There are about 50 functional VH germline genes which can be grouped into six subfamilies according to sequence homology VH 1 , VH2, VH3, VH4. VH5 and VI I 6 (Tomlinson et al, 1992, J. Mol. Biol. 227, 776-798; Matsuda & Honjo, 1996, Advan. Immunol. 62, 1 -29). About 40 functional VL kappa genes comprising seven subfamilies are known (Cox et al, 1994, Eur. J. Immunol. 24, 827-836; Barbie & Lefranc, 1998, Exp. Clin. Immunogenet. 15, 171 -183). Vkappa l . Vkappa2. Vkappa3. Vkappa4, Vkappa5, Vkappa6 and Vkappa7. Disclosed herein are heavy chains of antibodies of this invention that belong to the human VH3 subfamily and the light chains of antibodies of this invention that belong to the human Vkappa 1 subfamily, respectively. It is known that framework sequences of antibodies belonging to the same subfamily are closely related, e.g. antibodies comprising a human VI 13 subfamily member all share comparable stability (Honegger et al., 2009, Protein Eng Des Sei. 22(3): 121-134). It is well known in the art that CDRs from antibodies can be grafted on different frameworks while maintaining special features of the corresponding origin antibody. CDRs have been successfully grafted on frameworks belonging to a different species as well as on frameworks of the same species belonging to a different subfamily, in a further embodiment the antibody or antigen-binding fragment of the invention comprises at least one CDR sequence of an antibody of the invention as depicted in Table 31 and a human variable chain framework sequence.
In a preferred embodiment the aglycosyl antibody of the invention comprises a variable light chain or light chain antigen-binding region comprising the L-CDRl, L-CDR2 and L-CDR3 sequence of the variable light chain and a variable heavy chain or heavy chain antigen-binding region comprising the H- CDR1, H-CDR2 and H-CDR3 sequence of the variable heavy chain antibody of the invention as depicted in Table 31 and a human variable light and human variable heavy chain framework sequence.
In a more preferred embodiment the aglycosyl antibody of the invention comprises a variable light chain or light chain antigen-binding region comprising the L-CDRl, L-CDR2 and L-CDR3 sequence of the variable light chain and a variable heavy chain or heavy chain antigen-binding region comprising the H-CDR1, H-CDR2 and H-CDR3 sequence of the variable heavy chain antibody of the invention as depicted in Table 31 and a human VH3 subfamily framework sequence for the variable heavy chain and a human Vkappa 1 subfamily framework sequence for the variable light chain. In a more preferred embodiment the human VH3 subfamily framework sequence for the variable heavy chain is comprised in the group f VH3 subfamily framework sequence consisting of VH3-07, VH3-09, VI 13- 1 1. VH3- 13. VH3-15, VH3-20, VH3-21. VI I3-23. VH3-30, VH3-30.3, VH3-30.5, VI I3-33. VH3-43. VH3-48, VH3- 49, VH3-53, VH3-64. VH3-66, VH3-72, VH3-73, VH3-74 and VH3-d. in an even more preferred embodiment the human VH3 framework sequence has less than 16 or less than 15 amino acid exchanges compared to a human VH3-23 framework sequence. In a more preferred embodiment the human Vkappal subfamily framework sequence for the variable light chain is comprised in the group of Vkappa I subfamily framework sequence consisting of Vkappa 1-5, Vkappa 1-6, Vkappa 1-8, Vkappa 1D-8, Vkappa 1-9, Vkappa 1-12, Vkappa 1D-12, Vkappa 1-13, Vkappa I D- 13. Vkappa 1-16, Vkappa 1 D- 16. Vkappa 1-17, Vkappa 1D-17, Vkappa 1-27, Vkappa 1 -33. Vkappa 1 D-33. Vkappa 1-37, Vkappa lD-37, Vkappa 1-39, Vkappa I D-3 . Vkappa I D-42, Vkappa I D-43. In an even more preferred embodiment the human Vkappa 1 framework sequence has less than 15 or less than 13 amino acid exchanges compared to a human Vkappa 1-39 framework sequence.
In a more preferred embodiment the aglycosyl antibody of the invention comprises a variable light chain or light chain antigen-binding region comprising the L- DR I . L-CDR2 and I .-CDR3 sequence of the variable light chain and a variable heavy chain or heavy chain antigen-binding region comprising the H-CDR1, H-CDR2 and H-CDR3 sequence of the variable heavy chain antibody of the invention as depicted in Table 31 and a human VI I 3 subfamily framework sequence for the variable heavy chain and a human Vkappa 1-39 framework sequence for the variable light chain.
In a most preferred embodiment the aglycosyl antibody of the invention comprises a variable light chain or light chain antigen-binding region comprising the L-CDRl, L-CDR2 and L-CDR3 sequence of tfae variable light chain and a variable heavy chain or heavy chain antigen-binding region comprising the H-CDR1 , H-CDR2 and H-CDR3 sequence of the variable heavy chain antibody of the invention as depicted in Table 31 and a human VH3-3 framework sequence for the variable heavy chain and a human
Vkappa 1 -39 framework sequence for the variable light chain. In a preferred embodiment the variable light chain framework sequence belongs to the human
Vkappal subfamily and the variable heavy chain framework sequence belongs to the human VH3 subfamily. A VH3 subfamily or Vkappa I subfamily variable chain framework sequence may comprises sequence variations compared to the respective WT framework sequence to adopt the framework for insertion of the respective CDR sequence. In a further embodiment a VH3 subfamily or Vkappa I subfamily variable chain framework sequence comprising a sequence variation compared to the WT framework sequence is a VII 3 subfamily member or Vkappa ! subfamily member, respectively. Preferably, such a variant framework sequence has up to 15 sequence variations, more preferably up to 10 sequence variations, more preferably up to 5 sequence variations, most preferably up to 3 sequence variations. An antibody of the invention may be an lg( i (e.g. IgGl Ig( i2. IgG3, IgG4).
In a preferred embodiment the agiycosyl antibodies of the invention are monoclonal. In a further preferred embodiment the agiycosyl antibodies of the invention are human, humanized or chimeric.
In another aspect, the invention provides agiycosyl antibodies having an antigen-binding region that binds specifically to and/or has a high affinity for TWEAKR. An antibody is said to have a "high affinity" for an antigen if the affinity measurement is less than 250 nM (monovalent affinity of the antibody or aniigen-binding fragment). An inventive agiycosyl antibody can bind to human TWEAKR with an affinity of less than 250 nM, preferably less than 150 nM, more preferably less than 100 nM, more preferably less than 50 nM, more preferably less than 30 nM, more preferably less than 20 nM, determined as monovalent affinity to human TWEAKR. In another aspect, the invention provides antibodies having an antigen-binding region that binds specifically to TWEAKR and does not bind to other members of the TNF receptor superfamily (see Table 20).
It is another embodiment of the invention to provide antibodies which are internalized efficiently following binding to a TWEAKR expressing cell. An antibody of the invention might be co- administered with known medicaments, and in some instances the antibody might itself be modified. For example, an antibody could be conjugated to a cytotoxic agent, immunotoxin, toxophore or radioisotope to potentially further increase efficacy. An antibody internalizes "efficiently" when its time of half maximal internalization (t ½) as measured by granule count/cell into TWEAKR expressing tumor cells is shorter than 400 min or more preferably shorter than 300 min and still more preferably shorter than 200 min. Further preferred are antibodies or antigen-binding fragments with half maximal internalization times (t ½) of 100 minutes or less as determined by the protocol described in Example 7 and Figure 17.
Internalizable antibodies are suitable as targeting moiety of an antibody-drug conjugate (ADC). An antibody or antigen-binding fragment is suitable in an in vitro or in vivo method to deliver a compound, preferably a cytotoxic agent, into a TWEAKR expressing cell. The efficient internalization is shown with fluorescently labeled antibodies (Example 7). The efficient use as an antibody drug conjugate is exemplified with a Saporin-conjugated antibody (Example 7).
In some embodiments antibodies of the invention or nucleic acids encoding the same are isolated. An isolated biological component (such as a nucleic acid molecule or protein such as an antibody) is one that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, e.g., other chromosomal and extra-chromosomal ON A and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods as described for example in Sambrook et al., 1989 (Sambrook, J., Frits ch, E. F. and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. USA) and Robert K. Scopes et al. 1994 (Protein Purification, - Principles and Practice, Springer Science and Business Media LLC). The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
An antibody of the invention may be derived from a recombinant antibody library that is based on amino acid sequences that have been isolated from the antibodies of a large number of healthy volunteers e.g. using the n-CoDeR® technology the fully human CDRs are recombined into new antibody molecules. Or alternatively antibody libraries as the fully human antibody phage display library described in Hoet RM et a!.. Nat Biotechnol 2005;23(3):344-8) can be used to isolate TWEAKR- specific antibodies.
Peptide V ariants
Antibodies or antigen-binding fragments of the invention are not limited to the specific peptide sequences provided herein. Rather, the invention also embodies variants of these polypeptides. With reference to the instant disclosure and conventionally available technologies and references, the skilled worker will be able to prepare, test and utilize functional variants of the antibodies disclosed herein, while appreciating these variants having the ability to bind to TWEAKR fall within the scope of the present invention. A variant can include, for example, an antibody that has at least one altered complementary determining region (CDR) (hyper-variable) and/or framework (FR) (variable) domain/position, vis-a-vis a peptide sequence disclosed herein. To better illustrate this concept, a brief description of antibody structure follows. An antibody is composed of two peptide chains, each containing one (light chain) or three (heavy chain) constant domains and a variable region (VL, VH), the latter of which is in each case made up of four FR regions and three interspaced CDRs. The antigen-binding site is formed by one or more CDRs, yet the F R regions provide the structural framework for the CDRs and, hence, play an important role in antigen binding. By altering one or more amino acid residues in a CDR or FR region, the skilled worker routinely can generate mutated or diversified antibody sequences, which can be screened against the antigen, for new or improved properties, for example.
A further preferred embodiment of the invention is an antibody or antigen-binding fragment in which the VH and VL sequences are selected as shown in Table 31. The skilled worker can use the data in Table 31 to design peptide variants that are within the scope of the present invention. It is preferred that variants are constructed by changing amino acids within one or more CDR regions; a variant might also have one or more altered framework regions. Alterations also may be made in the framework regions. For example, a peptide FR domain might be altered where there is a deviation in a residue compared to a germiine sequence.
Alternatively, the skilled worker could make the same analysis by comparing the amino acid sequences disclosed herein to known sequences of the same class of such antibodies, using, for example, the procedure described by Knappik A., et al., JMB 2000, 296:57-86.
Furthermore, variants may be obtained by using one antibody as starting point for further optimization by diversifying one or more amino acid residues in the antibody, preferably amino acid residues in one or more CDRs, and by screening the resulting collection of antibody variants for variants with improved properties. Particularly preferred is diversification of one or more amino acid residues in CDR3 of VL and/or VH. Diversification can be done e.g. by synthesizing a collection of DMA molecules using trinucleotide mutagenesis (TRIM) technology (Virnekas B. et al., Nucl. Acids Res. 1994, 22 : 5600.). Antibodies or antigen-binding fragments thereof include molecules with modifications/variations including but not limited to e.g. modifications leading to altered hal - life (e.g. modification of the Fc part or attachment of further molecules such as PEG), altered binding affinity or altered A IX or CDC activity. Conservative Amino Acid Variants
Polypeptide variants may be made that conserve the overall molecular structure of an antibody peptide sequence described herein. Given the properties of the individual amino acids, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
For example, (a) n n polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophane, and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions typically may be made within groups (a)-(d). In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in a-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in β-pleated sheets. Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns. Some preferred substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I . Given the known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist readily can construct DNAs encoding the conservative amino acid variants. As used herein, "sequence identity" between two polypeptide sequences, indicates the percentage of amino acids that are identical between the sequences. "Sequence homology" indicates the percentage of amino acids that either is identical or that represent conservative amino acid substitutions.
DNA molecules of the invention
The present invention also relates to the DNA molecules that encode an antibody of the invention or antigen-binding fragment thereof. The DNA sequences used for the antibodies expressed are given in Table 32. These sequences are optimized for mammalian expression. DNA molecules of the invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants within the invention may be described by reference to their physical properties in hybridization. The skilled worker will recognize that DNA can be used to identify its complement and, since DNA is double stranded, its equivalent or homoiog, using nucleic acid hybridization techniques. It also will be recognized that hybridization can occur with less than 100% complementarity. However, given appropriate choice of conditions, hybridization techniques can be used to differentiate among DNA sequences based on their structural relatedness to a particular probe. For guidance regarding such conditions see, Sambrook et al., 1989 supra and Ausubel et al., 1995 (Ausubel, F. M ., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New Y rk : John Wiley and Sons).
Structural similarity between two polynucleotide sequences can be expressed as a function of
"stringency" of the conditions under which the two sequences will hybridize with one another. As used herein, the term "stringency" refers to the extent that the conditions disfavor hybridization. Stringent conditions strongly disfavor hybridization, and only the most structurally related molecules will hybridize to one another under such conditions. Conversely, non-stringent conditions favor hybridization of molecules displaying a lesser degree of structural relatedness. Hybridization stringency, therefore, directly correlates with the structural relationships of two nucleic acid sequences. The following relationships are useful in correlating hybridization and relatedness (where Tm is the melting temperature of a nucleic acid duplex): a. Tm = 69.3 + 0.41(%G+C)°C
b. The Tm of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatched base pairs.
c. (ΤΜ)μ2 - (Tm) μΐ = 18.5 1(¾ιομ2/μ1
where μΐ and μ2 are the ionic strengths of two solutions.
Hybridization stringency is a function of many factors, including overall DNA concentration, ionic strength, temperature, probe size and the presence of agents which disrupt hydrogen bonding. Factors promoting hybridization include high DNA concentrations, high ionic strengths, low temperatures, longer probe size and the absence of agents that disrupt hydrogen bonding. Hybridization typically is performed in two phases: the "binding" phase and the "washing" phase.
Functiona!!y Equivalent Variants
Yet another class of DNA variants within the scope of the invention may be described with reference to the product they encode. These functionally equivalent polynucleotides are characterized by the fact that they encode the same peptide sequences due to the degeneracy of the genetic code.
It is recognized that variants of DNA molecules provided herein can be constructed in several different ways. For example, they may be constructed as completely synthetic DNAs. Methods of efficiently synthesizing oligonucleotides in the range of 20 to about 150 nucleotides are widely available. See Ausubel et ah, section 2.11, Supplement 21 (1993). Overlapping oligonucleotides may be synthesized and assembled in a fashion first reported by Khorana et ah, J. Mo I. Biol. 72:209-217 (1971); see also Ausubel et ah, supra, Section 8.2. Synthetic DNAs preferably are designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector. As indicated, a method of generating variants is to start with one of the DNAs disclosed herein and then to conduct site-directed mutagenesis. See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typical method, a target DNA is cloned into a single-stranded DNA bacteriophage vehicle. Single- stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide alteration(s). The complementary strand is synthesized and the double stranded phage is introduced into a host. Some of the resulting progeny will contain the desired mutant, which can be confirmed using DNA sequencing. In addition, various methods are available that increase the probability that the progeny phage will be the desired mutant. These methods are well known to those in the field and kits are commercially available for generating such mutants. Antibody-Drug Conjugates (ADC)
The invention also provides antibody-drug conjugates (ADC, immunoconjugates) comprising an aglycosyl anti-TWEAKR antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes. In one embodiment, an immuno conj ugate is an antibody-drug conjugate (ADC) in which an aglycosyl antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235); an auri statin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof; an anthracycline such as daunomycin or doxorubicin; methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.
In another embodiment, an immunoconjugate comprises an aglycosyl antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alphas arcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (P API, P A I I, and PAP-S ), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
In another embodiment, an immunoconjugate comprises an aglycosyl antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include Th227, Ac225, At2", I!3 !, I125, Y90, Re186, Re188, Sin153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example Tc99m, or a spin label for nuclear magnetic resonance (NMR) imaging, such as iodine- 123 again, iodine-131 , indium- 1 1 1, fluorine- 19, carbon- 13, nitrogen- 1 . oxygen- 1 7, gadolinium, manganese or iron.
Recombinant DNA constructs and expression
The present invention further provides recombinant DNA constructs comprising one or more of the nucleotide sequences of the present invention (see Table 32). The recombinant constructs of the present invention are used in connection with a vector, such as a plasmid, phagemid, phage or viral vector, into which a DNA molecule encoding an antibody of the invention or antigen-binding fragment thereof or variant thereof is inserted.
An antibody provided herein can be prepared by recombinant expression of nucleic acid sequences encoding light and heavy chains or portions thereof in a host cell. To express an antibody a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the light and/or heavy chains or portions thereof such that the light and heavy chains are expressed in the host cell. Standard recombinant DNA methodologies are used to prepare and/or obtain nucleic acids encoding the heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor. N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al..
In addition, the nucleic acid sequences encoding variable regions of the heavy and/or light chains can be converted, for example, to nucleic acid sequences encoding full-length antibody chains, Fab fragments, or to scFv. The VL- or VH-encoding DNA fragment can be operatively linked, (such that the amino acid sequences encoded by the two DNA fragments are in-frame) to another DNA fragment encoding, for example, an antibody constant region or a flexible linker. The sequences of human heavy chain and light chain constant regions are known in the art (see e.g., Kabat, E. A., el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
In certain assays an expression of the antibodies of this invention as murine IgG is preferred, e.g. immunohistochemistry with human samples can be analyzed more easily by using murine antibodies. To create a polynucleotide sequence that encodes a scFv, the VH- and VL-encoding nucleic acids can be operatively linked to another fragment encoding a flexible linker such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al, Nature (1990) 348:552-554).
To express the antibodies standard recombinant DNA expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, DNA encoding the desired polypeptide can be inserted into an expression vector which is then transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells. Examples for prokaryotic host cells are e.g. bacteria, examples for eukaryotic host cells are yeast, insect or mammalian cells. In some embodiments, the DNAs encoding the heavy and light chains are inserted into separate vectors. In other embodiments, the DNA encoding the heavy and light chains is inserted into the same vector. It is understood that the design of the expression vector, including the selection of regulatory sequences is affected by factors such as the choice of the host cell, the level of expression of protein desired and whether expression is constitutive or inducible.
Therefore, an embodiment of the present invention are also host cells comprising the vector or a nucleic acid molecule, whereby the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.
Another embodiment of the present invention is a method of using the host cell to produce an antibody and antigen binding fragments, comprising culturing the host cell under suitable conditions and recovering said antibody. Therefore another embodiment of the present invention is the production of the antibodies according to this invention with the host cells of the present invention and purification of these antibodies to at least 95% homogeneity by weight.
Bacterial Expression
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include but are not limited to E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
Bacterial vectors may be, for example, bacteriophage-, piasmid- or phagemid-based. These vectors can contain a selectable marker and a bacterial ori in of replication derived from commercially available plasmids typically containing elements of the well-known cloning vector pBR322 (ATCC 37017). Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
Therefore, an embodiment of the present invention is an expression vector comprising a nucleic acid sequence encoding for the novel antibodies of the present invention. See Example 1 for an exemplary description. Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic host, including, for example, E. coli, Bacillus suhtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably, from E. coli cells.
