WO2016122702A1 - Multivalent molecules comprising dr5-binding domains - Google Patents

Multivalent molecules comprising dr5-binding domains Download PDF

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WO2016122702A1
WO2016122702A1 PCT/US2015/033099 US2015033099W WO2016122702A1 WO 2016122702 A1 WO2016122702 A1 WO 2016122702A1 US 2015033099 W US2015033099 W US 2015033099W WO 2016122702 A1 WO2016122702 A1 WO 2016122702A1
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seq
domain
amino acid
mab
acid sequence
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French (fr)
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Paul A. Moore
Leslie S. Johnson
Jonathan C. LI
Kalpana SHAH
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Macrogenics Inc
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Macrogenics Inc
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Priority to AU2015380455A priority Critical patent/AU2015380455A1/en
Priority to HK18106692.8A priority patent/HK1247215A1/zh
Priority to US15/545,871 priority patent/US10501552B2/en
Priority to RU2017129236A priority patent/RU2017129236A/ru
Priority to SG11201706024YA priority patent/SG11201706024YA/en
Priority to JP2017539274A priority patent/JP6602875B2/ja
Priority to EP15880591.1A priority patent/EP3250601A4/en
Priority to CN201580074115.8A priority patent/CN107250161A/zh
Application filed by Macrogenics Inc filed Critical Macrogenics Inc
Priority to CA2974807A priority patent/CA2974807A1/en
Priority to KR1020177023834A priority patent/KR20170105622A/ko
Publication of WO2016122702A1 publication Critical patent/WO2016122702A1/en
Priority to IL253668A priority patent/IL253668A0/en
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Multivalent Molecules Comprising DR5-Binding Domains ress- R ⁇ leresxes to Related A kcst siss;:
  • the present invention is directed to multivalent DR5 -Binding Molecules that comprise Binding Domain(s) of anti-DR5 antibodies, and particularly Binding Domain(s) of anti-human DR5 antibodies.
  • the DR5-Binding Molecules of the present invention include bivalent and tetravalent molecules having two, three or four DR5- Binding Domains each capable of binding human DR5.
  • the present invention is directed to multivalent DR5 -Binding Molecules that comprise diabodies, and more particularly, diabodies that comprise a covalently bonded complex of two or more polypeptide chains.
  • the invention particularly pertains to such multivalent DR5- Binding Molecules that comprise fragments of the anti-DR5 antibodies DR5 mAb 1 and/or DR5 mAb 2, and/or humanized and chimeric versions of such antibodies.
  • TNF Tumor Necrosis Factor
  • Fas ligand TNF
  • TNF-related apoptosis- inducing ligand TRAIL
  • TRAIL is a cytokine that is expressed by effector lymphocytes. TRAIL is expressed on the surface of immune effector cells such as natural killer cells, macrophages, dendritic cells and cytotoxic T cells in response to cytokines, particularly interferon- gamma that possesses a response element in the TRAIL gene promoter (Allen, J.E. et al. (2012) "Regulation Of The Human TRAIL Gene,” Cancer Biol. Ther. 13(12): 1143- 1151).
  • TRAIL natural killer
  • TRAIL-R1 also known as DR4
  • TRAIL-R2 also known as DR5
  • TRAIL-R3 DcRl
  • TRAIL-R4 DcR2
  • osteoprotegerin Choaudhari, B.R. et al. (2006) "Following the TRAIL to Apoptosis " Immunologic Res. 35(3):249-262; Carlo-Stella, C. et al. (2007) "Targeting TRAIL Agonistic Receptors for Cancer Therapy " Clin, Cancer 13(8):2313-2317; Allen, J.E. et al.
  • TRAIL-Rl (DR4) is expressed at very low levels in most human tissues including the spleen, thymus, liver, peripheral blood leukocytes, activated T cells, small intestine and some tumor cell lines.
  • TRAIL-R2 (DR5) is ubiquitously distributed both in normal and tumor cell lines but is more abundant in spleen, peripheral blood leukocytes, activated lymphocytes and hepatocytes (Abdulghani, J. et al. (2010) "TRAIL Receptor Signaling And Therapeutics " Expert Opin. Ther. Targets 14(10): 1091-1108).
  • DR4 and DR5 are single-pass type-I membrane proteins and are encoded by two genes located on chromosome 8p. DR4 and DR5 each contain extracellular regions that comprise Cysteine-Rich Domains (CRDs), a Transmembrane Domain, and a Death Domain located within the cytoplasmic portion of the receptors.
  • CRDs Cysteine-Rich Domains
  • DR5(L) long DR5
  • DR5(S) short DR5
  • DR4 and DR5 are able to transduce an apoptosis signal following TRAIL binding (van Roosmalen, I.A.M. et al. (2014) "Two Death-Inducing Human TRAIL Receptors To Target In Cancer: Similar Or Distinct Regulation And Function? “ Biochem. Pharamcol. 91 :447-456).
  • TRAIL When TRAIL binds to DR4 or DR5, the receptors homotrimerize, enabling the receptor's Death Domain to recruit the adaptor protein Fas-Associated Death Domain and the inactive, uncleaved form of caspase 8 (pro-caspase 8) or the uncleaved form of caspase 10 (pro-caspase 10).
  • the receptors, Fas-associated protein with Death Domain, and pro-caspase 8 or pro-caspase 10 together form the Death-Inducing Signaling Complex, (DISC).
  • DISC Death-Inducing Signaling Complex
  • caspase 8 then cleaves downstream substrates ultimately resulting in the cleavage and activation of effector caspase 3.
  • Activation of caspase 3 initiates a cascade of molecular activation events that ultimately leads to the production of death substrates (Schneider-Brachert, W. et al. (2013) "Membrane Trafficking of Death Receptors: Implications on Signalling " Int. J. Mol. Sci. 14:14475-14503; Falschlehner, C. et al.
  • the intrinsic pathway is mediated by the cleavage activation of the pro-apoptotic protein Bid, which then binds with other pro-apoptotic proteins to form a complex that mediates the release of cytochrome c from mitochondria.
  • Such release triggers a cascade of caspase release and activation leading to cell death (Kandasamy, K. et al. (2003) "Involvement Of Proapoptotic Molecules Bax And Bak In Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)- Induced Mitochondrial Disruption And Apoptosis: Differential Regulation Of Cytochrome C And Smac/DIABLO Release," Cancer Res.
  • TRAIL Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand
  • DR4 or DR5 DR preference
  • other tumor types do not (van Roosmalen, I.A.M. et al. (2014) "Two Death-Inducing Human TRAIL Receptors To Target In Cancer: Similar Or Distinct Regulation And Function? " Biochem. Pharamcol. 91 :447-456).
  • TRAIL is highly selective in its ability to recognize and kill damaged cells, while sparing normal cells, soluble recombinant TRAIL has been stated to have potential utility in the treatment of cancer (e.g. , colorectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma; sarcoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer and rectal cancer (see, Micheau, O. et al. (2013) "Death Receptors As Targets In Cancer," Br. J. Pharmacol. 169:1723-1744); Falschlehner, C.
  • cancer e.g. , colorectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma; sarcoma, non-Hodgkin's lymphoma,
  • Anti-DR4 and anti-DR5 monoclonal antibodies that might be capable of mimicking the signaling of TRAIL have been proposed as providing greater selectivity (Buchsbaum, D.J. et al. (2006) “TRAIL Receptor-Targeted Therapy,” Future Oncol. 2:493-508; Kelley, S. . et al. (2004) “Targeting Death Receptors In Cancer With Apo2L/TRAIL,” Curr. Opin. Pharmacol. 4:333-339; Papenfuss, . et al. (2008) “Death Receptors As Targets For Anti-Cancer Therapy," J. Cell. Mol. Med. 12:2566-2585; de Bruyn, M. et al. (2013) “Antibody-Based Fusion Proteins To Target Death Receptors In Cancer,” Cancer Lett. 332: 175-183).
  • mapatumumab Three Phase II clinical studies of mapatumumab, an anti-DR4 agonist antibody (Human Genome Sciences) have been reported to show a therapeutic effect in patients suffering from non-Hodgkin's lymphoma (NHL), colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) (Greco, F.A. et al. (2008) "Phase 2 Study Of Mapatumumab, A Fully Human Agonistic Monoclonal Antibody Which Targets And Activates The TRAIL Receptor-I, In Patients With Advanced Non-Small Cell Lung Cancer," Lung Cancer 61 :82-90; Trarbach, T. et al.
  • TRAIL- Rl Tumour Necrosis Factor Apoptosis-Inducing Ligand Receptor- 1
  • TRA-8/CS-1008 a humanized anti-DR5 antibody (Daiichi Sankyo (Tokyo, Japan)) is reported to have exhibited high antitumor activity against astrocytoma and leukemia cells in vitro and engrafted breast cancer cells in vivo (Buchsbaum, D.J. et al. (2003) "Antitumor Efficacy Of TRA-8 Anti-DR5 Monoclonal Antibody Alone Or In Combination With Chemotherapy And/Or Radiation Therapy In A Human Breast Cancer Model," Clin. Cancer Res. 9:3731-3741; Ichikawa, K. et al.
  • mDRA-6 (IgGl-k), a murine anti-human anti-DR5 monoclonal antibody (Henan University) has been reported to be able to induce the apoptosis of Jurkat cells via the TRAIL extrinsic pathway (Du, Y.-W. et al. (2011) ' Novel Agonistic Anti-Human Death Receptor 5 Monoclonal Antibody With Tumoricidal Activity Induces Caspase- And Mitochondrial-Dependent Apoptosis In Human Leukemia Jurkat Cells," Cancer Biother. Radiopharmaceut. 26(2): 143-152).
  • the chimeric DR-5 -targeting antibody LBY135 (Novartis) has been reported to have induced apoptosis in 50% of a panel of 40 human colon cancer cell lines with an IC50 of 10 nM or less and to have verified in vivo antitumor activity in human colorectal xenograft models in mice (Li, J. et al. (2008) "LBYI35, A Novel Anti-DR5 Agonistic Antibody Induces Tumor Cell-Specific Cytotoxic Activity In Human Colon Tumor Cell Lines And Xenografts ,” Drug Dev. Res. 69:69- 82; Sharma, S. et al.
