WO2016122701A1 - Anticorps anti-dr5 et molécules comprenant des domaines de liaison de dr5 de ceux-ci - Google Patents

Anticorps anti-dr5 et molécules comprenant des domaines de liaison de dr5 de ceux-ci Download PDF

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WO2016122701A1
WO2016122701A1 PCT/US2015/033082 US2015033082W WO2016122701A1 WO 2016122701 A1 WO2016122701 A1 WO 2016122701A1 US 2015033082 W US2015033082 W US 2015033082W WO 2016122701 A1 WO2016122701 A1 WO 2016122701A1
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amino acid
human
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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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    • 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
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2809—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 immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—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

  • the present invention is directed to the anti-DR5 antibody DR5 mAb 1 and DR5 mAb 2, and to humanized and chimeric versions of such antibodies.
  • the invention additionally pertains to DR5 -binding molecules that comprise fragments of such molecules, and to bispecific molecules, including diabodies, BiTEs, knobs/holes bispecific antibodies, etc., that comprise: (i) such DR5-binding fragments and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell.
  • 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-induced apoptosis is largely dependent on cellular damage recognition by, for example, the p53 tumor suppressor protein (Dimberg, L.Y. et al. (2013) "On The TRAIL To Successful Cancer Therapy? Predicting And Counteracting Resistance against TRAIL-Based Therapeutics," Oncogene 32: 1341-1350).
  • the dependence on p53 to elicit an apoptotic response poses a problem in cancer therapy, as loss of p53 occurs in more than half of all cancers cells because of inactivating mutations (Hollstein, M. et al. (1994) "Database Of p53 Gene Somatic Mutations In Human Tumors And Cell Lines " Nucleic Acids Res. 22:3551- 3555).
  • TRAIL is a type II protein with 281 amino acid residues and has homology with TNF-a and FasL (CD95L) (Chaudhari, B.R. et al. (2006) "Following the TRAIL to Apoptosis,” Immunologic Res. 35(3):249-262).
  • TRAIL consists of an extracellular TNF-like Domain, an extracellular stalk, a transmembrane helix, and a Cytoplasmic Domain.
  • TRAIL binds to two different types of receptors: death receptors (DR) that trigger TRAIL-induced apoptosis and decoy receptors inhibit this pathway.
  • DR death receptors
  • 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.
  • TRAIL may mediate cell death via an "intrinsic” pathway (Carlo-Stella, C. et al. (2007) “Targeting TRAIL Agonistic Receptors for Cancer Therapy " Clin, Cancer 13(8):2313-2317; Buchsbaum, D.J. et al. (2006) “TRAIL Receptor-Targeted Therapy,” Future Oncol. 2(4):493-508; Buchsbaum, D.J. et al. (2007) “TRAIL-Receptor-Antibodies as a Potential Cancer Treatment " Future Oncol. 3(4):405-409).
  • 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.
  • Bid pro-apoptotic protein
  • 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.K. et al. (2004) “Targeting Death Receptors In Cancer With Apo2L/TRAIL,” Curr. Opin. Pharmacol. 4:333-339; Papenfuss, K. 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 fromnon-Hodgkin's lymphoma (NHL), colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) (Greco, FA. et al. (2008) "Phase 2 Study Of Mapatumumab, A Fully Human Agonistic Monoclonal Antibody Which Targets And Activates The TRAIL Receptor-1, 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) "A 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) "LBY135, 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.
  • 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
  • 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).
  • soluble TRAIL preparations have been its relatively short in vivo half-life (approximately 30 minutes; Walczak, H. et al. (1999) "Tumoricidal Activity Of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand In Vivo," Nat. Med. 5: 157-163). Additionally, soluble recombinant TRAIL is capable of binding to TRAIL receptors (thus promoting cancer treatment) and to TRAIL decoy receptors (thus putative ly providing no therapeutic benefit). TRAIL may also have a role in cardiovascular disease (Martin- Ventura, J.L. et al.
  • 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 the anti-DR5 antibody DR5 mAb 1 and DR5 mAb 2, and to humanized and chimeric versions of such antibodies.
  • the invention additionally pertains to DR5 -binding molecules that comprise fragments of such molecules, and to bispecific molecules, including diabodies, BiTEs, knobs/holes bispecific antibodies, etc., that comprise: (i) such DR5-binding fragments and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell.
  • the invention provides an anti-human DR5 -binding molecule that comprises a Variable Light Chain Domain and a Variable Heavy Chain Domain, wherein:
  • the Variable Light Chain Domain comprises a CDRLI Domain, a CDRL2 Domain, and a CDRL3 Domain
  • the Variable Heavy Chain Domain comprises a CDRHI Domain, a CDRH2 Domain and a CDRH3 Domain
  • Chain CDRs of DR5 mAb 1 and respectively have the amino acid sequences: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and (2) the CDRHI Domain, CDRH2 Domain, and CDRH3 Domain are the Heavy Chain CDRs of DR5 mAb 1 , and respectively have the amino acid sequences: SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:ll; or
  • Chain CDRs of DR5 mAb 2 and, respectively have the amino acid sequences: SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; and (2) 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:19, SEQ ID NO:20, and SEQ ID NO:21.
  • the invention particularly concerns the embodiment of such anti-human DR5-binding molecule wherein: (1) the CDRLI Domain, CDRL2 Domain, and CDRL3 Domain are the Light Chain CDRs of DR5 mAb 1, and respectively have the amino acid sequences: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and
  • CDRHI Domain is the Heavy Chain CDRs of DR5 mAb 1 , and respectively have the amino acid sequences: SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:ll.
  • the invention further concerns the embodiments of such anti-human DR5- binding molecules wherein the Light Chain Variable Domain has the amino acid sequence of SEQ ID NO:3.
  • the invention further concerns the embodiments of such anti-human DR5- binding molecules wherein the Heavy Chain Variable Domain has the amino acid sequence of SEQ ID NO:8.
  • the invention further concerns the embodiments of such anti-human DR5- binding molecules wherein:
  • 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:14, SEQ ID NO:15, and SEQ ID NO:16; and
  • CDRHI Domain is the Heavy Chain CDRs of DR5 mAb 2, and respectively have the amino acid sequences: SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.
  • the invention further concerns the embodiments of such anti-human DR5- binding molecules wherein the Light Chain Variable Domain has the amino acid sequence of SEQ ID NO: 13.
  • the invention further concerns the embodiments of such anti-human DR5- binding molecules wherein the Heavy Chain Variable Domain has the amino acid sequence of SEQ ID NO: 18.
  • the invention further concerns the embodiments of all such anti-human DR5 -binding molecules wherein the molecule is an antibody, and especially wherein the molecule is a chimeric antibody or a humanized antibody.
  • the invention further concerns the embodiments of all such anti-human DR5 -binding molecules wherein the antibody, chimeric antibody or humanized antibody comprises a variant Fc Region that comprises one or more amino acid modifications that alter the affinity of the variant Fc Region for an FcyR, and especially wherein the modifications decrease the affinity of the variant Fc Region for FcyRIIB.
  • the invention further concerns the embodiments of all such anti-human DR5 -binding molecules wherein the modifications comprise at least one amino acid substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, wherein the numbering is that of the EU index as in Kabat.
  • the invention concerns anti-human DR5 -binding molecules wherein the modifications comprise:
  • the invention further concerns the embodiment wherein the anti-human DR5-binding molecule is a bispecific binding molecule, capable of simultaneously binding to human DR5 and to a second epitope, and particularly concerns the embodiment wherein the second epitope is an epitope of a molecule present on the surface of an effector cell (especially wherein the second epitope is an epitope of CD3, CD 16, CD 19, CD20, CD22, CD32, CD64, TCR, BCR, or NKG2D, and most particularly wherein the second epitope is an epitope of CD3).
  • the invention further concerns the embodiment of such anti-human DR5- binding molecules wherein the molecule is a diabody, the diabody being a covalently bonded complex that comprises two polypeptide chains or that comprises three polypeptide chains.
  • the invention additionally concerns the embodiment of such anti- human DR5 -binding molecules wherein the molecule comprises an Fc Region.
  • the invention additionally concerns the embodiment of such anti-human DR5 -binding molecules wherein the molecule comprises an Albumin-Binding Domain, and especially a deimmunized Albumin-Binding Domain.
  • the invention particularly concerns the embodiment of such anti-human DR5 -binding molecules wherein the molecule binds human DR5 and human CD3.
  • the invention particularly concerns the embodiment of such anti-human DR5-binding molecules wherein:
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 140 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 142;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 144 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 146;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 148 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 150;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 152 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 154;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 156 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 158;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 160 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 162;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 164 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 165; or
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 166 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 167.
  • the invention also concerns the embodiment wherein the anti-human DR5- binding molecule binds human DR5 and human CD3 and additionally comprises an Fc Region, and especially, wherein the DR5 -binding molecule comprises three polypeptide chains, wherein:
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 168;
  • the second polypeptide chain has the amino acid sequence of SEQ ID NO: 169; and the third polypeptide chain has the amino acid sequence of SEQ ID NO:170;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 171;
  • the second polypeptide chain has the amino acid sequence of SEQ ID NO: 172; and the third polypeptide chain has the amino acid sequence of SEQ ID NO:173;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 174;
  • the second polypeptide chain has the amino acid sequence of SEQ ID NO: 175; and the third polypeptide chain has the amino acid sequence of SEQ ID NO:176;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 177;
  • the second polypeptide chain has the amino acid sequence of SEQ ID NO: 178; and the third polypeptide chain has the amino acid sequence of SEQ ID NO:179;
  • the first polypeptide chain has the amino acid sequence of SEQ ID NO: 180;
  • the second polypeptide chain has the amino acid sequence of SEQ ID NO: 181; and the third polypeptide chain has the amino acid sequence of SEQ ID NO:182.
  • the invention further concerns the embodiments of all such anti-human DR5 -binding molecules wherein the molecule is a chimeric antigen receptor that comprises the Variable Light Chain Domain and the Variable Heavy Chain Domain and an intracellular domain selected from the group consisting of: 41 ⁇ 03 ⁇ , b2c- CD3C, CD28, CD28-4-lBB-CD3C, CD28-CD3C, CD28-Fc8RLy, CD28mut-CD3C, CD28-OX40-CD3C, CD28-OX40-CD3C, CD3C, CD4-CD3C, CD4-Fc8RIy, CD8-CD3C, FcsRLy, FcsRLyCAIX, Heregulin-CD3C, IL-13-CD3C, or Ly49H-CD3C.
  • the molecule is a chimeric antigen receptor that comprises the Variable Light Chain Domain and the Variable Heavy Chain Domain and an intracellular domain selected from the group consisting of: 41 ⁇ 03 ⁇ , b2
  • the invention further concerns the embodiments of all such anti-human DR5 -binding molecules wherein the molecule comprises an Fc Region, and wherein the Fc Region is a variant Fc Region that comprises one or more amino acid modifications that alter the affinity of the variant Fc Region for an FcyR, and more particularly, wherein the modifications comprise at least one amino acid substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, wherein the numbering is that of the EU index as in Kabat.
  • the invention further concerns the embodiments of such variant Fc Region-containing DR5 -binding molecules wherein the modifications comprise:
  • the invention further concerns the embodiments in which any of the above- described DR5 -binding molecules is used in the treatment of cancer. [0040] The invention further concerns the embodiments in which any of the above- described DR5 -binding molecules is detectably labled and is used in the diagnosis or prognosis of cancer.
  • the invention particularly concerns such use 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, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the group consisting
  • the invention particularly concerns such use in the treatment or diagnosis or prognosis of cancer, wherein the cancer is a 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 or a rectal cancer.
  • the cancer is a 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 or a rectal cancer.
  • the invention particularly concerns such use in the treatment or diagnosis or prognosis of cancer, wherein the 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 blastic plasmacytoid dendritic cell neoplasm
  • NHL non-Hod
  • Figure 1 provides a schematic of a covalently bonded diabody composed of two polypeptide chains, each having a Heterodimer-Promoting Domain. VL and VH Domains that recognize the same epitope are shown using the same shading.
  • Figure 2 provides a schematic of a covalently bonded diabody 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.
  • Figures 3A-3B provide schematics showing tetravalent diabodies composed of two pairs of polypeptide chains. The pairs are different, thus resulting in a bispecific molecule that is bivalent with respect to each of two epitopes, in which one is an epitope of DR5 and the other is an epitope of a molecule present on the surface of an effector cell.
  • 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. Only one pair of epitopes (shown with the same shading) is capable of binding to DR5.
  • Figure 3 A shows an Ig diabody, which contains antibody CH and CL domains.
  • Figure 3B shows an Fc diabody, which contains E-coil and K- coil heterodimer-promoting domains.
  • Figures 4A and 4B provide schematics of a covalently bonded diabody composed of three polypeptide chains. Two of the polypeptide chains possess a CH2 and CH3 Domain, such that the associated chains form an Fc Region that comprises all or part of an Fc Region. VL and VH Domains that recognize the same epitope are shown using the same shading.
  • 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).
  • Figures 7A-7B show the ability of DR5 mAb 1 to differentially bind to tumor cells.
  • Figure 7A 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.
  • Figure 7A, Panels A-F show the results of tissue incubated with labeled DR5 mAb 1 (5 ⁇ g/mL).
  • Figure 7A, Panels G- L show the results of tissue incubated with labeled isotype control mAb (5 ⁇ g/mL).
  • Figure 7B shows histological stains of tumorous colon (Panels A and C) and tumorous lung (Panels B and D).
  • Figure 7B, Panels A-B show the results of tissue incubated with labeled DR5 mAb 1 (5 ⁇ g/mL).
  • Figure 7B, Panels C-D show the results of tissue incubated with labeled isotype control mAb (5 ⁇ g/mL).
  • Figures 8A-8B show the ability of DR5 mAb 2 to differentially bind to tumor cells.
  • Figure 8 A 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.
  • the employed target tumor cells were: A549 adenocarcinomic human alveolar basal epithelial cells (Figure 10A), SKMES human lung cancer cells (Figure 10B), DU145 human prostate cancer cells (Figure IOC), A375 human malignant melanoma cells ( Figure 10D), and SKBR3 human HER2-overexpressing breast carcinoma cells (Figure 10E) and JIMT human breast carcinoma cells ( Figure 10F).