Mammalian Expression Preferred regulatory sequences for mammalian host cell expression include viral elements that direct hi h levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. 5,168,062 by Stinski, U.S. 4.5 1 0.245 by Bell et al. and U.S. 4,968,615 by Schaffher et al. The recombinant expression vectors can also include origins of replication and selectable markers (see e.g., U.S. 4.399.2 ! 6. 4.634.665 and U.S. 5,179,017). Suitable selectable markers include genes that confer resistance to drugs such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. For example, the dihydrofolate reductase I DI I FR ) gene confers resistance to methotrexate and the neo gene confers resistance to G418.
Trans fection of the expression vector into a host cell can be carried out using standard techniques such as electroporation, calcium-phosphate precipitation, and DEAE-dextran transfection. Suitable mammalian host cells for expressing the antibodies, antigen binding fragments thereof or variants thereof provided herein include Chinese Hamster Ovary (CHO cells) [including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601 -621], NSO myeloma cells, COS cells and SP2 cells.
A preferred system for transient expression in mammalian cells, using "Human Embryonic Kidney" (HEK) cells, is described in Tom et al., Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007. Briefly, a CMV-Promoter based expression plasmid is transfected into HEK293-6E cells. in some embodiments, the expression vector is designed such that the expressed protein is secreted into the culture medium in which the host cells are grown. The antibodies, antigen binding fragments thereof or variants thereof can be recovered from the culture medium using standard protein purification methods.
Purification Antibodies of the invention can be recovered and purified from recombinant cell cultures by well- known methods including, but not limited to ammonium sulfate or ethanol precipitation, acid extraction, Protein A chromatography, Protein G chromatography, anion or cation exchange chromatography, phospho-celiulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydro xylapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.
Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from an eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.
In preferred embodiments, the antibody is purified (1) to greater than 95% by weight of antibody as determined e.g. by the Lowry method, UV-Vis spectroscopy or by by SDS-C apillary Gel electrophoresis (for example on a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer device), and in further preferred embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated naturally occurring antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
Therapeutic Methods Therapeutic methods involve administering to a subject in need of treatment a therapeutically effective amount of an antibody contemplated by the invention. A "therapeutically effective" amount hereby is defined as the amount of an antibody that is of sufficient quantity to reduce proliferation of TWEAK R positive cell or to reduce size of a TWEAK expressing tumor in a treated area of a subject - either as a single dose or according to a multiple dose regimen, alone r in combination with other agents, which leads to the alleviation of an adverse condition, yet which amount is toxicologically tolerable. The subject may be a human or non-human animal (e.g. , rabbit, rat, mouse, dog, monkey or other lower-order primate).
It is an embodiment of the invention to provide an antibody for use as medicament.
It is an embodiment of the invention to provide an antibody for use as a medicament for the treatment of cancer. In a preferred embodiment the cancer is a solid tumor.
The inventive antibodies can be used as a therapeutic or a diagnostic tool in a variety of situations with aberrant TWEAKR-signaling, e.g. cell proliferative disorders such as cancer or fibrotic diseases. Disorders and conditions particularly suitable for treatment with an antibody of the inventions are solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, and their distant metastases. Those disorders also include lymphomas, sarcomas and leukemias.
Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers.
Examples of esophageal cancer include, but are not limited to esophageal cell carcinomas and Adeno carcinomas , as well as squamous cell carcinomas, Leiomyosarcoma, malignant melanoma, rhabdomyosarcoma and lymphoma.
Examples of gastric cancer include, but are not limited to intestinal type and diffuse type gastric adeno carcinoma .
Examples of pancreatic cancer include, but are not limited to ductal adenocarcinoma, adenosquamous carcinomas and pancreatic endocrine tumors. Examples of breast cancer include, but are not limited to triple negative breast cancer, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
Examples of cancers of the respiratory tract include, but are not limited to small-cell and non-small- cell lung carcinoma, as well as bronchial adenoma and pleuropuimonary blastoma.
Examples of brain cancers include, but are not limited to brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, glioblastoma, medul loblastoma, ependymoma, as well as neuroectodermal and pineal tumor.
Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal and vulvar cancer, as well as sarcoma of the uterus.
Examples of ovarian cancer include, but are not limited to serous tumour, endometrioid tumor, mucinous cystadenocarcinoma, granulosa cell tumor, Sertoii-Leydig cell tumor and arrhenoblastoma
Examples of cervical cancer include, but are not limited to squamous cell carcinoma, adenocarcinoma, adeno squamous carcinoma, small cell carcinoma, neuroendocrine tumour, glassy cell carcinoma and villoglandular adeno carcinoma .
Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, urethral, and hereditary and sporadic papillary renal cancers.
Examples of kidney cancer include, but are not limited to renal cell carcinoma, urothelial cell carcinoma, juxtaglomerular cell tumor (reninoma), angiomyolipoma, renal oncocytoma, Bellini duct carcinoma, clear-cell sarcoma of the kidney, mesoblastic nephroma and Wilms' tumor.
Examples of bladder cancer include, but are not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma and small cell carcinoma.
Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.
Examples of liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibroiamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.
Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head-and-neck cancers include, but are not limited to squamous cell cancer of the head and neck, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, salivary gland cancer, lip and oral cavity cancer, and squamous cell cancer.
Lymphomas include, but are not limited to AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.
Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lympho sarcoma, and rhabdomyosarcoma.
Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
In a preferred embodiment, the antibodies are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease. In a preferred embodiment, the antibodies of the invention are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease wherein the cancer is a solid cancer. In a preferred embodiment, the antibodies of the invention are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease comprised in a group consisting of gastric cancer, breast cancer, pancreatic cancer, colorectal cancer, kidney cancer, prostate cancer, ovarian cancer, cervical cancers, lung cancer, endometrial cancer, esophageal cancer, head and neck cancer, hepatocellular carcinoma, melanoma and bladder cancer. In a preferred embodiment, the antibodies are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease comprised in a group consisting of colorectal cancer, non small cell lung cancer, kidney cancer, melanoma, ovarian cancer, head and neck cancer and pancreatic cancer.
In addition, the inventive antibodies can also be used as a therapeutic or a diagnostic tool in a variety of other disorders wherein TWEAKR is involved such as, but not limited to fibrotic diseases such as intraalveolar fibrosis, silica-induced pulmonary fibrosis, experimental lung fibrosis, idiopathic lung fibrosis, renal fibrosis, as well as lymphangioleiomyomatosis, polycystic ovary syndrome, acne, psoriasis, cholesteatoma, cholesteatomatous chronic otitis media, periodontitis, solar lentigines, bowel disease, atherosclerosis or endometriosis. The disorders mentioned above have been well characterized in humans, but also exist with a similar etiology in other animals, including mammals, and can be treated by administering pharmaceutical compositions of the present invention. An antibody of the invention might be co-administered with known medicaments, and in some instances the antibody might itself be modified. For example, an antibody could be conjugated to a cytotoxic agent or radioisotope to potentially further increase efficacy.
Antibodies of the present invention may be administered as the sole pharmaceutical agent or in combination with one or more additional therapeutic agents where the combination causes no nacceptable adverse effects. This combination therapy includes administration of a single pharmaceutical dosage formulation which contains an antibody of the invention and one or more additional therapeutic agents, as well as administration of an antibody of the invention and each additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, an antibody of the invention and a therapeutic agent may be administered to the patient together in a single liquid composition, or each agent may be administered in separate dosage formulation.
Where separate dosage formulations are used, an antibody of the invention and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially). In particular, antibodies of the present invention may be used in fixed or separate combination with other anti-tumor agents such as alkylating agents, anti-metabolites, plant-derived anti-tumor agents, hormonal therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and/or biological response modifiers, anti-angiogenic compounds, and other anti-tumor drugs. In this regard, the following is a non-limiting list of examples of secondary agents that may be used in combination with the antibodies of the present invention:
Alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, thiotepa, ranimustine, nimustine, temozolomide, altretamine, apaziquone, brostallicin, bendamustine, carmustine, estramustine, fotemustine, glufosfamide, inafosfamide, bendamustin, and mitolactol; platinum-coordinated alkylating compounds include, but are not limited to, cisplatin, carboplatin, eptaplatin, lobaplatin, nedaplatin, oxaliplatin, and satraplatin;
Anti-metabolites include, but are not limited to, methotrexate, 6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil alone or in combination with leucovorin, tegafur, doxifluridine, carmofur, cytarabine, cytarabine ocfosfate, enocitabine, gemcitabine, fludarabin, 5-azacitidine, capecitabine, cladribine, clofarabine, decitabine, eflornithine, ethynylcytidine, cytosine arabinoside, hydroxyurea, melphalan, nelarabine, nolatrexed, ocfosfite, disodium premetrexed, pentostatin, pelitrexol, raltitrexed, triapine, trimetrexate, vidarabine, vincristine, and vinorelbine;
Hormonal therapy agents include, but are not limited to, exemestane, Lupron, anastrozole, doxercalciferol, fadrozole, formestane, 11 -beta hydro xysteroid dehydrogenase 1 inhibitors, 1 7 -alpha hydroxylase/ 17,20 lyase inhibitors such as abiraterone acetate, 5-alpha reductase inhibitors such as finasteride and epristeride, anti-estrogens such as tamoxifen citrate and fulvestrant, Trelstar, toremifene, raloxifene, lasofoxifene, letrozole, anti-androgens such as bicalutamide, flutamide, mifepristone, nilutamide, Casodex, and anti-progesterones and combinations thereof; Plant-derived anti-tumor substances include, e.g., tho e selected from mitotic inhibitors, for example epothilones such as sagopilone, ixabepilone and epothilone B, vinblastine, vinflunine, docetaxel, and Paclitaxel;
Cytotoxic topoisomerase inhibiting agents include, but are not limited to, aclarubicin, doxorubicin, amonafide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, Irinotecan, topotecan, edotecarin, epimbicin, etoposide, exatecan, gimatecan, lurtotecan, mitoxantrone, pirambicin, pixantrone, rubitecan, sobuzoxane, tafluposide, and combinations thereof;
Immunologicals include interferons such as interferon alpha, interferon alpha-2a, interferon alpha- lb. interferon beta, interferon gamma- la and interferon gamma-nl, and other immune enhancing agents such as L I -11.2 and other 11.2 derivatives, filgrastim, lentinan, sizofilan, TheraCys, ubenimex, aldesleukin, alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab, ozogamicin, ibritumomab, imiquimod, lenograstim, lentinan, melanoma vaccine (Corixa), molgramostim, sargramostim, tasonermin, tecleukin, thymalasin, tositumomab, Vimlizin, epratuzumab, mitumomab, oregovomab, pemtumomab, and Pi venge;
Biological response modifiers are agents that modify defense mechanisms of living organisms or biological responses such as survival, growth or differentiation of tissue cells to direct them to have antitumor activity; such agents include, e.g., krestin, lentinan, sizofiran, picibanil, ProMune, and ubenimex;
Anti-angiogenic compounds include, but are not limited to, acitretin, aflibercept, angiostatin, aplidine, asentar, axitinib, bevacizumab, brivanib alaninat, cilengtide, combreta statin, endostatin, fenretinide, halofuginone, pa/opanib. ranibizumab, rebimastat, recentin, regorafenib, removab, revlimid, sorafenib, squalamine, sunitinib, telatinib, thalidomide, ukrain, vatalanib, and vitaxin;
Antibodies include, but are not limited to, trastuzumab, cetuximab, bevacizumab, rituximab, ticilimumab, ipilimumab, lumiliximab, catumaxomab, atacicept, oregovomab, panitumumab and alemtu- zumab;
VEGF inhibitors such as, e.g., sorafenib, regorafenib, bevacizumab, sunitinib, recentin, axitinib, aflibercept, telatinib, brivanib alaninate, vatalanib, pa/opanib. and ranibizumab;
EGFR (HER1) inhibitors such as, e.g., cetuximab, panitumumab, vectibix, gefitinib, erlotinib, and Zactima; H ER2 inhibitors such as, e.g., lapatinib, tratuzumab, and pertuzumab; inTOR inhibitors such as, e.g., temsirolimus, sirolimus/Rapamycin, and everolimus; c-Met inhibitors;
PI3K inhibitors such as PI3K inhibitor 1 (2-3πιϊηο-Ν-[7-πΐ6Λοχγ-8-(3-ΓΓΐο ^Ηη-4-γ1ρΓοροχγ)- 2,3 -dihydroimidazo [ 1 ,2-c]quinazolin-5-yl]pyrimidine-5 -carboxamide dihydro chloride (see compound of Examples 1 and 2 WO 2012/136553, (which is incorporated herein by reference in its entirety) and AKT inhibitors;
CDK inhibitors such as roscovitine and flavopiridol;
Spindle assembly checkpoints inhibitors and targeted anti-mitotic agents such as PLK inhibitors, Aurora inhibitors (e.g. Hesperadin), checkpoint kinase inhibitors, and KSP inhibitors;
HDAC inhibitors such as, e.g., panobinostat, vorinostat, MS275, belinostat, and LBH589;
HSP90 and HSP70 inhibitors;
Proteasome inhibitors such as bortezomib and carfilzomib;
S erine/threonine kinase inhibitors including MEK inhibitors and Raf inhibitors such as sorafenib; Farnesyl transferase inhibitors such as, e.g., tipifarnib;
Tyrosine kinase inhibitors including, e.g., dasatinib, nilotibib, regorafenib, bosutinib, sorafenib, bevacizumab, sunitinib, cediranib, axitinib, aflibercept, telatinib, imatinib mesylate, brivanib alaninate, pazopanib, ranibizumab, vatalanib, cetuximab, panitumumab, vectibix, gefitinib, erlotinib, lapatinib, tratuzumab, pertuzumab, and c-Kit inhibitors; Vitamin D receptor agonists;
Bcl-2 protein inhibitors such as obatoclax, oblimersen sodium, and gossypol;
Cluster of differentiation 20 receptor antagonists such as, e.g., rituximab;
Ribonucleotide reductase inhibitors such as, e.g., gemcitabine;
Tumor necrosis factor related apoptosis inducing ligand receptor 1 agonists such as, e.g., mapatumumab; Tumor necrosis factor related apoptosis inducing ligand receptor 2 agonists such as e.g.. lexatumumab, conatumumab, CS-1008, PRO95780;
5 -Hydro xytryptamine receptor antagonists such as, e.g., rEV598, xaliprode, palonosetron hydrochloride, granisetron, Zindol, and AB-1001 ; Integrin inhibitors including alpha5-betal integrin inhibitors such as, e.g., E7820, JSM 6425, volociximab, and endo statin;
Androgen receptor antagonists including, e.g., nandrolone decanoate, fluoxymesterone, Android, Prost-aid, andromustine, bicalutamide, flutamide, apo-cyproterone, apo-flutamide, chlormadinone acetate, Androcur, Tabi, cyproterone acetate, and nilutamide; Aromatase inhibitors such as, e.g., anastrozole, letrozole, testolactone, exemestane, amino- glutethimide, and formestane;
Matrix metalloproteinase inhibitors;
Other anti-cancer agents including, e.g., alitretinoin, ampligen, atrasentan bexarotene, bortezomib, bosentan, calcitriol, exisulind, fotemustine, ibandronic acid, miltefosine, mitoxantrone, i -asparaginase. procarbazine, dacarbazine, hydroxy carbamide, pegaspargase, pentostatin, tazaroten, velcade, gallium nitrate, canfosfamide, darinaparsin, and tretinoin.
In a preferred embodiment, the antibodies of the present invention may be used in combination with chemotherapy (i.e. cytotoxic agents), anti-hormones and/or targeted therapies such as other kinase inhibitors (for example, EGFR inhibitors), mTOR inhibitors and angiogenesis inhibitors. In a preferred embodiment, the antibodies of the present invention may be used in combination with check-point inhibitors and antibodies to immunotherapy receptors which empower any ongoing anticancer immune response that might have been too weak or exhausted. Preferred are monoclonal antibodies (mAb) interfering with CTLA4, PD-1 (CD279, PDCD1), PD-L 1 (B7-H1 , CD274, PDCDILGI), PD-L2 (PDCD1LG2, B7-DC, CD273), CD276 (B7-H3), TNFRSF4 (OX-40, CD 1 34). CD27 (TNFRSF7), CD70 (CD27 Ligand), TNFRSF9 (CD137, CDwl37, 4- 1 B, CD 1 37. ILA), TNFRSF18 (GITR, AITR, CD357 ). KIR3 (KIR, NKAT3, NKB1 , CD158e), LAG3 (CD223 ). ΤΓΜ-3. Such antibodies include, but are not limited to, ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, RG-7446. BMS-936559, durvalumab, MSB-0010718C, rH ig 1 2B7. MGA-271, 124I-8H9, MEDI-6469, CDX- 1 1 27. varlilumab, AMG 172, ARGX-1 10, SGN-70A, urelumab, PF- 05082566, TRX-518, MK4166, lirilumab, BMS-986016. In a highly preferred embodiment the antibodies of the present invention are used in combination with anti-CTLA4, anti-PD-1 , and/ or anti- PD-L1 antibodies including but not limited to ipilimumab, nivolumab, pembrolizumab, BMS-936559, durvalumab, RG-7446.
The compounds of the present invention may also be employed in cancer treatment in conjunction with radiation therapy and/or surgical intervention. Furthermore, the antibodies of the invention may be utilized, as such or in compositions, in research and diagnostics, or as analytical reference standards, and the like, which are well known in the art.
Diagnostic Methods
An ti -TWEAKR antibodies can be used for detecting the presence of TWEAKR-expressing tumors. The presence of TWEAKR-containing cells or shed TWEAKR within various biological samples, including serum, and tissue biopsy specimens, may be detected with anti-TWEAKR antibodies. In addition, anti-TWEAKR antibodies may be used in various imaging methodologies such as immuno s cintigraphy with a "Tc (or other isotope) conjugated antibody. For example, an imaging protocol similar to the one described using a ' "in conjugated anti-PS MA antibody may be used to detect pancreatic or ovarian carcinomas (Sodee et al., Clin. Nuc. Med. 21 : 759-766, 1997). Another method of detection that can be used is positron emitting tomography by conjugating the antibodies of the invention with a suitable isotope (see Herzog et al., J. Nucl. Med. 34:2222-2226, 1993).
Pharmaceutical Compositions and Administration
To treat any of the foregoing disorders, pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. An antibody of the invention can be administered by any suitable means, which can vary, depending on the type of disorder being treated. Possible administration routes include parenteral (e.g., intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous), intrapulmonary and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration. In addition, an antibody of the invention might be administered by pulse infusion, with, e.g., declining doses of the antibody. Preferably, the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. The amount to be administered will depend on a variety of factors such as the clinical symptoms, weight of the individual, whether other drugs are administered. The skilled artisan will recognize that the route of administration will vary depending on the disorder or condition to be treated. An embodiment of the present invention are pharmaceutical compositions which comprise anti-
TWEAKR antibodies alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. A further embodiment are pharmaceutical compositions comprising a TWEAKR binding antibody and a further pharmaceutically active compound that is suitable to treat TWEAKR related diseases such as cancer. Any of these molecules can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers. In one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert.
The present invention also relates to the administration of pharmaceutical compositions. Such administration is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl-cellulose, hydro xypropylmethylcellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. I desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores can be provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, earbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e. dosage. Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, 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 oleate or triglycerides, or liposomes. Optionally, 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.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1 %-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
After pharmaceutical compositions comprising a compound of the invention formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of anti-TWEAKR antibodies such labeling would include amount, frequency and method of administration. Kits
The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
Therapeutically Effective Dose
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose, i.e. treatment of a particular disease state characterized by TWEAKR expression.
The determination of an effective dose is well within the capability of those skilled in the art.