  • lexatumumab an anti-DR5 agonist antibody
  • Human Genome Sciences Human Genome Sciences
  • drozitumab Kang, Z. et al. (2011) “Drozitumab, A Human Antibody To Death Receptor 5, Has Potent Antitumor Activity against Rhabdomyosarcoma With The Expression Of Caspase-8 Predictive Of Response,” Clin. Cancer Res. 17(10):3181- 3192; Zinonos, I. et al.
  • Anti-DR antibodies are disclosed in United States Patents No. 8,790,663; 8,715,668; 8,703,712; 8,461,311; 8,409,570; 8,372,396; 8,329,180; 8,173,128; 8,097,704; 8,067,001; 8,030,023; 8,029,783; 7,981,421; 7,897,730; 7,893,216; 7704502 and 7,476,383; in United States Patent Publications No.
  • Bispecific antibody molecules having an scFv Domain capable of binding to a tumor antigen and a soluble TRAIL (sTRAIL) or Fas (CD95) Ligand (FasL) Domain capable of binding to a death receptor or to Fas, have also been proposed (see, Wajant, H. et al. (2013) " 'Engineering Death Receptor Ligands For Cancer Therapy," Cane. Lett. 332: 163-174).
  • sTRAIL soluble TRAIL
  • Fas CD95
  • FasL Fas
  • FasL Fas
  • Such genetic fusion of a tumor-selective antibody fragment to sTRAIL and sFasL yielded highly selective anticancer therapeutics with favorable anticancer features.
  • the employed fusion proteins were twice the size of non- targeted soluble ligands.
  • Bispecific antibody molecules capable of binding to DR5 are disclosed in United States Patent Publications No. 2014/0370019; 2014/0308288; 2013/0243780; 2012/0184718 and 2009/0175854; in European Patent Publication Nos. EP 1790663; EP 2059533; EP 2684896 and EP 2350641; and in WIPO Publications No. WO 2014/159562; WO 2014/161845; WO 2014/050779; WO 2014/009358 and WO 2013/148877.
  • TRAIL has been proposed as a potential therapeutic for the treatment of bacterial pathogens (Benedict, C.A. et al. (2012) "TRAIL: Not Just For Tumors Anymore! “ J. Exp. Med. 209(11): 1903-1906).
  • TRAIL may also have a role in the structural changes in asthmatic airways because it is expressed by various inflammatory cells including eosinophils (Chaudhari, B.R. et al. (2006) “Following the TRAIL to Apoptosis " Immunologic Res. 35(3):249-262).
  • TRAIL resistance may reflect the presence of defects in the TRAIL receptors of the tumor cells, or increased expression of inhibitors that are very selective for death receptors such as FLIP or the decoy receptors TRAIL-R3 and TRAIL-R4. See, Abdulghani, J. et al. (2010) ("TRAIL Receptor Signaling And Therapeutics,” Expert Opin. Ther. Targets 14(10):1091-1108).
  • TRAIL-based therapeutics have typically been proposed only as agents to be provided in concert with other chemotherapeutic agents (Buchsbaum, D.J. et al. (2006) “TRAIL Receptor-Targeted Therapy,” Future Oncol. 2(4):493-508).
  • the present invention is directed to multivalent DR5 -Binding Molecules that comprise Binding Domain(s) of anti-DR5 antibodies, and particularly Binding Domain(s) of anti-human DR5 antibodies.
  • the DR5-Binding Molecules of the present invention include bivalent and tetravalent molecules having two, three or four DR5- Binding Domains each capable of binding human DR5.
  • the present invention is directed to multivalent DR5 -Binding Molecules that comprise diabodies, and more particularly, diabodies that comprise a covalently bonded complex of two or more polypeptide chains.
  • the invention particularly pertains to such multivalent DR5- Binding Molecules that comprise fragments of the anti-DR5 antibodies DR5 mAb 1 and/or DR5 mAb 2, and/or humanized and chimeric versions of such antibodies.
  • the invention provides a multivalent DR5 -Binding Molecule that is a bispecific binding molecule, capable of simultaneously binding to two different epitopes of human Death Receptor 5 (DR5), wherein the multivalent DR5-Binding Molecule comprises four antigen-binding domains each capable of binding human DR5.
  • the invention also provides a multivalent DR5 -Binding Molecule that is a monospecific binding molecule, capable of binding to an epitope of human DR5, wherein the multivalent DR5 -Binding Molecule comprises four antigen-binding domains each capable of binding human DR5.
  • the invention particularly concerns the embodiment of all such multivalent DR5 -Binding Molecules capable of simultaneously binding to two, three, or four human DR5 polypeptides.
  • the invention further concerns the embodiments of such multivalent DR5- Binding Molecules, wherein the multivalent DR5 -Binding Molecule is an Fc Region- containing diabody, the diabody being a covalently bonded complex that comprises two pairs of polypeptides, wherein each pair comprises a first polypeptide chain and a second polypeptide chain.
  • the invention further concerns the embodiments of such multivalent DR5- Binding Molecules, wherein:
  • the first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a variable light chain (VL) Domain of a monoclonal antibody capable of binding to a first D 5 epitope (VLl);
  • VH variable heavy chain
  • the CDRLI Domain, CDRL2 Domain, and CDRL3 Domain are the Light Chain CDRs of DR5 mAb 2, and, respectively have the amino acid sequences: SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81, and the CDRHI Domain, CDRH2 Domain, and CDRH3 Domain are the Heavy Chain CDRs of DR5 mAb 2, and respectively have the amino acid sequences: SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO:85; or
  • the VLl has the amino acid sequence of SEQ ID NO: 70, and the VH1 has the amino acid sequence of SEQ ID NO:74; or
  • the VLl has the amino acid sequence of SEQ ID NO: 78, and the VH1 has the amino acid sequence of SEQ ID NO:82; or
  • the VLl has the amino acid sequence of SEQ ID NO: 86, and the VH1 has the amino acid sequence of SEQ ID NO:90; or
  • the VLl has the amino acid sequence of SEQ ID NO: 94, and the VH1 has the amino acid sequence of SEQ ID NO:98; and wherein:
  • VH2 has the amino acid sequence of SEQ ID NO:8; or
  • the VL2 has the amino acid sequence of SEQ ID NO: 13, and the VH2 has the amino acid sequence of SEQ ID NO: 18; or (iii) the VL2 has the amino acid sequence of SEQ ID NO:23, and the VH2 has the amino acid sequence of SEQ ID NO:31; or
  • VL2 has the amino acid sequence of SEQ ID NO: 25
  • VH2 has the amino acid sequence of SEQ ID NO:31
  • VL2 has the amino acid sequence of SEQ ID NO: 27, and the VH2 has the amino acid sequence of SEQ ID NO:31;
  • the VL2 has the amino acid sequence of SEQ ID NO: 70, and the VH1 has the amino acid sequence of SEQ ID NO:74; or
  • the VL2 has the amino acid sequence of SEQ ID NO: 86, and the VH1 has the amino acid sequence of SEQ ID NO:90; or
  • the VL2 has the amino acid sequence of SEQ ID NO: 94, and the VH2 has the amino acid sequence of SEQ ID NO:98.
  • the invention further concerns the embodiments of such multivalent DR5- Binding Molecules, wherein the VLl and the VL2 do not have the same amino acid sequence, and wherein the VH1 and the VH2 do not have the same amino acid sequence.
  • the invention further concerns the embodiments of such multivalent DR5- Binding Molecules, wherein the multivalent DR5 -Binding Molecule is an Fc Region- containing diabody, the diabody being a covalently bonded complex that comprises two pairs of polypeptides wherein:
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 120, and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 118; or
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 134 or SEQ ID NO: 138, and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 136; or
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 140 or SEQ ID NO: 144, and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 142; or
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 152 or SEQ ID NO: 156, and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 154; or
  • the invention further concerns methods of promoting cell death comprising exposing a cell to any of the above described multivalent DR5-Binding Molecules. In particular, where the cell is a tumor cell.
  • the invention further concerns such methods of promoting cell death further comprising exposing the cell to a histone deacetylase inhibitor.
  • the invention further concerns the embodiments in which any of the above- described multivalent DR5 -Binding Molecules is used in the treatment of cancer.
  • the invention further concerns the embodiments in which any of the above-described multivalent DR -binding molecules is used in combination with a histone deacetylase inhibitor in the treatment of cancer.
  • the invention further concerns the embodiments in which any of the above- described multivalent DR5 -Binding Molecules is detectably lab led and is used in the diagnosis or prognosis of cancer.
  • the invention particularly concerns such use of any of the above described multivalent DR5 -Binding Molecules in the treatment or diagnosis or prognosis of cancer, wherein the cancer is characterized by the presence of a cancer cell selected from the group consisting of a cell of: an adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma,
  • the invention particularly concerns such use of any of the above described multivalent DR5 -Binding Molecules in the treatment or diagnosis or prognosis of cancer, wherein the cancer is exerciserectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma; sarcoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer or a rectal cancer.
  • the invention particularly concerns such use of any of the above described multivalent DR5 -Binding Molecules in the treatment or diagnosis or prognosis of cancer, wherein the cancer is acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute B lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphomas (NHL), including mantel cell leukemia (MCL), and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, or Burkitt's lymphoma.
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • B-ALL acute B lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • HCL
  • Figure 1 provides a schematic of a representative covalently bonded diabody molecule having two epitope binding sites composed of two polypeptide chains, each having an E-coil or K-coil Heterodimer-Promoting Domain. VL and VH Domains that recognize the same epitope are shown using the same shading.
  • the epitopes are different epitopes of the same antigen resulting in a bispecific molecule that is monovalent for each epitope, but is bivalent with respect to the antigen (e.g., DR5).
  • the epitopes are the same epitope (e.g., the same VL Domain CDRs and VH Domain CDRs are used on each chain) resulting in a monospecific molecule that is bivalent.
  • Figure 2 provides a schematic of a representative covalently bonded diabody molecule having two epitope binding sites composed of two polypeptide chains, each having a CH2 and CH3 Domain, such that the associated chains form an Fc Region that comprises all or part of a naturally occurring Fc Region.