  • 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.
  • the present invention is directed to the anti-DR5 antibody DR5 mAb 1 and DR5 mAb 2, and to humanized and chimeric versions of such antibodies.
  • the invention additionally pertains to DR5 -binding molecules that comprise fragments of such molecules, and to bispecific molecules, including diabodies, BiTEs, knobs/holes bispecific antibodies, etc., that comprise: (i) such DR5-binding fragments and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell.
  • 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 Domain 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 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., IgG, IgE, IgM, IgD, IgA and IgY), class ⁇ e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2 ) or subclass.
  • IgGi IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2
  • subclass e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2
  • 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) The Use Of Antibodies In The Treatment Of Infectious Diseases," Singapore Med. J. 50(7):663-666). Nearly
  • 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 bispecific molecules of the invention as well as a chimeric antibody, a humanized antibody, and/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), three Constant Domains (CHI, CH2 and CH3), and a Hinge Domain located between the CHI and CH2 Domains.
  • VL Variable Domain
  • CL Constant Domain
  • VH Variable 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 1 10 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).
  • the Variable 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 CDRHI Domain, CDRH2 Domain, and CDRH3 Domain.
  • 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 particularly encompasses 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 Domains 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 humanized variants of the 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 that is based upon the structure and /or sequence of a human immunoglobulin.
  • the anti-human DR5 antibodies of the present invention thus include humanized, chimeric or caninized derivatives of antibodies DR5 mAb 1 or DR5 mAb 2.
  • the polynucleotide sequence of the variable domains of such antibodies may be used for genetic manipulation to generate such derivatives and to improve the affinity, or other characteristics of such antibodies.
  • 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.
  • 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 Domain 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). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, or six) 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):
  • amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG3 is (SEQ ID NO:198):
  • amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG4 is (SEQ ID NO:199):
  • the numbering of the residues in an IgG heavy chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, NH1 , MD (1991), expressly incorporated herein by references.
  • the "EU index as in Kabat” refers to the numbering of the human IgGl EU antibody.
  • Amino acids from the Variable Domains of the mature heavy and light chains of immunoglobulins are designated by the position of an amino acid in the chain. Kabat described numerous amino acid sequences for antibodies, identified an amino acid consensus sequence for each subgroup, and assigned a residue number to each amino acid.
  • Rabat's numbering scheme is extendible to antibodies not included in his compendium by aligning the antibody in question with one of the consensus sequences in Kabat by reference to conserved amino acids.
  • This method for assigning residue numbers has become standard in the field and readily identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, an amino acid at position 50 of a human antibody light chain occupies the equivalent position to an amino acid at position 50 of a mouse antibody light chain.
  • Polymorphisms have been observed at a number of different positions within antibody constant regions (e.g., Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as numbered by the EU index as set forth in Kabat), and thus slight differences between the presented sequence and sequences in the prior art can exist. Polymorphic forms of human immunoglobulins have been well- characterized.
  • Gm Glm (1, 2, 3, 17) or Glm (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (bl, c3, b3, bO, b3, b4, s, t, gl, c5, u, v, g5)
  • Glm 1, 2, 3, 17
  • Glm a, x, f, z
  • G2m G2m (23) or G2m (n)
  • G3m 5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28
  • G3m bl, c3, b3, bO, b3, b4, s, t, gl, c5, u, v, g5)
  • Lefranc, et al. "The human IgG subclasses: molecular analysis of structure, function and regulation.” Pergamon, Oxford, pp. 43
  • the antibodies of the present invention may be incorporate any allotype, isoallotype, or haplotype of any immunoglobulin gene, and are not limited to the allotype, isoallotype or haplotype of the sequences provided herein.
  • the C-terminal amino acid residue (bolded above) of the CH3 Domain may be post-translationally removed. Accordingly, the C-terminal residue of the CH3 Domain is an optional amino acid residue in the LAG-3 -binding molecules of the invention.
  • the present invention specifically encompasses DR5-binding molecules lacking the C-terminal residue of the CH3 Domain. Also specifically encompassed by the instant invention are such molecules comprising the C-terminal residue of the CH3 Domain.
  • Activating and inhibitory signals are transduced through the Fc Receptors (FcyRs) following their ligation to an Fc Region.
  • FcyRs Fc Receptors
  • TAMs immunoreceptor tyrosine-based activation motifs
  • ITIMS immunoreceptor tyrosine-based inhibitory motifs
  • ITAM-containing FcyR complexes include FcyRI, FcyRIIA, FcyRIIIA, whereas ITIM-containing complexes only include FcyRIIB.
  • Human neutrophils express the FcyRIIA gene.
  • FcyRIIA clustering via immune complexes or specific antibody cross-linking serves to aggregate ITAMs along with receptor-associated kinases which facilitate ITAM phosphorylation.
  • ITAM phosphorylation serves as a docking site for Syk kinase, activation of which results in activation of downstream substrates (e.g., PI3K). Cellular activation leads to release of proinflammatory mediators.
  • the FcyRIIB gene is expressed on B lymphocytes; its extracellular domain is 96% identical to FcyRIIA and binds IgG complexes in an indistinguishable manner.
  • the presence of an ITIM in the cytoplasmic domain of FcyRIIB defines this inhibitory subclass of FcyR. Recently the molecular basis of this inhibition was established.
  • an antibody to bind an epitope of an antigen depends upon the presence and amino acid sequence of the antibody's VL and VH Domains. Interaction of an antibody light chain and an antibody heavy chain and, in particular, interaction of its VL and VH Domains forms one of the two epitope-binding sites of a natural antibody. Natural antibodies are capable of binding to only one epitope species (i.e., they are monospecific), although they can bind multiple copies of that species (i.e., exhibiting bivalency or multi valency).
  • the binding domains of the present invention bind to epitopes in an "immunospecific" manner.
  • an antibody, diabody or other epitope binding molecule is said to "immunospecifically” bind a region of another molecule (i.e., an epitope) if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with that epitope relative to alternative epitopes.
  • an antibody that immunospecifically binds to a viral epitope is an antibody that binds this viral epitope with greater affinity, avidity, more readily, and /or with greater duration than it immunospecifically binds to other viral epitopes or non- viral epitopes.
  • 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.
  • antibodies can be enhanced by generating multispecific antibody-based molecules that can simultaneously bind two separate and distinct antigens (or different epitopes of the same antigen) and/or by generating antibody-based molecule having higher valency (i.e., more than two binding sites) for the same epitope and/or antigen.
  • a wide variety of recombinant bispecific antibody formats have been developed (see, e.g., PCT Publication Nos.
  • linker peptides either to fuse a further binding protein ⁇ e.g., an scFv, VL, VH, etc.) to, or within the antibody core (IgA, IgD, IgE, IgG or IgM), or to fuse multiple antibody binding portions ⁇ e.g., two Fab fragments or scFvs).
  • Alternative formats use linker peptides to fuse a binding protein ⁇ e.g.
  • an scFv, VL, VH, etc) to an a dimerization domain such as the CH2-CH3 Domain or alternative polypeptides (WO 2005/070966, WO 2006/107786A WO 2006/107617A, WO 2007/046893).
  • a dimerization domain such as the CH2-CH3 Domain or alternative polypeptides (WO 2005/070966, WO 2006/107786A WO 2006/107617A, WO 2007/046893).
  • WO 2005/070966, WO 2006/107786A WO 2006/107617A, WO 2007/046893 WO 2005/070966, WO 2006/107786A WO 2006/107617A, WO 2007/046893.
  • such approaches involve compromises and trade-offs.
  • WO 2013/174873, WO 2011/133886 and WO 2010/136172 disclose that the use of linkers may cause problems in therapeutic settings, and teaches a trispecific antibody in which the CL and CHI Domains are switched from their respective natural positions and the VL and VH Domains have been diversified (WO 2008/027236; WO 2010/108127) to allow them to bind to more than one antigen.
  • the molecules disclosed in these documents trade binding specificity for the ability to bind additional antigen species.
  • PCT Publications Nos. WO 2013/163427 and WO 2013/119903 disclose modifying the CH2 Domain to contain a fusion protein adduct comprising a binding domain. The document notes that the CH2 Domain likely plays only a minimal role in mediating effector function.
  • PCT Publications Nos. WO 2010/028797, WO2010028796 and WO 2010/028795 disclose recombinant antibodies whose Fc Regions have been replaced with additional VL and VH Domains, so as to form trivalent binding molecules.
  • PCT Publications Nos. WO 2003/025018 and WO2003012069 disclose recombinant diabodies whose individual chains contain scFv Domains.
  • PCT Publications No. WO 2013/006544 discloses multivalent Fab molecules that are synthesized as a single polypeptide chain and then subjected to proteolysis to yield heterodimeric structures. Thus, the molecules disclosed in these documents trade all or some of the capability of mediating effector function for the ability to bind additional antigen species.
  • a diabody is based on the antibody derivative known as a single-chain Variable Domain fragment (scFv).
  • scFv Single-chain Variable Domain fragment
  • Such molecules are made by linking light and/ or heavy chain Variable Domains 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. (1988) "Single- Chain Antigen-Binding Proteins," Science 242:423-426).
  • 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.
  • non-monospecific diabodies provides a significant advantage over antibodies: including but not limited to, the capacity to co-ligate and co-localize cells that express different epitopes.
  • Bispecific diabodies thus have wide- ranging applications including therapy and immunodiagnosis. Bispecificity allows for great flexibility in the design and engineering of the diabody in various applications, providing enhanced avidity to multimeric antigens, the cross-linking of differing antigens, and directed targeting to specific cell types relying on the presence of both target antigens.
  • diabody molecules known in the art Due to their increased valency, low dissociation rates and rapid clearance from the circulation (for diabodies of small size, at or below ⁇ 50 kDa), diabody molecules known in the art have also shown particular use in the field of tumor imaging (Fitzgerald et al. (1997) “Improved Tumour Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris, " Protein Eng. 10: 1221). Of particular importance is the co-ligating of differing cells, for example, the cross-linking of cytotoxic T cells to tumor cells (Staerz et al.
  • bispecific diabodies can be used to co-ligate receptors on the surface of different cells or on a single cell. Co-ligation of different cells and/or receptors is useful to modulation effector functions and/or immune cell signaling.
  • Diabody epitope-binding domains may also be directed to a surface determinant of a B cell, such as CD19, CD20, CD22, CD30, CD37, CD40, and CD74 (Moore, P. A. et al. (2011) “Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma, ' " Blood 117(17):4542-4551; Cheson, B.D. et al. (2008) "Monoclonal Antibody Therapy For B- Cell Non-Hodgkin 's Lymphoma," N. Engl. J. Med. 359(6):613-626; Castillo, J.
  • effector cell activation is triggered by the binding of an antigen bound antibody to an effector cell via Fc-FcyR interaction; thus, in this regard, diabody molecules may exhibit Ig-like functionality independent of whether they comprise an Fc Region (e.g., as assayed in any effector function assay known in the art or exemplified herein (e.g., ADCC assay)).
  • the diabody By cross-linking tumor and effector cells, the diabody not only brings the effector cell within the proximity of the tumor cells but leads to effective tumor killing (see e.g., Cao et al. (2003) "Bispecific Antibody Conjugates In Therapeutics, " Adv. Drug. Deliv. Rev. 55: 171- 197).
  • non-monospecific diabodies require the successful assembly of two or more distinct and different polypeptides (i.e., such formation requires that the diabodies be formed through the heterodimerization of different polypeptide chain species). This fact is in contrast to monospecific diabodies, which are formed through the homodimerization of identical polypeptide chains. Because at least two dissimilar polypeptides (i.e., two polypeptide species) must be provided in order to form a non- monospecific diabody, and because homodimerization of such polypeptides leads to inactive molecules (Takemura, S. et al.
  • bispecific diabodies composed of non- covalently associated polypeptides are unstable and readily dissociate into nonfunctional monomers (see, e.g., Lu, D. et al. (2005) "A Fully Human Recombinant IgG- Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity," J. Biol. Chem. 280(20): 19665-19672).
  • DART® Dual Affinity Re-Targeting Reagents
  • Such diabodies comprise two or more covalently complexed polypeptides and involve engineering one or more cysteine residues into each of the employed polypeptide species that permit disulfide bonds to form and thereby covalently bond two polypeptide chains.
  • cysteine residues For example, the addition of a cysteine residue to the c-terminus of such constructs has been shown to allow disulfide bonding between the polypeptide chains, stabilizing the resulting heterodimer without interfering with the binding characteristics of the bivalent molecule.
  • 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.
  • the second polypeptide contains (in the N-terminal to C -terminal direction): (i) a First Domain that comprises a binding region of a Light Chain Variable Domain of the second immunoglobulin (VL2), (ii) a Second Domain that comprises a binding region of a Heavy Chain Variable Domain of the first immunoglobulin (VH1), and (iii) a Third Domain that contains a cysteine residue (or a cysteine-containing domain) and, a complementary Heterodimerization-Promoting Domain that complexes with the Heterodimerization- Promoting Domain of the first polypeptide chain in order to promote heterodimerization with the first polyptide chain.
  • the cysteine residue (or a cysteine-containing domain) of the third domain of the second polypeptide chain serves to promote the covalent bonding of the second polypeptide chain to the first polypeptide chain of the diabody.
  • Such molecules are stable, potent and have the ability to simultaneously bind two or more antigens.
  • the Third Domains of the first and second polypeptides each contain a cysteine residue, which serves to bind the polypeptides together via a disulfide bond.
  • Figure 1 provides a schematic of such a diabody, which utilizes E-coil/K-coil heterodimerization domains and a cysteine containing linker for covalent bonding.
  • one or both of the polypeptides may additionally possesses the sequence of a CH2-CH3 Domain, such that complexing between the two diabody polypeptides forms an Fc Region that is capable of binding to the Fc receptor of cells (such as B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils and mast cells).
  • 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.
  • Fc-Region-containing DART® diabodies may comprise three polypeptide chains.
  • the first polypeptide of such a diabody contains three Domains: (i) a VL1- containing Domain, (ii) a VH2-containing Domain and (iii) a Domain containing a CH2-CH3 sequence.