Determining a therapeutically effective amount of the novel antibody of this invention largely will depend on particular patient characteristics, route of administration, and the nature of the disorder being treated. General guidance can be found, for example, in the publications of the International Conference on Harmonization and in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528 (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. C o.. 1990). More specifically, determining a therapeutically effective amount will depend on such factors as toxicity and efficacy of the medicament. Toxicity may be determined using methods well known in the art and found in the foregoing references. Efficacy may be determined utilizing the same guidance in conjunction with the methods described below in the Examples.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., neoplastic ceils, or in animal models, usually mice, rabbits, dogs, pigs or monkeys. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to that amount of antibody or antigen-binding fragment thereof, that ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, ED50 LD50. Pharmaceutical compositions that exhibit large therapeutic indices are pre erred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease state, e.g., tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered for example every 3 to 4 days, every week, once every two weeks, or once every three weeks, depending on half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 2 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature. See U.S. Pat. No. 4,657,760; 5,206,344; or 5,225.212. Those skilled in the art will employ different formulations for polynucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. Preferred specific activities for a radiolabelled antibody may range from 0.1 to 10 mCi/mg of protein (Riva et al, Clin. Cancer Res. 5:3275-3280, 1999; Ulaner et al, 2008 Radiology 246(3):895-902)
A further preferred embodiment of the Invention is: 1. An isolated anti-TWEAKR antibody comprising a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region, which specifically binds to the D at position 47 (D47) of TWEAK R (SEQ ID NO: 169).
2. The antibody according to embodiment 1 which comprises an amino acid substitution at amino acid positions 297 and / or 299 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
3. The antibody according to embodiment 2 which comprises an amino acid substitution of N297A or N297Q (EU index as in Kabat).
4. The antibody according to anyone of the preceding embodiments wherein the antibody binds an Fc gamma receptor, preferentially FcyRIIB with a KD value which is more than 10 fold higher than that of the parent antibody comprising an unmodified Fc region.
5. The antibody according to anyone of the preceding embodiments wherein the antibody binds a human FcyRIIB with a KD value larger than 200 μΜ. Tfae antibody according to anyone of the preceding embodiments wherein the antibody specifically binds to the D at position 47 (D47) of TWEAKR (SEQ ID NO: 169), when the antibody loses more than 80% of its ELISA signal on TPP-2614 compared to TPP-2203.
The antibody according to anyone of the preceding embodiments wherein the antibody is an agonistic antibody.
The antibody according to anyone of the preceding embodiments, which comprises:
a variable heavy chain comprising:
(a) a heavy chain CDR1 encoded by an amino acid sequence comprising the formula PYPMX (SEQ ID NO: 171), wherein X is I or M;
(b) a heavy chain CDR2 encoded by an amino acid sequence comprising the formula YISPSGGXTHYADSVKG (SEQ ID NO: 172), wherein X is S or K; and
(c) a heavy chain CDR3 encoded by an amino acid sequence comprising the formula
GGDTYFDYFDY (SEQ ID NO: 173); and a variable light chain comprising:
(a) a light chain CDR1 encoded by an amino acid sequence comprising the formula RASQSISXYL (SEQ I D NO: 174), wherein X is G or S;
(b) a light chain CDR2 encoded by an amino acid sequence comprising the formula XASSLQS (SEQ I D NO: 175), wherein X is Q, A, or N; and
(c) a light chain CDR3 encoded by an amino acid sequence comprising the formula QQSYXXPXIT (SEQ I D NO: 176), wherein X at position 5 is T or S, and X at position 6 is T or S, and X at position 8 is G, or F.
The antibody according to anyone of the preceding embodiments comprising: a. a variable heavy chain comprising the variable heavy chain CDR1 sequence as presented by SEQ I D NO: 6, the variable heavy chain CDR2 sequence as presented by SEQ I D NO: 7, and the variable heavy chain CDR3 sequence as presented by SEQ I D NO: 8, and a variable light chain comprising the variable light chain CDR1 sequence presented by SEQ I D NO: 3, the variable light chain CDR2 sequence presented by SEQ I D NO: 4, and the variable light chain CDR3 sequence presented by SEQ I D NO: 5, or a variable heavy chain comprising the variable heavy chain CDR1 sequence as presented by SEQ ID NO: 16, the variable heavy chain CDR2 sequence as presented by SEQ I NO: 17, the variable heavy chain CDR3 sequence as presented by SEQ ID NO: 18, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 13, the variable light chain CDR2 sequence presented by SEQ ID NO: 14, and the variable light chain CDR3 sequence presented by SEQ ID NO: 15, or a variable heavy chain comprising the variable heavy chain CDRl sequence as presented by SEQ ID NO: 26, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 27, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:28, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 23, the variable light chain CDR2 sequence presented by SEQ ID NO: 24. and the variable light chain CDR3 sequence presented by SEQ I D NO:25, or a variable heavy chain comprising the variable heavy chain CDRl sequence as presented by SEQ ID NO: 36, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 37, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:38, and a variable light chain comprising the variable light chain CDR l sequence presented by SEQ ID NO: 33, the variable light chain CDR2 sequence presented by SEQ ID NO: 34. and the variable light chain CDR3 sequence presented by SEQ ID NO:35, or a variable heavy chain comprising the variable heavy chain CDRl sequence as presented by SEQ ID NO: 46, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 47, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:48, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 43, the variable light chain CDR2 sequence presented by SEQ ID NO: 44, and the variable light chain CDR3 sequence presented by SEQ ID NO:45, or a variable heavy chain comprising the variable heavy chain CDRl sequence as presented by SEQ ID NO: 56, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 57, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:58, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 53, the variable light chain CDR2 sequence presented by SEQ ID NO: 54, and the variable light chain CDR3 sequence presented by SEQ ID NO:55, or a variable heavy chain comprising the variable heavy chain CDR1 sequence as presented by SEQ ID NO: 66, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 67, the variable heavy chain CDR3 sequence as presented by SEQ I NO:68, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 63, the variable light chain CDR2 sequence presented by SEQ ID NO: 64, and the variable light chain CDR3 sequence presented by SEQ ID NO: 65, or a variable heavy chain comprising the variable heavy chain CDR l sequence as presented by SEQ ID NO: 76, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 77, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:78, and a variable light chain comprising the variable light chain CDR l sequence presented by SEQ ID NO: 73, the variable light chain CDR2 sequence presented by SEQ ID NO: 74, and the variable light chain CDR3 sequence presented by SEQ ID NO: 75, or a variable heavy chain comprising the variable heavy chain CDR l sequence as presented by SEQ ID NO: 86, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 87, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:88, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 83, the variable light chain CDR2 sequence presented by SEQ ID NO: 84, and the variable light chain CDR3 sequence presented by SEQ I D NO: 85, or a variable heavy chain comprising the variable heavy chain CDRl sequence as presented by SEQ ID NO: 96, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 97, the variable heavy chain CDR3 sequence as presented by SEQ ID NO:98, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 93, the variable light chain CDR2 sequence presented by SEQ ID NO: 94, and the variable light chain CDR3 sequence presented by SEQ ID NO: 5, or a variable heavy chain comprising the variable heavy chain CDRl sequence as presented by SEQ ID NO: 106, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 107, the variable heavy chain CDR3 sequence as presented by SEQ I D NO: 108, and a variable light chain comprising the variable light chain CDRl sequence presented by SEQ ID NO: 103, the variable light chain CDR2 sequence presented by SEQ ID NO: 104, and the variable light chain CDR3 sequence presented by SEQ ID NO: 105 or 1. a variable heavy chain comprising the variable heavy chain CDR1 sequence as presented by SEQ ID NO: 1 16, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 1 1 7. the variable heavy chain CDR3 sequence as presented by SEQ I D NO: l 18, and a variable light chain comprising the variable light chain CDR1 sequence presented by SEQ I D NO: 1 13, the variable light chain CDR2 sequence presented by SEQ I D NO: 114, and the variable light chain CDR3 sequence presented by SEQ I D NO: l 15,. The antibody according to anyone of the preceding embodiments comprising: a. a variable heavy chain sequence as presented by SEQ I D NO: 10 and a variable light chain sequences as presented by SEQ I D NO:9, or b. a variable heavy chain sequence as presented by SEQ I D NO:20 and a variable light chain sequences as presented by SEQ I D NO: 19, or c. a variable heavy chain sequence as presented by SEQ ID NO:30 and a variable light chain sequences as presented by SEQ I D NO: 29, or d. a variable heavy chain sequence as presented by SEQ I D NO:40 and a variable light chain sequences as presented by SEQ I D NO: 39, or e. a variable heavy chain sequence as presented by SEQ I D NO:50 and a variable light chain sequences as presented by SEQ ID NO:49, or f. a variable heavy chain sequence as presented by SEQ I D NO: 60 and a variable light chain sequences as presented by SEQ I D NO: 9, or g. a variable heavy chain sequence as presented by SEQ I D NO: 70 and a variable light chain sequences as presented by SEQ I D NO:69, or h. a variable heavy chain sequence as presented by SEQ I D NO:80 and a variable light chain sequences as presented by SEQ I D NO: 79, or i. a variable heavy chain sequence as presented by SEQ I D NO: 90 and a variable light chain sequences as presented by SEQ I D NO: 89, or j. a variable heavy chain sequence as presented by SEQ ID NO: 100 and a variable light chain sequences as presented by SEQ I D NO:99, or k. a variable heavy chain sequence as presented by SEQ I D NO: 1 10 and a variable light chain sequences as presented by SEQ I D NO: 109, or 1. a variable heavy chain sequence as presented by SEQ I D NO: 120 and a variable light chain sequences as presented by SEQ ID NO: l 19. The antibody according to any one of the preceding embodiments, which is an IgG antibody, preferably a human IgGl . The antibody according to anyone of the preceding embodiments comprising a heavy chain sequence as presented by SEQ ID NO: 2 1 3 and a light chain sequences as presented by SEQ ID NO: l . The antibody according to any one of the preceding embodiments, which is a monoclonal antibody. The antibody according to any one of the preceding embodiments, which is a human, humanized or chimeric antibody. An antibody-drug conjugate, comprising an antibody according to any one of the embodiments 1 to 14. An isolated nucleic acid sequence that encodes the antibody according to any one of the embodiments 1 to 14. A vector comprising a nucleic acid sequence according to embodiment 16. An isolated cell expressing an antibody according to any one of the embodiments 1 to 14 and /or comprising a nucleic acid according to embodiment 16 or a vector according to embodiment 17. An isolated cell according to embodiment 18, wherein said cell is a prokaryotic or a eukaryotic cell. A method of producing an antibody according to any one of the embodiments 1 - 14 comprising culturing of a cell according to embodiment 19 and purification of the antibody. An antibody according to any one of the embodiments 1 - 14 or an antibody-drug conjugate according to embodiment 15 for use as a medicament. An antibody according to any one of the embodiments 1 - 14 for use as a diagnostic agent. An antibody according to any one of the embodiments 1 - 14 or an antibody-drug conjugate according to embodiment 1 5 for use as a medicament in the treatment of cancer. A pharmaceutical composition comprising an antibody according to any one of the embodiments 1 14 or an antibody-drug conjugate according to embodiment 1 5. 25. A combination of a pharmaceutical composition according to embodiment 24 and one or more therapeutically active compounds.
26. A combination according to claim 25, wherein a therapeutically active compound is an anti- CTLA4, an anti-PD-1, or an anti-PD-Ll antibody 27. A method for treating a disorder or condition associated with the undesired presence of TWEAK R. comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to embodiment 24 or a combination according to embodiment 25 or embodiment 26.
EXAMPLES
The present invention is further described by the following examples. The examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplifications, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.
All examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques of the following examples can be carried out as described in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In the examples in addition to aglycosyl anti-TWEAKR antibodies (like TPP-2658) antibodies are described (like TPP-2090) which can be transformed via mutations into the corresponding aglycosyl antibodies. As an example the antibody TPP-2090 is provided which has been transformed into the aglycosyl antibody TPP-2658 by mutation of N297 into Alanine. In order to transform such glycosylated antibodies into aglycosyl antibodies mutation of N297 to Alanine or Glutamine in the Fc region (using Kabat EU numbering) is preferred. Highly preferred is a mutation of N297 to Alanine.
EXAMPLE 1 : Antibody Generation from Dyax antibody library
A fully human antibody phage display library (Hoet RM et al., Nat Biotechnol 2005;23(3):344-8) was used to isolate TWEAKR-specific, human monoclonal antibodies of the present invention by protein panning (Hoogenboom H.R., Nat Biotechnol 2005;23(3): 1 105-16) with dimeric Fc-fused extracellular domains of human and murine TWEAK as immobilized target.
Table 1 : List of recombinant antigens used for antibody selection
Figure imgf000066_0001
The antigens were biotinylated using an approximately 2-fold molar excess of biotin-LC-NHS (Pierce; Cat. No. 21347) according to manufacturer's instructions and desalted using Zeba desalting columns (Pierce; Cat. No. 89889). Washed Magnetic beads (Dynabeads) were incubated o/n with 200 nM of biotinylated human antigen at 4 °C and blocked for lh at 4 °C with blocking buffer (PBS with 3% BSA, 0.05% Tween-20). The blocked Fab-phage library was added to the blocked TWEAKR-beads (Dynabeads streptavidin M280 - Invitrogen 112-06D) and incubated for 30 min at room temperature. After stringent washing (3 x in blocking buffer and 9 x in PBS (150 inM NaCl; 8 mM Na2HP04; 1.5 mM KH2PO4; adjusted to pH = 7.4-7.6) with 0.05% Tween-20) Fab-phages binding specifically to biotinylated TWEAKR-beads (Dynabeads streptavidin M280 - Invitrogen 112-06D) were resuspended in PBS and for amplification directly used for infection of Escherichia coli strain TGI . In selection round two murine TWEAKR (200 nM) was used to select for cross-reactive binders and in selection round three the concentration of human TWEAKR was decreased (100 nM) to augment the selection pressure for high affinity binders.
1 1 different Fab-phages were identified and the corresponding antibodies were re-cloned into a mammalian IgG expression vector which provides the missing CH2-CH3 domains not present in the soluble Fab. The resulting IgGs were transiently expressed in mammalian cells as described in Tom et al., Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves D 11 roc her. Scion Publishing Ltd, 2007. Briefly, a CMV-Promoter based expression plasmid was transfected into HEK293-6E cells and incubated in Fembach -Flasks or Wave-Bags. Expression was at 37 °C for 5 to 6 days in F 17 Medium (Invitrogen). 1 % Ultra-Low IgG FCS (Invitrogen) and 0.5 mM Valproic acid (Sigma) were supplemented 24 h post transfection. The antibodies were purified by Protein A chromatography and further characterized by their binding affinity to soluble monomeric TWEAKR in ELISA and Biacore analysis as described in Example 2.
Table 2: List of recombinant antigen used for affinity measurement
Figure imgf000067_0001
To determine the cell binding characteristics of ami -TWEAKR antibodies, binding was tested by flow cytometry to a panel of cell lines (HT29, HS68, HS578). Cells were suspended in dilutions of the antibodies (5 μg/ml) in FACS buffer, and incubated on ice for lh. In the following a secondary antibody (PE goat anti-human IgG, Dianova #109-1 15-098) was added. After incubation for lh on ice cells were analyzed by flow cytometry using a FACS-Array (BD Biosciences).
NF-kappaB reporter gene assays were performed to assess the agonistic activity of all 1 1 identified antibodies (human IgGl). HEK293 cells were transiently transfected with a NF-kappaB reporter construct (BioCat, cat. No. LR-00 1 -PA ) using 293fectin according to manufacturer's instruction. White poly-lysine coated 384well plates (BD) were seeded with transfected cells in F 17 media (serum- free; Invitrogen) at 37 °C, 5% C02. On the next day cells were stimulated with purified antibodies at different concentrations for 6h and subsequently a luciferase assay was carried out following standard procedures. Internalization is followed by fluorescence labeling of anti-TWEAKR antibodies (CypHer 5E mono
NHS ester; GE Healthcare). Prior to treatment HT29 cells (2 x l OVwell) were seeded in 100 μΐ media in 96-MTP plates (fat, black, clear bottom No 4308776, Applied Biosystems). After 18 h incubation at 37 °C / 5% ( ( the media (Table No 21) was changed and labeled anti-TWEAKR antibodies were added in different concentrations (10, 5, 2.5, 1 , 0.1 μ^ητΐ). The chosen incubation time was 0, 0.25, 0.5, 1 , 1.5, 2, 3, 6 and 24h. Fluorescence measurement was performed with an InCeli analyzer 1000 (GE Healthcare).
The antibody with the strongest in vitro efficacy (TPP-883) was selected for further potency and affinity maturation.
TPP-883
SEQ I D NO .71
AQDIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFSGSGSGTDFTLTI S SLQPEDFATYYCQQSYS S PGITFGPGTKVE IKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS S TLTLSKADYEKHKLYACEVTHQGLS SPVTKSFNRGEC
SEQ ID NO.72
EVQLLESGGGLVQPGGSLRLSCAASGFTFS PYPMMWVRQAPGKGLEWVSYI S PSGGKTHY ADSVKGRFT I SRDNSKNTLYLQMNSLRAEDTAVYYCARGGDGYFDYFDYWGQGTLVTVS S ASTKGPSVFPIAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS S GLYSLS SWTVPS S SLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT I SKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ I D N0.71) and heavy (SEQ ID NO.72) chains of TPP-883; CDRs of both the heavy and light chain are underlined.