  • VL and VH Domains that recognize the same epitope are shown using the same shading.
  • the epitopes are different epitopes of the same antigen resulting in a bispecific molecule that is monovalent for each epitope, but is bivalent with respect to the antigen (e.g. , DR5).
  • the epitopes are the same epitope (e.g., the same VL Domain CDRs and VH Domain CDRs are used on each chain) resulting in a monospecific molecule that is bivalent.
  • Figure 3 provides a schematic showing a representative tetravalent diabody molecule composed of two pairs of polypeptide chains (i.e. , four polypeptide chains in all).
  • One polypeptide of each pair possesses a CH2 and CH3 Domain, such that the associated chains form an Fc Region that comprises all or part of a naturally occurring Fc Region.
  • VL and VH Domains that recognize the same epitope are shown using the same shading.
  • the two pairs of polypeptide chains may be same.
  • the resulting molecule is bispecific and bivalent with respect to each bound epitope.
  • the resulting molecule is monospecific and tetravalent with respect to a single epitope.
  • the two pairs of polypeptides may be different.
  • the resulting molecule is tetraspecific and monovalent with respect to each bound epitope.
  • the resulting molecule is tetravalent with respect to the antigen (e.g., DR5).
  • the epitopes are the same epitope (e.g., the same 3 CDRLS and the same 3 CDRHS domains are used on each chain), the resulting molecule is monospecific and tetravalent.
  • the resulting molecule is monospecific and tetravalent with respect to a single epitope.
  • the two pairs of polypeptides may be different.
  • the resulting molecule is tetraspecific and monovalent with respect to each bound epitope.
  • the epitopes are all epitopes of the same antigen, the resulting molecule is tetravalent with respect to the antigen (e.g., DR5).
  • Figure 5 shows the ability of anti-human DR5 monoclonal antibodies DR5 mAb 1 and DR5 mAb 2 to bind to human DR5 and to the DR5 of cynomolgus monkey.
  • FIG. 6 Panels A-H, show the kinetics of binding of DR5 mAb 1 (Panels A and E), DR5 mAb 2 (Panels B and F), DR5 mAb 3 (Panels C and G) and DR5 mAb 4 (Panels D and H) for human DR 5 (Panels A-D) and for cynomolgus monkey DR5 (Panels E-H).
  • Figure 8A shows histological stains of normal colon (Panel A), kidney (Panel B), lung (Panel C), heart (Panel D), liver (Panel E) and pancreas (Panel F) tissue incubated with labeled DR5 mAb 2 (5 ⁇ g/mL).
  • Figure 8B shows histological stains of tumorous colon (Panels A and B) and tumorous lung (Panels C and D).
  • Figure 8B, Panels A and C show the results of tissue incubated with labeled DR5 mAb 2 (5 ⁇ g/mL).
  • Figure 8B, Panels B and D show the results of tissue incubated with labeled isotype control mAb (5 ⁇ g mL).
  • Figures 9A-9K show the ability of the DR5 mAb 2 x CD3 mAb 2 diabody to mediate the cytotoxicity of 7860 renal cell adenocarcinoma cells (Figure 9A), A498 kidney carcinoma cells (Figure 9B), AsPC 1 pancreatic adenocarcinoma cells (Figure 9C), LNCap androgen-sensitive human prostate adenocarcinoma cells ( Figure 9D), SW48 colorectal adenocarcinoma cells ( Figure 9E), A549 adenocarcinomic human alveolar basal epithelial cells (Figure 9F), SKMES human lung cancer cells (Figure 9G), DU145 human prostate cancer cells ( Figure 9H), A375 human malignant melanoma cells ( Figure 91), SKBR3 human HER2-overexpressing breast carcinoma cells (Figure 9J) and JIMT human breast carcinoma cells (Figure 9K).
  • Such target cells were incubated in the presence of peripheral blood mononuclear cells (PBMC) for 24 hours at an effector to target cell ratio of 20:1 or 30:1.
  • PBMC peripheral blood mononuclear cells
  • the percentage cytotoxicity of the target cells was determined by measuring the release of lactate dehydrogenase (LDH) into the media by damaged cells.
  • Figures 10A-10F show the unexpected superiority of DR5 mAb 1 and DR5 mAb 2. Superiority was assessed by comparing the ability of DR5 x CD3 diabodies having the VL and VH Domains of DR5 mAb 1, DR5 mAb 2, DR5 mAb 3, or DR5 mAb 4, to mediate the cytotoxicity of tumor cells.
  • Figure 11 shows the ability of DR5 mAb 2 x CD3 mAb 2 diabody and its humanized derivatives: hDR5 mAb 2 (2.2) x CD3 mAb 2, hDR5 mAb 2 (2.3) x CD3 mAb 2, hDR5 mAb 2 (2.4) x CD3 mAb 2, or hDR5 mAb 2 (2.5) x CD3 mAb 2 to simultaneously bind to DR5 and to CD3.
  • Figure 12 shows the ability of DR5 mAb 2 x CD3 mAb 2 diabody and its humanized derivatives: hDR5 mAb 2 (2.2) x CD3 mAb 2, hDR5 mAb 2 (2.3) x CD3 mAb 2, hDR5 mAb 2 (2.4) x CD3 mAb 2, or hDR5 mAb 2 (2.5) x CD3 mAb 2 to mediate the cytotoxicity of Colo205 colorectal carcinoma cells.
  • Figure 13 shows the growth inhibition curves of COLO205 cells treated with DR5 mAb I, DR5 mAb 2, cross-linked DR5 mAb 1, cross-linked DR5 mAb 2, or the combination of DR5 mAb 1 and DR5 mAb 2 without cross-linking.
  • Cross-linked DR5 mAb 1, cross-linked DR5 mAb 2, and the combination of DR5 mAb 1 and DR5 mAb 2 without cross-linking are able to inhibit the growth of COLO205 cells.
  • Figure 14A-14C show that both cross-linked DR5 mAb 1 and cross-linked DR5 mAb 2 induce apoptosis as measured by increased production of nucleosomes (Figure 14A), increased cleaved PARP (Figure 14B), and increased active caspase 3 ( Figure 14C).
  • Figure 15A-15B show that a representative tetravalent DR5-Binding Molecule (a bispecific E-coil/K-coil-Fc Region-containing diabody tetravalent for DR5 designated "DR5 mAb 2 x DR5 mAb 1 Fc diabody") does not bind normal tissues.
  • Figure 15A shows histological stains of normal colon (Panels A and G), liver (Panels B and H), lung (Panels C and I), pancreas (Panels D and J), kidney (Panels E and K) and heart (Panels F and L) tissue.
  • FIG 15B Top Panel shows the results of tissue incubated with a labeled tetravalent DR5 -Binding Molecule (DR5 mAb 2 x DR5 mAb 1 Fc diabody at 0.625 ⁇ g/mL).
  • Figure 15 B Bottom Panel show the results of tissue incubated with labeled control diabody (4-4-20 x CD3 mAb 2 at 0.625 ⁇ g/mL).
  • Figure 16 show that a representative tetravalent DR5 -Binding Molecule (a bispecific E-coil/K-coil-Fc Region-containing diabody tetravalent for DR5) strongly binds tumorous tissues.
  • Figure 16 shows histological stains of tumorous breast (Panels A and E), tumorous colon (Panels B and F), tumorous lung (Panels C and G), and tumorous prostate tissue (Panels D and H).
  • Panels A-D show the results of tissue incubated with a labeled tetravalent DR5 -Binding Molecule (DR5 mAb 2 x DR5 mAb 2 Fc diabody at 0.625 ⁇ g/mL).
  • Panels E-H show the results of tissue incubated with labeled control diabody (4-4-20 x CD3 mAb 2 at 0.625 ⁇ g/mL).
  • Figure 17A-17C shows the growth inhibition curves of COLO205 (Figure 17A), A498 (Figure 17B) and S MES ( Figure 17C) cells treated with six different representative tetravalent DR5 -Binding Molecules (monospecific or bispecific E- coil/ -coil-Fc Region-containing diabodies tetravalent for DR5) including two comprising Fc Region variants with reduce binding to FcyRs and reduced effector function activity. All the tetravalent DR -Binding Molecules have potent cytotoxicity in these cells and were more potent than the TRAIL-His positive control.
  • Figure 18 shows that representative tetravalent DR5-Binding Molecules (monospecific or bispecific E-coil/K-coil-Fc Region-containing diabodies tetravalent for DR5) induce apoptosis as measured by increased production of nucleosomes.
  • Figure 19 shows the growth inhibition curves of COLO205 cells treated with different DR5 -Binding Molecules including three different representative tetravalent DR5 -Binding Molecules (monospecific or bispecific E-coil/K-coil-Fc Region-containing diabodies tetravalent for DR5); and two different anti-DR5 antibodies (DR5 mAb 8 (KMTR2); and DR5 mAb 4 (conatumumab) with and without cross-linking).
  • Each DR5 -Binding Molecule tested comprised an Fc Region variant with reduce binding to FcyRs and reduced effector function activity.
  • Each DR5- Binding Molecule tested comprised an Fc Region variant with reduced binding to FcyRs and reduced effector function activity. All the tetravalent DR5 -Binding Molecules have potent cytotoxicity in these cells and were more potent than the anti- DR5 antibodies DR5 mAb 4 and DR5 mAb 8.
  • Tumor volume is shown as a group mean ⁇ SEM.
  • the present invention is directed to multivalent DR5 -Binding Molecules that comprise Binding Domain(s) of anti-DR5 antibodies, and particularly Binding Domain(s) of anti-human DR5 antibodies.
  • the DR5-Binding Molecules of the present invention include bivalent and tetravalent molecules having two, three or four DR5- Binding Domains each capable of binding human DR5.
  • the present invention is directed to multivalent DR5 -Binding Molecules that comprise diabodies, and more particularly, diabodies that comprise a covalently bonded complex of two or more polypeptide chains.
  • the invention particularly pertains to such multivalent DR5- Binding Molecules that comprise fragments of the anti-DR5 antibodies DR5 mAb 1 and/or DR5 mAb 2, and/or humanized and chimeric versions of such antibodies.