  • the second polypeptide of such DART® diabodies contains: (i) a VL2-containing Domain, (ii) a VH1 -containing Domain and (iii) a Domain that promotes heterodimerization and covalent bonding with the diabody' s first polypeptide chain.
  • the third polypeptide of such DART® diabodies comprises a CH2-CH3 sequence.
  • the first and second polypeptides are bonded to one another through a disulfide bond involving cysteine residues in their respective third Domains.
  • the first and third polypeptide chains complex with one another to form an Fc Region that is stabilized via a disulfide bond.
  • Alternative constructs are known in the art for applications where a tetravalent molecule is desirable but an Fc is not required including, but not limited to, tetravalent tandem antibodies, also referred to as "TandAbs" (see, e.g. United States Patent Publications Nos. 2005-0079170, 2007-0031436, 2010-0099853, 2011-020667 2013-0189263; European Patent Publication Nos.
  • the preferred DR5 -binding molecules of the present invention include antibodies, diabodies, BiTEs, etc. capable of binding to a continuous or discontinuous ⁇ e.g., conformational) portion (epitope) of human DR5.
  • the DR5 -binding molecules of the present invention will preferably also exhibit the ability to bind to the DR5 molecules of one or more non-human species, especially, murine, rodent, canine, and primate species.
  • the amino acid sequence of human DR5 precursor (NCBI Sequence NP_003833.4) (SEQ ID NO:2) is:
  • residues 1-55 are a signal sequence
  • residues 57-94 are a first Cysteine -Rich Domain (CRD)
  • residues 97-137 are a second Cysteine-Rich Domain (CRD)
  • residues 138-178 are a third Cysteine-Rich Domain (CRD)
  • residues 211-231 are the Transmembrane Domain
  • residues 232-440 are the Cytoplasmic Domain (containing the receptor's Death Domain).
  • the preferred anti-human DR5-binding molecules of the present invention possess the VL and/or VH Domains of murine anti-human DR5 monoclonal antibodies "DR5 mAb 1" and/or "DR5 mAb 2,” and more preferably possess 1, 2 or all 3 of the CDRLS of the VL Domain and/or 1 , 2 or all 3 of the CDRHS of the VH Domain of such anti-human DR5 monoclonal antibodies.
  • the 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.
  • preferred anti-human DR5-binding molecules include antibodies having variant Fc Regions, bispecific (or multispecific) antibodies, chimeric or humanized antibodies, BiTes, diabodies, chimeric antigen receptors (CARs), etc.
  • CDRLI of DR5 mAb 1 (SEQ ID NO:4): RASKSVSSSGYSYMH CDR L 2 of DR5 mAb 1 (SEQ ID NO:5): LSSNLDS CDR L 3 of DR5 mAb 1 (SEQ ID NO:6): QHSRDLPPT
  • VL Domain of DR5 mAb 1 is preferably encoded by a polynucleotide
  • CDRLS are shown in underline):
  • 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
  • CDRLS are shown in underline):
  • 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-3 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
  • hDR5 mAb 2 VH-2 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: RASQDV TAVA (SEQ ID NO: 196).
  • FcyRI CD64
  • FcyRIIA CD32A
  • FcyRIII CD 16
  • FcyRIIB CD32B
  • the amino acid sequence of an exemplary IgGl Fc Region SEQ ID NO:l is presented above.
  • Modification of the Fc Region normally leads to an altered phenotype, for example altered serum half-life, altered stability, altered susceptibility to cellular enzymes or altered effector function. It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example. Reduction or elimination of effector function is desirable in certain cases, for example in the case of antibodies whose mechanism of action involves blocking or antagonism, but not killing of the cells bearing a target antigen.
  • Increased effector function is generally desirable when directed to undesirable cells, such as tumor and foreign cells, where the FcyRs are expressed at low levels, for example, tumor-specific B cells with low levels of FcyRIIB (e.g., non-Hodgkins lymphoma, CLL, and Burkitt's lymphoma).
  • FcyRIIB e.g., non-Hodgkins lymphoma, CLL, and Burkitt's lymphoma.
  • molecules of the invention with conferred or altered effector function activity are useful for the treatment and/or prevention of a disease, disorder or infection where an enhanced efficacy of effector function activity is desired.
  • the DR5-binding molecules of the present invention comprise an Fc Region that possesses one or more modifications (e.g., substitutions, deletions, or insertions) to the sequence of amino acids of a wild-type Fc Region (SEQ ID NO:l), which reduce the affinity and avidity of the Fc Region and, thus, the molecule of the invention, for one or more FcyR receptors.
  • the molecules of the invention comprise an Fc Region that possesses one or more modifications to the amino acids of the wild-type Fc Region, which increase the affinity and avidity of the Fc Region and, thus, the molecule of the invention, for one or more FcyR receptors.
  • the molecules comprise a variant Fc Region wherein said variant confers or mediates increased ADCC activity and/or an increased binding to FcyRIIA, relative to a molecule comprising no Fc Region or comprising a wild-type Fc Region.
  • the molecules comprise a variant Fc Region wherein said variant confers or mediates decreased ADCC activity (or other effector function) and/or an increased binding to FcyRIIB, relative to a molecule comprising no Fc Region or comprising a wild-type Fc Region.
  • the invention encompasses DR5 -binding molecules comprising a variant Fc Region, which variant Fc Region does not show a detectable binding to any FcyR, relative to a comparable molecule comprising the wild-type Fc Region.
  • the invention encompasses DR5 -binding molecules comprising a variant Fc Region, which variant Fc Region only binds a single FcyR, preferably one of FcyRIIA, FcyRIIB, or FcyRIIIA.
  • any such increased affinity and/or avidity is preferably assessed by measuring in vitro the extent of detectable binding to the FcyR or FcyR-related activity in cells that express low levels of the FcyR when binding activity of the parent molecule (without the modified Fc Region) cannot be detected in the cells, or in cells which express non-FcyR receptor target antigens at a density of 30,000 to 20,000 molecules/cell, at a density of 20,000 to 10,000 molecules/cell, at a density of 10,000 to 5,000 molecules/cell, at a density of 5,000 to 1,000 molecules/cell, at a density of 1,000 to 200 molecules/cell or at a density of 200 molecules/cell or less (but at least 10, 50, 100 or 150 molecules/cell).
  • the DR5 -binding molecules of the present invention may comprise altered affinities for an activating and/or inhibitory Fey receptor.
  • the DR5- binding molecule comprises a variant Fc Region that has increased affinity for FcyRIIB and decreased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Region.
  • the DR5 -binding molecule of the present invention comprise a variant Fc Region, which has decreased affinity for FcyRIIB and increased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Region.
  • the DR5 -binding molecules of the present invention comprise a variant Fc Region that has decreased affinity for FcyRIIB and decreased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Region.
  • the DR5 -binding molecules of the present invention comprise a variant Fc Region, which has unchanged affinity for FcyRIIB and decreased (or increased) affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Region.
  • the DR5-binding molecules of the present invention comprise a variant Fc Region having an altered affinity for FcyRIIIA and/or FcyRIIA such that the immunoglobulin has an enhanced effector function, e.g., antibody dependent cell mediated cytotoxicity.
  • effector cell functions include antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting, Clq binding, and complement dependent cell mediated cytotoxicity.
  • the alteration in affinity or effector function is at least 2-fold, preferably at least 4-fold, at least 5 -fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, or at least 100-fold, relative to a comparable molecule comprising a wild-type Fc Region.
  • the variant Fc Region immunospecifically binds one or more FcRs with at least 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, or 250% greater affinity relative to a molecule comprising a wild-type Fc Region.
  • Such measurements can be in vivo or in vitro assays, and in a preferred embodiment are in vitro assays such as ELISA or surface plasmon resonance assays.
  • the DR5-binding molecules of the present invention comprise a variant Fc Region wherein said variant agonizes at least one activity of an FcyR receptor, or antagonizes at least one activity of an FcyR receptor.
  • the molecules comprise a variant that antagonizes one or more activities of FcyRIIB, for example, B cell receptor-mediated signaling, activation of B cells, B cell proliferation, antibody production, intracellular calcium influx of B cells, cell cycle progression, FcyRIIB -mediated inhibition of FcsRI signaling, phosphorylation of FcyRIIB, SHIP recruitment, SHIP phosphorylation and association with She, or activity of one or more downstream molecules (e.g., MAP kinase, INK, p38, or Akt) in the FcyRIIB signal transduction pathway.
  • FcyRIIB for example, B cell receptor-mediated signaling, activation of B cells, B cell proliferation, antibody production, intracellular calcium influx of B cells, cell cycle progression, FcyRIIB -mediated inhibition of FcsRI signaling, phosphorylation of FcyRIIB, SHIP recruitment, SHIP phosphorylation and association with She, or activity of one or more downstream molecules (e.g
  • the DR5 -binding molecules of the present invention comprise a variant that agonizes one or more activities of FcsRI, for example, mast cell activation, calcium mobilization, degranulation, cytokine production, or serotonin release.
  • 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.
  • This type of variant Fc Region may be used alone, or in combination with an amino acid modification, to affect Fc-mediated effector function and/or binding activity.
  • 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 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 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):
  • 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.
  • any amino acid modifications e.g. , substitutions
  • any amino acid modifications at any of positions 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301 , 303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331 , 333, 334, 335, 337, 338, 339, 340, 359, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439.
  • 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 at any of positions 252, 254, 265, 268, 269, 270, 278, 289, 292, 293, 294, 295, 296, 298, 300, 301 , 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 or 439.
  • 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:
  • Still more particularly preferred variants include one or more modifications selected from Groups 1-105:
  • a multivalent DR5 binding molecule of the invention will comprise a variant Fc Region having at least one modification in the Fc Region.
  • the variant Fc Region comprises at least one substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, wherein said numbering is that of the EU index as in Kabat.
  • the variant Fc Region comprises:
  • the variant Fc Region comprises substitutions of:
  • 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 Models," Immunol. Lett. 82:57-65; Presta, L.G. et al. (2002) “Engineering Therapeutic Antibodies For Improved Function," 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 a parent antibody.
  • 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 antibody, 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 antibody 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.
  • N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue.
  • O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine.
  • the molecules of the invention may comprise one or more glycosylation sites, including N-linked and O-linked glycosylation sites. Any glycosylation site for N-linked or O-linked glycosylation known in the art may be used in accordance with the instant invention.
  • 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. 106-116, 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 adding or deleting a glycosylation site.
  • Methods for modifying the carbohydrate content of antibodies (and molecules comprising antibody domains) are well known in the art and encompassed within the invention, see, e.g., U.S. Patent No. 6,218,149; EP 0 359 096 Bl; U.S. Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No. 2003/0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety.
  • 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 J . 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).
  • the DR5-binding molecules of the present invention may be monospecific single-chain diabodies such as scFvs or Chimeric Antigen Receptors (CARs).
  • scFvs are made by linking light and/ or heavy chain Variable Domains by using a short linking peptide.
  • First-generation CARs typically had the intracellular domain from the CD3 ⁇ - chain, which is the primary transmitter of signals from endogenous TCRs.
  • Second-generation CARs possessed additional intracellular signaling domains from various costimulatory protein receptors ⁇ e.g., CD28, 41BB, ICOS, etc) to the cytoplasmic tail of the CAR in order to provide additional signals to the T cell.
  • Third-generation CARs combine multiple signaling domains, such as CD3z- CD28-41BB or CD3z-CD28-OX40, in order to further augment potency (Tettamanti, S. et al. (2013) "Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CDI23-Specific Chimeric Antigen Receptor," Br. J. Haematol. 161 :389-401 ; Gill, S. et al.
  • the Chimeric Antigen Receptors of the present invention comprise an scFv fused to an intracellular domain of a receptor.
  • the Variable Light Chain and Variable Heavy Chain Domains of the scFv are selected from any of the anti-human DR5 antibodies disclosed herein.
  • the intracellular domain of the receptor is selected from the intracellular domain of any of: 41BB-CD3C, b2c-CD3C, CD28, CD28-4-lBB-CD3C, CD28-CD3C, CD28-Fc8RLy, CD28mut-CD3C, CD28-OX40-CD3C, CD28-OX40- CD3C, CD3C, CD4-CD3C, CD4-Fc8RLy, CD8-CD3C, FcsRLy, FcsRLyCAIX, Heregulin- CD3C, IL-13-CD3C, or Ly49H-CD3C (Tettamanti, S. et al.
  • One embodiment of the present invention relates to bispecific monovalent diabodies that are capable of binding to a "first epitope" and a "second epitope,” wherein the first epitope is an epitope of human DR5 and the second is an epitope of a molecule (e.g., CD3, CD 16, CD 19, CD20, CD22, CD32, CD64, T cell receptor (TCR), the B cell receptor (BCR), NKG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • TCR T cell receptor
  • BCR B cell receptor
  • NKG2D NKG2D, etc.
  • the second epitope is not an 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 DR5 and the second epitope.
  • the first polypeptide chain of such an embodiment of bispecific monovalent diabodies comprises, in the N-terminal to C-terminal direction, an N-terminus, the VL Domain of a monoclonal antibody capable of binding to either the first or second epitope (i.e., either VLDRS or VLE P ito P e 2), a first intervening spacer peptide (Linker 1), a VH Domain of a monoclonal antibody capable of binding to either the second epitope (if such first polypeptide chain contains VLDRS) or the first epitope (if such first polypeptide chain contains VLEpitope 2), a cysteine-containing second intervening spacer peptide (Linker 2), a Heterodimer-Promoting Domain and a C-terminus ( Figure 1).
  • VLl and VHl denote respectively, the Variable Light Chain Domain and Variable Heavy Chain Domain that bind the "first” epitope.
  • VL2 and VH2 denote respectively, the Variable Light Chain Domain and Variable Heavy Chain Domain that bind the "second" epitope.
  • the second polypeptide chain of this embodiment of bispecific monovalent 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 DR5 or the second epitope (i.e., either VLDRS 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 VLDRS) or to DR5 (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 a spacer peptide (Linker 2) optionally containing a cyst
  • 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 a first antigen (i.e., either DR5 or an antigen that contains the second epitope).