Maturation was done by a first mutation gathering round followed by recombination of the most affinity- and potency- increasing amino acid changes. For mutation gathering NNK (N = AGCT, K = G or T) randomizations at the following individual amino acid positions were generated by site directed mutagenesis using synthetic oligonucleotides including NNK codon-diversification (continuous amino acid nomenclature, compare Figure 25): S35, S36. Y37 and N39 in CDR-Ll ; A51 , S53, S54, Q56 and S57 in CDR-L2; S92, Y93, S94, S95, G97 and 198 in CDR-L3; P31, Y32, P33, M34 and M35 in CDR- HI ; Y50, S52, P53, S54, G56, K57 and 1159 in CDR-H2; G99, G100, D101 , G102, Y103, F 104, D105 and Y106 in CDR-H3. The DNA of all single NNK saturation mutagenesis libraries were re-cloned in a mammalian IgG expression vector for potency maturation and in a phagemid vector for affinity maturation, respectively. Affinity maturation was done by phage panning. Washed magnetic beads (Dynabeads) were incubated o/n with 10 nM, 1 nM, 100 pM and 10 pM of biotinylated human antigen at 4°C and blocked for lh at 4°C with blocking buffer (PBS with 3% BSA, 0.05% Tween-20). The blocked Fab-phage library was added with 10000-fold, 1000-fold and 100-fold excess compared to the theoretical library complexity to the blocked TWEAKR-Dynabeads and incubated for 30min at room temperature. Thus in total, 12 strategies were followed (4 antigen concentrations x 3 Fab-phage titers). After stringent washing (3 x in blocking buffer and 9 x in PBS with 0.05% Tween-20) Fab-phages binding specifically to biotinylated TWEAKR-Dynabeads (Dynabeads streptavidin M280 - Invitrogen 1 12-06D) were resusp ended in PBS and for amplification directly used for infection of Escherichia coli strain TGI . in selection round two the concentration of human TWEAKR-Fc was decreased (1 nM, 100 pM, 10 pM and 1 pM) and the same Fab-phage titer was used for all 12 strategies (4.4 x 10"). For soluble Fab expression the phagemid vector was digested with the restriction endonuclease Mlul to remove the gene!II membrane anchor sequence required for Fab display on phage and religated. 96 variants of each of the 12 selection pools were expressed as soluble Fabs and tested in an ELISA format. Therefore, 2.5 nM biotinylated TWEAKR-Fc antigen were coated and binding of soluble Fabs was detected by Anti-c-Myc antibody (Abeam ab62928). 7 single substitutions variants (continuous amino acid nomenclature, compare Figure 25) were detected with improved binding to TWEAKR-Fc (Seq I D No 138): S36G of CDR-L 1. A51Q and S57K of CDR-L2, S94T and G97F of CDR-L3, M35I of CDR- HI and G102T of CDR-H3. For potency maturation HEK293 cells were rransfected with an NF-kappaB reporter (BioCat, cat. No. LR-0051 -PA). White poly-lysine coated 384weli plates (BD) were seeded with rransfected cells in F17 media (serum-free; Invitrogen) and individual variants of the NNK- diversified positional antibody (human IgGl) libraries were transiently expressed in mammalian cells. On the next day NF-kappaB reporter cells were stimulated with the expressed single NNK mutagenesis antibody variants for 6h and subsequently a luciferase assay was carried out following standard procedures. 1 single substitution variant was detected with improved agonistic activity: G102T of C DRIB. This variant was also obtained from affinity maturation and showed also there the greatest affinity enhancement. After mutation gathering by affinity and potency screening all 7 beneficial single substitutions were recombined (library complexity: 128 variants) in one recombination library. To this end, oligonucleotides were synthesized to introduce selected mutations or the corresponding wild type amino acid at each selected position. Library construction was performed using sequential rounds of overlap extension PCR. The final PCR product was ligated into a bacterial soluble Fab expression vector and 528 variants were randomly selected (~ 4fold oversampling) for equilibrium ELISA screening with soluble Fabs as described before. Finally, 7 variants were selected based on enhanced affinity compared to the best single substitution variant, G102T. The corresponding DNA of these were re-cloned in a mammalian IgG expression vector and tested for functional activity in the afore mentioned NF-kappaB reporter ceil assay. Finally, the obtained sequences were compared with human germline sequences and deviations without significant impact on affinity and potency were adjusted. Antibodies depicted in Table 31 were obtained by antibody library screening and by affinity and/or potency maturation.
TPP-2658
SEQ ID NO.1 :
DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYQASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPFITFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.213 :
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMIWVRQAPGKGLEWVSYISPSGGSTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA
S YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVE ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ ID NO. l) and heavy ( SEQ ID NO.213) chains of TPP-2658; CDRs of both the heavy and light chain are underlined.
TPP-2090
SEQ ID NO.1 :
DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYQASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPFITFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNFYPREAKVQ KVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.2 :
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMIWVRQAPGKGLEWVSYISPSGGSTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN YVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ ID NO. I) and heavy (SEQ I D NO.2) chains of TPP-2090; CDRs of both the heavy and light chain are underlined.
TPP-2149
SEQ ID NO.11
DIQMTQSPATLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYQASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPFITFGPGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO.12
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMIWVRQAPGKGLEWVSYISPSGGKTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ ID NO. l l) and heavy (SEQ ID NO.12) chains of TPP-2149; CDRs of both the heavy and light chain are underlined.
TPP-2093 SEQ ID NO.21
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYQASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPFITFGQGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.22
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVSYISPSGGSTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ I N0.21) and heavy ( SEQ I D NO.22) chains of TPP-2093; CDRs of both the heavy and light chain are underlined.
TPP-2148
SEQ ID NO.31
DIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYQASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPFITFGPGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO.32
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVSYISPSGGKTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ ID N0.31) and heavy (SEQ ID NO.32) chains of TPP-2148; CDRs of both the heavy and light chain are underlined.
TPP-2084 SEQ ID NO.41
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPGITFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.42
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVSYISPSGGSTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ ID N0.41) and heavy (SEQ ID NO.42) chains of TPP-2084; CDRs of both the heavy and light chain are underlined.
ΤΡΡ-2Θ77
SEQ ID NO.51 DIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPGITFGPGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.52 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVSYISPSGGKTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQD LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ ID N0.51) and heavy (SEQ I NO.52) chains of TPP-2077; CDRs of both the heavy and light chain are underlined. TPP-1538
SEQ ID NO.61
AQDIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPGITFGPGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPM WVRQAPGKGLEWVSYISPSGGKTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQD LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ ID NO.61) and heavy (SEQ ID NO.62) chains of TPP-1538; CDRs of both the heavy and light chain are underlined.
TPP-1854
SEQ ID NO.81 AQDIQMTQSPATLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYNASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPFITFGPGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.82
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMIWVRQAPGKGLEWVSYISPSGGKTHY
ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS NSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ I D NO.81) and heavy (SEQ I D NO.82) chains of TPP-1854; CDRs of both the heavy and light chain are underlined.
TPP-1853
SEQ ID NO.91
AQDIQMTQSPATLSASVGDRVTITCRASQSISSYLN YQQKPGKAPKLLIYNASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPGITFGPGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO.92
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVSYISPSGGKTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS NSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ ID N0.91) and heavy (SEQ ID NO.92) chains of TPP-1853; CDRs of both the heavy and light chain are underlined.
TPP-1857
SEQ ID NO.101
AQDIQMTQSPATLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYNASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPGITFGPGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.102
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMM VRQAPGKGLEWVSYISPSGGKTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ ID NO.101) and heavy (SEQ ID NO.102) chains of TPP-1857; CDRs of both the heavy and light chain are underlined. TPP-1858
SEQ ID NO. Ill
AQDIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPFITFGPGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO.112
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVSYISPSGGKTHY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGDTYFDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS NSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN YVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQD LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG
Amino acid sequences of the light (SEQ ID NO. l l l) and heavy (SEQ I NO.112) chains of TPP-1858; CDRs of both the heavy and light chain are underlined.
EXAMPLE 2: Biochemical characteristics of the antibody Determination of binding affinities by Biacore analysis:
Binding affinities of anti-TWEAKR antibodies were determined by surface plasmon resonance analysis on a Biacore T100 instrument (GE Healthcare Biacore, Inc.). Antibodies were immobilized onto a CM 5 sensor chip through an indirect capturing reagent, anti-human IgG(Fc). Reagents from the "Human Antibody Capture Kit" (BR-1008-39, GE Healthcare Biacore, Inc.) were used as described by the manufacturer. Anti-TWEAKR antibodies were injected at a concentration of 10 μg/ml at 10 μΐ/min for 10 sec. Table 3: List of recombinant antigen (TWEAKR) used for affinity measurement
Figure imgf000079_0001
Figure imgf000079_0002
Table 5: List of commerciall available antibodies used for affinity measurements
Figure imgf000079_0003
Various concentrations (200 nM, 100 nM, 50 nM, 25 n M, 1 2.5 n , 6.25 nM, 3.12 nM, 1.56nM) of purified recombinant human TWEAKR protein (TPP-2305, SEQ ID NO: 168) were injected in HEPES- EP buffer (GE Healthcare Biacore, Inc.) over immobilized anti-TWEAKR antibodies at a flow rate of 60 μΐ/min for 3 minutes and the dissociation was allowed for 5 minutes. Sensorgrams were generated after in-line reference cell correction followed by buffer sample subtraction. The dissociation equilibrium constant (KD) was calculated based on the ratio of association ( ) and dissociation rate (koff) constants, obtained by fitting sensorgrams with a first order 1 : 1 binding model. Table 6: Monovalent KD values of anti-TWEAKR antibodies measured by Biacore with TWEAKR kon (1/ s) k0ff (1/s) KD (nM)
TPP-883 4.40E+06 9.10E-01 205.9
TPP-1538 4.20E+06 l . lOE-01 27.6
TPP-2077 3.00E+06 8.60E-02 28.9
TPP-2084 4.20E+06 l . lOE-01 27.6
TPP-2148 5.10E+06 1.30E-01 24.5
TPP-2093 4.10E+06 9.00E-02 22.1
TPP-2149 8.40E+06 l .OOE-01 12.1
TPP 2090 9.10E+06 l . lOE-01 12.4 kon (1/Ms) k0ff (1/s) D (nM)
PDL-192(TPP-1104) 1.00E+07 3.80E-02 3.7
136.1(TPP-2194) 3.84E+07 3.24E-02 0.8
18.3.3(TPP-2193) 1.64E+07 2.85E-02 1.7
P4A8(TPP-1324) 1.20E+06 2.70E-03 2.3
P3G5(TPP-2195) 2.3 IE+06 1.22E-03 0.5
P2D3(TPP-2196) 1.32E+06 5.64E-04 0.4
ITEM-1 3.80E+06 l . iOE-02 2.9
ITEM-4 2.80E+06 2.00E-03 0.7
The antibodies of the disclosure were determined to bind TWEAK R with moderate affinity (KD valus of 10 200 nM) whereas some antibodies used for comparison (e.g. PDL-192(TPP-1 104),
136.1(TPP-2194), 18.3.3(TPP-2193), P4A8(TPP-1324), P3G5(TPP-2195), P2D3(TPP-2196), ITEM-1 , ITEM-4) show high affinity binding (0.7 - 3.7 nM). Sequences of variable domains of antibodies PDL- 192, 136.1, 18.3.3, P4A8, P3G5 and P2D3 were obtained from patents WO2009/020933 and WO2009/140177 and sequences encoding the constant region of human IgGl and murine IgG2 were added, resulting in full length IgGs PDL-192(TPP-1 104), 136.1(TPP-2194), 18.3.3(TPP-2193), P4A8(TPP-1324), P3G5(TPP-2195), P2D3(TPP-2196). The range of affinities measured in this study for other previously described antibodies is well in line with published data: For PDL-192, 18.3.3 and 136.1 , KD values of 5.5, 0.2 and 0.7 nM were published (WO2009/020933); for P4A8 2.6 nM (WO2009/140177). For comparison, the native ligand TWEAK binds TWEAKR with a KD value of 0.8 - 2.4 nM (Immunity. 2001 Nov; 15(5):837-46; Biochem J. 2006 Jul 15;397(2):297-304; Arterioscler Thromb Vase Biol. 2003 Apr l ;23(4):594-600). As a result the antibodies of the disclosure (TPP-883, TPP-1538, TPP-2077, TPP-2084, TPP-2148,
TPP-2093, TPP-2149 and TPP-2090) bind TWEAKR with moderate affinity (KD 10 - 200 nM) .
Analysis of species cross reactivity by Biacore analysis:
For the analysis of species cross reactivity, human, rat, murine, dog, pig and macaca fascicuiaris TWEAKR were expressed and purified as human Fc fragment fusion proteins and immobilized onto a CM5 sensor chip using amine coupling via a standard EDC/NHS-mediated chemistry (BR- 1006-33, GE
Healthcare Biacore, Inc.).
Table 7: List of recombinant proteins used in ELISA for profiling interspecies binders
Nomenclature Description SEQ ID NO
TPP-1846 MAC-TNFRSF12Aaa28-80-hIgGl -Fc 133
TPP-1779 RAT-TNFRSF12Aaa28-80-hIgGl -Fc 134
TPP-1778 PIG-TNFRSF12Aaa28-80-hIgGl -Fc 135
TPP-1777 DOG-TNFRSF 12Aaa28-80-hIgGl -Fc 136
TPP-599 HUMAN-TNFRSF 12Aaa28-80-hIgGl -Fc 138
TPP-601 MURINE-TNFRSF 12Aaa28-80-hIgGl -Fc 137 Various concentrations (200 nM, 100 nM, 50 nM, 25 nM, I 2.5 nM, 6.25 nM, 3.12 nM, 1.56 nM) of anti-TWEAKR antibodies were injected in HEPES-EP buffer (GE Healthcare Biacore, Inc.) over the immobilized TWEAKR species at a flow rate of 60 μΐ/min for 3 minutes and the dissociation was allowed for 5 minutes. Sensorgrams were generated after in-line reference cell correction followed by buffer sample subtraction. The dissociation equilibrium constant (KD) was calculated based on the ratio of association (koni) and dissociation rated (kofn) constants, obtained by fitting sensorgrams with a bivalent analyte model using Biavaluation Software. The species cross reactivity of anti-TWEAKR antibodies has been determined in "avidity mode" with immobilized bivalent antigen which does not provide "absolute" KD values, but gives good comparative data. Table 8: KD values (nM) of anti-TWEAKR antibodies to different species measured by Biacore
Figure imgf000081_0001
As a result the antibodies of the disclosure (TPP-1538, TPP-2077, TPP-2084 and TPP-2090) show affinity to ail tested species (human, rat, murine, dog, pig and macaca fascicularis TWEAKR).
Characterization of the binding epitope of TPP-2090 by N- and C-terminal truncation variants of the TWEAKR ectodomain:
The alignment of the TWEAKR cysteine rich domain (aa 34-68) of different species (Figure 1) shows that it is well conserved throughout all 6 analyzed species. PDL-192 binds dependent of R56 (WO2009/020933 : Figure 2B) and therefore does not bind to rat, pig and mouse TWEAKR. TPP-2090 binds dependent of the conserved amino acid 047 and therefore binds to all depicted species. In a first approach to characterize the binding epitope of the aforementioned antibodies, a - and C- terminal truncation mutant of the TWEAKR ectodomain was generated and tested for its ability to bind to the different anti-TWEAKR antibodies. N-terminally, amino acids 28 to 33 and C -terminally amino acids 69 to 80 were deleted, thus the cysteine rich domain with disulfide bridges between Cys36-Cys49, Cys52-Cys67 and Cys55-Cys64 remains intact (compare Figure 2). Both constructs, the full ectodomain 28-80 including the N- and C -terminus and the truncated ectodomain 34-68 were expressed and purified as Fc fusion proteins TPP-2202 and TPP-2203, respectively.
To analyze for binding, 1 μg ml of the respective dimeric TWEAKR-Fc construct were coated and, 0.3 μg/ml and 0.08 μg/ml of biotinylated IgG were used as soluble binding partner. Detection was done with Streptavidin-HRP and Ampiex-Red substrate. IgGs were biotinylated using an approximately 2- fold molar excess of biotin-LC-NHS (Pierce; Cat. No. 21347) according to manufacturer's instructions and desalted using Zeba desalting columns (Pierce; Cat. No. 89889). At all applied concentrations of the soluble ligand, the antibodies of the present disclosure show saturated binding to both constructs, whereas antibodies P4A8(TPP-1324), P3G5(TPP-2195) and ITEM-4 show saturated binding only to the full length ectodomain and impaired binding to the N- and C -terminally truncated construct (Figure 3 & Figure 4). This indicates that the binding epitope of the antibodies of the present invention is located within the cysteine rich domain between amino acid 34-68. To analyze whether the N-terminus or the C- terminus of the TWEAK ectodomain is needed for P4A8(TPP-1324) and P3G5(TPP-2195) binding a monomeric ectodomain with the C -terminal deletion of amino acids 69 to 80 was generated. The binding of P4A8(TPP-1324) and P3G5(TPP-2195) to the C-terminally truncated TWEAKR ectodomain is also impaired whereas the antibodies of the present disclosure show saturated binding (Figure 5). Table 9: List of recombinant antigens used in ELISA analysis for epitope profiling
Figure imgf000082_0001
Table 10: List of antibodies used in ELISA anal sis for e ito e rofilin
Figure imgf000082_0002
Thus, the binding epitope of TPP-2090, TPP-2084, PDL-192(TPP-1 104) and 136.1 (TPP-2194) is located within the cysteine rich domain and the binding epitope of P4A8 (TPP- 1324) and P3G5(TPP- 2195) is located at least partially outside of the cysteine rich domain.
Effect of TWEAKR-Fc Muteins on antibody affinity:
To define the binding characteristics of the antibodies of the disclosure in more detail certain muteins of TWEAKR that have been proposed to be relevant for binding of known agonistic antibodies were tested (WO2009/140177). Therefore, the full ectodomain (amino acids 28-80) with the following single amino acid substitutions were expressed and purified as Fc fusion proteins: T33Q; S40R; W42A; M50A; R56P; H60K; 1.650.
Table 11: List of recombinant proteins used in ELISA analysis for mutein binding
Nomenclature Description SEQ ID NO
TPP- 1990 hTNFRSF12Aaa28-80-L65Q-hIgGl-Fc 142
TPP- 1 989 hTNFRSF12Aaa28-80-H60K-MgGl -Fc 143
TPP-2683 hTNFRSF 1 2 Aaa28-8()-R56P-hIgG 1 -Fc 144
TPP- 1988 hTNFRSF12Aaa28-80-M50A- gGl -Fc 145
TPP- 1 985 hTNFRSF 12Aaa28-80-W42A-hIgGl -Fc 146 Nomenclature Description SEQ ID NO
TPP-1987 hTNFRSF 1 2Aaa2N-80-S40R-hlgG 1 -Fc 147
TPP-I 986 hTNFRSF12Aaa28-80-T33Q-hIgGI-Fc 148
TPP-599 hTNFRSF 12Aaa28-80- gGl -Fc 138
To collect dose-response data, the different TWEAKR-Fc muteins were coated with a low concentration (62 ng ml) in a 384-well Maxisorb ELI SA plate and a serial 2fold dilution of biotinylated lg( i starting with a concentration of 100 nM was used as soluble binding partner. Detection was done with Streptavidin-HRP and Amplex Red. The tested igGs were TPP-2090 and TPP-2084, PDL-192, 136.1 and 18.3.3 from WO2009/020933, P4A8 and P3G5 from WO2009/140177, and ITEM - ! and ITEM-4 from Nakayama et al [Biochem Biophys Res Com 306: 819-825].
Figure imgf000083_0002
Table 13: List of commercially available antibodies used in ELI SA for mutein binding
Figure imgf000083_0003
IgGs were biotinylated using an approximately 2-fold molar excess of biotin-LC-NHS (Pierce; Cat.
No. 21347) according to manufacturer's instructions and desalted using Zeba desalting columns (Pierce; Cat. No. 89889). The dose-response data were fitted and IC50s determined. To visualize the results a table was generated, "-" indicates IC50s above 50 nM, "+" indicates IC50s in the range of 1 to 150 pM.
Figure imgf000083_0001
As published before, IT EM -4 shows impaired binding to the H60K mutein [WO2009/140177: Figure 23 F] and PDL-192 to the R56P mutein [WO2009/020933 : Figure 22B]. In contrast to published data, ITEM- ! shows impaired binding to R56P and all antibodies to W42A [WO2009/140177: Figure 23E, Figure 23F]. This difference can be explained by the method chosen; the extreme low coating concentration favors the discrimination of off-rate impairments since it minimizes avidity effects. As none of the analyzed antibodies shows unimpaired binding to the W42A mutein, this substitution seems to cause rather structural changes and not a direct alteration of the binding epitope.
In contrast to ITEM- ! , ITEM-4, PDL-192, 136.1 and 18.3.3, the antibodies of the present disclosure bind independent of all but W42A substitutions. Alanine scan of Cysteine Rich Domain:
To delineate the binding site of the antibodies an alanine scan of the cysteine rich domain (amino acids 34-68) was performed. In Figure 4 it could be shown that N- and C -terminal truncation variants of the full length ectodomain of TWEAKR do not impair binding of the antibodies. Therefore the binding epitope is localized within the cysteine rich domain. The following substitutions were introduced in the TWEAKR(34-68)-Fc construct: S37A, R38A, S40A, S41 A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A and L65A.