  • the antibodies of the present invention are immunoglobulin molecules capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the Variable Region of the immunoglobulin molecule.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • antibody refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies, and and epitope-binding fragments of any of the above.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type ⁇ e.g.
  • antibodies have been shown to be useful as therapeutic agents. The last few decades have seen a revival of interest in the therapeutic potential of antibodies, and antibodies have become one of the leading classes of biotechnology-derived drugs (Chan, C.E. et al. (2009) "73 ⁇ 4e Use Of Antibodies In The Treatment Of Infectious Diseases," Singapore Med. J. 50(7):663-666). Nearly 200 antibody-based drugs have been approved for use or are under development.
  • the term "monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an antigen. Monoclonal antibodies are highly specific, being directed against a single epitope (or antigenic site).
  • the term "monoclonal antibody” encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') 2 Fv), single-chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity and the ability to bind to an antigen. It is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e.g. , by hybridoma, phage selection, recombinant expression, transgenic animals, etc).
  • the term includes whole immunoglobulins as well as the fragments etc. described above under the definition of "antibody.”
  • Methods of making monoclonal antibodies are known in the art. One method which may be employed is the method of Kohler, G. et al. (1975) "Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity " Nature 256:495-497 or a modification thereof.
  • monoclonal antibodies are developed in mice, rats or rabbits.
  • the antibodies are produced by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations that contain the desired epitope.
  • the immunogen can be, but is not limited to, primary cells, cultured cell lines, cancerous cells, proteins, peptides, nucleic acids, or tissue.
  • Cells used for immunization may be cultured for a period of time (e.g. , at least 24 hours) prior to their use as an immunogen.
  • Cells may be used as immunogens by themselves or in combination with a non-denaturing adjuvant, such as Ribi (see, e.g., Jennings, V.M. (1995) "Review of Selected Adjuvants Used in Antibody Production,” ILAR J. 37(3): 119-125).
  • a non-denaturing adjuvant such as Ribi (see, e.g., Jennings, V.M. (1995) "Review of Selected Adjuvants Used in Antibody Production," ILAR J. 37(3): 119-125).
  • Ribi non-denaturing adjuvant
  • cells should be kept intact and preferably viable when used as immunogens. Intact cells may allow antigens to be better detected than ruptured cells by the immunized animal.
  • the immunogen may be administered multiple times at periodic intervals such as, bi weekly, or weekly, or may be administered in such a way as to maintain viability in the animal ⁇ e.g. , in a tissue recombinant).
  • existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for a desired pathogenic epitope can be sequenced and produced recombinantly by any means known in the art.
  • such an antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation.
  • the sequence encoding the antibody of interest may be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use.
  • the polynucleotide sequence of such antibodies may be used for genetic manipulation to generate the multispeciflc ⁇ e.g., bispecific, trispecific and tetraspecific) molecules of the invention as well as an affinity optimized antibody, a chimeric antibody, a humanized antibody, or a caninized antibody, to improve the affinity, or other characteristics of the antibody.
  • the general principle in humanizing an antibody involves retaining the basic sequence of the antigen-binding portion of the antibody, while swapping the non-human remainder of the antibody with human antibody sequences.
  • Natural antibodies are composed of two Light Chains complexed with two Heavy Chains. Each light chain contains a Variable Domain (VL) and a Constant Domain (CL). Each heavy chain contains a Variable Domain (VH), and three Constant Domains (CHI, CH2 and CH3), and a Hinge Domain located between the CHI and CH2 Domains.
  • VL Variable Domain
  • CL Constant Domain
  • CHI Constant Domain
  • CH2 and CH3 Three Constant Domains
  • Hinge Domain located between the CHI and CH2 Domains.
  • the basic structural unit of naturally occurring immunoglobulins e.g., IgG
  • the amino-terminal (“N") portion of each chain includes a Variable Domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal ("C") portion of each chain defines a constant region, with light chains having a single constant domain and heavy chains usually having three constant domains and a hinge region.
  • the structure of the light chains of an IgG molecule is n-VL-CL-c and the structure of the IgG heavy chains is n-VH-CHl-H-CH2-CH3-c (where H is the hinge region, and n and c represent, respectively, the N-terminus and the C-terminus of the polypeptide).
  • Varaible Domains of an IgG molecule consist of the complementarity determining regions (CDR), which contain the residues in contact with epitope, and non-CDR segments, referred to as framework segments (FR), which in general maintain the structure and determine the positioning of the CDR loops so as to permit such contacting (although certain framework residues may also contact antigen).
  • CDR complementarity determining regions
  • FR framework segments
  • the VL and VH Domains have the structure n-FRl-CDRl-FR2-CDR2- FR3-CDR3-FR4-C.
  • Polypeptides that are (or may serve as) the first, second and third CDR of an antibody Light Chain are herein respectively designated CDRLI Domain, CDRL2 Domain, and CDRL3 Domain.
  • polypeptides that are (or may serve as) the first, second and third CDR of an antibody Heavy Chain are herein respectively designated CDRRI Domain, CDRH2 Domain, and CDRR3 Domain.
  • CDRRI Domain CDRRI Domain
  • CDRH2 Domain CDRH2 Domain
  • CDRR3 Domain CDRR3 Domain
  • the terms CDRLI Domain, CDRL2 Domain, CDRL3 Domain, CDRHI Domain, CDRH2 Domain, and CDRH3 Domain are directed to polypeptides that when incorporated into a protein cause that protein to be able to bind to an specific epitope regardless of whether such protein is an antibody having light and heavy chains or a diabody or a single-chain binding molecule (e.g., an scFv, a BiTe, etc.), or is another type of protein.
  • the invention also encompasses multivalent DR5 -Binding Molecules comprising single-chain Variable Domain fragments ("scFv") of the anti-DR5 antibodies of this invention.
  • Single-chain Variable Domain fragments are made by linking Light and/ or Heavy chain Variable Domain by using a short linking peptide.
  • Bird et al. (1988) ( ⁇ 'Single-Chain Antigen-Binding Proteins," Science 242:423-426) describes example of linking peptides which bridge approximately 3.5 nm between the carboxy terminus of one Variable Domain and the amino terminus of the other Variable Domain. Linkers of other sequences have been designed and used (Bird et al.
  • Linkers can in turn be modified for additional functions, such as attachment of drugs or attachment to solid supports.
  • the single-chain variants can be produced either recombinantly or synthetically.
  • an automated synthesizer can be used for synthetic production of scFv.
  • a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli.
  • Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides.
  • the resultant scFv can be isolated using standard protein purification techniques known in the art.
  • the invention also particularly encompasses multivalent DR5 -Binding Molecules comprising humanized variants of the anti-DR5 antibodies of the invention.
  • humanized antibody refers to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and /or sequence of a human immunoglobulin.
  • the antigen-binding site may comprise either complete variable domains fused onto constant domains or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the Variable Domains.
  • Antigen-binding sites may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign Variable Domains remains (LoBuglio, A.F. et al. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response " Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224).
  • Variable Domains of both heavy and light chains contain three complementarity determining regions (CDRs) which vary in response to the antigens in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs.
  • CDRs complementarity determining regions
  • FRs framework regions
  • the Variable Domains can be "reshaped” or “humanized” by grafting CDRs derived from non-human antibody on the FRs present in the human antibody to be modified.
  • humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies).
  • humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which differ in sequence relative to the original antibody.
  • a number of "humanized” antibody molecules comprising an antigen- binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent or modified rodent V regions and their associated complementarity determining regions (CDRs) fused to human constant domains (see, for example, Winter et al. (1991) "Man-made Antibodies " Nature 349:293-299; Lobuglio et al. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response " Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224 (1989), Shaw et al.
  • CDRs complementarity determining regions
  • Fey Receptors Fey Receptors
  • Fc Region is a domain that is recognized by cellular Fc Receptors (FcyRs).
  • Fc Region is used to define a C-terminal region of an IgG heavy chain.
  • the amino acid sequence of the CH2-CH3 Domain of an exemplary human IgGl is (SEQ ID NO:l):
  • binding does not necessarily require (although it can include) exclusive binding.
  • reference to binding means “specific” binding. Two molecules are said to be capable of binding to one another in a “physiospecific” manner, if such binding exhibits the specificity with which receptors bind to their respective ligands.
  • Each of the two polypeptides of the simplest bispecific DART® diabody comprises three Domains.
  • the first polypeptide comprises (in the N-terminal to C- terminal direction): (i) a First Domain that comprises a binding region of a Light Chain Variable Domain of a first immunoglobulin (VL1), (ii) a Second Domain that comprises a binding region of a Heavy Chain Variable Domain of a second immunoglobulin (VH2), and (iii) a Third Domain that contains a cysteine residue (or a cysteine- containing domain) and a Heterodimer-Promoting Domain that serves to promote heterodimerization with the second polypeptide of the diabody and to covalently bond the diabody' s first and second polypeptides to one another.
  • Exemplary multivalent DR5 -Binding Molecules of the present invention also includes monospecific molecules (e.g. , bispecific antibodies, monospecific diabodies, etc.) possessing at least two, preferably at least four, binding sites which bind the same DR5 epitope. Such molecules are monospecific with respect to said DR5 epitope, and are at least bivalent, preferably tetravalent, with respect to DR5. It will be noted that where more than two binding sites that bind the same DR5 epitope are present, the multivalent DR5-Binding Molecule will remain monospecific with respect to the epitope but will exhibit a higher valency for DR5.
  • monospecific molecules e.g. , bispecific antibodies, monospecific diabodies, etc.
  • amino acid sequences of particular anti-DR5 -Binding Molecules, and polynucleotides encoding the same are provided below.
  • the present invention also encompasses minor variations of these sequences including, for example amino acid substitutions of the C-terminal and/or N- terminal amino acid residues which may be introduced to facilitate subcloning.