  • a first antigen i.e., either DR5 or an antigen that contains 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 specific for a second antigen (i.e., either an antigen that contains the second epitope or DR5).
  • 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 an epitope of DR5 and to the second epitope (i.e., they comprise VLDRS/VHDRS and
  • 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): GGGS GGGG.
  • the cysteine-containing second intervening spacer peptide will contain 1, 2, 3 or more cysteines.
  • a preferred cysteine-containing spacer peptide has the sequence is SEQ ID NO:34: GGCGGG.
  • Linker 2 does not comprise a cysteine and a Cysteine-Containing Heterodimer-Promoting Domain, as described below is used.
  • both a cysteine-containing Linker 2 and a cysteine-containing Heterodimer-Promoting Domain are used.
  • the Heterodimer-Promoting Domains may be GVE PKS C (SEQ ID NO:35) VE PKS C ( SEQ ID NO:36) or AE PKS C (SEQ ID NO:200) on one polypeptide chain and GFNRGEC (SEQ ID NO:37) or FNRGEC (SEQ ID NO:38) on the other polypeptide chain (US2007/0004909).
  • 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. It is immaterial which coil is provided to the first or second polypeptide chains, provided that a coil of opposite charge is used for the other polypeptide chain.
  • 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.
  • the positively charged amino acid is preferably lysine and/or the negatively charged amino acid is preferably glutamic acid. It is possible for only a single Heterodimer-Promoting Domain to be employed (since such domain will inhibit homodimerization and thereby promote heterodimerization), however, it is preferred for both the first and second polypeptide chains of the diabodies of the present invention to contain Heterodimer-Promoting Domains.
  • 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).
  • Heterodimer-Promoting Domain in which one of the four tandem "K- coil" helical domains of SEQ ID NO:40 has been modified to contain a cysteine residue: KVAACKE -KVAALKE -KVAALKE -KVAALKE (SEQ ID NO:42).
  • a diabody in order to improve the in vivo pharmacokinetic properties of diabodies, 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.
  • Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin possesses several small molecule binding sites that permit it to non-covalently bind to other proteins and thereby extend their serum half-lives.
  • the Albumin-Binding Domain 3 (ABD3) of protein G of Streptococcus strain G148 consists of 46 amino acid residues forming a stable three-helix bundle and has broad albumin-binding specificity (Johansson, M.U. et al. (2002) “Structure, Specificity, And Mode Of Interaction For Bacterial Albumin- Binding Modules " J. Biol. Chem. 277(10):8114-8120.
  • 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:
  • LAEAKVLANR ELDKYGVS DY YKNL I S 66 NAKS 7 o VEGVKAL IA79E I LAAL P are particularly preferred as such deimmunized Albumin-Binding Domains exhibit substantially wild-type binding while providing attenuated MHC class II binding.
  • the first polypeptide chain of such a diabody having an Albumin-Binding Domain contains a third linker (Linker 3) preferably positioned C-terminally to the E-coil (or K-coil) Domain of such polypeptide chain so as to intervene between the E-coil (or K- coil) Domain and the Albumin-Binding Domain (which is preferably a deimmunized Albumin-Binding Domain).
  • Linker 3 is SEQ ID NO:46:
  • One embodiment of the present invention relates to bispecific diabodies comprising an Fc Region capable of simultaneously binding to DR5 and a second epitope (e.g., CD3).
  • a second epitope e.g., CD3
  • IgG CH2-CH3 Domains to one or both of the diabody polypeptide chains such that the complexing of the diabody chains results in the formation of an Fc Region, increases the biological half-life and/or alters the valency of a diabody,.
  • Incorporating IgG CH2-CH3 Domains onto both of the diabody polypeptides will permit a two-chain bispecific Fc-Region-containing diabody to form ( Figure 2).
  • Figure 3A shows diabodies possessing the Constant Light (CL) Domain and the Constant Heavy CHI Domain, however fragments of such domains as well as other polypeptides may alternatively be employed as Heterodimer-Promoting Domains (see., e.g., Figure 3B, United States Patent Publications No. 2013-0295121; 2010-0174053 and 2009- 0060910; European Patent Publication No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publications No. WO 2012/162068; WO 2012/018687; WO 2010/080538).
  • CHI domain a peptide having the amino acid sequence GVE PKS C (SEQ ID NO:35) or VE PKS C ( SEQ ID NO:36), derived from the hinge domain of a human IgG
  • VE PKS C SEQ ID NO:36
  • CL domain a peptide having the amino acid sequence derived from the hinge domain of a human IgG
  • C-terminal 6 amino acids of the human kappa light chain GFNRGEC (SEQ ID NO:37) or FNRGEC (SEQ ID NO:38).
  • Additional or alternative linkers that may be employed in the Fc Region- containing diabody molecules of the present invention include: AS KG (SEQ ID NO:47), DKTHTC P PC P (SEQ ID NO:48), LE PKS S (SEQ ID NO:49), and AP S S S PME (SEQ ID NO:50), GGC, and GGG.
  • SEQ ID NO:49 may be used in lieu of GGG or GGC for ease of cloning. Additionally, SEQ ID NO:49 may be immediately followed by SEQ ID NO:47 to form an alternate linker (LE PKS S DKTHTC P PC P; SEQ ID NO:51).
  • a representative peptide containing four-chain diabody is shown in Figure 3A.
  • a representative coil domain containing four-chain diabody is shown in Figure 3B.
  • the Fc Region-containing diabodies may comprise three polypeptide chains.
  • the first polypeptide of such a diabody contains three Domains: (i) a VL1 -containing Domain, (ii) a VH2-containing Domain and (iii) a Domain containing a CH2-CH3 sequence.
  • the second polypeptide of such diabodies contains: (i) a VL2-containing Domain, (ii) a VH1 -containing Domain and (iii) a Domain that promotes heterodimerization and covalent bonding with the diabody 's first polypeptide chain.
  • the third polypeptide of such diabodies comprises a CH2-CH3 sequence.
  • the first and second polypeptide chains of such diabodies associate together to form a VL1/VH1 binding site that is capable of binding to the first epitope, as well as a VL2/VH2 binding site that is capable of binding to the second epitope.
  • the first and second polypeptides are bonded to one another through a disulfide bond involving cysteine residues in their respective Third Domains.
  • the first and third polypeptide chains complex with one another to form an Fc Region that is stabilized via a disulfide bond.
  • Such diabodies have enhanced potency.
  • Figures 4A and 4B illustrate the structures of such diabodies.
  • Such Fc-Region-containing DART® diabodies may have either of two orientations (Table 2): Table 2
  • 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 lack the C-terminal amino acid residue.
  • the Fc Region of the bispecific monovalent Fc 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).
  • FcyR(s) FcyR(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.).
  • Fc Region modifications identified as altering effector function are known in the art, including modifications that increase binding to activating receptors (e.g., FcyRIIA (CD16A) and reduce binding to inhibitory receptors (e.g., FcyRIIB (CD32B) (see, e.g., Stavenhagen, J.B. et al. (2007) "Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors," Cancer Res. 57(18):8882- 8890).
  • Exemplary variants of human IgGl Fc Regions with reduced binding to CD32B and/or increased binding to CD16A contain F243L, R292P, Y300L, V305I or P296L substitutions. These amino acid substitutions may be present in a human IgGl Fc Region in any combination.
  • the human IgGl Fc Region variant contains a F243L, R929P and Y300L substitution.
  • the human IgGl Fc Region variant contains a F243L, R292P, Y300L, V305I and P296L substitution.
  • CH2-CH3 Domains of the polypeptide chains of the Fc Region-containing diabodies of the present invention to exhibit decreased (or substantially no) binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD 16a) or FcyRIIIB (CD 16b) (relative to the binding exhibited by the wild-type IgGl Fc Region (SEQ ID NO:l).
  • Fc variants and mutant forms capable of mediating such altered binding are described above.
  • the CH2-CH3 domains of the polypeptide chains of the Fc Region- containing diabodies of the present invention comprise an IgG Fc Region that exhibits reduced ADCC effector function.
  • the CH2-CH3 Domain of the first and/or third polypeptide chains of such diabodies include any 1, 2, or 3, of the substitutions: L234A, L235A, D265A, N297Q andN297G.
  • the human IgGl Fc Region variant contains an N297Q substitution, an N297G substitution, L234A and L235A substitutions or a D265A substitution, as these mutations abolish FcR binding.
  • the Fc Region-containing diabodies of the present invention comprise an IgG2 Fc Region or an IgG4 Fc Region.
  • 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) “73 ⁇ 4e 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) “73 ⁇ 4e 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 Heter
  • 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
  • steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., "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.
  • the "knob” is engineered into the CH2-CH3 Domains of the first polypeptide chain and the "hole” is engineered into the CH2-CH3 Domains of the third polypeptide chain of diabodies comprising three polypeptide chains.
  • the "knob” will help in preventing the first polypeptide chain from homodimerizing via its CH2 and/or CH3 Domains.
  • the third polypeptide chain preferably contains the "hole” substitution it will heterodimerize with the first polypeptide chain as well as homodimerize with itself.
  • a similar strategy may be utilized for diabodies comprising four chains where the "knob” is engineered into the CH2-CH3 Domains of the second polypeptide chain and the "hole” is engineered into the CH2-CH3 Domains of the fourth polypeptide chain.
  • 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 the third polypeptide chain is preferably mutated by amino acid substitution at position 435 (H435R).
  • 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):
  • a preferred sequence for the CH2 and CH3 Domains of the second polypeptide chain of an Fc Region-containing diabody of the present invention having two polypeptide chains (or the third polypeptide chain of an Fc Region-containing diabody having three polypeptide chains) will have the "hole-bearing" sequence (SEQ ID NO:53):
  • NO:53 include a substitution at position 234 with alanine and 235 with alanine, and thus form an Fc Region exhibit decreased (or substantially no) binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD 16a) or FcyRIIIB (CD 16b) (relative to the binding exhibited by the wild-type Fc Region (SEQ ID NO: 1).
  • 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
  • a "knob-bearing” CH2-CH3 Domain e.g., SEQ ID NO: 52
  • the second polypeptide chain of an Fc Region- containing diabody of the present invention having two polypeptide chains (or the third polypeptide chain of an Fc Region-containing diabody having three or four polypeptide chains).
  • DR5 mAb 3 drozitumab
  • 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”
  • KMTR2 designated herein as "DR5 mAb 8"
  • CDRLI of DR5 mAb 5 (SEQ ID NO:71): KASQDVGTAVA CDR L 2 of DR5 mAb 5 (SEQ ID NO:72): WASTRHT CDR L 3 of DR5 mAb 5 (SEQ ID NO:73): QQYSSYRT [00168]
  • the amino acid sequence of the VH Domain of tigatumumab ("DR5 mAb5 ') (SEQ ID NO:74) is shown below (CDRH residues are shown underlined):
  • KVQLQQSGAE LVKPGASVKL SCKASGYTFT DYTIHWVKQR SGQGLEWI GW FYPGGGYIKY NEKFKDRATL TADKS SNTVY MELSRLT SEG SAVYFCARHE EGIYFDYWGQ GTTLTVS S
  • KVQLQQSGAE LVKPGASVKL SCKASGYTFT DYTIHWVKQR SGQGLEWIGW FYPGGGYIKY NEKFKDRATL TADKSSNTVY MELSRLTSED SAVYFCARHE EGIYFDYWGQ GTTLTVSS
  • CD2 is is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells. CD2 enhances NK cell cytotoxicity, possibly as a promoter of NK cell nanotube formation (Mace, E.M. et al. (2014) "Cell Biological Steps And
  • Anti-CD2 antibody Lo-CD2a was employed as a reference antibody.
  • the amino acid sequence of the VL Domain of anti-CD2 antibody (Lo-CD2a; ATCC Accession No: 11423) is (SEQ ID NO: 102)
  • the second epitope that is bound by the DR5- binding molecules of the present invention will be an epitope of CD3.
  • 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 CD35 chain, and two CD3s chains.
  • TCR T cell receptor
  • bispecific anti-human CD3 x anti-human DR5-binding molecules were produced.
  • An anti-human CD3 x anti-human DR5-binding molecules were produced.
  • CD3 antibody used for such constructs is designated herein as "CD3 mAb 2."
  • the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104) is shown below (CDRL residues are shown underlined):
  • CD3 mAb 2 SEQ ID NO: 105
  • RSSTGAVTTSNYAN CDR L 2 of CD3 mAb 2 SEQ ID NO: 106
  • GTNKRAP CDR L 3 of CD3 mAb 2 SEQ ID NO: 107): ALWYSNLWV
  • CDRH3 of CD3 mAb 2 (SEQ ID NO:lll): HGNFGNSYVSWFAY [00180]
  • a variant VH Domain was employed for CD3 mAb 2.
  • the variant VH Domain possesses a D65G substitution, thus having the amino acid sequence shown below (SEQ ID NO: 112) (CDRH residues are shown underlined):
  • substitution causes the CDRH2 to have the amino acid sequence (SEQ ID NO:113) RIRSKYNNYATYYADSVKg.
  • the substituted position (D65G) is shown in double underline.
  • a second anti-CD3 antibody used herein is antibody Muromonab-CD3
  • OKT3 (Xu et al. (2000) “In Vitro Characterization Of Five Humanized OKT3
  • Interleukin-10 A Possible Cause Of Lymphoproliferative Disorders Associated With
  • CD 16 is the FcyRIIIA receptor.
  • CD 16 is expressed by neutrophils, eosinophils, natural killer (NK) cells, and tissue macrophages that bind aggregated but not monomeric human IgG (Peltz, G.A. et al. (1989) "Human Fc Gamma Bill: Cloning, Expression, And Identification Of The Chromosomal Locus Of Two Fc Receptors For IgG " Proc. Natl. Acad. Sci. (U.S.A.) 86(3): 1013-1017; Bachanova, V. et al. (2014) "NK Cells In Therapy Of Cancer," Crit. Rev. Oncog.
  • CD 19 antigen is a type I transmembrane glycoprotein belonging to the immunoglobulin Ig superfamily.