Table 15: List of TWEAKR mutein constructs for alanine scan of cysteine rich domain
Nomenclature description SEQ ID NO
TPP-2203 TweakR-ECD-34-68-hIgGFc-His 140
TPP-2625 TweakR-ECD-34-68-hIgGFc-His-L65A 149
TPP-2624 TweakR-ECD-34-68-hIgGFe-His-F63A 150
TPP-2623 TweakR-ECD-34-68-hIgGFc-His-D62A 151
TPP-2622 TweakR-ECD-34-68-hIgGFc-His-S61A 152
TPP-2621 TweakR-ECD-34-68-hIgGFc-His-H60A 1 53
TPP-2620 TweakR-ECD-34-68-hIgGFc-His-P59A 154
TPP-2619 TweakR-ECD-34-68-hIgGFc-His-R58A 1 55
TPP-2618 TweakR-ECD-34-68-hIgGFc-His-R56A 156
TPP-2617 TweakR-ECD-34-68-hIgGFc-His-S54A 157
TPP-2616 TweakR-ECD-34-68-hIgGFc-His-D51A 158
TPP-2615 TweakR-ECD-34-68-hIgGFc-His-K48A 159
TPP-2614 TweakR-ECD-34-68-hIgGFc-His-D47A 160
TPP-2613 TweakR-ECD-34-68-hIgGFc-His-D45A 161
TPP-2612 TweakR -EC D-34-6S-hI g( i Fc-H is-S43 A 162
TPP-261 1 TweakR-ECD-34-68-hIgGFc-His-W42A 163
TPP-2610 TweakR-ECD-34-68-hIgGFc-His-S41A 164
TPP-2609 TweakR-ECD-34-68-hIgGFc-His-S40A 165
TPP-2608 TweakR-ECD-34-68-hIgGFc-His-R38A 166
TPP-2607 TweakR-ECD-34-68-hIgGFc-His-S37A 167 Tfaese TWEAKR(34-68)-Fc muteins were expressed in HEK293 cells. To collect dose-response data, I "( is were coated at a concentration of 1 μ^ιηΐ in a 384-well Maxisorp ELISA plate and a serial 2fold dilution of the TWEAKR mutein containing supernatant was used as soluble binding partner. Detection was done with anti-HIS-HRP and Amplex Red. The tested IgGs were TPP-2090, PDL-192 from WO2009/020933 and ITEM- 1 (purchased from Abeam).
Table 16: List of antibodies used for alanine scan of c steine rich domain
Figure imgf000085_0001
To assess the relevance of each TWEAKR mutein for binding to different IgGs a correlation blot at a certain mutein concentration was prepared. Exempiarily, in Figure 6 the correlation blot for the 8 fold diluted supernatant of the TWEAKR expression broth is shown with PDL-192(TPP-1104) on the X axis and TPP-2090 on the Y axis. The blot shows that binding of TPP-2090 was impaired by the substitution D47A and binding of PDL-192(TPP-1 104) by substitution R56A. The identified dependencies on certain TWEAKR amino acids for antibody interaction correlate with the agonistic activity that has been determined for these antibodies. The native ligand TWEAK shows efficient activation of TWEAKR and binds dependent of Leucin 46 in the cysteine rich domain of TWEAKR (Pellegrini et al., FEBS 280: 1818-1829). P4A8 shows very low agonistic activity and at least partially interacts with domains outside of the cysteine rich domain of TWEAKR (Figure 4). PDL-192 shows moderate agonistic activity and binds dependent of R56 to the cysteine rich domain but opposite to the TWEAK ligand site. T - 2090 and TWEAK bind dependent on D47 and L46, respectively, and therefore bind to a similar binding site (Figure 7).
To support the evidence of a common epitope TPP-2090, TPP-2149, TPP-2093, TPP-2148, TPP- 2084, TPP-2077, TPP- 1 538. TPP-883, TPP- 1 54. TPP-1853, TPP- ! 857. TPP- 1 858 were tested. Ail antibodies, which have been tested, specifically bind to the D at position 47 (D47) of TWEAKR (see figure 6C). Again PD1.- 1 2Π Ρ- 1 1 04 ) is still capable of binding to D47A mutein of TWEAKR. In conclusion, the antibodies of this disclosure (e.g. TPP-2090) bind to TWEAKR dependent on
D47.
The identified dependencies on certain TWEAKR amino acids for antibody interaction correlate with the agonistic activity that has been determined for these antibodies. The native ligand TWEAK shows efficient activation of TWEAKR and binds dependent on Leucin 46 in the cysteine rich domain of TWEAKR (Pellegrini et al.. FEBS 280: 1818-1829). P4A8 shows very low agonistic activity and at least partially interacts with domains outside of the cysteine rich domain of TWEAKR. PDL-192 shows moderate agonistic activity and binds dependent of R56 to the cysteine rich domain but opposite to the TWEAK ligand site. Antibodies of this disclosure (see Figure 6C) bind dependent on D47, and TWEAK binds dependent on L46, and binds to a similar but distinguishable binding site (Figure 7). Therefore the antibodies of this disclosure which show a strong agonistic activity bind to a novel epitope (D47 dependent) for antibodies which is connected to very strong agonistic activity. Interestingly, Michaelson et al (see page 369, left column in Michaelson JS et al., MAbs. 2011 Jul-Aug;3(4):362-75) gave an explanation why all agonistic antibodies examined by them have weaker agonistic activity compared to the natural ligand TWEAK. In their conclusion, the decreased efficacy might be a function of the dimeric binding interaction of an antibody with TWEAKR wherein TWEAK presumably engages in a trimeric interaction. Therefore, it is a surprising finding that an antibody of the invention, though in a dimeric interaction with TWEAKR has even higher agonistic activity. This surprising effect is coupled to the specific binding property of the antibodies of this disclosure, hence specific binding to D47 of TWEAKR.
Characterization of antibodies of the disclosure by epitope competition experiments:
To understand the difference of antibodies of this disclosure and other known anti-TWAEKR antibodies competition experiments were performed. This investigation of overlapping binding motifs for several anti-TWEAKR antibodies has been performed by surface plasmon resonance analysis on a
Biacore T100 instrument (GE Healthcare Biacore, Inc.).
Table 17: List of antibodies used for com etition ex eriments
Figure imgf000086_0001
Table 18: List of commerciall available antibodies used for com etition ex eriments
Figure imgf000086_0002
Table 1 : List of recombinant antigen used for competition experiments
Figure imgf000086_0003
All antibodies were immobilized directly onto a CM5 sensor chip using the "Amine coupling Kit" ( BR- 1 006-33. GE Healthcare Biacore, Inc.). Reagents have been used as described by the manufacturer. For saturation of the 1 st antibody (immobilized antibody) with antigen, 200 nM TWEAKR (TPP-2305) in HEPES-EP buffer (GE Healthcare Biacore, Inc.) was injected at 30 μΐ/min for 120 sec. Subsequently 200 nM of the 2nd antibody ("competing antibody") in HEPES-EP buffer were injected into the flow cell at 30 μΐ/min for 120 sec. Generally, sensorgrams were generated after in-line reference cell correction followed by sample buffer subtraction. The qualitative competition data (Figure 8) has been generated by thorough manual inspection of the sensorgrams using Biavaluation Software. A lack of a second binding event after injection of the 2nd antibody indicated clear competition within a respective antibody pair. Non competing antibody pairs showed clear binding signal over background after 2nd antibody injection. In addition, self-competition (1 st & 2nd antibody identical) was monitored as an internal system control. Overall, a matrix of nine versus nine antibodies was included into this analysis. In general anti-TWEAKR antibodies could be clustered into three distinct "competition groups"
(Figure 9). One group contains exclusively TPP-2084 and TPP-2090, both showing competition to all other tested members. These other members could be split into two separate sets of antibodies, which do not show any competition between each other. Therefore "full" competition with all tested anti- TWEAKR antibodies is unique for TPP-2084 and TPP-2090. This supports the findings described above that both tested antibodies of the disclosure bind to a new and unique epitope.
Selectivity assessment of the antibodies of the disclosure:
The antibody TPP-2090 was also tested for binding to other members of the TNF receptor superfamily to assess its selectivity. The TNF receptor superfamily shows very high sequence divergence as depicted in Figure 10. Most similar to TWEAKR are TNFRSF13C and TNFRSF 17 with only about 30% sequence identity. The epitope region itself (cysteine rich domain) has no match in any of the other TN RSF members (BLAST E -Value = 0.7 for best hit). The ectodomains of all 29 known TNF receptor superfamily members were purchased as Fc fusion proteins (Table 20) and 1 μg/ml were coated in a Maxisorp EL IS A plate. To collect dose-response data a serial 3 fold dilution of biotinylated IgG starting with a concentration of 2 μΜ was used as soluble binding partner. Detection was done with anti-hlgGl -HRP and Amplex Red. The tested I G was TPP-2090. As depicted in Figure 11 TPP-2090 binds already at a very low concentration of 300 pM in saturation to TWEAKR whereas also at a very high concentration of 75 nM it does not bind to all other 28 TNF receptor superfamily members.
Thus, TPP-2090 binds selectively to TWEAKR.
Table 20: List of recombinant proteins used in ELI SA for selectivity profiling
Protein Nomenclature Origin Cat.No. (R&D Systems)
TWEAKR (TNFRSF 12) 1 Human 1610-TW-050
Apo-3 (TNFRSF25) 3 Human 943-D3-050 Protein Nomenclature Origin Cat.No. (R&D Systems)
Trail-R ! (TNFRSFiOA) 4 Human 347-DR-lOO/CF
Trail-R2 (TNFRSF10B) 5 Human 631-T2-100/CF
CD-385 (TNFRSF21) 6 Human 144-DR-100
CD95 (TNFRSF6) 7 Human 326-FS-050/CF
Rank (TNFSF1 1) 8 Human 390-T -Ol O/CF
TNF-R1 (TNFRSF1A) 9 Human 636-R1-025/CF
TNF-R2 (TNFRSFI B) 10 Human 1089-R2-025/CF
BAFF-R (TNFRSF13C) 11 Human 1 162-BR-050
DcR3 (TNFRSF6B) 12 Human 142-DC-lOO
BCMA (TNFRSF17) 13 Human 193-BC-050
TACI (TNFRSF13B) 14 Human 174-TC-050
OX40 (TNFRSF4) 15 Human 3388-OX-050
CD30 (TNFRSF8) 16 Human 6126-CD-l OO
CD27 (TNFRSF7) 17 Human 382-CD-lOO
CD40 (TNFRSF5) 18 Human 1493-CD-050
Osteoprotegerin (TNFRSF1 IB) 19 Human 805-OS-l OO/CF
EDAR 20 Human 157-ER-100
GITR (TNFRSF18) 21 Human 689-GR-100
HVEM (TNFRSF 14) 22 Human 356-HV-lOO/CF
NGF R (TNFRSF16) 23 Human 367-NR-050/CF
Trail R3 (TNFRSF10C) 24 Human 630-TR-lOO/CF
Lymphotioxin β R 25 Human 629-LR-100
Trail R 4 (TNFRSF10D) 26 Human 633-TR-100
EDA2R (TNFRSF27) 27 Human 1093-XD-050
TROY (TNFRSF19) 28 Human 1548-TR-100
RELT (TNFRSFI 9L) 29 Human 1385-RT-050
4- IBB (TNFRSF9) 30 Human 838-4B-100
EXAMPLE 3: Binding of anti-TWEAKR antibodies to eel! surface of cancer ceil lines
To determine the binding characteristics of the anti-TWEAKR antibodies on mouse and human cancer cell lines, binding was tested by flow cytometry to a panel of cell lines. Adherent cells were washed twice with PBS without ( a and Mg (Biochrom #L1825: aqueous solution containing 8000 mg/l NaCl, 200 mg/l KCl, 1 150 mg/l Na2HP04, and 200 mg/l KH2P04) and detached by enzyme-free PBS based cell dissociation buffer (Invitrogen). Cells were suspended at approximately 105 cells/well in FACS buffer (PBS without Ca/Mg, containing 3% FCS, Biochrom). Cells were centrifuged (250 g, 5 min, 4 °C) and supernatant discarded. Cells were resuspended in dilutions of the antibodies of interest (10 μg/ml in 80 μΐ if not indicated otherwise) in FACS buffer, and incubated on ice for 1 h. In the following cells were washed once with 100 μΐ cold FACS buffer and 80 μΐ secondary antibody diluted at 1 : 150 (PE goat anti-human IgG, Dianova #109-1 15-098, or PE Goat Anti-Mouse IgG, Jackson Immuno Research #1 15-1 15-164) was added. After incubation for lh on ice cells were again washed with cold FACS buffer, resuspended in 100 μΐ FACS buffer and analyzed by flow cytometry using a FACS-Array (BD Biosciences). Results are calculated as Geo Mean of fluorescence detected by the antibody of interest subtracted by background fluorescence as measured by detection with the secondary antibody alone. Values are scored according to the following system:
Geo Mean - Geo Mean of secondary antibody alone >10: +, >100: ++, >1000: +++, 10000: ++++, close to category border in (). The sources of the cell lines are given in Table 21.
As shown in Table 21 , ail anti-TWEAKR antibodies of this disclosure used at a concentration of 10μg/ml bind a broad range of tumor cells expressing TWEAKR of murine (4T1 , Lewis Lung) and human (all other cell lines included in the table) origin representing a variety of tumor entities.
Table 21 : Binding of anti-TWEAKR antibodies (10 μg/ml) to different cell lines by scoring of FACS analysis: TPP-1538 and TPP-2090 bind to a broad panel of murine and human tumor cell lines representing a variety of tumor indications.
Tumor Entitv Cell Line Source Media* TPP-1538 TPP-2090
NSCLC A549 DSMZ ACC107 1 ++ ++
EKVX NCI 60-Panel, lot 502463 2 ++ n.d.
NCI-H322 ECACC 951 11734 2 +(+) ++
Calu-6 ATCC HTB-56 2 ++ n.d.
NCI-H520 ATCC HTB-182 2 - n.d.
NCI -H 1975 ATCC CRL-5908 2 ++(+) n.d.
NCI -H460 ATCC HTB-177 1 ++ ++
SCLC NCI-H69 ATCC HTB-1 19 2 - n.d.
CRC WiDr ATCC CCL-218 4 ++(+) +++
HT-29 DSMZ ACC299 1 ++ ++(+)
Lovo DSMZ ACC350 2 (+ ) n.d.
SW-480 DSMZ ACC3 I 3 2 ++ n.d.
HNSCC A253 ATCC HTB-41 11 +(+) n.d.
H SC -3 JCRB #JCRB0623 4 +(+) n.d.
SCC4 DSMZ ACC618 10 +++ +++
Fadu ATCC HTB-43 4 ++ ++(+)
RCC 786-0 ATCC CRL- 1932 1 +++(+) ++++
PancCA BxPC3 ATCC CRL-1687 2 ++(+) n.d.
As-PCl ATCC CRL-1682 2 +(+) n.d.
MiaPaca2 ATCC CRL-1420 9 + n.d.
OvCa SK-OV-3 ATCC HTB-77 3 ++(+) +++
BreastCA MDA-MB-231 ATCC HTB-26 1 ++ n.d.
MDA-MB-453 DSMZ ACC-65 1 + n.d.
Melanoma A375 ATCC CRL-1619 3 ++ ++
GastricCA NCI-N87 ATCC CRL-5822 2 ++ n.d.
Esophageal CA Kyse-180 DSMZ ACC379 2 (+ ) n.d.
Hematological CA Jurkat ATCC TI B- 1 52 2 n.d.
Kasumi-2 DSMZ ACC526 2 - n.d.
Bladder CA Scaber ATCC HTB-3 8 ++ ++
HCC SK-Hepl DSMZ ACC 141 7 ++(+) +++
Huh7 JCRB JCRB0403 6 n.d. ++
HepG2 ATCC HB-8065 3 n.d. ++
Hep3B2.1-7 ATCC HB-8064 4 n.d. ++(+)
PLC-PRF5 ATCC CRL8024 2 n.d. ++ Tumor Entity Cell Line Source Media* TPP-1538 TPP-2090
Prostate CA PC 3 DSMZ ACC465 1 ++ ++(+)
Neuroblastoma SKNAS ATCC CRL-2 1 37 5 n.d. +(+)
Murine C A cell lines Lewis Lung ATCC CRL-1642 3 + n.d.
4T1 ATCC CRL-2539 2 +(+) n.d.
(Geo Mean-Geo Mean of secondary antibody alone >10: +, >100: ++, >1000: +++, >10000: ++++, close to category border in ())
*List of growth media for cancer cell lines from Table 21 :
I . DM EM / Ham's F12; (Biochrom;# FG 4815, with stable Glutamin), 10% FCS 2. RPMI 1640; (Biochrom;# FG 1215, with stable Glutamin), 10% FCS
3. DM EM; (Biochrom;# FG 0435, with stable Glutamin) ,10% FCS
4. M EM Earle's; (Biochrom;# FG 0325. with stable Glutamin), 10% FC S
5. DM EM; (Biochrom;# FG 0435, with stable Glutamin) I . - A I a n y 1 - 1. -Glutamin; (2mM extra for final 4mM, Biochrom, # K 0302)
Non Essentiell Amino Acids; (final: lx, Biochrom; # K 0293), 10% FCS
6. DM EM; (Biochrom;# FG 0445,high glucose with stable Glutamin) ,10% FCS
7. RPM I 1640; (Biochrom;# FG 1215, with stable Glutamin), 20% FCS
8. M EM Earle's; (Biochrom;# F 0315), L-Alanyl-L-Glutamine; (final: 2m M, Biochrom; # K 0302) Non Essentiell Amino Acids; (final: lx, Biochrom; # K 0293), 10% FC S 9. DM EM / Ham's F12; (Biochrom;# FG 4815, with stable Glutamin), Horse Serum (final: 2.5%);
(Biochrom; # S 9135) 10% FCS
10. DM EM / Ham's F12; (Biochrom;# FG 4815, with stable Glutamin), Hydrocortison; (final:
40ng/mL, Biochrom; # K 3520), 10% FCS
I I . McCoy's 5A; (Biochrom;# F 1015) L-Alanyl-L-Glutamine; (1.5mM extra, Biochrom; # K 0302), 10% FCS
Overall, it has to be noted, that the maximal cellular binding of the antibodies by FACS analysis is moderate compared to other known antibodies. As shown in Table 23 and Figure 12, the amount of antibody bound to the different cells as detected by FACS analysis is lower as compared to other known antibodies (PDL-192(TPP-1 104), P4A8(TPP-1324)) at 10 μg/ml, a concentration where cellular binding of the antibody as detected by FACS analysis has reached its plateau (data not shown). Table 22: List of antibodies used for FACS anal sis
Figure imgf000091_0001
Table 23: Binding of different anti-TWEAKR antibodies 10 μg/ml to a panel of cell lines by scoring of
FACS analysis. GeoMean of Fluorescence measures by detection with a specific antibody minus GeoMean measured with the secondary antibody only is shown.
Figure imgf000091_0002
EXAMPLE 4: Induction of Caspase-3/7 activation in different TVV EAKR expressing ceil lines
To determine the level of apoptosis induction, Caspase-3/7 activation was measured after treatment of cancer cells with TWEAK or agonistic anti-TWEAKR antibodies. Therefore HT-29 cells were plated at a density of 4000 cells/75 μΐ/well in 96 well plates in assay medium (DMEM/Ham'sF12, Biochrom #FG4815 + 10% FCS + 100 ng/ml I F gamma (R&D Systems #285-IF)). 24h later cells were incubated with antibodies to the TWEAK R (see Table 24), recombinant human TWEAK ( R& D. #1090-TW- 025/CF, E. coli derived recombinant soluble human TWEAK, Arg93-His249 of accession # Q4A W9, Entrez Gene ID 8742, with an N-terminal Met and 6-His tag) or corresponding isotype control IgG at various concentrations as indicated. After 24h incubation with the antibodies, Caspase 3/7 activity was determined by adding 100 μΐ/well Caspase 3/7 Solution (Promega, #G8093) to the cells, incubation for one hour and reading of luminescence on a VICTOR V ( Perk in Elmer).