  • the VL Domain of DR5 mAb 1 is preferably encoded by a polynucleotide (SEQ ID NO: 7) having the sequence shown below (polynucleotides encoding the CDRL residues are shown in underline): gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctcgggca gagggccacc atctcatgca gggccagcaa agtgtcagt tcctctggct atagttatat gcactggtac caacagaaac caggacagcc acccaaagtc ctcatctttc tttcatccaa cctagattct ggggtccctg ccaggttcag tggcaggg tctgggacag acttcaccct caacatccat
  • VH Domain of D 5 mAb 1 SEQ ID NO:8
  • CDRH residues are shown underlined
  • the C -terminal amino acid may be substituted with alanine to facilitate subcloning of this VH Domain:
  • the VH Domain of DR5 mAb 1 is preferably encoded by a polynucleotide
  • VL Domain of DR5 mAb 2 is preferably encoded by a polynucleotide
  • VH Domain of DR5 mAb 2 is preferably encoded by a polynucleotide
  • the above-described murine anti-human DR5 antibody DR5 mAb 2 was humanized in order to demonstrate the capability of humanizing an anti-human DR5 antibody so as to decrease its antigenicity upon administration to a human recipient.
  • the humanization yielded four humanized VL Domains designated herein as "hDR5 mAb 2 VL-2,” “hDR5 mAb 2 VL-3,” “hDR5 mAb 2 VL-4,” and "hDR5 mAb 2 VL-
  • hDR5 mAb 2 VH-2 one humanized VH Domain, designated herein as "hDR5 mAb 2 VH-2.” Any of the humanized VL Domains may be paired with the humanized VH Domain. Accordingly, any antibody comprising one of the humanized VL Domains paired with the humanized VH Domain is referred to generically as “hDR5 mAb 2,” and particular combinations of humanized VL/VH Domains are referred to by reference to the VL Domain.
  • hDR5 mAb 2 VL-2 is preferably encoded by a polynucleotide (SEQ ID NO: 1]
  • hDR5 mAb 2 VL-4 is preferably encoded by a polynucleotide (SEQ ID NO: 1]
  • hDR5 mAb 2 VL-5 is preferably encoded by a polynucleotide (SEQ ID NO: 1
  • the CDRLI of the VL Domain of hDR5 mAb 2 VL-3, hDR5 mAb 2 VL-4 and hDR5 mAb VL-5 has the amino acid sequence RASQDVNTAVA (SEQ ID NO:165).
  • hDR5 mAb 2 VH-2 is preferably encoded by a polynucleotide (SEQ ID NO: 1
  • DR5 mAb 1 and DR5 mAb 2 a number of additional anti-human DR5 antibodies are known in the art including: drozitumab (designated herein as “DR5 mAb 3"), conatumumab (designated herein as “DR5 mAb 4"), tigatumumab (designated herein as “DR5 mAb 5"), LBY135-1 (designated herein as “DR5 mAb 6”), LBY135-2 (designated herein as “DR5 mAb 7”) and KMTR2 (designated herein as "DR5 mAb 8").
  • the multivalent DR5-Binding Molecules of the instant invention may comprise the CDRs of the VL and/or VH Domains from one or more of DR5 mAb I , DR5 mAb 2, hDR5 mAb2, DR5 mAb 3, DR5 mAb 4, DR5 mAb 5, DR5 mAb 6, DR5 mAb 7, and DR5 mAb 8.
  • the multivalent DR5-Binding Molecules of the instant invention may comprise at least one antigen-binding portion from one or more of DR5 mAb 1, DR5 mAb 2, hDR5 mAb2, DR5 mAb 3, DR5 mAb 4, DR5 mAb 5, DR5 mAb 6, DR5 mAb 7, and DR5 mAb 8.
  • the multivalent DR5 -Binding Molecules of the instant invention comprise at least one antigen-binding portion from DR5 mAb 1 and/or DR5 mAb 2.
  • CDRL2 of DR5 mAb 3 (SEQ ID NO:56): GA NRPS CDRL3 of DR5 mAb 3 (SEQ ID NO:57): NSADS SGNHW
  • CDRL3 of DR5 mAb 4 (SEQ ID NO:65): QQFGSSPWT
  • TKVEIK CDRLI of DR5 mAb 5 (SEQ ID NO:71): KASQDVGTAVA
  • CD LI of DR5 mAb 7 (SEQ ID NO:87): KASQDVNTAIA
  • CDRL3 of DR5 mAb 7 (SEQ ID NO:89): QQHYTTPFT
  • the molecules comprise an Fc Region comprising regions from two or more IgG isotypes (e.g., IgGl , IgG2, IgG3 and IgG4).
  • IgG isotypes exhibit differing physical and functional properties including serum half- life, complement fixation, FcyR binding affinities and effector function activities (e.g., ADCC, CDC, etc.) due to differences in the amino acid sequences of their hinge and/or Fc Regions, for example as described in Flesch and Neppert (1999) J. Clin. Lab. Anal. 14: 141-156; Chappel et al. (1993) J. Biol. Chem.
  • the amino acid modification and IgG hinge/Fc Region may display similar functionality (e.g., increased affinity for FcyRIIA) and may act additively or, more preferably, synergistically to modify the effector functionality in the molecule of the invention, relative to a molecule of the invention comprising a wild-type Fc Region.
  • the amino acid modification and IgG Fc Region may display opposite functionality (e.g., increased and decreased affinity for FcyRIIA, respectively) and may act to selectively temper or reduce a specific functionality in the molecule of the invention, relative to a molecule of the invention not comprising an Fc Region or comprising a wild-type Fc Region of the same isotype.
  • the multivalent DR5 -Binding Molecules of the present invention comprise a variant Fc Region, wherein said variant Fc Region comprises at least one amino acid modification relative to a wild-type Fc Region, such that said molecule has an altered affinity for an FcR, provided that said variant Fc Region does not have a substitution at positions that make a direct contact with FcyR based on crystallographic and structural analysis of Fc-FcR interactions such as those disclosed by Sondermann et al. (2000) Nature 406:267-73.
  • the molecules of the invention comprise variant Fc Regions comprise modification of at least one residue that does not make a direct contact with an FcyR based on structural and crystallographic analysis, e.g. , is not within the Fc-FcyR binding site.
  • Variant Fc Regions are well known in the art, and any known Fc variant may be used in the present invention to confer or modify the effector function exhibited by a molecule of the invention comprising an Fc Region (or portion thereof) as functionally assayed, e.g., in an NK dependent or macrophage dependent assay.
  • Fc Region variants identified as altering effector function are disclosed in the Antibody Engineering Technology Art, and any suitable variant disclosed therein may be used in the present molecules.
  • the multivalent DR5-Binding Molecules of the present invention comprise a variant Fc Region, having one or more amino acid modifications in one or more regions, which modification(s) alter (relative to a wild- type Fc Region) the Ratio of Affinities of the variant Fc Region to an activating FcyR (such as FcyRIIA or FcyRIIIA) relative to an inhibiting FcyR (such as FcyRIIB):
  • multivalent DR5-Binding Molecules of the present invention that possess a variant Fc Region (relative to the wild-type Fc Region) in which the Fc variant has a Ratio of Affinities greater than 1.
  • Such molecules have particular use in providing a therapeutic or prophylactic treatment of a disease, disorder, or infection, or the amelioration of a symptom thereof, where an enhanced efficacy of effector cell function (e.g. , ADCC) mediated by FcyR is desired, e.g., cancer or infectious disease.
  • an Fc variant having a Ratio of Affinities less than 1 mediates decreased efficacy of effector cell function.
  • Table 1 lists exemplary single, double, triple, quadruple and quintuple mutations by whether their Ratio of Affinities is greater than or less than 1.
  • any amino acid modifications e.g., substitutions
  • any amino acid modifications e.g., substitutions
  • any amino acid modifications at any of positions 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301 , 303, 305, 307, 309, 331 , 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 and preferably one or more of the following residues: H280, Q280, Y280, G290, S290, T290, Y290, N294, K295, P296, D298, N298, P298, V298, 1300 or L300.
  • the variant Fc Region has any of the following residues: A256, N268, Q272, D286, Q286, S286, A290, S290, A298, M301 , A312, E320, M320, Q320, R320, E322, A326, D326, E326, N326, S326, K330, T339, A333, A334, E334, H334, L334, M334, Q334, V334, K335, Q335, A359, A360 or A430.
  • any amino acid modifications e.g., substitutions
  • any amino acid modifications e.g., substitutions
  • any amino acid modifications e.g., substitutions
  • any amino acid modifications e.g., substitutions
  • any amino acid modifications e.g., substitutions
  • any of the following residues A255, A256, A258, A267, A268, N268, A272, Q272, A276, A280, A283, A285, A286, D286, Q286, S286, A290, S290, M301, E320, M320, Q320, R320, E322, A326, D326, E326, S326, K330, A331, Q335, A337 or A430.
  • Preferred variants include one or more modifications at any of positions: 228, 230, 231, 232, 233, 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 271, 273, 275, 281, 284, 291, 296, 297, 298, 299, 302, 304, 305, 313, 323, 325, 326, 328, 330 or 332.
  • Particularly preferred variants include one or more modifications selected from groups A-AI:
  • the invention encompasses the use of any Fc variant known in the art, such as those disclosed in Jefferis, B.J. et al. (2002) "Interaction Sites On Human IgG-Fc For FcgammaR: Current Model ,” Immunol. Lett. 82:57-65; Presta, L.G. et al. (2002) “Engineering Therapeutic Antibodies For Improved Function,' 1 Biochem. Soc. Trans. 30:487-90; Idusogie, E.E. et al. (2001) "Engineered Antibodies With Increased Activity To recruit Complement " J. Immunol. 166:2571-75; Shields, R.L. et al.
  • the molecules of the invention further comprise one or more glycosylation sites, so that one or more carbohydrate moieties are covalently attached to the molecule.
  • the molecules of the invention with one or more glycosylation sites and/or one or more modifications in the Fc Region confer or have an enhanced antibody mediated effector function, e.g., enhanced ADCC activity, compared to to the unmodified molecule.
  • the invention further comprises molecules comprising one or more modifications of amino acids that are directly or indirectly known to interact with a carbohydrate moiety of the Fc Region, including but not limited to amino acids at positions 241, 243, 244, 245, 245, 249, 256, 258, 260, 262, 264, 265, 296, 299, and 301.