  • CD 19 is expressed on follicular dendritic cells and B cells. It is considered a pan B-cell marker expressed throughout B-cell development but with threefold higher expression in mature cells as compared to immature B-cells (Raufi A. et al. (2013) "Targeting CD 19 In B-Cell Lymphoma: Emerging Role Of SAR3419 " Cancer Manag. Res. 5:225-233).
  • Many CD 19 antibodies have been described (e.g., MD1342, , MEDI-551, etc.) (Mei, H.E. et al.
  • amino acid sequence of the VH Domain of anti-CD 19 antibody HD37 is (SEQ ID NO: 121) (CDRH residues are shown underlined):
  • CD20 is a B-cell-specific differentiation antigen that is expressed on mature B cells and in most B-cell non-Hodgkin's lymphomas but not on early B-cell progenitors or later mature plasma cells (Maloney, D.G. (2012) Anti-CD20 Antibody Therapy for B-Cell Lymphomas," N. Engl. J. Med. 366:2008-2016).
  • Rituximab is an illustrative anti-human CD20 antibody.
  • the amino acid sequence of the VL Domain of anti-CD20 antibody (rituximab) is (SEQ ID NO: 122) (CDRL residues are shown underlined):
  • CD22 is a sugar binding transmembrane protein found on the surface of mature B cells and to a lesser extent on some immature B cells (WO 2011/032633; Poe, J.C. et al. (2012) "0022 And Siglec-G In B Cell Function And Tolerance” Trends Immunol.33(8):413-420; Chen, W.C. et al. (2012) “Targeting B Lymphoma With Nanoparticles Bearing Glycan Ligands Of CD22" Leuk. Lymphoma 53(2):208-210; Walker, J.A. (2008) “CD22: An Inhibitory Enigma," Immunology 123(3):314-325; Coleman, M. et al. (2003) “Epratuzumab: Targeting B-Cell Malignancies Through CD22," Clin. Cancer Res.9(10 Pt 2):3991S-3994S).
  • epratuzumab is (SEQ ID NO: 124) (CDRL residues are shown underlined):
  • CD32B is the FcyRIIB receptor. CD32B is widely expressed, and is present on all hematopoietic cells, including monocytes, macrophages, B cells, NK cells, neutrophils, mast cells, and platelets.
  • a preferred sequence for the VL Domain of an antibody that binds to human CD32B is (SEQ ID NO: 126) (CDRL residues are shown underlined):
  • GTKLELK A preferred sequence for the VH Domain of an antibody that binds to human
  • CD32B is (SEQ ID NO: 127) (CDRH residues are shown underlined):
  • CD64 is the FcyRI receptor and is expressed on monocytes/macrophages, dendritic cells, and activated granulocytes. The expression can be upregulated by IFN- ⁇ stimulation. CD64 binds IgG immune complex. CD64 plays a role in antigen capture, phagocytosis of IgG/antigen complexes, and antibody dependent cellular cytotoxicity (WO 2006/002438).
  • CD64 is (SEQ ID NO: 128) (CDRL residues are shown underlined):
  • CD64 is (SEQ ID NO: 129) (CDRH residues are shown underlined):
  • the BCR is composed of a membrane immunoglobulin which, together with non-covalently associated a and ⁇ subunits of CD79 ("CD79a” and “CD79b,” respectively), forms the BCR complex.
  • CD79a and CD79b are signal transducing subunits that contain a conserved immunoreceptor tyrosine -based activation motif ("ITAM") required for signal transduction (Dylke, J. et al. (2007) Role Of The Extracellular And Transmembrane Domain Of Ig-Alpha/Beta In Assembly Of The B Cell Antigen Receptor (BCR) " Immunol. Lett. 112(l):47-57; Cambier, J.C.
  • ITAM immunoreceptor tyrosine -based activation motif
  • Phosphorylated ITAMs recruit additional effectors such as PI3K, PLC- ⁇ and members of the Ras/MAPK pathway. These signaling events are responsible for both the B cell proliferation and increased expression of activation markers (such as MHCII and CD86) that are required to prime B cells for their subsequent interactions with T-helper (“Th”) cells.
  • activation markers such as MHCII and CD86
  • a preferred sequence for the VL Domain of an antibody that binds to the human B Cell Receptor (CD79) is (SEQ ID NO: 130) (CDRL residues are shown underlined):
  • a preferred sequence for the VH Domain of an antibody that binds to the human B Cell Receptor (CD79) is (SEQ ID NO: 131) (CDRH residues are shown underlined):
  • the second epitope that is bound by the DR5- binding molecules of the present invention will be an epitope of the T cell Receptor (TCR).
  • T cell Receptor is natively expressed by CD4+ or CD8+ T-cells, and permits such cells to recognize antigenic peptides that are bound and presented by class I or class II MHC proteins of antigen-presenting cells.
  • Recognition of a pMHC (peptide-MHC) complex by a TCR initiates the propagation of a cellular immune response that leads to the production of cytokines and the lysis of the antigen-presenting cell (see, e.g., Armstrong, K.M. et al.
  • CD3 is the receptor that binds to the TCR (Thomas, S. et al. (2010) “Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer,” Immunology 129(2): 170-177; Guy, C.S. et al. (2009) “Organization Of Proximal Signal Initiation At The TCR. CD3 Complex " Immunol. Rev. 232(1):7-21; St. Clair, E.W. (Epub 2009 Oct 12) "Novel Targeted Therapies For Autoimmunity " Curr. Opin. Immunol.
  • Antibodies that specifically bind to the T-cell Receptor include the anti-TCR antibody BMA 031 (EP 0403156; Kurrle, R. et al. (1989) "BMA 031 - A TCR-Specific Monoclonal Antibody For Clinical Application," Transplant Proc. 21(1 Pt 1): 1017- 1019; Nashan, B. et al. (1987) "Fine Specificity Of A Panel Of Antibodies against The TCR/CD3 Complex," Transplant Proc. 19(5):4270-4272; Shearman, C.W. et al.
  • TKLEIK The amino acid sequence of a VH Domain of anti-TCR antibody BMA 031 is (SEQ ID NO: 133) (CDRH residues are shown underlined):
  • the second epitope that is bound by the DR5- binding molecules of the present invention will be an epitope of the NKG2D receptor.
  • the NKG2D receptor is expressed on all human (and other mammalian) Natural Killer cells (Bauer, S. et al. (1999) Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA " Science 285(5428):727-729; Jamieson, A.M. et al.
  • binding ligands include the histocompatibility 60 (H60) molecule, the product of the retinoic acid early inducible gene-1 (RAE-1), and the murine UL16- binding proteinlike transcript 1 (MULT1) (Raulet D.H. (2003) “Roles Of The NKG2D Immunoreceptor And Its Ligands " Nature Rev. Immunol. 3:781-790; Coudert, J.D. et al. (2005) "Altered NKG2D Function In NK Cells Induced By Chronic Exposure To Altered NKG2D Ligand-Expressing Tumor Cells " Blood 106: 1711-1717).
  • H60 histocompatibility 60
  • RAE-1 retinoic acid early inducible gene-1
  • MULT1 murine UL16- binding proteinlike transcript 1
  • Antibodies that specifically bind to the NKG2D Receptor include KYK-2.0 (Kwong, KY et al. (2008) “Generation, Affinity Maturation, And Characterization Of A Human Anti- Human NKG2D Monoclonal Antibody With Dual Antagonistic And Agonistic Activity " J. Mol. Biol. 384: 1143-1156; and PCT/US09/54911).
  • GGTQLTVL [00211] The amino acid sequence of the VH Domain of anti-NKG2D antibody
  • KYK-1.0 is (SEQ ID NO:135) (CDRH residues are shown underlined):
  • the anti-fiuorescein antibody 4-4-20 (Gruber, M. et al. (1994) Efficient Tumor Cell Lysis Mediated By A Bispecific Single Chain Antibody Expressed In Escherichia coli," J. Immunol. 152(11):5368-5374; Bedzyk, W.D. et al. (1989) "Comparison Of Variable Region Primary Structures Within An Anti-Fluorescein Idiotype Family " J. Biol. Chem. 264(3): 1565-1569) was used in control diabodies.
  • the amino acid sequences of the variable light and variable heavy Domains of anti- fiuorescein antibody 4-4-20 are as follows:
  • An exemplary bispecific diabody composed of two polypeptide chains was constructed having the VL and VH Domains of anti-human DR5 antibody DR5 mAb 1 and the VL and VH Domains of CD3 mAb 2.
  • the diabody was designated "DR5 mAb
  • amino acid residues 1-111 correspond to the amino acid sequence of the VL Domain of DR5 mAb 1 (SEQ ID NO:3)
  • residues 112-119 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 120-244 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112)
  • residues 245-249 correspond to the AS KG linker (SEQ ID NO:47)
  • residues 250-277 correspond to a cysteine- containing E-coil Domain (SEQ ID NO:41).
  • NO: 140 is SEQ ID NO: 141:
  • CD3 mAb 2 diabody is (SEQ ID NO:142):
  • amino acid residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-239 correspond to the amino acid sequence of the VH Domain of DR5 mAb 1 (SEQ ID NO:8), except that the C-terminal serine residue of SEQ ID NO:8 has been replaced with an alanine residue)
  • residues 240-244 correspond to an AS KG linker (SEQ ID NO: 47)
  • residues 245-272 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:
  • An exemplary bispecific diabody composed of two polypeptide chains was constructed having the VL and VH Domains of anti-human DR5 antibody DR5 mAb 2 and the VL and VH Domains of CD3 mAb 2.
  • the diabody was designated "DR5 mAb
  • amino acid residues 1-107 correspond to the amino acid sequence of the VL Domain of DR5 mAb 2 (SEQ ID NO: 13), residues 108-115 correspond to intervening spacer peptide (Linker 1) (SEQ ID NO:33), residues 116- 240 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112), residues 241-245 correspond to an AS KG linker (SEQ ID NO: 47) and residues 246-273 correspond to a cysteine-containing E- coil Domain (SEQ ID NO:41).
  • a polynucleotide that encodes SEQ ID NO: 144 is SEQ ID NO:145:
  • amino acid sequence of the second polypeptide chain of the DR5 mAb 2 x CD3 mAb 2 diabody is (SEQ ID NO:146):
  • amino acid residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-237 correspond to the amino acid sequence of the VH Domain of DR5 mAb 2 (SEQ ID NO: 18)
  • residues 238-242 correspond to an AS KG linker (SEQ ID NO: 47)
  • residues 243-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • a polynucleotide that encodes SEQ ID NO: 146 is SEQ ID NO:147:
  • An exemplary bispecific diabody composed of two polypeptide chains was constructed having the VL and VH Domains of humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH Domains of CD3 mAb 2.
  • the amino acid sequence of the hDR5 mAb 2 VL-2 polypeptide (SEQ ID NO:23) was employed.
  • the diabody was designated "hDR5 mAb 2 (2.2) x CD3 mAb 2 diabody.”
  • the amino acid sequence of the first polypeptide chain of this diabody is (SEQ ID NO:148):
  • 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
  • residues 116-240 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112)
  • residues 241-245 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 246-273 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:41).
  • SEQ ID NO:148 is SEQ ID NO:149:
  • CD3 mAb 2 diabody is (SEQ ID NO: 150):
  • amino acid residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 (SEQ ID NO:31)
  • residues 238-242 correspond to an AS KG linker (SEQ ID NO: 47)
  • residues 243-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • a polynucleotide that encodes SEQ ID NO: 150 is SEQ ID NO:151:
  • An exemplary bispecific diabody composed of two polypeptide chains was constructed having the VL and VH Domains of humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH Domains of CD3 mAb 2.
  • the amino acid sequence of the hDR5 mAb 2 VL-3 polypeptide (SEQ ID NO:25) was employed.
  • the diabody was designated "hDR5 mAb 2 (2.3) x CD3 mAb 2 diabody.”
  • the amino acid sequence of the first polypeptide chain of this 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-3 (SEQ ID NO:25), residues 108-115 correspond to intervening spacer peptide (Linker 1) (SEQ ID NO:33), residues 116-240 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112), residues 241-245 correspond to an AS KG linker (SEQ ID NO:47) and residues 246-273 correspond to the cysteine- containing E-coil (SEQ ID NO:41).
  • a polynucleotide that encodes SEQ ID NO: 152 is SEQ ID NO: 153:
  • CD3 mAb 2 diabody is (SEQ ID NO: 154):
  • amino acid residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104), residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33), residues 119-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 (SEQ ID NO:31), residues 238-242 correspond to an AS KG linker (SEQ ID NO: 47), and residues 243-270 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:
  • An exemplary bispecific diabody composed of two polypeptide chains was constructed having the VL and VH Domains of humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH Domains of CD3 mAb 2.
  • the amino acid sequence of the hDR5 mAb 2 VL-4 polypeptide (SEQ ID NO:27) was employed.
  • the diabody was designated "hDR5 mAb 2 (2.4) x CD3 mAb 2 diabody.”
  • the amino acid sequence of the first polypeptide chain of this diabody is (SEQ ID NO: 156):
  • 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
  • Linker 1 SEQ ID NO:33
  • residues 116-240 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112)
  • residues 241-245 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 246-273 correspond to a cysteine- containing E-coil Domain (SEQ ID NO:41).
  • a polynucleotide that encodes SEQ ID NO:33 amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution
  • residues 241-245 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 246-273 correspond to a cysteine- containing E-coil Domain (SEQ ID NO:41).
  • CD3 mAb 2 diabody is (SEQ ID NO: 158):
  • amino acid residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 (SEQ ID NO:31)
  • residues 238-242 correspond to an AS KG linker (SEQ ID NO: 47)
  • residues 243-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • a polynucleotide that encodes SEQ ID NO: 158 is SEQ ID NO:159:
  • An exemplary bispecific diabody composed of two polypeptide chains was constructed having the VL and VH Domains of humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH Domains of CD3 mAb 2.
  • the amino acid sequence of the hDR5 mAb 2 VL-5 polypeptide (SEQ ID NO:29) was employed.
  • the diabody was designated "hDR5 mAb 2 (2.5) x CD3 mAb 2 diabody.”
  • the amino acid sequence of the first polypeptide chain of this diabody is (SEQ ID NO: 160):
  • 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
  • Linker 1 SEQ ID NO:33
  • residues 116-240 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112)
  • residues 241-245 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 246-273 correspond to a cysteine- containing E-coil Domain (SEQ ID NO:41).