As shown in Figure 13 and Table 25, incubation with the antibodies of the disclosure lead to a stronger maximal induction of Caspase 3/7 as compared to the antibodies described in the art (PDL- 192(TPP-1104), P4A8(TPP-1324), 136.1 (TPP-2194)) and also as compared to 300 ng/ml recombinant human TWEAK (Table 25). Thus, the antibodies described herein, are superior to the previously described antibodies to induce Caspase 3/7 in HT-29 cells.
To determine whether the strong efficacy of the antibodies also holds true in other ceil lines than HT-29, the capacity to induce Caspase 3/7 by anti-TWEAKR antibodies as compared to recombinant human TWEAK was evaluated in a panel of cell lines. For this analysis the following conditions were used: WiDr cells were plated at 3000 cells/well and incubated for 48h in the presence of TWEAK or the described antibodies, A 253 cells were plated at 2500 cells/well and incubated for 24h, NCI-H322 cells were plated at 5000 cells/well and incubated for 48h and 786-0 cells were plated at 2500 cells/well and incubated for 48h. The cells were plated in the media as described in Table 21 , for A253, NCI-H322 and 786-0 cells lOOng/ml I FN gamma (R&D Systems #285-IF) was added. 24 h after plating antibodies at 100 μg/ml or TWEAK at 300 ng/ml (100 ng/ml TWEAK for NCI-H322 cells) were added and the cells were further incubated for the time periods indicated above. At the end of the incubation time Caspase 3/7 activity was determined as described for HT-29 cells. The fold induction of Caspase 3/7 was calculated as compared to untreated cells. As shown in Table 25, all tested antibodies including TPP-2658, the aglycosylated variant of TPP-
2090, showed an increased Caspase 3/7 induction in HT-29 ceils as compared to other known antibodies and reach a stronger activity as compared to 300 ng ml TWEAK ligand. This strong efficacy to induce apoptosis in cancer cells (as measured by Caspase 3/7 activation) was also seen in WiDr, A253, NCI- H322 and 786-0 cells, where the tested antibodies of this disclosure induced higher fold-changes as compared to other antibodies and 300 ng/ml TWEAK in most experiments.
Table 24: List of antibodies used f r Caspase induction assay
Figure imgf000092_0001
Table 25: Fold induction of Caspase 3/7 in different cancer cells after incubation with 100 μg/ml anti- TWEAKR antibodies or recombinant human TWEAK (300 ng/ml or *100 ng/ml). Results from 1 -3 representative experiment carried out in triplicates are shown, including standard deviations. Tested antibodies show enhanced induction of Caspase 3/7 in different cell types as compared to known antibodies or recombinant TWEAK.
HT-29 WiDr A253 NCI-H322 786-0
TWEAK 2.31±0.29 1.22±0.02 1.53±0.13 1.39*±0.01 1.45±0.05
TPP-1538 2.44+0. ! 2 1.53±0.1 1 n.d. 1.49±0.07 1.81±0.09
TPP-1853 2.53±0.03 n.d. n.d. 1.94±0.10 1.88±0.21 Ηΐ-29 WiDr A253 NCI-H322 786-0
TPP-1854 2.70±0.14 n.d. n.d. 1.98±0.04 2.1 1±0.19
TPP-1857 2.37±0. Ι4 n.d. n.d. 1.89±0.04 1.76±0.04
ΤΡΡ-1858 2.41±0.16 n.d. n.d. 2.0U0.12 2.22±0.14
ΤΡΡ-2084 2.59±0.14 n.d. 1.60±0. i0 1.58±0.23 1.61±0.13
ΤΡΡ-2090 2.84±0.31 1.73±0.14 1.75±0.18 1.95±0.14 1.56±0.04
ΤΡΡ-2093 2.54±0.04 n.d. n.d. n.d. 1.43±0.17
Ρ4Α8(ΤΡΡ-Ι324) 1.49±0.24 1.12±0.07 1.38±0.10 1.02±0.01 1.10±0.05
PDL-192(TPP-1104) 1.89±0.17 I .15±0.04 1.30±0.08 1.40±0.3 1.16±0.06
136.1(ΤΡΡ-2194) 1.81±0.02 n.d. n.d. n.d. n.d.
ΤΡΡ-2658 2.66±0.09 n.d. n.d. n.d. n.d.
EXAMPLE 5: Inhibition of proliferation by agonistic anti-TWEAKR antibodies in cancer ceil lines
To investigate whether the efficacy of the antibodies of this disclosure to induce Caspase 3/7 is also reflected by an efficacious inhibition of proliferation of different cancer cell lines, anti-proliferative activity was measured after incubation with the antibodies of the disclosure as compared to reference antibodies or TWEAK ligand.
Therefore cells were plated in 96 well plates in 75 μΐ assay medium (growth media from Table 21 , plus 100 ng/ml I FN gamma for 786-0 cells) at the following cell numbers: WiDr cells 3000 cells/well, 786-0 cells 2500 cells/well. 24h later cells were incubated with anti-TWEAKR antibodies (see Table 26), recombinant human TWEAK or isotype control IgG (not shown) at the indicated concentrations (antibodies from 0.03-300 μg/ml, TWEAK 100 or 300 ng/ml). At the time of antibody addition cell viability was determined in sister plates (time point zero): therefore 75 μΐ/well CTG solution (Promega Cell Titer Glo solution (catalog # G755B and G756B) was added to the ceils, incubated for 10 minutes and luminescence was read on a Victor V ( erk in Elmer). 96h after incubation with the agents, cell viability was determined in the assay plates by addition of 100 μΐ/'well CTG solution, 10 min incubation and reading of luminescence. Proliferation in control wells was calculated by subtracting time zero values from the luminescence values in the untreated control wells. % of cell proliferation was calculated in the compound treated wells as compared to the untreated control wells. The resulting dose response curves representing cell proliferation in the treated cells as compared to control cells are shown in Figure 14A (WiDr cells) and Figure 14B (786-0 cells). All tested antibodies of the disclosure inhibited proliferation of Wi Dr cells by 50-60% and of 786-0 cells by 70-80%, which was significantly more than the proliferation inhibition reached by other known antibodies, e.g. PDL- 192(TPP-1104) and P4A8(TPP-1324). Thus, the antibodies of the current disclosure are the most efficacious anti-proliferative anti-TWEAKR antibodies described to date.
To evaluate, whether this strong anti-proliferative activity can also be observed in a broader cell panel, in addition LOVO. NCI-H1975, SW-480 (all 3000 cells/well), HT-29 (4000 cells/well), A253 and SK-OV3 (both 2500 cells/well) cells were incubated with 100 μ^ηιΐ anti-TWEAKR antibodies or TWEAK ligand for the time periods as indicated in Table 27. All cells were seeded in the growth media indicated in Table 21 and for all cells except for WiDr cells 100 ng/ml IFNgamma was added to the assay medium when seeding the cells. The percentage of growth inhibition for treated cells as compared to proliferation in untreated control cells was measured and calculated as described above.
As shown in Table 27 the antibodies of the current disclosure are more efficacious as compared to other known antibodies in inhibiting proliferation of various cancer cell lines at 100 μg/ml. In most experiments the antibodies of the current disclosure also show equal or stronger efficacy as compared to 100-300 ng/ml TWEAK ligand. Thus, the antibodies described in this invention are unique in their activity to induce apoptosis and proliferation inhibition in a broad panel of cancer cell lines. Table 27a shows the results of a separate experiment in WiDr cells with or without of 100 ng/ml IFNgamma added to the medium under the same conditions as described above. TPP-2658, the aglycosylated variant of TPP-2090, shows anti-proliferative activity comparable to TPP-2090 and human recombinant TWEAKR ligand after 96h incubation. No activity was found with the isotype control antibody.
Table 26: List of antibodies used for roliferation assa
Figure imgf000094_0002
Table 27: % Inhibition of proliferation induced by incubation with 100 μg/mί anti-TWEAKR antibodies or TWEAK ligand (*100 ng/ml, **300 ng/ml). Incubation time in the presence of the agents is indicated as time of assay in [h]. Results from 1 -3 representative experiments carried out in triplicates are shown.
Antibodies show stronger inhibition of cancer cell proliferation as compared to known antibodies (PDL- 192(TPP-1104), P4A8(TPP-1324)) and equal or stronger activity as compared to recombinant TWEAK.
Figure imgf000094_0001
Table 27a: Inhibition of proliferation (IC50) of WiDr cells induced by incubation with anti-TWEAKR antibodies or TWEAK ligand. Incubation time was 96h. Results from a representative experiment carried out in triplicates are shown. TPP-2658 demonstrates comparable potency to TPP-2090 and recombinant human TWEAK in the same nanomolar range.
Figure imgf000095_0001
EXAMPLE 6: Cytokine secretion induced by anti-TWEAKR antibodies from cancer ceils and xenograft tumors
Next, it was of interest to investigate whether the strong agonistic activity of the antibodies of this disclosure is also seen in the induction of cytokine secretion from cancer cells.
Therefore A375 cells were plated at 2500 cells/well in 96 well plates in growth medium DM EM (Biochrom;# FG 0435. with stable Glutamin), 10% PC'S. 24h later cells were incubated with anti- TWEAKR antibodies, recombinant human TWEAK at various concentrations as indicated or corresponding isotype control IgG. 24h after start of the incubation with the antibodies, cell supernatant or dilutions thereof were added to the Capture Elisa Plate of the human CXCL8/IL-8 ELISA Kit (R&D Systems DY208) and incubated over night at 4 °C by shaking 300 rpm. On the next day, samples were analyzed by using the human CXCL8/IL-8-ELISA Kit (R&D Systems DY208) according to the manufacturer's instructions. Optical density was measured at 450 nm (Tecan Spectra, Rainbow) together with background correction. To calculate absolute levels of IL-8 a standard curve using recombinant human IL-8 protein was applied according to the manufacturer's recommendations (R&D Systems).
As shown in Figure 15 and 15a, the antibodies including TPP-2658, the aglycosylated variant of TPP-2090, showed increased induction of IL-8 release as compared to other antibodies previously known. At 100 μg/ml the antibodies of this disclosure TPP-1538/-1854/-2084/-2090 reached 134/129/113/103% of the activation as compared to 300 ng/ml TWEAK ligand respectively. In contrast, the antibodies used for comparison, PDL-192(TPP-1 104)/P4A8(TPP- 1324)/! 36.1 (TPP-2194), reached only 66/29/93%o, respectively. Thus, the antibodies show the strongest activity with regard to induction of IL-8 secretion as compared to previously known antibodies and 300 ng/ml TWEAK ligand.
To investigate, whether the observed cytokine secretion is also of relevance in xenograft tumors in mice, human and murine cytokines in serum/plasma from tumor bearing (A375, Wi Dr) as well as tumor free mice were investigated. 5x106 A375 cells in Matrigel/PBS (1 :1) or 5x106 Wi Dr cells in Matrigel/Medium (1 : 1) were subcutaneously inoculated in immunodeficient female N RI nude mice. Parallel to A375-bearing mice, non-tumor mice were investigated. Treatment started 7d after inoculation with established tumors of about 40 mm2. Mice were treated by a single intravenous injection of TPP- 1538 (10 mg/kg) or TPP-2090 (3 mg/kg) both diluted in PBS into the tail vein. Mice were then sacrificed at given time-points (0, 6h, 24h for A375-bearing mice and 0, 7h, 24h, 72h, 168h, 240h for WiDr-b earing mice) to harvest serum/plasma samples. Blood was collected after decapitation and serum was prepared by 30 minutes clotting with subsequent centrifugation at lOOOxg.
Cytokines were quantified using Luminex® bead immunoassays. Human cytokines were determined with Human Cytokine Magnetic 2 -pi ex panel (Invitrogen®, Cat-No. LHC0009M), comprising IL-Ι β, I L- I RA. I I .-2. I L-2 R. IL-4, I L-5. IL-6, 11. -7, IL-8, IL-10, IL-12 (p40), IL-13, I L- 1 5. IL-17, TNF-a, IFN-a, IFN-γ, GM-CSF, MIP- l a, ΜΙΡ-Ιβ, I - 1 0. MIG, Eotaxin, RANTES, and MCP-1). Murine cytokines were determined with Mouse Cytokine Magnetic 20-piex panel (Invitrogen®, Cat-No. LHC0006M) comprising FGF basic, GM-CSF, IFN-γ, IL-l a, IL-Ιβ, 1 1.-2. IL-4, IL-5, IL-6, IL-10, I L- 1 . I L- 1 2 (p40/p70), IL-17, I P- 1 0. KC, MCP-1 , M IG. MIP-l a, TNF-a und VEGF). The assays were conducted according to the manufacturer's instructions and measured by the Luminex reader io- PI ex 200 (Bio-Rad GmbH). Cytokine concentrations were interpolated from standard curves, as part of the assay procedure, by the operating software Bio-Plex Manger (Bio-Rad GmbH).
As shown in Figure 16A, human IL-8 is released from WiDr xenograft by a single treatment with TPP-2090 3 mg/kg in a time dependent manner. In addition, induction of secretion of human MCP-1 , I P- 1 0 and I L- 1 5 was observed after treatment with TPP-2090 (not shown).
To further investigate whether the cytokine induction observed in the plasma of tumor bearing mice after treatment with agonistic anti-TWEAK.ll antibodies of the disclosure was indeed tumor specific, a similar investigation was carried out in A375 tumor bearing and tumor free mice and human as well as murine cytokines were measured.
As shown in Figure 16B, human IL-8 is released from A375 xenografts in tumor bearing mice 6 h after treatment with TPP-1538 at 10 mg/kg. In addition, increased levels of human MCP- 1 . IP-10 and IL-IRA were observed (not shown). In contrast, in the plasma of treated tumor free mice no increased secretion of human cytokines was detected (Figure 16B and not shown). I n addition, no increase of murine cytokines including the murine IL-8 analogue KC was detected in the plasma of neither tumor bearing nor tumor free mice after treatment with TPP-1538 (data not shown). To summarize, the antibodies of the present disclosure potently induce secretion of cytokines from cancer cells and xenografts in vivo in a tumor specific manner. EX AM PLE 7: Internalization of anti-TWEAKR antibodies and usability for drug conjugate approaches
To investigate, whether anti-TWEAKR antibodies of the current disclosure are potentially usable for the generation of antibody drug conjugates (ADC)s, the internalization capacity of the antibodies was investigated.
To visualize this process the TWEAK R. specific antibodies TPP-1538 and TPP-2090 and an isotype control antibody were selected. The antibodies were conjugated in the presence of a twofold molar excess of CypHer 5E mono NHS ester (batch 357392, GE Healthcare) at pH 8.3. After the conjugation the reaction mixture was dialyzed (slide- A-Lyser Dialysis Cassettes MWCD 1 OkD, Fa. Pierce) overnight at 4 °C to eliminate excess dye and to adjust the pH-value. Afterwards the protein solution was concentrated (VIVASPIN 500, Fa Sartorius Stedim Biotec). In addition to the pH-dependent fluorescent dye CypHer5E the pH-independent dye Alexa 488 was used. The dye load of the antibody was determined with a spectrophotometer (Fa. NanoDrop). The dye load of TPP-1538, TPP-2090 and an isotype control antibody were in a similar range. The affinity of the labeled antibodies was tested in a cell binding-assay to ensure that labeling did not alter the binding to TWEAK R. These labeled antibodies were used in the following internalization assays. Prior to treatment cells (2xl04/well) were seeded in 100 μΐ medium in a 96-MTP (fat, black, clear bottom No 4308776, Fa. Applied Biosy stems). After 18 h incubation at 37 °C/5%CC>2 medium was changed and labeled anti-TWEAKR antibodies TPP-1538 and TPP-2090 were added in various concentrations (10, 5, 2.5, 1 , 0.3, 0.1 μg/ml). The identical treatment was carried out with the isotope control antibody (negative control). The incubation time was chosen to be 0, 0.5 h, 1 h, 2 h, 3 h. 6 h and 24 h. The fluorescence measurement was performed with the InC ell Analyzer 1000 (Fa. GE Healthcare). Granule counts per cell and total fluorescence intensity were measured in a kinetic fashion.
A highly specific and significant internalization of TPP-1538 and TPP-2090 was observed in endogenous TWEAKR expressing cancer ceil lines 786-0 (renal cancer) and HT-29 (colon cancer).
This internalization was target dependent as uptake could only be demonstrated using the anti- TWEAKR antibodies while no internalization was observed with the isotype control antibodies. During the first 6h the anti-TWEAKR antibodies showed a 20-40-fold increase of antibody internalization compared to isotype controls. Isotype control antibodies showed a minor internalization after a long exposure (>24 h).
Internalization of anti-TWEAKR antibodies labeled with Alexa 488 upon binding reveals that more than 50% of internalized antibodies seem to follow the endocytotic pathway. in Figure 17, evaluation of the time course of specific internalization of TPP-1538 and TPP-2090 upon binding to endogenous TWEAKR expressing cells is shown. Internalization of antibodies (1 μg/ml) was investigated on renal cancer cell line 786-0. Granule counts per cell were measured in a kinetic fashion. Rapid internalization could be observed for TPP-1538 and TPP-2090, whereas the isotype control hlgGl did not internalize. Additionally, a significantly improved internalization efficacy was seen with TPP-2090.The higher efficacy of TPP-2090 could be verified in internalization assays performed using a variety of cancer cells with different receptor levels (not shown).
Additionally, the activity of anti-TWEAKR antibodies to inhibit proliferation of cells when incubated in the presence of saporin-conjugated secondary antibodies was evaluated in Hum-Zap assays. Therefore 786-0 cells were plated at 2500ΰ6ΐΐ8/75μ1Λνβ11 in 96 well plates in growth medium (DMEM/Ham ' sF 12, Biochrom #FG4815 + 10% FCS). 24h later 40nM antibodies (TPP-1538, TPP-2090 or isotype control antibody) were incubated in the presence or absence of 4 n M saporin-conjugated secondary antibodies (Hum Zap. Advanced Targeting Systems Cat #IT-22, Lot 59-83) for 15 min at room temperature. After the incubation time 25 μΐ of the reaction mix was added to the cells, resulting in a final concentration of 10 nM antibody in the sample wells. At the time of the antibody addition cell viability was determined in sister plates (time point zero). Therefore, 75 μΐ/well CTG solution (Promega Cell Titer Glo solution (catalog # G755B and G756B) was added to the cells, incubated for 10 minutes and luminescence was read on a Victor V ( Perkin Elmer).
48 h after start of incubation with the antibody/Zap complex, 100 μΐ ννβΐΐ CTG solution was added to all test wells, incubated for 10 minutes and luminescence was read on a VICTOR V.
As shown in Figure 18, incubation of 786-0 cells with anti-TWEAKR antibodies at 10 nM in the presence of saporin-conjugated secondary antibodies almost completely inhibited cell proliferation, whereas under the same experimental conditions (absence of IF gamma, 48h incubation time only) no anti-proliferative activity was observed in the absence of saporin-conjugated secondary antibodies or with isotype control antibodies.