  • Amino acids that directly or indirectly interact with a carbohydrate moiety of an Fc Region are known in the art, see, e.g. , Jefferis et al., 1995 Immunology Letters, 44: 111-7, which is incorporated herein by reference in its entirety.
  • the invention encompasses molecules that have been modified by introducing one or more glycosylation sites into one or more sites of the molecules, preferably without altering the functionality of the molecules, e.g., binding activity to target antigen or FcyR.
  • Glycosylation sites may be introduced into the variable and/or constant region of the molecules of the invention.
  • "glycosylation sites” include any specific amino acid sequence in an antibody to which an oligosaccharide (i.e. , carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach. Oligosaccharide side chains are typically linked to the backbone of an antibody via either N-or O-linkages.
  • An exemplary N-linked glycosylation site that is useful in accordance with the methods of the present invention is the amino acid sequence: Asn-X-Thr/Ser, wherein X may be any amino acid and Thr/Ser indicates a threonine or a serine.
  • a site or sites may be introduced into a molecule of the invention using methods well known in the art to which this invention pertains (see for example, IN VITRO MUTAGENESIS, RECOMBINANT DNA: A SHORT COURSE, J. D. Watson, et al. W.H. Freeman and Company, New York, 1983, chapter 8, pp. 106-1 16, which is incorporated herein by reference in its entirety.
  • An exemplary method for introducing a glycosylation site into a molecule of the invention may comprise: modifying or mutating an amino acid sequence of the molecule so that the desired Asn-X-Thr/Ser sequence is obtained.
  • the invention encompasses methods of modifying the carbohydrate content of a molecule of the invention by deleting one or more endogenous carbohydrate moieties of the molecule.
  • the invention encompasses shifting the glycosylation site of the Fc Region of an antibody, by modifying positions adjacent to 297.
  • the invention encompasses modifying position 296 so that position 296 and not position 297 is glycosylated.
  • Effector function can also be modified by techniques such as by introducing one or more cysteine residues into the Fc Region, thereby allowing interchain disulfide bond formation in this region to occur, resulting in the generation of a homodimeric antibody that may have improved internalization capability and/or increased complement-mediated cell killing and ADCC (Caron, P.C. et al. (1992) "Engineered Humanized Dimeric Forms Of IgG Are More Effective Antibodies " J. Exp. Med. 176:1191-1195; Shopes, B. (1992) "A Genetically Engineered Human IgG Mutant With Enhanced Cytolytic Activity " ⁇ . Immunol. 148(9):2918-2922.
  • Homodimeric antibodies with enhanced antitumor activity may also be prepared using heterobifunctional cross- linkers as described in Wolff, E.A. et al. (1993) "Monoclonal Antibody Homodimers: Enhanced Antitumor Activity In Nude Mice " Cancer Research 53:2560-2565.
  • an antibody can be engineered which has dual Fc Regions and may thereby have enhanced complement lysis and ADCC capabilities (Stevenson, G.T. et al. (1989) "A Chimeric Antibody With Dual Fc Regions (bisFabFc) Prepared By Manipulations At The IgG Hinge " Anti-Cancer Drug Design 3:219-230).
  • Multivalent DR5-Binding Molecules Comprising Diabodies Lacking Fc Regions
  • One embodiment of the present invention relates to multivalent DR5- Binding Molecules comprising or consisting of bispecific diabodies that are capable of binding to a first epitope ("Epitope 1") and a second epitope (“Epitope 2”), wherein the first epitope is an epitope of human DR5 and the second epitope is a different epitope of DR5.
  • such diabodies comprise, and most preferably are composed of, a first polypeptide chain and a second polypeptide chain, whose sequences permit the polypeptide chains to covalently bind to each other to form a covalently associated complex that is capable of simultaneously binding to a first DR5 epitope and the second DR5 epitope.
  • such diabodies may bind the first and second epitope on a single DR5 polypeptide (i.e., bind intramolecularly), or they may bind the first epitope on a one DR5 polypeptide and the second epitope on another DR5 polypeptide (i.e., bind intermolecularly).
  • such diabodies cross-link DR5 molecules that are arrayed on the surface of a cell.
  • the first polypeptide chain of such bispecific diabodies comprises, in the N-terminal to C-terminal direction, an N-terminus, the VL Domain of a first monoclonal antibody capable of binding to either the first or second epitope (i.e., either VLEpitope 1 or VLE P ito P e 2), a first intervening spacer peptide (Linker 1), a VH Domain of a second monoclonal antibody capable of binding to either the second epitope (if such first polypeptide chain contains VLEpitope 1) or the first epitope (if such first polypeptide chain contains VLEpitope 2), a second intervening spacer peptide (Linker 2) optionally containing a cysteine residue, a heterodimer-promoting Domain and a C- terminus ( Figure 1).
  • VL1 and VH1 denote respectively, the Variable Light Chain Domain and Variable Heavy Chain Domain of the first monoclonal antibody.
  • VL2 and VH2 denote respectively, the Variable Light Chain Domain and Variable Heavy Chain Domain of the second antibody.
  • the second polypeptide chain of this embodiment of bispecific diabodies comprises, in the N-terminal to C-terminal direction, an N-terminus, a VL Domain of a monoclonal antibody capable of binding to either the first or the second epitope (i.e., either VLEpitope 1 or VLEpitope 2, and being the VL Domain not selected for inclusion in the first polypeptide chain of the diabody), an intervening linker peptide (Linker 1), a VH Domain of a monoclonal antibody capable of binding to either the second epitope (if such second polypeptide chain contains VLEpitope 1) or to the first epitope (if such second polypeptide chain contains VLEpitope 2), a spacer peptide (Linker 2) optionally containing a cysteine residue, a heterodimer-promoting Domain, and a C-terminus ( Figure 1).
  • Linker 1 an intervening linker peptide
  • Linker 2 optionally containing a cysteine residue, a hetero
  • the VL Domain of the first polypeptide chain interacts with the VH Domain of the second polypeptide chain to form a first functional antigen-binding site that is specific for DR5 (/. e. , either the first or the second epitope).
  • the VL Domain of the second polypeptide chain interacts with the VH Domain of the first polypeptide chain in order to form a second functional antigen-binding site that is alsospecific for DR5 (i.e., either the second epitope or the first epitope).
  • the selection of the VL and VH Domains of the first and second polypeptide chains is coordinated, such that the two polypeptide chains of the diabody collectively comprise VL and VH Domains capable of binding to both a first epitope of DR5 and to a second epitope of DR5 (i.e., they comprise VLE P ito P e i/ VLE P ito P e ⁇ and VLE P it ope 2/VHE P it ope 2).
  • the length of the intervening linker peptide is selected to substantially or completely prevent the VL and VH Domains of the polypeptide chain from binding to one another.
  • the VL and VH Domains of the first polypeptide chain are substantially or completely incapable of binding to one another.
  • the VL and VH Domains of the second polypeptide chain are substantially or completely incapable of binding to one another.
  • a preferred intervening spacer peptide has the sequence (SEQ ID NO:33): GGGSGGGG.
  • the Heterodimer-Promoting Domains of such diabodies are formed from one, two, three or four tandemly repeated coil domains of opposing charge that comprise a sequence of at least six, at least seven or at least eight charged amino acid residues (Apostolovic, B. et al. (2008) "pH-Sensitivity of the E3/K3 Heterodimeric Coiled Coil," Biomacromolecules 9:3173-3180; Arndt, K.M. et al.
  • Helix-stabilized Fv (hsFv) Antibody Fragments Substituting the Constant Domains of a Fab Fragment for a Heterodimeric Coiled-coil Domain," J. Molec. Biol. 312:221-228; Arndt, K.M. et al. (2002) “Comparison of In Vivo Selection and Rational Design of Heterodimeric Coiled Coils," Structure 10:1235-1248; Boucher, C. et al. (2010) “Protein Detection By Western Blot Via Coiled-Coil Interactions," Analytical Biochemistry 399:138-140; Cachia, P.J. et al.
  • Such repeated coil domains may be exact repeats or may have substitutions.
  • the Heterodimer-Promoting Domain of the first polypeptide chain may comprise a sequence of eight negatively charged amino acid residues and the Heterodimer-Promoting Domain of the second polypeptide chain may comprise a sequence of eight negatively charged amino acid residues.
  • the positively charged amino acid may be lysine, arginine, histidine, etc. and/or the negatively charged amino acid may be glutamic acid, aspartic acid, etc.
  • one of the Heterodimer-Promoting Domains will comprise four tandem "E-coil” helical domains (SEQ ID NO:39: EVAALEK- EV AALEK - EV AALEK - EV AALEK), whose glutamate residues will form a negative charge at pH 7, while the other of the Heterodimer-Promoting Domains will comprise four tandem "K-coil” domains (SEQ ID NO:40: KVAALKE -KVAALKE -KVAALKE - KVAALKE), whose lysine residues will form a positive charge at pH 7.
  • Heterodimer-Promoting Domain in which one of the four tandem "E-coil" helical domains of SEQ ID NO:39 has been modified to contain a cysteine residue: E V AACEK - EV AALEK - EV AALEK - EV AALEK (SEQ ID NO:41).
  • a diabody may be modified to contain a polypeptide portion of a serum-binding protein at one or more of the termini of the diabody. Most preferably, such polypeptide portion of a serum-binding protein will be installed at the C-terminus of the diabody.
  • a particularly preferred polypeptide portion of a serum-binding protein for improving the in vivo pharmacokinetic properties of a diabody is the Albumin-Binding Domain (ABD) from streptococcal protein G, and more preferably, the Albumin-Binding Domain 3 (ABD3) of protein G of Streptococcus strain G148 (SEQ ID NO:43): LAEAKVLANR ELDKYGVS DY YKNL I DNAKS AEGVKAL I DE I LAAL P.
  • 64A/65A/71A+66S 64A/65A/71A+66D; 64A/65A/71A+66E; 64A/65A/79A+66S;
  • Variant deimmunized ABD having the amino acid sequence:
  • Monospecific diabodies may readily be generated from homodimerization of polypeptide chains comprising, in the N-terminal to C-terminal direction, an N- terminus, the VL Domain of a monoclonal antibody capable of binding to an epitope of DR5 a first intervening spacer peptide (Linker 1), a VH Domain of a monoclonal antibody capable of binding to the epitope of DR5.