  • a polynucleotide that encodes SEQ ID NO:33 amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution
  • residues 241-245 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 246-273 correspond to a cysteine- containing E-coil Domain (SEQ ID NO:41).
  • NO:160 is SEQ ID NO:161:
  • CD3 mAb 2 diabody is (SEQ ID NO: 162):
  • amino acid residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 (SEQ ID NO:31)
  • residues 238-242 correspond to an AS KG linker (SEQ ID NO: 47)
  • residues 243-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • a polynucleotide that encodes SEQ ID NO: 162 is SEQ ID NO:163:
  • a diabody composed of two polypeptide chains was constructed using the VL and VH Domains of DR5 mAb 3 and CD3 mAb 2.
  • the amino acid sequence of the first polypeptide chain of the diabody had the sequence (SEQ ID NO:
  • amino acid residues 1-108 correspond to the VL Domain of DR5 mAb 3 (SEQ ID NO:54)
  • residues 109-116 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 117-241 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112)
  • residues 242-246 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 247-275 correspond to a cysteine-containing K- coil Domain (SEQ ID NO: 42).
  • amino acid sequence of the second polypeptide chain of the diabody had the sequence (SEQ ID NO: 165) (CDRL and CDRH residues are shown underlined):
  • amino acid residues 1-110 correspond to the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-239 correspond to the amino acid sequence of the VH Domain of DR5 mAb 3 (SEQ ID NO:58)
  • residues 240-244 correspond to an ASTKG linker (SEQ ID NO:47)
  • residues 245-272 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • a diabody composed of two polypeptide chains was constructed using the VL and VH Domains of DR5 mAb 4 and CD3 mAb 2.
  • the amino acid sequence of the first polypeptide chain of the diabody had the sequence (SEQ ID NO: 166) (CDRL and CDRH residues are shown underlined):
  • amino acid residues 1-108 correspond to the VL Domain of DR5 mAb 4 (SEQ ID NO:62)
  • residues 109-116 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 117- 241 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO:96)
  • residues 242-246 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 247-275 correspond to a cysteine-containing E- coil Domain (SEQ ID NO:41).
  • amino acid sequence of the second polypeptide chain of the diabody had the sequence (SEQ ID NO: 167) (CDRL and CDRH residues are shown underlined):
  • amino acid residues 1-110 correspond to the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-240 correspond to the amino acid sequence of the VH Domain of DR5 mAb 4 (SEQ ID NO:66)
  • residues 241-245 correspond to an ASTKG linker (SEQ ID NO:47)
  • residues 246-273 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • hDR5 mAb 2 (2.2) x CD3 mAb 2 Fc Region-Containing Diabodies
  • An exemplary bispecific Fc Region-containing diabody composed of three polypeptide chains was constructed having the VL and VH Domains of humanized anti- human DR5 antibody hDR5 mAb 2 and the VL and VH Domains of CD3 mAb 2 and employing the amino acid sequence of the hDR5 mAb 2 VL-2 polypeptide (SEQ ID NO:23).
  • the diabody was designated "hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc ver 1 diabody.”
  • the amino acid sequence of the first polypeptide chain of this diabody is (SEQ ID NO: 168):
  • 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 intervening spacer peptide (Linker 1) (SEQ ID NO:33), residues 116-240 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112), residues 241-245 correspond to an AS KG linker (SEQ ID NO:47), residues 246-273 correspond to a cysteine- containing E-coil Domain (SEQ ID NO:41), residues 274-276 correspond to the linker GGG, residues 277-286 correspond to a linker (DKTHTCPPCP; SEQ ID NO:48) derived from an IgG hinge domain and residues 287-503 correspond to a "knob-bearing" CH2- CH3 sequence (SEQ ID NO:52).
  • the second polypeptide correspond to the amino acid sequence of the VL Domain of
  • residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119- 237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 (SEQ ID NO:31)
  • residues 238-242 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 243-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • the third polypeptide chain of the hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc diabody has the amino acid sequence (SEQ ID NO: 170):
  • amino acid residues 1-10 correspond to the linker DKTHTCPPCP (SEQ ID NO:48) derived from an IgG hinge domain and residues 11- 227 correspond to a "hole-bearing" CH2-CH3 sequence (SEQ ID NO:53).
  • SEQ ID NO:48 linker DKTHTCPPCP
  • residues 11- 227 correspond to a "hole-bearing" CH2-CH3 sequence (SEQ ID NO:53).
  • a second exemplary bispecific Fc Region-containing diabody composed of three polypeptide chains was constructed having the VL and VH Domains of humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH Domains of CD3 mAb 2.
  • the amino acid sequence of the hDR5 mAb 2 VL-2 polypeptide (SEQ ID NO:23) was employed for this construct.
  • the diabody was designated "hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc ver 2 diabody.”
  • the amino acid sequence of the first polypeptide chain of this diabody is (SEQ ID NO: 171):
  • amino acid residues 1-10 correspond to the linker DKTHTCPPCP (SEQ ID NO:48) derived from an IgG hinge domain
  • residues 11-227 correspond to the "knob-bearing" CH2-CH3 sequence (SEQ ID NO:52)
  • residues 228- 235 is a linker
  • residues 236-342 correspond to the amino acid sequence of the VL Domain of hDR5 mAb 2 VL-2 (SEQ ID NO:23)
  • residues 343-350 correspond to intervening spacer peptide (Linker 1) (SEQ ID NO:33)
  • residues 351-475 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 (SEQ ID NO: 108)
  • residues 476-481 is the cysteine-containing spacer peptide GGCGGG (Linker 2) (SEQ ID NO:34)
  • residues 482-510 correspond to the K-coil (SEQ ID NO:40).
  • the second polypeptide chain of the hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc ver 2 diabody has the amino acid sequence (SEQ ID NO: 172):
  • amino acid residues 1-110 correspond to the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 (SEQ ID NO:31)
  • residues 238-242 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 243-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • the third polypeptide chain of the hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc ver 2 diabody has the amino acid sequence (SEQ ID NO: 173):
  • amino acid residues 1-10 correspond to the linker DKTHTCPPCP (SEQ ID NO:48) derived from an IgG hinge domain and residues 11- 227 correspond to a "hole-bearing" CH2-CH3 sequence (SEQ ID NO:53).
  • SEQ ID NO:48 linker DKTHTCPPCP
  • residues 11- 227 correspond to a "hole-bearing" CH2-CH3 sequence (SEQ ID NO:53).
  • a third exemplary bispecific Fc Region-containing diabody composed of three polypeptide chains was constructed having the VL and VH Domains of humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH Domains of
  • CD3 mAb 2 VL-3 polypeptide The amino acid sequence of the hDR5 mAb 2 VL-3 polypeptide (SEQ ID NO: 1
  • the diabody was designated "hDR5 mAb
  • 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 intervening spacer peptide (Linker 1) (SEQ ID NO:33), residues 116-240 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112), residues 241-245 correspond to an AS KG linker (SEQ ID NO:47), residues 246-273 correspond to a cysteine- containing E-coil Domain (SEQ ID NO:41), residues 274-276 correspond to the linker GGG, residues 277-286 correspond to a linker (DKTHTCPPCP; SEQ ID NO:48) derived from an IgG hinge domain and
  • the second polypeptide chain of the hDR5 mAb 2 (2.3) x CD3 mAb 2 w/Fc diabody has the amino acid sequence (SEQ ID NO: 175):
  • residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119- 237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 (SEQ ID NO:31)
  • residues 238-242 correspond to an ASTKG linker (SEQ ID NO:47)
  • residues 243-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • the third polypeptide chain of the hDR5 mAb 2 (2.3) x CD3 mAb 2 w/Fc diabody has the amino acid sequence (SEQ ID NO: 176):
  • NVFSCSVMHE ALHNRYTQKS LSLSPGK [00266]
  • amino acid residues 1-10 correspond to the linker DKTHTCPPCP (SEQ ID NO:48) derived from an IgG hinge domain and residues 11- 227 correspond to a "hole-bearing" CH2-CH3 sequence (SEQ ID NO:53).
  • hDR5 mAb 2 (2.4) x CD3 mAb 2 w/Fc ver 1 Diabody
  • a fourth exemplary bispecific Fc Region-containing diabody composed of three polypeptide chains was constructed having the VL and VH Domains of humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH Domains of
  • CD3 mAb 2 The amino acid sequence of the hDR5 mAb 2 VL-4 polypeptide (SEQ ID NO: 1
  • the diabody was designated "hDR5 mAb
  • 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 116-240 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112), residues 241-245 correspond to an AS KG linker (SEQ ID NO:47), residues 246-273 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:41), residues 274-276 correspond to the linker GGG, residues 277-286 correspond to a linker (DKTHTCPPCP; SEQ ID NO:48) derived from an IgG hinge domain and residues 287-503 correspond to a "knob-bearing" CH2-CH3 sequence (SEQ ID NO:52). [00
  • residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119- 237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 (SEQ ID NO:31)
  • residues 238-242 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 243-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • the third polypeptide chain of the hDR5 mAb 2 (2.4) x CD3 mAb 2 w/Fc diabody has the amino acid sequence (SEQ ID NO: 179):
  • amino acid residues 1-10 correspond to the linker DKTHTCPPCP (SEQ ID NO:48) derived from an IgG hinge domain and residues 11- 227 correspond to a "hole-bearing" CH2-CH3 sequence (SEQ ID NO:53).
  • DKTHTCPPCP linker DKTHTCPPCP
  • residues 11- 227 correspond to a "hole-bearing" CH2-CH3 sequence (SEQ ID NO:53).
  • a fifth exemplary bispecific Fc Region-containing diabody composed of three polypeptide chains was constructed having the VL and VH Domains of humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH Domains of CD3 mAb 2.
  • the amino acid sequence of the hDR5 mAb 2 VL-5 polypeptide (SEQ ID NO:29) was employed for this construct.
  • the diabody was designated "hDR5 mAb 2 (2.5) x CD3 mAb 2 w/Fc ver 1 diabody.”
  • the amino acid sequence of the first polypeptide chain of this diabody is (SEQ ID NO: 180):
  • 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 108-115 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 116-240 correspond to the amino acid sequence of the VH Domain of CD3 mAb 2 having the D65G substitution (SEQ ID NO: 112)
  • residues 241-245 correspond to an AS KG linker (SEQ ID NO:47)
  • residues 246-273 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:41)
  • residues 274-276 correspond to the linker GGG
  • residues 277-286 correspond to a linker (DKTHTCPPCP; SEQ ID NO:48) derived from an IgG hinge domain and residues 287-503 correspond to a "knob-bearing" CH2-CH3 sequence (SEQ ID NO:52):
  • the second polypeptide chain of the hDR5 mAb 2 (2.5) x CD3 mAb 2 w/Fc diabody has the amino acid sequence (SEQ ID NO:181):
  • residues 1-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-110 correspond to the amino acid sequence of the VL Domain of CD3 mAb 2 (SEQ ID NO: 104)
  • residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • I l l - 237 correspond to the amino acid sequence of the VH Domain of hDR5 mAb 2 (SEQ ID NO:31), residues 238-242 correspond to an AS KG linker (SEQ ID NO:47), and residues 243-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • the third polypeptide chain of the hDR5 mAb 2 (2.5) x CD3 mAb 2 w/Fc diabody has the amino acid sequence (SEQ ID NO: 182):
  • amino acid residues 1-10 correspond to the linker DKTHTCPPCP (SEQ ID NO:48) derived from an IgG hinge domain and residues 11- 227 correspond to a "hole-bearing" CH2-CH3 sequence (SEQ ID NO:53).
  • BiTes (Bispecific T cell Engagers) are a form of diabody in which a single polypeptide chain molecule is designed to possess two antigen-binding domains, one of which binds to a T cell antigen and the second of which binds to an antigen present on the surface of a target (WO 05/061547; Baeuerle, P et al. (2008) “BiTE®: A New Class Of Antibodies That recruit T Cells," Drugs of the Future 33: 137-147; Bargou, et al. 2008) "Tumor Regression in Cancer Patients by Very Low Doses of a T Cell- Engaging Antibody Science 321 : 974-977).
  • the sequences of four illustrative anti- DR5 x anti-CD3 BiTe molecules are presented below. a. DR5 mAb 5 x OKT3 BiTE (DR5 VHVL- OKT3
  • a first anti-DR5 x anti-CD3 BiTe molecule may be constructed in a VH-VL orientation using the VL and VH Domains of DR5 mAb 5 and the VL and VH Domains of OKT3.
  • the BiTe would have an orientation of DR5 VHVL - OKT3 VHVL.
  • the amino acid sequence of the BiTe is shown below (SEQ ID NO: 183) (CDRL and CDRH residues are shown underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYVMSWVRQA PGKGLEWVAT ISSGGSYTYY PDSVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCARRG DSMITTDYWG QGTLVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCKAS QDVGTAVAWY QQKPGKAPKL LIYWASTRHT GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQQYSSYRT FGQGTKVEIK SGGGGSQVQL QQSGAELARP GASVKMSCKA SGYTFTRYTM HWVKQRPGQG LEWIGYI PS RGYTNYNQKF KDKATLTTDK SSSTAYMQLS
  • amino acid residues 1-119 correspond to the VH Domain of DR5 mAb 5 (SEQ ID NO:74)
  • amino acid residues 120-134 are the linker GGGGSGGGGSGGGGS (SEQ ID NO:184)
  • residues 135-240 correspond to the VL Domain of DR5 mAb 5 (SEQ ID NO:70)
  • residues 241-246 are the linker SGGGGS (SEQ ID NO: 185)
  • residues 247-438 correspond to the VH Domain of OKT3 (SEQ ID NO:115)
  • residues 439-453 are the linker GGGGSGGGGSGGGGS (SEQ ID NO: 184)
  • residues 454-560 correspond to the VL Domain of OKT3 (SEQ ID NO:114).
  • DR5 mAb 5 x OKT3 BiTE DR5 VLVH- OKT3
  • a second anti-DR5 x anti-CD3 BiTe molecule may be constructed in a VLVH orientation using the VL and VH Domains of DR5 mAb 5 and the VL and VH Domains of OKT3.