To summarize, anti-TWEAKR antibodies of the present disclosure show rapid internalization and targeted delivery of conjugated payloads and are thus well suitable for the generation and use as ADC's.
EXAMPLE 8: Anti-Tumor efficacy of anti-TWEAKR antibodies in xenograft models in vivo
To investigate, whether anti-TWEAKR antibodies show anti-tumor activity in vivo xenograft tumors derived from different cancer cell lines or patient derived tumor models were tested for their sensitivity against tumor growth inhibition by agonistic anti-TWEAKR antibodies in mono- or combination therapy. Before start of the in vivo experiments expression of TWEAKR in the selected xenograft models was evaluated by immunohistochemistry. Therefore, frozen sections (5 μΜ) of the corresponding xenografts were fixed with acetone for 5 min at 4 °C and blocked against unspecific protein binding and peroxidase activity. Tissue sections were incubated with rabbit anti-TWEAKR antibody (Fnl4, Epitomics, 3488-1) at room temperature for 60 min, followed by peroxidase labeled anti rabbit polymer (DAKO, K401 1) incubation for 30 min. Sections were developed with diaminobenzidine and finally counters tained with hematoxylin. Only models that were positive for expression of the TWEAKR were used for in vivo experiments
For the investigation of the anti-tumor activity of the anti-TWEAKR antibodies in vivo, nude mice bearing xenografts from different human tumor cell lines or patient-derived tumors were treated by repeated intravenous injections.
Tumor cell lines were cultivated as described in the parts above and 100 μΐ containing cell line specific numbers of tumor cells inoculated subcutaneously (s.c.) into female athymic nude mice (NMRI nu/nu, 6-8 weeks, 20-25 g, Taconic). Mice were housed under standardized pathogen free conditions and treated according to the animal welfare guidelines.
After tumor growth to a size of approximately 40 mm2 mice were randomized into control and treatment groups with a respective group size of n=8-10. Mice were treated with various doses of anti- TWEAKR antibodies diluted in PBS by intravenous injection (i.v.) into the tail vein with a twice per week schedule (q4dx3 : applications twice per week, three applications in total; q4dx8 : applications twice per week, eight applications in total). Combination therapy partners such as Regorafenib (10 mg kg daily, per os) and the PI3K- inhibitor 1 (10 mg kg. BID, 2 d on, 5 d off (applications twice daily on two consecutive days, followed by five days without treatment), i.v.) were diluted in their respective formulations whereas the standard of care therapies Irinotecan (5 mg/kg, 4 d on, 3 d off (applications once daily on four consecutive days followed by three days without treatment) i.v.) and Paclitaxel (16 mg/kg, q7dx4 (applications once per week, four applications in total), i.v.) were diluted in 0.9% NaCi. Animals injected with PBS served as the control (vehicle) group. The applied volume of the compounds was 5 ml/kg body weight per mouse.
Tumor growth was monitored 2-3 times per week by caliper measurement (length x width in mm) as well as body weight (in g). At the end of study tumors were dissected, weighted and used for the calculation of tumor-to-control (T/C) ratios (mean tumor weight of treated animals divided by mean tumor weight of control/vehicle animals).
In the human renal ceil cancer model 786-0 (positive TWEAKR expression) efficacy of the anti- TWEAKR antibodies TPP-2084 and TPP-2090 was tested at three different low doses against the i so type control antibody. 2xl06 tumor cells in 100% Matrigel were s.c. inoculated in female nude mice. After 7d established tumors with a size of about 40 mm2 were treated with 0.3 mg'kg, 1 mg kg and 3 mg kg of antibodies (i.v., q4dx3 : applications twice per week, three applications in total). At day 40, comparison of tumor weights after dissection showed dose-dependent efficacy of TPP-2084 and TPP- 2090 which was highest at 3 mg/kg (Figure 19). A clear differentiation against the i so type and vehicle group (treated with PBS) could be demonstrated. No loss of bodyweight was observed in any of the groups. Tumor-to-control ratios listed in Table 28 demonstrate good efficacy in the 786-0 model and in further tumor models (A375, A253, SK-OV-3, Bx-PC3, treated with 3-10 mg/kg anti-TWEAKR antibodies TPP-1538, TPP-2094 or TPP-2090 in a q7dx3 (applications once per week, three applications in total) or q4dx3 (applications twice per week, three applications in total) schedule with the exception of MDA-MB-231 where more intense dosing schedules of anti-TWEAKR antibodies might be required to reach monotherapy efficacy.
Table 28: Final Tumor-to-Control (T/C) ratios in 786-0 and further tumor models after treatment with TPP-1538, TPP-2084 or TPP-2090. Anti-TWEAKR antibodies show strong anti-tumor activity in a variety of xenograft models from different solid tumor indications.
Figure imgf000100_0001
T/C: tumor-to- control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
Figure 20 shows the efficacy of the anti-TWEAKR antibody TPP-2090 in the human colon cancer xenograft WiDr (which represents a subclone of the HT-29 tumor cell line) in monotherapy and combination therapy with Irinotecan and Regorafenib. 5xl06 WiDr cells in Matrigel/Medium (1 : 1) were s.c. inoculated in immunodeficient NMRI nude mice. Treatment started 7d after inoculation with established tumors of about 40 mm2. Even 3 mg 'kg of TPP-2090 (i.v., q4dx7: applications twice per week, seven applications in total) in monotherapy was strongly effective to control tumor growth. Combination of 3 mg/kg TPP-2090 with either Irinotecan ( 5 mg'kg, i.v., 4d on, 3d off) or Regorafenib (10 mg kg, p.o.. daily) resulted in additive efficacy with tumor regression. All therapeutic regimens were well tolerated by the mice (max. 4% initial and reversible body weight loss). Final T/C values are listed in Table 29.
TPP-2090 was also investigated in other colorectal tumor models such as SW480 and the patient- derived tumor model Co5682 with similar good results (Table 29). A dose of 10 mg/kg TPP-2090 was effective in monotherapy in SW480 to control tumor growth (T/C 0.49) and to lead to tumor regression in combination with 5 mg kg Irinotecan (T/C 0.22) or 10 mg kg Regorafenib (T/C 0.37). In Co5682 xenografts 3 mg/kg of TPP-2090 showed synergistic efficacy with tumor regression in combination with Irinotecan (T/C 0.23) and tumor stasis in combination with Regorafenib (T/C 0.27). Table 29: Final Tiimor-to-Control (T/C) ratios of two colon cancer cell lines WiDr and SW480 and one patient-derived colon cancer xenograft Co5682 after treatment with TPP-2090 and combination partners based on tumor weights at study end.
Figure imgf000101_0001
T/C: tumor-to-control ratio based on final tumor weight after dissection, Tx: therapy, combo: combination therapy Figure 21 shows the efficacy of the anti-TWEAKR antibody TPP-2090 in the human non-small-cell lung cancer xenograft NCI-H322 in monotherapy and combination therapy with Paclitaxel. 5x106 NCI- H322 cells in Matrigei were s.c. inoculated in immunodeficient NMRI nude mice. Treatment started 14d after inoculation with established tumors of about 45 mm2. At a dose of 5 mg kg TPP-2090 (i.v., q4dx8) was strongly effective in monotherapy demonstrating tumor regression. Combination of 10 mg k TPP- 2090 with Paclitaxel (16 mg/kg, i.v., q7dx4) resulted in slight additive efficacy. All therapeutic regimens were well tolerated by the mice (max. 3% reversible body weight loss). Final T/C values are listed in Table 30.
Again, TPP-2090 was also investigated in other lung cancer models such as NCI-H 1 975 and the patient-derived tumor models Lu7343 and Lu7433 with comparable results (Table 30). A dose of 3 mg/kg TPP-2090 showed additive effects in NCI-H 1 975 in combination with 16mg/kg Paclitaxel resulting in tumor regression (T/C 0.08). Additive effects were also achieved with the same dose (3 mg/kg) of the aglycosylated variant TPP-2658 in combination with paclitaxel (16 mg/kg) in NCI- H 1975 xenografts. Similarly, in the patient-derived NSCLC models Lu7343 and Lu7433 combination of 3 mg/kg TPP-2090 with 10 mg/kg of the PI3K-inhibitor 1 led to tumor control or regression (T/C 0.18- 0.36) in an additive efficacious manner.
PI3K-inhibitor 1 is (2-amino-N-[7-methoxy-8-(3-mo holin-4-ylpropoxy)-2,3-dihydroimidazo[l ,2- c]quinazolin-5-yl]pyiimidine-5-carboxamide dihydrochloride.
Table 30: Final Tumor-to-Control (T/C) ratios of two NSCLC cell lines NCI-H322 and H 1 975 and two patient-derived lung cancer xenografts Lu7343 and Lu7433 after treatment with TPP-2090 and combination partners based on tumor weights at study end. T/C (final)
Tumor Model ΤΡΡ-2Θ90 mono Tx Combo TPP-2090 Combo TPP-2090
+ Paclitaxel + PI3K-inhibitor 1
NCI-H322 0.17 0.14 -
NCI-H1975 0.48 0.08
Lu7343 0.65 - 0.18
Lu7433 0.53 0.36
T/C: tumor-to-control ratio based on final tumor weight after dissection, Tx: therapy, combo: combination therapy
Ail described in vivo examples demonstrate the strong efficacy of the anti-TWEAKR antibody TPP-2090 in a broad panel of cell line-derived and patient-derived human tumor models (all with TWEAK R. positive expression) in monotherapy as well as in combination therapy. TPP-2090 was well tolerated by the mice at all doses used.
EXAMPLE 9: Mode of action of anti-TWEAKR antibodies in xenograft models
To investigate the mode of action of anti -TWEAKR antibodies in vivo, tumors from WiDr xenografts were taken at study end as described in Example 8 and investigated by immunohistochemistry and Western Blot Analysis.
Frozen sections (5 μιη) of WiDr xenografts (tumors from 3 individual animals per group) were stained immunohistochemically for the proliferation marker protein Ki67 (see EXAMPLE 8 for details of in vivo experiment). Sections were fixed with freshly prepared 4% paraformaldehyde for 20 min at 4 °C and blocked against unspecific protein binding and peroxidase activity. Murine anti Ki67 antibody (DAKO, M7240) was labeled with biotin according to manufacturer's instruction (DAKO, K3954) and incubated at room temperature for 60 min with the tissue sections, followed by Extra Vidin-p er oxidas e (DAKO, K3468) incubation for 30 min. Sections were developed with diaminobenzidine and finally counters tained with hematoxylin. For quantification, entire tumor sections were scanned and analyzed using AR IOL automated microscopy version 3.2 (Applied Imaging, San Jose CA, USA). Representative images of PBS (i.v., q4dx7) and TPP-2090 (10 mg/kg, i.v., q4dx7) treated xenografts stained for Ki67 are shown in Figure 22 A and B respectively. The quantification using the ARIOL image system, revealed 355+A59 Ki67 positive cells / mm2 in the group treated with 10 mg/kg TPP-2090 (i.v. q4dx7), and 863 +/-90 Ki67 positive cells /' mm2 in the vehicle treated group. Thus, in line with the observed reduction in tumor volume, treatment with agonistic anti-TWEAKR antibodies leads to a reduction of the proliferation marker Ki67 in xenograft tumors.
In addition Wi Dr xenograft tumors (see Example 8 for details of in vivo experiment), snap frozen at study end were analyzed by Western Blot to evaluate effects of the antibody treatment on Stat-1 and NF- kappaB signaling pathways. Tumors of 4 individual animals per group were cut in slices of around 5mm diameter and each slice deposited in a 2ml Eppendorf tube together with a precooled 5mm steel bull (Qiagen) and 500 μΐ lysis buffer (50 rriM Hepes pH 7.2, 150 mM NaCl, ImM MgCi2, 10 mM Na4P207, 100 mM NaF, 10% Glycerin, 1.5% Triton X-100, freshly added Complete Protease Inhibitor cocktail (Roche No. 1873580001), 4 mM Na3VC¼, pH adjusted to 7.4 with NaOH)). Samples were lysed for 3 min at 300 Hz in a Tissuelyzer (Qiagen) followed by incubation on ice for 30min. In the following, samples were centrifuged for 10 min at 13000 rpm at 4 °C in a Micro-centrifuge (Eppendorf) and supernatants from one original tumor pooled back together. Protein levels in the tumor lysates were determined by using the BCA protein assay kit (Novagen, lysates 1 :50 diluted in ¾0). Samples were diluted to a final concentration of 4mg/ml and 10 μΐ of sample were mixed with 3.08 μΐ of (10*) Sample Reducing agend, 10 μΐ H20 and 7,68 μΐ (4*) NuPAGE Sample Buffer (Invitrogen). Samples corresponding to 40 μg of protein were applied to NuPage 4-12% SOS page gels from Invitrogen and run for 2 h 45 min at 120 V. Blotting was carried out by an iBlot system (Invitrogen) according to the manufacturer's recommendations. Membranes were blocked for 2h at room temperature in 5% BLOT QuickBlocker in PBST (Invitrogen), followed by incubation with primary antibodies over night at 4 °C. Primary antibodies were as follows: Phospho-Statl #9167S, Stat-1 #9172, both Cell Signaling Technology, dilution 1 : 1000; TWEAKR Fn l 4 #3488-1 Epitomics, dilution 1 : 10000; NF-kappaB2 pl 00/p52 #4882S, Ceil Signaling Technology, dilution 1 : 1000 in in 3% BLOT QuickBlocker in PBST. On the next day membranes were washed three times in PBST, followed by incubation with secondary antibodies (Peroxidase-conjugated donkey anti-rabbit IgG # NA934, GE Healthcare 1 : 10000 in 3% BLOT QuickBlocker/PBST) for 2h at room temperature. Subsequently, membranes were washed four times for 10 min with PBST and signals were detected by chemoluminescence after incubation with ECL reagent. To detect the loading control, membranes were stripped with stripping solution Re-Blot Plus strong solution #2504, Milipore (1 : 10 in Milipore-H20) for 15 min shaking at room temperature, followed by blocking and detection with anti-GAPDH antibody (clone6C5, # MAB374, Millipore 1 : 10000 in 3% QuickBlocker/PBST) and secondary antibody (Peroxidase-conjugated goat anti-mouse IgG, Jackson Immunoresearch #1 15-035-003, 1 : 10000 in 3% BLOT Quickbiocker/PBST).
Representative Blots from tumors of 2 animals per group treated with TPP-2090 3 mg kg side by side with tumors from vehicle treated animals are shown in Figure 23. Treatment with TPP-2090 leads to strong increase of total and phosphorylated Stat-1 levels as well as a strong activation of NF-kappaB2 as indicated by the appearance of the p52 fragment. Thus, the NF-kappaB2 as wells as Stat-1 pathways are activated by agonistic anti-TWEAKR antibodies in xenograft tumors and this activation is potentially involved in the anti-tumor activity of the corresponding antibodies. Table 31: Protein se uences of the antibodies:
Figure imgf000104_0002
Figure imgf000104_0001
Figure imgf000105_0001
EXAMPLE 10: Anti-tumor efficacy of anti-TWEAKR antibody TPP-2090 in further human colorectal cancer models in vivo
Animal studies were conducted as described in example 8 for further human colorectal cancer tumor cell lines Colo205 and LoVo and for further human colorectal cancer patient-derived models
Co7553, Co5896, Co5676, Co5841 , CXF 1 103 and CXF 533. Standard dosing schedule was 10 mg/kg of TPP-2090 twice weekly for 4 weeks in monotherapy or in combination with regorafenib or the standard of cares (SoCs) irinotecan (5-15 mg/kg i.p., 4 d on, 3 d off), oxaliplatin (3-8 mg/kg i.p., twice weekly), 5-fhioroiiracil (50-100 mg/kg i.p., once weekly) and cetuximab (15 mg/kg i.p., twice weekly) . TPP-2090 and cetuximab were formulated in PBS, which was also used as the vehicle in the control group, and the SoCs were formulated in 0.9% NaCl. The formulation of regorafenib is described in example 8.
The monotherapeutic efficacy of TPP-2090 in these human colorectal cancer patient-derived and cell line based models was moderate with final Tumor-to-Control (T/C) ratios in the range of 0.48-1.07. The combinations of TPP-2090 with SoCs (in particular 5-FU and irinotecan) resulted in significant additive and synergistic effects (see Table 33-35 ). In cases where the monotherap eutic efficacy of T - 2090 in these models were limited more intense dosing schedules of anti-TWEAKR antibodies might be required to reach higher monotherapy efficacy, as has been shown in example 8 for colorectal cancer.
Table 33: Final Tumor-to-Control (T/C) ratios of colorectal cancer models treated with TPP-2090
Combination TPP-2090 with:
Tumor-
TPP-2090 Irinotecan Oxaliplatin
model
MonoTx
Mono Combi Benefit of Combi Mono Combi Benefit of Combi
Response
Colo205 1.07 0.47 0.27 synergistic effect 0.89 1.20 no benefit
LoVo 1 .25 0.52 0.64 no benefit 0.76 0.8 no benefit
Co7553 0.79 0.1 1 0.15 no benefit 0.78 0.59 additive effect Combination TPP-2090 with:
Tumor-
TPP-2090 Irinotecan Oxaliplatin
model
MonoTx
Mono Combi Benefit of Combi Mono Combi Benefit of Combi
Response
Co5896 0.91 n.d. n.d. n.d. n.d. n.d. n.d.
Co5676 (*) 0.48 0.37 0.27 additive effect n.d. n.d. n.d.
Co5841 0.56 0.17 0.1 no benefit 0.81 0.58 no benefit
CXF1103 0.87 0.66 0.39 additive effect 1.29 0.79 no benefit
CXF533 0.88 n.d. n.d. n.d. 1.08 0.85 no benefit
T/C: tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
n.d.: not determined
Table 34: Final Ί umor-to-Control (T/C) ratios of colorectal cancer models treated with TPP-2090
Figure imgf000106_0001
T/C: tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
n.d.: not determined
Table 35: Tumor-to-Control (T/C) ratios of colorectal cancer models treated with TPP-2090 in combination with cetuximah
Figure imgf000106_0002
T/C: tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
n.d.: not determined EXAMPLE 11 : Anti-tumor efficacy of anti-TWEAKR antibody TPP-20 0 in human bladder cancer models in vivo
Animal studies were conducted as described in example 8 for the human bladder cancer cell lines SCaBER and KU-19-19 and for the human bladder cancer patient-derived models BXF1352 and BXF1228. Standard dosing schedule was 10 mg/kg of TPP-2090 twice weekly for 4 weeks in monotherapy or in combination with the standard of cares (SoCs) gemcitabine (200 mg kg i.p.. once weekly) and cisplatin (3 mg/kg i.p.. once weekly). TPP-2090 was formulated in PBS, which was also used as the vehicle in the control group, and the standard of cares (SoCs) were formulated in 0.9% NaCl.