  • the length of the intervening linker peptide (Linker 1 which separates such VL and VH Domains) is selected to substantially or completely prevent the VL and VH Domains of the polypeptide chain from binding to one another.
  • the polypeptide chains may optionally comprise a cysteine-containing peptide which can form a covalent disulfide linkage between the pair of polypeptides.
  • diabodies of the invention may comprise four different chains.
  • the first and third polypeptide chains of such a diabody contain three domains: (i) a VL1 -containing Domain, (ii) a VH2-containing Domain, (iii) Heterodimer-Promoting Domain and (iv) a Domain containing a CH2-CH3 sequence.
  • VH2 Domain of said first polypeptide chain and said VL 1 Domain of said second polypeptide chain form an Antigen-Binding Domain capable of specific binding to a second epitope of DR5;
  • the Fc Region (i.e., CH2-CH3 domains of an IgG heavy chain) may be a variant Fc Region having altered affinity for an FcyR and/or altered effector function and/or altered serum half-life.
  • the Fc Region is a variant lacking the C -terminal residue.
  • Diahodx DH5 Binding Molecules Comprising , K-( oil l leterodimer-
  • [Linker 1] is SEQ ID NO:33;
  • VH2 Domain comprises the VH Domain from an anti-DR5 antibody
  • Linker 2 comprises the amino acids “GGG , " or "GGC,” or is selected from
  • Linker 3 comprises the amino acids “GGG , " or "GGC,” or is selected from
  • Heterodimer-Promoting Domain is an E-coil Domain (SEQ ID NO:39 or
  • [Heterodimer-Promoting Domain] of the first polypeptide chain and the [Heterodimer-Promoting Domain] of the second polypeptide chain are not both E-coil Domains or both K-coil Domains.
  • the Fc Region of the Fc Region-containing diabodies of the present invention may be either a complete Fc Region (e.g. , a complete IgG Fc Region) or only a fragment of a complete Fc Region.
  • the Fc Region of the Fc Region- containing diabodies of the present invention may possess the ability to bind to one or more Fc receptors (e.g., FcyR(s)), more preferably such Fc Region will cause altered binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD 16a) or FcyRIIIB (CD 16b) (relative to the binding exhibited by a wild-type Fc Region) or will substantially eliminate the ability of such Fc Region to bind to inhibitory receptor(s).
  • the Fc Region of the Fc Region-containing diabodies of the present invention may include some or all of the CH2 Domain and/or some or all of the CH3 Domain of a complete Fc Region, or may comprise a variant CH2 and/or a variant CH3 sequence (that may include, for example, one or more insertions and/or one or more deletions with respect to the CH2 or CH3 Domains of a complete Fc Region).
  • Such Fc Regions may comprise non-Fc polypeptide portions, or may comprise portions of non- naturally complete Fc Regions, or may comprise non-naturally occurring orientations of CH2 and/or CH3 Domains (such as, for example, two CH2 Domains or two CH3 Domains, or in the N-terminal to C-terminal direction, a CH3 Domain linked to a CH2 Domain, etc.).
  • IgGl comprising the L234A/L235A substitutions is (SEQ ID NO: 102):
  • a CH2-CH3 domain which inherently exhibits decreased (or substantially no) binding to FcyRIIIA (CD 16a) and/or reduced effector function (relative to the binding exhibited by the wild-type IgGl Fc Region (SEQ ID NO:l)) is utilized.
  • the Fc Region-containing diabodies of the present invention comprise an IgG2 Fc Region (SEQ ID NO: 164) or an IgG4 Fc Region (SEQ ID NO: 103), optionally lacking the C-terminal amino acid residues.
  • an IgG4 Fc Region in utilized also encompasses the introduction of a stabilizing mutation such as S228P, as numbered by the EU index as set forth in Kabat (Lu et al, (2008) "The Effect Of A Point Mutation On The Stability Of Igg4 As Monitored By Analytical Ultracentrifugation," J Pharmaceutical Sciences 97:960-969) to reduce the incidence of strand exchange.
  • a stabilizing mutation such as S228P, as numbered by the EU index as set forth in Kabat (Lu et al, (2008) "The Effect Of A Point Mutation On The Stability Of Igg4 As Monitored By Analytical Ultracentrifugation," J Pharmaceutical Sciences 97:960-969) to reduce the incidence of strand exchange.
  • Other stabilizing mutations known in the art may be introduced into an IgG4 Fc Region (Peters, P et al., (2012) "Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased
  • the Fc Region lacks the C-terminal amino acid residue.
  • the CH2 and/or CH3 Domains of such polypeptide chains need not be identical in sequence, and advantageously are modified to foster complexing between the two polypeptide chains.
  • an amino acid substitution preferably a substitution with an amino acid comprising a bulky side group forming a "knob", e.g., tryptophan
  • the hole e.g., a substitution with glycine
  • Such sets of mutations can be engineered into any pair of polypeptides comprising CH2-CH3 Domains that form an Fc Region.
  • Methods of protein engineering to favor heterodimerization over homodimerization are well known in the art, in particular with respect to the engineering of immunoglobulin-like molecules, and are encompassed herein (see e.g., Ridgway et al. (1996) “ 'Knobs-Into-Holes ' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization, " Protein Engr. 9:617-621, Atwell et al. (1997) "Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library, " J. Mol. Biol.
  • a preferred knob is created by modifying a native IgG Fc Region to contain the modification T366W.
  • a preferred hole is created by modifying a native IgG Fc Region to contain the modification T366S, L368A and Y407V.
  • the protein A binding site of the CH2 and CH3 Domains of one chain is preferably mutated by amino acid substitution at position 435 (H435R) on the third polypeptide containing the "hole” substitutions.
  • H435R amino acid substitution at position 435
  • a preferred sequence for the CH2 and CH3 Domains of the first polypeptide chain of an Fc Region-containing diabody of the present invention will have the "knob- bearing" sequence (SEQ ID NO:52):
  • the first polypeptide chain will have a "knob-bearing" CH2-CH3 sequence, such as that of SEQ ID NO:52.
  • a "hole-bearing" CH2-CH3 Domain e.g., SEQ ID NO:53
  • "knob-bearing" CH2-CH3 Domain e.g., SEQ ID NO:53
  • CD3 is a T cell co-receptor composed of four distinct chains (Wucherpfennig, K.W. et al. (2010) “Structural Biology Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling," Cold Spring Harb. Perspect. Biol. 2(4):a005140; pages 1-14).
  • the complex contains a CD3y chain, a CD36 chain, and two CD3s chains. These chains associate with a molecule known as the T cell receptor (TCR) in order to generate an activation signal in T lymphocytes.
  • TCR T cell receptor
  • TCRs do not assemble properly and are degraded (Thomas, S. et al.
  • OKT3 (Xu et al. (2000) “In Vitro Characterization Of Five Humanized OKT3
  • multivalent DR5 -Binding Molecules possessing at least two, and preferably, at least four DR5 binding sites may have a variety of structures.
  • structures comprising the antigen-binding portions of immunoglobulins, including, but not limited to, IgG-based bispecific antibodies, and molecules comprising diabodies are preferred.
  • Specific, non-limiting, examples of multivalent DR -Binding Molecules comprising diabodies are provided.
  • alternative structures including those disclosed above (see, e.g., Figures 1-4) or otherwise apparent to one of skill in the art are encompassed by the instant invention.
  • Exemplary bispecific Fc Region-Containing diabodies tetravalent for DR5 composed of two pairs of polypeptide chains are constructed having the VL and VH Domains of anti-human DR5 antibody DR5 mAb 1 and the VL and VH Domains of DR5 mAb 2.
  • One Fc Region-Containing diabody designated "DR5 mAb 1 x DR5 mAb 2 Fc diabody,” contains a wild-type IgGl Fc Region.
  • the amino acid sequence of the first polypeptide chain of this Fc Region-Containing diabody is (SEQ ID NO:116):
  • residues 244-271 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:47) residues 244-271 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:47) residues 244-271 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:47) residues 244-271 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:47) residues 244-271 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:47) residues 244-271 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:47) residues 244-271 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:47) residues 244-271 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:47) residues 244-271 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:47) residues
  • residues 272-277 correspond to a LEPKSS linker (SEQ ID NO: 49), residues
  • 278-287 correspond to a linker (DKTHTCPPCP; SEQ ID NO:48) derived from an IgGl hinge domain, and residues 288-503 correspond to a wild-type IgGl Fc Region (SEQ ID NO:48).
  • SEQ ID NO:116 is SEQ ID NO:117:
  • Fc Region-containing diabody designated "DR5 mAb 1 x DR5 mAb 2 Fc diabody (AA)," is identical to DR5 mAb 1 x DR5 mAb 2 Fc diabody except the Fc Region is a variant having a L234A/L235A double mutation (underlined) which reduces/eliminates binding to FcyRIIIA and reduces/eliminates effector functions.
  • the amino acid sequence of the first polypeptide chain of this Fc Region-Containing diabody is (SEQ ID NO:120):
  • a polynucleotide that encodes SEQ ID NO: 120 is SEQ ID NO: 121:
  • the CH2-CH3 region of IgG2 or IgG4 may be used.
  • amino acid residues 288-504 of SEQ ID NOs:116 or 120 will be replaced with SEQ ID NO: 164 (CH2-CH3 of IgG2) or SEQ ID NO: 103 (CH2-CH3 of IgG4), optionally lacking the C-terminal amino acid residue.
  • Exemplary bispecific Fc Region-Containing diabodies tetravalent for DR5 composed of two pairs of polypeptide chains are constructed having the VL and VH Domains of anti-human DR5 antibody DR5 mAb 2 and the VL and VH Domains of DR5 mAb 1.
  • One Fc Region-Containing diabody designated "DR5 mAb 2 x DR5 mAb 1 Fc diabody,” contains a wild-type IgGl Fc Region.