  • the BiTe would have an orientation of DR5 VLVH - OKT3 VLVH.
  • the amino acid sequence of the BiTe is shown below (SEQ ID NO: 186) (CDRL and CDRH residues are shown underlined):
  • a third anti-DR5 x anti-CD3 BiTe molecule may be constructed in a VHVL orientation using the VL and VH Domains of DR5 mAb 5 and the VL and VH Domains of OKT3.
  • the BiTe would have an orientation of OKT3 VHVL - DR5 VHVL.
  • the amino acid sequence of the BiTe is shown below (SEQ ID NO: 188) (CDRL and CDRH residues are shown underlined):
  • amino acid residues 1-192 correspond to the VH Domain of OKT3 (SEQ ID NO: 115)
  • residues 193-207 are the linker GGGGSGGGGSGGGGS (SEQ ID NO:184)
  • residues 208-314 correspond to the VL Domain of OKT3 SEQ ID NO:114
  • residues 315-320 are the linker SGGGGS (SEQ ID NO:185)
  • residues 321-439 correspond to the VH Domain of DR5 mAb 5 (SEQ ID NO:73)
  • residues 440-454 are the linker GGGGSGGGGSGGGGS (SEQ ID NO:184)
  • residues 455-560 correspond to the VL Domain of DR5 mAb 5 (SEQ ID NO:64).
  • OKT3 x DR5 mAb 5 BiTE OKT3 VLVH- DR5
  • a fourth anti-DR5 x anti-CD3 BiTe molecule may be constructed in a VLVH orientation using the VL and VH Domains of DR5 mAb 5 and the VL and VH Domains of OKT3.
  • the BiTe would have an orientation of OKT3 VLVH - DR5 VLVH.
  • the amino acid sequence of the BiTe is shown below (SEQ ID NO: 189) (CDRL and CDRH residues are shown underlined):
  • amino acid residues 1-107 correspond to the VL Domain of OKT3 (SEQ ID NO:114)
  • residues 108-122 are the linker GGGGS GGGGSGGGGS (SEQ ID NO:184)
  • residues 123-314 correspond to the VH Domain of OKT3 (SEQ ID NO:115)
  • residues 315-319 are the linker GGGGS (SEQ ID NO: 187)
  • residues 320-425 correspond to the VL Domain of DR5 mAb 5 (SEQ ID NO:70)
  • residues 426-440 are the linker GGGGS GGGGS GGGGS (SEQ ID NO:184)
  • residues 441-560 correspond to the VH Domain of DR5 mAb 5 (SEQ ID NO:74).
  • a diabody composed of two polypeptide chains may be constructed using the VL and VH Domains of DR5 mAb 6 and an anti-CD 16 antibody.
  • the amino acid sequence of the first polypeptide chain of the diabody may have the sequence (SEQ ID NO: 190) (CDRL and CDRH residues are shown underlined): DIAMTQSHKF MSTLVGDRVS ITCKASQDV TAIAWYQQKP GQSPKLLIYW ASTRHTGVPD RFYGSGSGTD YTLTISSMEA EDAATYYCQQ WSSNPLTFGA GTKLELKRAG GGSGGGGQVT LKESGPGILQ PSQTLSLTCS FSGFSLRTSG MGVGWIRQPS GKGLEWLAHI WWDDDKRYNP ALKSRLTISK DTSSNQVFLK IASVDTADTA TYYCAQINPA WFAYWGQGTL VTVSA
  • amino acid residues 1-109 correspond to the VL Domain of DR5 mAb 6 (SEQ ID NO:78)
  • residues 110-117 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 118-235 correspond to the amino acid sequence of the VH Domain of the CD 16 antibody (SEQ ID NO: 117).
  • amino acid sequence of the second polypeptide chain of the diabody may have the sequence (SEQ ID NO: 191) (CDRL and CDRH residues are shown underlined):
  • amino acid residues 1-111 correspond to the VL Domain of the CD16 antibody (SEQ ID NO:116)
  • residues 112-119 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 120-237 correspond to the amino acid sequence of the VH Domain of DR5 mAb 6 (SEQ ID NO:82).
  • a diabody composed of two polypeptide chains may be constructed using the VL and VH Domains of DR5 mAb 7 and an anti-NKG2D antibody.
  • the anti- NKG2D antibody binds the Natural Killer Group 2D (NKG2D) receptor.
  • the NKG2D receptor is expressed on all human (and other mammalian) Natural Killer cells (Bauer, S . et al. ( 1999) Activation OfNK Cells AndT Cells By NKG2D, A Receptor For Stress- Inducible MICA," Science 285(5428):727-729; Jamieson, A.M. et al.
  • Such diabodies have the ability to localize a NK cell (by binding such NK cell to the NKG2D-binding portion of the diabody) to the location of a cancer cell (by binding such cancer cell to the DR5-binding portion of the diabody).
  • the localized NK cell can then mediate the killing of the cancer cell in a process termed herein "redirected" killing.
  • NKG2D-binding portion of the diabody one could provide a TCR (T cell Receptor)-binding portion, so as to localize a T cell (by binding such T cell to the TCR-binding portion of the diabody) to the location of the DR5 -expressing cancer cell and accomplish redirected killing of the cancer cell.
  • amino acid sequence of the first polypeptide chain of the diabody may have the sequence (SEQ ID NO: 192) (CDRL and CDRH residues are shown underlined):
  • amino acid residues 1-104 correspond to the VL Domain of DR5 mAb 7 (SEQ ID NO:86)
  • residues 105-112 correspond to the intervening spacer peptide GGGSGGGG (Linker 1) (SEQ ID NO:33)
  • residues 113-233 correspond to the amino acid sequence of the VH Domain of the anti-NKG2D antibody (SEQ ID NO: 137)
  • residues 234-238 correspond to an AS KG linker(SEQ ID NO:47)
  • residues 239-266 correspond to a cysteine-containing E-coil Domain (SEQ ID NO:41).
  • amino acid sequence of the second polypeptide chain of the diabody may have the sequence (SEQ ID NO: 193) (CDRL and CDRH residues are shown underlined):
  • amino acid residues 1-110 correspond to the VL Domain of the anti-NKG2D antibody (SEQ ID NO: 136)
  • residues 111-118 correspond to the intervening spacer peptide GGGS GGGG (Linker 1) (SEQ ID NO:33)
  • residues 119-236 correspond to the amino acid sequence of the VH Domain of DR5 mAb 7 (SEQ ID NO:90)
  • residues 237-241 correspond to an AS TKG linker (SEQ ID NO:47)
  • residues 242-270 correspond to a cysteine-containing K-coil Domain (SEQ ID NO:42).
  • DR5 -Binding Molecles comprise three Light Chain (VL) CDRs and three Heavy Chain (VH) CDRs for each binding domain
  • VL Light Chain
  • VH Heavy Chain
  • the invention also includes DR5- Binding Molecles that possess:
  • An anti-DR5 polypeptide, and other DR5 agonists, antagonists and modulators can be created from the polynucleotides and/or sequences of the DR5 mAb 1 or DR5 mAb 2 antibodies by methods known in the art, for example, synthetically or recombinantly.
  • One method of producing such peptide agonists, antagonists and modulators involves chemical synthesis of the polypeptide, followed by treatment under oxidizing conditions appropriate to obtain the native conformation, that is, the correct disulfide bond linkages. This can be accomplished using methodologies well known to those skilled in the art (see, e.g., Kelley, R. F. et al.
  • Polypeptides of the invention may be conveniently prepared using solid phase peptide synthesis (Merrifield, B. (1986) "Solid Phase Synthesis," Science 232(4748):341-347; Houghten, R.A. (1985) "General Method For The Rapid Solid- Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen-Antibody Interaction At The Level Of Individual Amino Acids," Proc. Natl. Acad. Sci. (U.S.A.) 82(15):5131- 135; Ganesan, A. (2006) “Solid-Phase Synthesis In The Twenty-First Century," Mini Rev. Med. Chem. 6(1):3-10).
  • Fully human antibodies having one or more of the CDRs of DR5 mAb 1 or DR5 mAb 2 or which compete with DR5 mAb 1 or DR5 mAb 2 for binding to human DR5 or a soluble form thereof may be obtained through the use of commercially available mice that have been engineered to express specific human immunoglobulin proteins.
  • Transgenic animals that are designed to produce a more desirable (e.g. , fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XENOMOUSETM (Abgenix, Inc., Fremont, CA) and HUMAB-MOUSE® and TC MOUSETM (both from Medarex, Inc., Princeton, NJ).
  • antibodies may be made recombinantly and expressed using any method known in the art.
  • Antibodies may be made recombinantly by first isolating the antibodies made from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g. , CHO cells). Another method that may be employed is to express the antibody sequence in plants ⁇ (e.g., tobacco) or transgenic milk. Suitable methods for expressing antibodies recombinantly in plants or milk have been disclosed (see, for example, Peeters et al. (2001) "Production Of Antibodies And Antibody Fragments In Plants," Vaccine 19:2756; Lonberg, N. et al.
  • the antibodies or protein of interest may be subjected to sequencing by Edman degradation, which is well known to those of skill in the art.
  • the peptide information generated from mass spectrometry or Edman degradation can be used to design probes or primers that are used to clone the protein of interest.
  • An alternative method of cloning the protein of interest is by "panning" using purified DR5 or portions thereof for cells expressing an antibody or protein of interest that possesses one or more of the CDRs of DR5 mAb 1 or DR5 mAb 2 or that competes with DR5 mAb 1 or DR5 mAb 2 for binding to human DR5.
  • the "panning" procedure may be conducted by obtaining a cDNA library from tissues or cells that express DR5, overexpressing the cDNAs in a second cell type, and screening the transfected cells of the second cell type for a specific binding to DR5 in the presence or absence of DR5 mAb 1 or DR5 mAb 2.
  • Detailed descriptions of the methods used in cloning mammalian genes coding for cell surface proteins by “panning” can be found in the art (see, for example, Aruffo, A. et al. (1987) "Molecular Cloning Of A CD28 cDNA By A High-Efficiency COS Cell Expression System," Proc. Natl. Acad. Sci.
  • Vectors containing polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; and infection ⁇ e.g., where the vector is an infectious agent such as vaccinia virus).
  • electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances
  • microprojectile bombardment e.g., where the vector is an infectious agent such as vaccinia virus.
  • infection ⁇ e.g., where the vector is an infectious agent such as vaccinia virus.
  • the choice of introducing vectors or polynucleotides will often depend on features of the host cell.
  • Any host cells capable of overexpressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest.
  • suitable mammalian host cells include but are not limited to COS, HeLa, and CHO cells.
  • the host cells express the cDNAs at a level of about 5-fold higher, more preferably 10-fold higher, even more preferably 20-fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells.
  • Screening the host cells for a specific binding to DR5 is effected by an immunoassay or FACS. A cell overexpressing the antibody or protein of interest can be identified.
  • the invention includes polypeptides comprising an amino acid sequence of the antibodies of this invention.
  • the polypeptides of this invention can be made by procedures known in the art.
  • the polypeptides can be produced by proteolytic or other degradation of the antibodies, by recombinant methods (i.e., single or fusion polypeptides) as described above or by chemical synthesis.
  • Polypeptides of the antibodies, especially shorter polypeptides up to about 50 amino acids, are conveniently made by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available.
  • an anti-DR5 polypeptide could be produced by an automated polypeptide synthesizer employing the solid phase method.
  • the invention includes modifications to DR5 mAb 1 or DR5 mAb 2 antibodies and their polypeptide fragments that bind to DR5 and the agonists, antagonists, and modulators of such molecules, including functionally equivalent antibodies and fusion polypeptides that do not significantly affect the properties of such molecules as well as variants that have enhanced or decreased activity. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or use of chemical analogs.
  • Amino acid residues that can be conservatively substituted for one another include but are not limited to: glycine/alanine; serine/threonine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; lysine/arginine; and phenylalanine/tyrosine.
  • These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation.
  • the amino acid substitutions would be conservative, i.e., the substituted amino acid would possess similar chemical properties as that of the original amino acid.
  • conservative substitutions are known in the art, and examples have been provided above.
  • Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the Variable Domain. Changes in the Variable Domain can alter binding affinity and/or specificity. Other methods of modification include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, for attachment of labels for immunoassay, such as the attachment of radioactive moieties for radioimmunoassay. Modified polypeptides are made using established procedures in the art and can be screened using standard assays known in the art.
  • the invention encompasses fusion proteins comprising one or more of the polypeptides or DR5 mAb 1 or DR5 mAb 2 antibodies of this invention.
  • a fusion polypeptide is provided that comprises a light chain, a heavy chain or both a light and heavy chain.
  • the fusion polypeptide contains a heterologous immunoglobulin constant region.
  • the fusion polypeptide contains a light chain Variable Domain and a heavy chain Variable Domain of an antibody produced from a publicly-deposited hybridoma.
  • an antibody fusion protein contains one or more polypeptide domains that specifically bind to DR5 and another amino acid sequence to which it is not attached in the native molecule, for example, a heterologous sequence or a homologous sequence from another region.
  • compositions comprising the DR5-binding molecules of the present invention (e.g., diabodies comprising antigen-binding domains from anti-DR5 antibodies, such as DR5 mAb 1 and DR5 mAb 2, or their humanized derivatives), polypeptides derived from such molecules, polynucleotides comprising sequences encoding such molecules or polypeptides, and other agents as described herein.
  • DR5-binding molecules of the present invention e.g., diabodies comprising antigen-binding domains from anti-DR5 antibodies, such as DR5 mAb 1 and DR5 mAb 2, or their humanized derivatives
  • polypeptides derived from such molecules polynucleotides comprising sequences encoding such molecules or polypeptides, and other agents as described herein.
  • the DR5-binding molecules of the present invention have the ability to act as agonist agents, mimicking TRAIL, and thus leading to the activation of DR5.
  • the monospecific antibodies, DR5 mAb 1 and DR5 mAb 2, their humanized derivatives, and their DR5-binding fragments may be used as surrogates for TRAIL so as to promote the death of tumor cells that express DR5. Since DR5 is ubiquitously distributed in tumor cell lines, the monospecific DR5- binding molecules of the present invention provide a general therapy for cancer.