Strong monotherapeutic efficacy of TPP-2090 was found in SCaBER xenograft model. The combination of TPP-2090 in the human bladder cancer patient-derived bladder cancer models BXF1352 and BXF1228 with SoCs (Cisplatin and Gemcitabine) resulted in significant synergistic effects (see
Table 36). in cases where the monotherapeutic efficacy of TPP-2090 in these bladder cancer models were limited more intense dosing schedules of anti-TWEAKR antibodies might be required to reach higher monotherapy efficacy. Table 36: Tumor-to-Control (T/C) ratios of human bladder cancer models treated with TPP-2090 in monotherapy or combination with cisplatin or gemcitabine
Figure imgf000107_0001
T/C: tumor-to-control ratio based on final tumor weight
n.d.: not determined
EXAMPLE 12: Anti-tumor efficacy of anti-TWEAKR antibody TPP-2090 in further human cancer models in vivo
Animal studies were conducted as described in example 8 for further human cancer cell lines of different indications. Standard dosing schedule was 10 mg/kg of TPP-2090 twice weekly for 2-3 weeks in monotherapy. TPP-2090 was formulated in PBS, which was also used as the vehicle in the control group. Strong monotherapeutic efficacy of TPP-2090 was found in SCC4 (head & neck cancer) and A375
(melanoma) xenografts, and moderate efficacy in BxPC3 (pancreatic cancer) xenografts (see Table 37). In cases where the monotherapeutic efficacy of TPP-2090 in certain xenograft models were limited (ACHN (renal cell cancer), PA- ! (ovarian cancer), NCI-292 (non- small cell lung cancer) and U87MG (glioblastoma)) more intense dosing schedules of anti-TWEAKR antibodies might be required to reach higher monotherapy efficacy.
Table 37: Tumor-to-control (T/C) ratios values of further human cancer models treated with TPP- 20 0 in monotherapy
Figure imgf000108_0001
T/C: tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
EXAMPLE 13: Further mode of action of anti-TWEAKR antibodies in xenograft models
To evaluate if the anti-tumor efficacy of TPP-2090 is dependent on antibody-dependent cellular cytotoxicity (ADCC) or agonistic activity alone is already sufficient, xenografts studies in SCID beige mice (Janvier) were conducted, and the aglycosyl variant of TPP-2090, namely TPP-2658, was investigated in NMRi nude mice.
Binding of TPP-2090 and TPP-2658 to human FcgR2B/C (CD32b/c, R&D Systems, Inc., catalog number 1875-CD), cynomolgus (Macaca fascicularis) FcgR2B (CD32b, Sino Biological Inc., catalog number 90014-C08H) and murine FcgR2B (CD32b, R&D Systems, Inc., catalog number 1460-CD) were measured by using surface plasmon resonance (SPR) assays. Experiments were performed using a Biacore T200 instmment (GE Healthcare Biacore, Inc.) equipped with Series S Sensor Chips CM5 (GE Healthcare Biacore, Inc.). Binding assays were carried out at 25 °C with assay buffer HBS-EP+ (10 mM HEPES pH 7.4, 150 nM Nad. 0.05% SP20; GE Healthcare Biacore, Inc.). Polyhistidine-tagged FcyR proteins were captured with an anti-His capture antibody covalently immobilized to the chip surface via amine coupling chemistry. Reagents for amine coupling ( 1 -ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanolamine-HCi pH 8.5) were used from the Amine Coupling Kit (GE Healthcare, product code BR-1000-50). anti-His capture antibody was obtained from (Molecular Probes, Inc., catalog number P21215) and immobilization buffer (10 mM sodium acetate pH 4.0) was used from the Human Antibody Capture Kit (GE Healthcare, BR- 1008-39). The sensor chip surface was activated with a freshly prepared solution of 0.2 M EDC and 0.05 M NHS passed over the chip surface for 420 s at a flow rate of 10 μΐ/min, followed by an injection of anti-His capture antibody (dissolved to 10 μg/mί in immobilization buffer) for 180 s at a flow rate of 5 μΐ/min. Excess of activated groups were blocked with a 1 molar solution of ethanolamine injected at a flow rate of 10 μΐ/min for 420 s. TPP-2090 and TPP-2658 were used as analyte. FcyR variants were captured at a flow rate of 10 μΐ/min to a final response of - 26 RU prior to each analyte injection. For analysis of binding various concentrations between 0.64 and 25 μΜ of human IgG in assay buffer (see above) were injected over the captured FcyR variants at a flow rate of 30 μΐ/min for 3 minutes and the dissociation was monitored for 5 minutes. Obtained sensorgrams were double-referenced, i.e. in-line reference cell correction followed by buffer sample subtraction. Data evaluation was performed with the Biacore T200 Evaluation Software Package. SPR results clearly indicate that the aglyco form of TPP2090, namely TPP-2658 abolishes binding to the tested FcyR variants under current experimental conditions. This is in agreement with previous studies (cf. Mimura et al. 2001, JBC, doi: 10.1074/jbc,M107478200). Tabie 42: SPR analysis of FCgR interactions: TPP-2658 does not bind to FCgRs
Figure imgf000109_0001
"+" denotes that binding was detected at several concentrations
"-" denotes that no binding was detected under current experimental conditions, i.e. at antibody concentrations up to 25 μΜ (this means KD > 200 μ ).
Moreover, influence of deglycosylation of TPP-2090 on TWEAKR binding was quantitatively analyzed. Here, TPP-2090 and TPP-2658 were captured with an anti-hlgG capture antibody (Human Antibody Capture Kit, GE Healthcare, BR-1008-39) covalently immobilized to the chip surface via amine coupling chemistry as described above. Purified recombinant human TWEAKR-ECD protein (Fitzgerald Industries International, MA, USA, catalog number 30R-AT080) was used as analyte. After capture of TPP-2090 and TPP-2658 at 5 μg/ml in assay buffer at a flow rate of 10 μΐ/min for 60 seconds to a final response of -220 RU. TWEAKR-ECD protein was injected at concentrations ranging from
1.56 200 nM in assay buffer HBS-EP+ (see above) over the captured antibodies at a flow rate of 60 μΐ/min for 3 minutes. Dissociation was monitored for 20 minutes. Obtained experimental sensorgrams were double-referenced and affinities {K values) were derived by a steady-state affinity analysis implemented in the Biacore T200 Evaluation Software package. As listed in Table 43. no differences between TPP-2090 and TPP-2658 were found with regard to binding affinity to soluble human TWEAKR-ECD.
Tabie 43: SPR analysis of TWEAK interactions: deglycosylation does not influence binding affinity to soluble human TWEAKR-ECD
Affinity (An) to TWEAKR-ECD
in nM
TPP-2090 13
TPP-2658 13 Animal studies were conducted as described in example 8 in WiDr (human colorectal cancer) and SCaBER (human bladder cancer) xenografts in SCID beige mice with tumor growth of control groups comparable to those in NMRI nude mice of previous studies. Standard dosing schedule was 10 mg/kg of TPP-2090 i.v. (formulated in PBS) twice weekly for 2 weeks in monotherapy.
A similar strong monotherapeutic efficacy of TPP-2090, including its aglycosyl variant TPP-2658, in NK-cell lacking SCID beige mice xenograft models (WiDr and SCaBER) was found as seen in NMRI nudes mice. This indicates an in vivo mode of action independent from ADCC (see Table 38).
Tab!e 38: Tumor-to-Control (T/C) ratios of WiDr and SCaBER tumors in SCID beige mice treated with TPP-2090 in monotherapy (NMRI mice for comparison)
Figure imgf000110_0001
T/C: tumor-to-control ratio based on final tumor weight after dissection or based on measurement of tumor area (*).
In vitro analysis showed that HT29 cell binding of TPP-2090 resulted in dose-dependent ADCC of target cells by NK92V effector cells while the aglycosyl TPP-2658 was not capable of inducing ADCC (Table 40). lxl O4 HT-29 target cells were dispensed and the tested antibodies were added in a final concentration of 25 μg ml; 5 μg ml; 1 μg/ml; 0.2 μg/ml and 0.04 μg/ml. After a preincubation time of 30 min effector cells were added (5x104 NK92V effector cells). After 4 h incubation at 37 °C HT29 cell lysis was determined with the Cytotoxicity Detection Kit - LDH (Roche)", maximum release was obtained from cells solubilized in 1% Triton X-100, negative controls were not preincubated with an antibody. The following formula was used for calculation of % HT29 lysis: [Ext (sample) - Ext (negative) x 100] / [Ext (Maximum release) - Ext (negative)]. TPP-2090 resulted in dose-dependent A DCC of target cells by NK92V effector cells and is dependent on N297 glycosylation.
Whereas in vivo a similar effect was found when an aglycosyl variant of TPP-2090, namely TPP- 2658, was used in either a Wi Dr- or A375 -xenograft model (see Table 39). The variant TPP-2658 showed equally strong monotherapeutic efficacy as the TPP-2090 in both models indicating an ADCC- independent mode of action.
Combination treatment with check-point inhibitors:
Preclinical evidence suggests that monoclonal antibodies can be used in combinations which provide evidence for synergistic effects in many models. This includes antibodies to check-point inhibitors and to immunotherapy receptors. As an example TPP-2658 was combined with anti-CLLA4, anti PD-1 , anti-PD-Ll antibodies binding to the murine proteins (mouse surrogates). Tfae anti-CLLA4 (BE0131 -R005mg) and anti PD-1 antibody (BE0146-R005mg) were purchased from BioXcell. The endotoxin concentration was confirmed to be below 2 EU/mg by standard methology and antibodies were used directly for in vivo experiemnts.
For anti-PD-Ll the variable domain of anti-PD-Ll antibody RG7446 from patent US8217149 was fused with mlgGl CH1-CH3 sequences to optain a full length chimeric IgG with the following sequence: anti-PD-L1 -mlgGl Kappa_RG7 46 light chain (SEQ ID NO:214):
DIQMTQS PS SLSASVGDRVT I TCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGT DFTLT I S SLQPEDFATYYCQQYLYHPATFGQGTKVE IKRADAAPTVS IFPPS SEQLTSGGASWCFLNN FYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVK SFNRNEC anti-PD-L1 -mlgGl Kappa_RG7446 heavy_chain (SEQ ID NO:215):
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLE VAWI S PYGGSTYYADSVKGRFT I SADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTN SMVTLGCLVKGYFPEPVTVTWNSGSLS SGVHTFPAVLQSDLYTLS S SVTVPS STWPSETVTCNVAHPAS STKVDKKIVPRDCGCKPC I CTVPEVS SVFI FPPKPKDVLT ITLTPKVTCVWDI SKDDPEVQFS FVDD VEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT I SKTKGRPKAPQVYT I PPPKEQMAKDKVSLTCMI TDFFPEDI TVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEA GNTFTCSVLHEGLHNHHTEKSLSHSPGK
The anti-PD-Ll antibody was expressed as described above in the chapter antibody generation.
I n addition, strong monotherapeuti c efficacy of TPP-2658 was observed in a syngeneic mouse CRC model (CT26) in immunecompetent mice of strain Balb/cAnN (see Table 39). The study was conducted as described in example 8 with the exception that treatment of the mice started already 3 days after tumor cell inoculation before tumor establishment and without randomization. The tumor take rate, however, was 100%. Within this experiment, an additive effect of the combination of TPP-2658 with the immune-checkpoint inhibitor anti CTLA4 antibody was found which led to the complete remission of tumors in 8 of 8 tumor bearing mice.
In comparison, the respective monotherapies with TPP-2658 and anti-CTLA4 led to 3 of 8 (38% CR) and 5 of 8 (63%) complete tumor regression. An additive effect was also observed for the combination of TPP-2658 ith the anti-PD-1 inhibitor which led to complete tumor regression in 5 of 8 mice (63% CR) in contrast to 0% CR for the anti-PD-1 monotherapy (table 39a).
An additive effect of combining TPP-2658 with the immune-checkpoint inhibitor anti-PD-Ll was observed in MC38 mouse colon tumor (experimental setting identical to the described CT26 experiment above). Compounds were applied i.v. in a q4dx3 schedule. In contrast to the respective monotherapies a - I l l - benefit of the combination regarding the T/C values based on either final tumor weights or finale tumor areas was detected (Table 39a).
Table 39: Tumor-to-Control (T/C) ratios of WiDr and A375-xenografts treated with TPP-2658 (TPP-2090 for comparison)
Figure imgf000112_0001
Tabie 39a: Combination (Combo) effects in syngeneic MC38 and CT26 tumors treated with TPP- 2658 and immune-checkpoint inhibitors (anti-PD-Ll, -CTLA4- und -PD-1 [mouse surrogates])
Figure imgf000112_0002
T/C: Tumor-to-Controi ratios
CR: compiete tumor regression (rate in %)
Table 40: In vitro ADCC assay with HT-29 target cells and NK92V effector cells for testing antibody TPP-2090 (MgGl) and TPP-2658 (aglycosylated counterpart of TPP-2090 - gGl N297A):
Lysis [%] for TPP-2658
Antibody concentration [ug/ml] Lysis [%] for TPP-2090
(aglycosylated)
25 16.8 -1 .1
5 16.4 -1 .3
1 15.1 -1 .3
0.2 9.8 -1 .4
0.04 6.3 -0.8 EXAMPLE 14: Comparison of toxicologic side effects of TWEAKR antibodies in cynomolgus monkeys
To evaluate tolerability of TWEAKR antibodies of this disclosure TPP-2090 and its aglycosyl variant TPP-2658 were investigated and compared at a 10 ing'kg dose intravenously (i.v.) applied weekly for four times in female and male cynomolgus monkeys (n=2-4). The liver-specific enzymes alanin-aminotransferase (ALAT) and glutamatedehydrogenase (GLDH) as well as the pancreatic enzymes lipase and amylase and the kidney markers creatinine (CREA) and urea were determined in blood samples of the monkeys as laboratory parameters. Increase in these liver and pancreas enzymes in the blood indicate cellular damages of these particular organs. Similarly, increased creatinine and urea values in the blood indicate kidney impairment. Histopathology (grade 1 -5 according to the commonly used computer-assisted scoring system; grade 1 : minor abnormalities, grade 2: slight abnormalities, grade 3: moderate abnormalities, grade 4: marked abnormalities, grade 5: severe abnormalities) of sacrificed animals was focused on endothelia of the heart, pancreas and liver tissues. Table 41 shows that at a weekly dose of 10 mg/kg, drastically enhanced tolerability and reduced alterations in laboratory parameters and tissue histopathology were found for TPP-2658 compared to TPP-2090.
Although the Tweak R is expressed in liver, pancreas and kidneys only at a very low level, T - 2090 induced toxicities due to its agonistic mode of action strongly potentiated by FcyR-mediated cross- linking by immune cells. In contrast, TPP-2658, lacking FcyR binding ability drastically reduced these toxicities to minor grades.
No enhancement of ALAT, amylase, urea and CREA levels were observed under TPP-2658 treatment at all. GLDH levels were reduced by TPP-2658 compared to TPP-2090 whereas lipase levels remained the same.
Histopathologically, high grade hyperplasia of liver bile ducts induced by TPP-2090 was totally absent at the same dose of TPP-2658. Existence of granuloma in the liver (grade 2) was regarded as unspecific event. Hyperplasia of pancreatic cells was reduced to a low grade of 2 by TPP-2658 compared to grade 5 induced by TPP-2090. Whereas TPP-2090 induced vasculitis (inflammation of endothelial cells) in the heart it was not the case with TPP-2658
Table 41 : Laboratory and histopatho!ogica! parameters of cynomolgus monkeys (n=2-4) treated with either ΤΡΡ-2Θ9Θ or TPP-2658 Lv. at a weekly dose of 10 mg/kg for four times.
Parameter ΤΡΡ-2Θ90 TPP-2658
Tolerability, Mortality / premature sacrifice (3rd Tolerated over 4 weeks
overall week onwards) Parameter TPP-20 0 TPP-2658
ALAT 2 - 10 x fold increase compared to No change compared to normal levels
normal levels
GLDH 3 - 75 x fold increase compared to 0 /' 5 x fold increase compared to normal levels normal levels
Lipase 2 - 90 x fold increase compared to 16 / 2 x fold increase compared to normal levels normal levels
Amylase 2 - 17 x fold increase compared to No change compared to normal levels
normal levels
CREA 1 1.5 - 20 x fold increase compared No change compared to normal levels
to normal levels
Urea 3 - 10 x fold increase compared to No change compared to normal levels
normal levels
Heart vasculitis/degeneration of coronary No signs of abnormalities
arteries (up to grade 5)
Liver bile duct hyperplasia / inflammation Granuloma (grade 2),
(up to grade 5) Lymph infiltration (grade 1)
Pancreas degeneration / ductal hyperplasia in ductal hyperplasia, periductal fibrosis, lymph exocrine part (up to grade 5) infiltration (all grade 2, one animal)
Kidneys Tubular degeneration. Tubular proliferation (Gr 2-3)
Glomerular injury (Gr 4-5 )

Claims

CLAIMS:
1. An isolated anti-TWEAKR antibody comprising a mutated Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region and which comprises: a variable heavy chain comprising the variable heavy chain CDR1 sequence as presented by SEQ ID NO: 6, the variable heavy chain CDR2 sequence as presented by SEQ ID NO: 7, and the variable heavy chain CDR3 sequence as presented by SEQ ID NO: 8, and a variable light chain comprising the variable light chain CDR1 sequence presented by SEQ I NO: 3, the variable light chain CDR2 sequence presented by SEQ I D NO: 4, and the variable light chain CDR3 sequence presented by SEQ I D NO: 5.
2. The antibody according to claim 1 which comprises an amino acid substitution of N297A or N297Q of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
3. The antibody according to claim 1 or claim 2 wherein the antibody binds an Fc gamma receptor, preferentially FcyRIIB, with a KD value which is more than 10 fold higher than that of the parent antibody comprising an unmodified Fc region.
4. The antibody according claim 1 or claim 2 wherein the antibody binds a human FcyRIIB with a KD value larger 50 μΜ.
5. The antibody according to any one of the preceding claims which specifically binds to the D at position 47 (D47) of TWEAKR as depicted in SEQ I D NO: 169.
6. The antibody according to any one of the preceding claims wherein the antibody is an agonistic antibody.
7. The antibody according to any one of the preceding claims comprising: a variable heavy chain sequence as presented by SEQ ID NO: 10 and a variable light chain sequences as presented by SEQ ID NO:9.
8. The antibody according to any one of the preceding claims, which is an IgG antibody, preferably a human IgGl .
9. The antibody according to anyone of the preceding claims comprising heavy chain sequence as presented by SEQ I D NO: 2 1 3 and a light chain sequences as presented by SEQ I D NO:l .
10. The antibody according to any one of the preceding claims, which is a monoclonal antibody.
1 1. The antibody according to any one of the preceding claims, which is a human, humanized or chimeric antibody.
12. An antibody-drug conjugate, comprising an antibody according to any one of claims 1 to 1 1.
13. An isolated nucleic acid sequence that encodes the antibody according to any one of claims 1 to 1 1.
14. A vector comprising a nucleic acid sequence according to claim 13.
15. An isolated cell expressing an antibody according to any one of the claims 1 to 11 and /or comprising a nucleic acid according to claim 13 or a vector according to claim 14.
16. An isolated cell according to claim 15, wherein said cell is a prokaryotic or a eukaryotic cell.
17. A method of producing an antibody according to any one of the claims 1 to 11 comprising culturing of a cell according to claim 16 and purification of the antibody.
18. An antibody according to any one of claims 1 to 11 or an antibody-drug conjugate according to claim 12 for use as a medicament.
19. An antibody according to any one of claims 1 to 1 1 for use as a diagnostic agent.
20. An antibody according to any one of claims 1 to 11 or an antibody-drug conjugate according to claim 12 for use as a medicament for the treatment of cancer.
21. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 1 1 or an antibody-drug conjugate according to claim 12.
22. A combination of a pharmaceutical composition according to claim 21 and one or more therapeutically active compounds.
23. A combination according to claim 22, wherein a therapeutically active compound is an anti- CTLA4. an anti-PD-1 , or an anti-PD-Ll antibody.
24. A method for treating a disorder or condition associated with the undesired presence of TWEAKR, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to claim 21 or a combination according to claim 22 or claim 23.
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