  • the amino acid sequence of the first polypeptide chain of this Fc Region-Containing diabody is (SEQ ID NO:122):
  • a polynucleotide that encodes SEQ ID NO:138 is SEQ ID NO:139:
  • Exemplary monospecific Fc Region-Containing diabodies tetravalent for DR5 composed of two pairs of polypeptide chains are constructed having the VL Domain of anti-human DR5 antibody hDR5 mAb 2 VL-2 and the VH Domain of anti- human DR5 antibody hDR5 mAb 2 VH-2.
  • the first Fc Region-Containing diabody designated "hDR5 mAb 2.2 x hDR5 mAb 2.2 Fc diabody,” contains a wild-type IgGl Fc Region.
  • the amino acid sequence of the first polypeptide chain of this Fc Region- Containing diabody is (SEQ ID NO: 140):
  • residues 116-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 VH-2 (SEQ ID NO:31), residues 235-239 correspond to an ASTKG linker (SEQ ID NO:47) residues 240-267 correspond to a cysteine-containing E-coil
  • residues 274-283 correspond to a linker (DKTHTCPPCP; SEQ ID NO:48) derived from an IgGl hinge domain, and residues 284-499 correspond to a wild-type
  • SEQ ID NO: 14 A polynucleotide that encodes SEQ ID NO: 140 is SEQ ID NO: 141:
  • amino acid sequence of the second polypeptide chain of hDR5 mAb 2.2 x hDR5 mAb 2.2 Fc diabody is (SEQ ID NO: 142):
  • amino acid residues 1-107 correspond to the amino acid sequence of the VL Domain of hDR5 mAb 2 VL-2 (SEQ ID NO:23), residues 108-115 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33), residues 116-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 VH-2 (SEQ ID NO:31), residues 235-239 correspond to an ASTKG linker (SEQ ID NO:47) residues 240-267 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • a polynucleotide that encodes SEQ ID NO:142 is SEQ ID NO: 143:
  • Region-Containing diabody is (SEQ ID NO: 144):
  • a polynucleotide that encodes SEQ ID NO: 144 is SEQ ID NO: 145:
  • the second polypeptide chain of hDR5 mAb 2.2 x hDR5 mAb 2.2 Fc diabody (AA) is also SEQ ID NO: 142 (encoded by SEQ ID NO: 143), described in detail above.
  • the CH2-CH3 region of IgG2 or IgG4 may be used.
  • amino acid residues 284-500 of SEQ ID NOs:140 or 144 will be replaced with SEQ ID NO: 164 (CH2-CH3 of IgG2) or SEQ ID NO: 103 (CH2-CH3 of IgG4), optionally lacking the C-terminal amino acid residue.
  • Exemplary monospecific Fc Region-Containing diabodies tetravalent for DR5 composed of two pairs of polypeptide chains are constructed having the VL Domain of anti-human DR5 antibody hDR5 mAb 2 VL-3 and the VH Domain of anti- human hDR5 antibody hDR5 mAb 2 VH-3.
  • the first Fc Region-Containing diabody designated "hDR5 mAb 2.3 x hDR5 mAb 2.3 Fc diabody,” contains a wild-type IgGl Fc Region.
  • the amino acid sequence of the first polypeptide chain of this Fc Region- Containing diabody is (SEQ ID NO: 146):
  • amino acid residues 1-107 correspond to the amino acid sequence of the VL Domain of hDR5 mAb 2 VL-3 (SEQ ID NO:25), residues
  • residues 116-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 VH-2 (SEQ ID NO:31), residues 235-239 correspond to an ASTKG linker (SEQ ID NO:47) residues 240-267 correspond to a cysteine-containing E-coil
  • residues 274-283 correspond to a linker (DKTHTCPPCP; SEQ ID NO:48) derived from an IgGl hinge domain, and residues 284-499 correspond to a wild-type
  • SEQ ID NO:l lacking the C-terminal amino acid residue.
  • amino acid residues 1-107 correspond to the amino acid sequence of the VL Domain of hDR5 mAb 2 VL-3 (SEQ ID NO:25), residues 108-115 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33), residues 116-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 VH-2 (SEQ ID NO:31), residues 235-239 correspond to an ASTKG linker (SEQ ID NO:47) residues 240-267 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • a polynucleotide that encodes SEQ ID NO:148 is SEQ ID NO: 149:
  • Fc Region-containing diabody designated "hDR5 mAb 2.3 x hDR5 mAb 2.3 Fc diabody (AA),” is identical to hDR5 mAb 2.3 x hDR5 mAb 2.3 Fc diabody except the Fc Region is a variant having a L234A/L235A double mutation (underlined) which reduces/eliminates binding to FcyRIIIA and reduces/eliminates effector functions.
  • the amino acid sequence of the first polypeptide chain of this Fc Region-Containing diabody is (SEQ ID NO: 150): DIQMTQSPSF LSASVGDRVT ITCRASQDVN TAVA YQQKP GKAPKLLIY ASTRHTGVPD RFSGSGSGTD FTLTISSLQP EDVATYYCQQ HYITPWTFGG GTKLEIKGGG SGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYTFTEYILH WVRQAPGQGL EWMGWFYPGN NNIKYNEKFK DRVTITADKS TSTVYMELSS LRSEDTAVYY CARHEQGPGY FDYWGQGTLV TVSSASTKGE VAACEKEVAA LEKEVAALEK EVAALEKLEP KSSDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFN YVDGVEVHNA KTK
  • a polynucleotide that encodes SEQ ID NO: 150 is SEQ ID NO: 151 :
  • the second polypeptide chain of hDR5 mAb 2.3 x hDR5 mAb 2.3 Fc diabody (AA) is also SEQ ID NO: 148 (encoded by SEQ ID NO: 149), described in detail above.
  • SEQ ID NO: 149 encoded by SEQ ID NO: 149
  • the CH2-CH3 region of IgG2 or IgG4 may be used.
  • amino acid residues 284-500 of SEQ ID NOs:146 or 150 will be replaced with SEQ ID NO: 164 (CH2-CH3 of IgG2) or SEQ ID NO: 103 (CH2-CH3 of IgG4), optionally lacking the C-terminal amino acid residue.
  • Exemplary monospecific Fc Region-Containing diabodies tetravalent for DR5 composed of two pairs of polypeptide chains are constructed having the VL Domain of anti-human DR5 antibody hDR5 mAb 2 VL-4 and the VH Domain of anti- human hDR5 antibody hDR5 mAb 2 VH-4.
  • the first Fc Region-Containing diabody designated "hDR5 mAb 2.4 x hDR5 mAb 2.4 Fc diabody,” contains a wild-type IgGl Fc Region.
  • the amino acid sequence of the first polypeptide chain of this Fc Region- Containing diabody is (SEQ ID NO: 152
  • amino acid residues 1-107 correspond to the amino acid sequence of the VL Domain of hDR5 mAb 2 VL-4 (SEQ ID NO:27), residues 108-115 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33), residues 116-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 VH-2 (SEQ ID NO:31), residues 235-239 correspond to an ASTKG linker (SEQ ID NO:47) residues 240-267 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:41), residues 268-273 correspond to a LEPKSS linker (SEQ ID NO: 49), residues 274-283 correspond to a linker (DKTHTCPPCP; SEQ ID NO:48) derived from an IgGl hinge domain, and residues 284-499 correspond to a wild-type IgGl Fc Region (SEQ ID NO:
  • amino acid sequence of the second polypeptide chain of hDR5 mAb 2 x hDR5 mAb 2 Fc diabody is (SEQ ID NO: 154):
  • amino acid residues 1-107 correspond to the amino acid sequence of the VL Domain of hDR5 mAb 2 VL-4 (SEQ ID NO: 17)
  • residues 108-115 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 116-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 VH-2 (SEQ ID NO:31)
  • residues 235-239 correspond to an ASTKG linker (SEQ ID NO:47)
  • residues 240-267 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • a polynucleotide that encodes SEQ ID NO:154 is SEQ ID NO: 155:
  • hDR5 mAb 2.4 x hDR5 mAb 2.4 Fc diabody (AA) is identical to hDR5 mAb 2.4 x hDR5 mAb 2.4 Fc diabody except the Fc Region is a variant having a L234A/L235A double mutation
  • Region-Containing diabody is (SEQ ID NO: 156):
  • a polynucleotide that encodes SEQ ID NO: 156 is SEQ ID NO: 157:
  • the second polypeptide chain of hDR5 mAb 2 x hDR5 mAb 2 Fc diabody is also SEQ ID NO: 154 (encoded by SEQ ID NO: 155), described in detail above.
  • the CH2-CH3 region of IgG2 or IgG4 may be used.
  • amino acid residues 284-500 of SEQ ID NOs:152 or 156 will be replaced with SEQ ID NO: 164 (CH2-CH3 of IgG2) or SEQ ID NO: 103 (CH2-CH3 of IgG4), optionally lacking the C-terminal amino acid residue.
  • Exemplary monospecific Fc Region-Containing diabodies tetravalent for DR5 composed of two pairs of polypeptide chains are constructed having the VL Domain of anti-human DR5 antibody hDR5 mAb 2 VL-5 and the VH Domain of anti- human hDR5 antibody hDR5 mAb 2 VH-2.
  • the first Fc Region-Containing diabody designated "hDR5 mAb 2.5 x hDR5 mAb 2.5 Fc diabody,” contains a wild-type IgGl Fc Region.
  • the amino acid sequence of the first polypeptide chain of this Fc Region- Containing diabody is (SEQ ID NO: 158):
  • amino acid residues 1-107 correspond to the amino acid sequence of the VL Domain of hDR5 mAb 2 VL-5 (SEQ ID NO:29), residues
  • residues 116-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 VH-2 (SEQ ID NO:31), residues 235-239 correspond to an ASTKG linker (SEQ ID NO:47) residues 240-267 correspond to a cysteine-containing E-coil
  • residues 274-283 correspond to a linker (DKTHTCPPCP; SEQ ID NO:48) derived from an IgGl hinge domain, and residues 284-499 correspond to a wild-type
  • SEQ ID NO: 158 is SEQ ID NO: 159:

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US10501552B2 (en) 2019-12-10
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US20180016344A1 (en) 2018-01-18
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