  • the cancers that may be treated by such molecules include cancers 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, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, a gallbladder or bile duct
  • the monospecific DR5-binding molecules of the present invention may be used in the treatment of 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.
  • the bispecific DR5-binding molecules of the present invention augment the cancer therapy provided by DR5 by promoting the redirected killing of tumor cells that express the second specificity of such molecules (e.g., CD3, CD 16, CD 19, NKG2D, etc.).
  • Such DR5 -binding molecules are particularly useful for the treatment of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including blastic crisis of CML and Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplasia syndrome (MDS), acute B lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), including Richter's syndrome or Richter's transformation of 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, and Burkitt's lymphoma.
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • the DR5 -binding molecules of the present invention may be detectably labeled and used in the diagnosis of cancer or in the imaging of tumors and tumor cells.
  • compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms.
  • Such compositions comprise a prophylactically or therapeutically effective amount of the DR5 -binding molecules of the present invention, or a combination of such agents and a pharmaceutically acceptable carrier.
  • compositions of the invention comprise a prophylactically or therapeutically effective amount of the DR5 -binding molecules of the present invention and a pharmaceutically acceptable carrier.
  • the invention particularly encompasses such pharmaceutical compositions in which the DR5 -binding molecule is: a DR5 mAb 1, a DR5 mAb 2 antibody, a humanized DR5 mAb 1, a humanized DR5 mAb 2 antibody, or a DR5- binding fragment of any such antibody, or a bispecific DR5 diabody (especially, a DR5 x CD3 bispecific monovalent Fc diabody).
  • a bispecific DR5 diabody especially, a DR5 x CD3 bispecific monovalent Fc diabody.
  • such molecules that comprise the 3 CDRLS and the 3 CDRHS of DR5 mAb 1, or the 3 CDRLS and the 3 CDRHS of DR5 mAb 2.
  • compositions that additionally include a second therapeutic antibody (e.g., tumor-specific monoclonal antibody) that is specific for a particular cancer antigen, and a pharmaceutically acceptable carrier.
  • a second therapeutic antibody e.g., tumor-specific monoclonal antibody
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include, but are not limited to those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with a DR5 -binding molecule of the present invention (and more preferably, a DR5 mAb 1 or DR5 mAb 2 antibody, a humanized DR5 mAb 1 or humanized DR5 mAb 2 antibody, (or a DR5 -binding fragment of any such antibody) or in which the DR5 -binding molecule is a bispecific DR5 diabody (especially, a DR5 x CD3 bispecific monovalent Fc diabody).
  • a DR5 -binding molecule of the present invention and more preferably, a DR5 mAb 1 or DR5 mAb 2 antibody, a humanized DR5 mAb 1 or humanized DR5 mAb 2 antibody, (or a DR5 -binding fragment of any such antibody) or in which the DR5 -binding molecule is a bispecific DR5 diabody (especially, a
  • Such molecules that comprise the 3 CDRLS and the 3 CDRHS of DR5 mAb 1, or the 3 CDRLS and the 3 CDRHS of DR5 mAb 2, alone or with such pharmaceutically acceptable carrier.
  • one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • kits that can be used in the above methods.
  • a kit can comprise any of the DR5 -binding molecules of the present invention.
  • the kit can further comprise one or more other prophylactic and/or therapeutic agents useful for the treatment of cancer, in one or more containers; and/or the kit can further comprise one or more cytotoxic antibodies that bind one or more cancer antigens associated with cancer.
  • the other prophylactic or therapeutic agent is a chemotherapeutic.
  • the prophylactic or therapeutic agent is a biological or hormonal therapeutic.
  • compositions of the present invention may be provided for the treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder or infection by administering to a subject an effective amount of a fusion protein or a conjugated molecule of the invention, or a pharmaceutical composition comprising a fusion protein or a conjugated molecule of the invention.
  • such compositions are substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side effects).
  • the subject is an animal, preferably a mammal such as non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., monkey such as, a cynomolgus monkey, human, etc.).
  • a mammal such as non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., monkey such as, a cynomolgus monkey, human, etc.).
  • the subject is a human.
  • compositions of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fusion protein, receptor-mediated endocytosis (See, e.g., Wu et al. (1987) "Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System, " J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • Methods of administering a molecule of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes).
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous
  • epidural e.g., intranasal and oral routes
  • mucosal e.g., intranasal and oral routes.
  • the DR5 -binding molecules of the present invention are administered intramuscularly, intravenously, or subcutaneously.
  • the compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • Administration can be systemic or local.
  • pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • inhaler or nebulizer e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the invention also provides that the DR5 -binding molecules of the present invention are packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the molecule.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of the molecule.
  • such molecules are supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g. , with water or saline to the appropriate concentration for administration to a subject.
  • the DR5 -binding molecules of the present invention are supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 ⁇ g, more preferably at least 10 ⁇ g, at least 15 ⁇ g, at least 25 ⁇ g, at least 50 ⁇ g, at least 100 ⁇ g, or at least 200 ⁇ g.
  • the lyophilized DR5 -binding molecules of the present invention should be stored at between 2 and 8°C in their original container and the molecules should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted.
  • such molecules are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the molecule, fusion protein, or conjugated molecule.
  • such DR5 -binding molecules when provided in liquid form are supplied in a hermetically sealed container in which the molecules are present at a concentration of least 1 ⁇ g/ml, more preferably at least 2.5 ⁇ g/ml, at least 5 ⁇ g/ml, at least 10 ⁇ g/ml, at least 50 ⁇ g/ml, or at least 100 ⁇ g/ml.
  • composition of the invention which will be effective in the treatment, prevention or amelioration of one or more symptoms associated with a disorder can be determined by standard clinical techniques.
  • dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the dosage administered to a patient is preferably determined based upon the body weight (kg) of the recipient subject.
  • the dosage administered is typically from at least about 0.3 ng/kg per day to about 0.9 ng/kg per day, from at least about 1 ng/kg per day to about 3 ng/kg per day, from at least about 3 ng/kg per day to about 9 ng/kg per day, from at least about 10 ng/kg per day to about 30 ng/kg per day, from at least about 30 ng/kg per day to about 90 ng/kg per day, from at least about 100 ng/kg per day to about 300 ng/kg per day, from at least about 200 ng/kg per day to about 600 ng/kg per day, from at least about 300 ng/kg per day to about 900 ng/kg per day, from at least about 400 ng/kg per day to about 800 ng/kg per day, from at least about 500
  • the patient is administered a treatment regimen comprising one or more doses of such prophylactically or therapeutically effective amount of a DR5 -binding molecule of the present invention, wherein the treatment regimen is administered over 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.
  • the treatment regimen comprises intermittently administering doses of the prophylactically or therapeutically effective amount of the DR5 -binding molecules of the present invention (for example, administering a dose on day 1 , day 2, day 3 and day 4 of a given week and not administering doses of the prophylactically or therapeutically effective amount of the DR5 -binding molecule (and particularly, a DR5 mAb 1 , a DR5 mAb 2 antibody, a humanized DR5 mAb 1 , a humanized DR5 mAb 2 antibody, or a DR5 -binding fragment of any such antibody, or a bispecific DR5 diabody (especially, a DR5 x CD3 bispecific monovalent Fc diabody).
  • intermittently administering doses of the prophylactically or therapeutically effective amount of the DR5 -binding molecules of the present invention for example, administering a dose on day 1 , day 2, day 3 and day 4 of a given week and not administering doses
  • preferably encompassed is the administration (on day 5, day 6, and day 7 of the same week) of such molecules that comprise the 3 CDRLS and the 3 CDRHS of DR5 mAb 1 , or the 3 CDRLS and the 3 CDRHS of DR5 mAb 2) on day 5, day 6 and day 7 of the same week).
  • administration on day 5, day 6, and day 7 of the same week
  • such molecules that comprise the 3 CDRLS and the 3 CDRHS of DR5 mAb 1 , or the 3 CDRLS and the 3 CDRHS of DR5 mAb 2) on day 5, day 6 and day 7 of the same week.
  • there are 1 , 2, 3, 4, 5 or more courses of treatment may be the same regimen or a different regimen.
  • the administered dose escalates over the first quarter, first half or first two-thirds or three-quarters of the regimen(s) (e.g., over the first, second, or third regimens of a 4 course treatment) until the daily prophylactically or therapeutically effective amount of the DR5 -binding molecule is achieved.
  • Table 3 provides 5 examples of different dosing regimens described above for a typical course of treatment.
  • the dosage and frequency of administration of a DR5 -binding molecule of the present invention may be reduced or altered by enhancing uptake and tissue penetration of the molecule by modifications such as, for example, lipidation.
  • the dosage of a DR5-binding molecule of the invention administered to a patient may be calculated for use as a single agent therapy.
  • the molecule may be used in combination with other therapeutic compositions and the dosage administered to a patient are lower than when said molecules are used as a single agent therapy.
  • compositions of the invention may be administered locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • an implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • care must be taken to use materials to which the molecule does not absorb.
  • compositions of the invention can be delivered in a vesicle, in particular a liposome ⁇ See Langer (1990) "New Methods Of Drug Delivery, " Science 249: 1527- 1533); Treat et al, in LIPOSOMES IN THE THERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 3 17-327).
  • compositions of the invention can be delivered in a controlled-release or sustained-release system. Any technique known to one of skill in the art can be used to produce sustained-release formulations comprising one or more of the DR5 -binding molecule(s) of the invention. See, e.g., U.S. Patent No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al. (1996) "Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A Sustained-Release Gel, " Radiotherapy & Oncology 39: 179-189, Song et al.

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Abstract

La présente invention concerne l'anticorps anti-DR5 mAb 1 et mAb 2, et des versions humanisées et chimériques de tels anticorps. L'invention concerne en outre des molécules de liaison de DR5 qui comprennent des fragments de telles molécules, et des molécules bispécifiques, comprenant des diabodies, des BiTes, des anticorps bispécifiques knob/hole, etc., qui comprennent : (i) de tels fragments de liaison de DR5 et (ii) un domaine capable de se lier à un épitope d'une molécule présente sur la surface d'une cellule effectrice.
PCT/US2015/033082 2015-01-26 2015-05-29 Anticorps anti-dr5 et molécules comprenant des domaines de liaison de dr5 de ceux-ci WO2016122701A1 (fr)

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EP3582806A4 (fr) * 2017-02-20 2020-12-16 Dragonfly Therapeutics, Inc. Protéines se liant à her2, nkg2d et cd16
CN112437672A (zh) * 2018-05-07 2021-03-02 蜻蜓疗法股份有限公司 结合nkg2d、cd16和肿瘤相关抗原的蛋白质
EP3579848A4 (fr) * 2017-02-08 2021-03-03 Dragonfly Therapeutics, Inc. Protéines de fixation multi-spécifiques destinées à l'activation de cellules tueuses naturelles et leurs utilisations thérapeutiques pour traiter le cancer
EP3630181A4 (fr) * 2017-05-23 2021-03-17 Dragonfly Therapeutics, Inc. Protéine de liaison nkg2d, cd16 et antigène associé à une tumeur
EP3583133A4 (fr) * 2017-02-20 2021-04-14 Dragonfly Therapeutics, Inc. Protéines de liaison à gd2, nkg2d et cd16
EP3630169A4 (fr) * 2017-05-23 2021-04-21 Dragonfly Therapeutics, Inc. Protéine se liant au nkg2d, cd16 et antigène associé à une tumeur
EP3668893A4 (fr) * 2017-08-16 2021-08-04 Dragonfly Therapeutics, Inc. Protéines se liant à nkg2d, cd16 et à l'egfr, hla-e, ccr4, ou pd-l1
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EP3583133A4 (fr) * 2017-02-20 2021-04-14 Dragonfly Therapeutics, Inc. Protéines de liaison à gd2, nkg2d et cd16
US11884732B2 (en) 2017-02-20 2024-01-30 Dragonfly Therapeutics, Inc. Proteins binding HER2, NKG2D and CD16
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EP3630183A4 (fr) * 2017-05-23 2021-03-03 Dragonfly Therapeutics, Inc. Protéine de liaison à nkg2d, cd16 et ror1 ou ror2
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US11566067B2 (en) 2017-11-10 2023-01-31 Ngm Biopharmaceuticals, Inc. Methods of lowering triglyceride levels with an ANGPTL8-binding antibody or antigen-binding fragment thereof
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US11884733B2 (en) 2018-02-08 2024-01-30 Dragonfly Therapeutics, Inc. Antibody variable domains targeting the NKG2D receptor
EP3749347A4 (fr) * 2018-02-08 2021-11-03 Dragonfly Therapeutics, Inc. Polythérapie de cancer impliquant des protéines de liaison multi-spécifiques qui activent des cellules tueuses naturelles
US11939384B1 (en) 2018-02-08 2024-03-26 Dragonfly Therapeutics, Inc. Antibody variable domains targeting the NKG2D receptor
EP3790585A4 (fr) * 2018-05-07 2022-05-11 Dragonfly Therapeutics, Inc. Protéine de liaison au nkg2d, au cd16 et à un antigène associé à une tumeur
CN112437672A (zh) * 2018-05-07 2021-03-02 蜻蜓疗法股份有限公司 结合nkg2d、cd16和肿瘤相关抗原的蛋白质
JP2021523140A (ja) * 2018-05-07 2021-09-02 ドラゴンフライ セラピューティクス， インコーポレイテッド Ｎｋｇ２ｄ、ｃｄ１６、及び腫瘍関連抗原に結合するタンパク質
WO2021216739A1 (fr) * 2020-04-23 2021-10-28 Baylor College Of Medicine Surmonter le micro-environnement tumoral pour une thérapie cellulaire par ciblage de cellules suppressives dérivées de myéloïde par l'intermédiaire d'un récepteur spécifique de trail-r2
US11883432B2 (en) 2020-12-18 2024-01-30 Century Therapeutics, Inc. Chimeric antigen receptor system with adaptable receptor specificity
WO2024083021A1 (fr) * 2022-10-20 2024-04-25 北京三诺佳邑生物技术有限责任公司 Combinaison d'anticorps se liant spécifiquement à trail ou fasl, et anticorps bispécifique

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