WO2018119166A1 - Adam9-binding molecules, and methods of use thereof - Google Patents

Adam9-binding molecules, and methods of use thereof Download PDF

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Publication number
WO2018119166A1
WO2018119166A1 PCT/US2017/067770 US2017067770W WO2018119166A1 WO 2018119166 A1 WO2018119166 A1 WO 2018119166A1 US 2017067770 W US2017067770 W US 2017067770W WO 2018119166 A1 WO2018119166 A1 WO 2018119166A1
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domain
seq
adam9
binding
molecule
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English (en)
French (fr)
Inventor
Deryk T. Loo
Juniper A. SCRIBNER
Bhaswati Barat
Gundo Diedrich
Leslie S. Johnson
Ezio Bonvini
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Macrogenics Inc
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Macrogenics Inc
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Priority to US16/471,449 priority Critical patent/US11242402B2/en
Priority to RU2019119078A priority patent/RU2783619C2/ru
Priority to EP25206714.5A priority patent/EP4653464A2/en
Priority to JP2019534220A priority patent/JP7128819B2/ja
Priority to BR112019012950A priority patent/BR112019012950A2/pt
Priority to EP17882545.1A priority patent/EP3558368B1/en
Priority to KR1020197020727A priority patent/KR102630036B1/ko
Priority to CN201780079587.1A priority patent/CN110167591B/zh
Priority to CA3048211A priority patent/CA3048211A1/en
Application filed by Macrogenics Inc filed Critical Macrogenics Inc
Priority to AU2017382234A priority patent/AU2017382234B2/en
Priority to IL267589A priority patent/IL267589B2/en
Priority to MX2019007404A priority patent/MX2019007404A/es
Publication of WO2018119166A1 publication Critical patent/WO2018119166A1/en
Priority to ZA2019/03967A priority patent/ZA201903967B/en
Anticipated expiration legal-status Critical
Priority to US17/553,941 priority patent/US20220324995A1/en
Priority to US18/915,947 priority patent/US20250115676A1/en
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    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
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Definitions

  • the present invention is directed to molecules, such as monospecific antibodies and bispecific, trispecific or multispecific binding molecules, including diabodies, BiTEs, and antibodies that are capable of specifically binding to "Disintegrin and Metalloproteinase Domain-containing Protein 9" ("ADAM9").
  • ADAM9 "Disintegrin and Metalloproteinase Domain-containing Protein 9"
  • the invention particularly concerns such binding molecules that are capable of exhibiting high affinity binding to human and non-human ADAM9.
  • the invention further particularly relates to such molecules that are thereby cross-reactive with human ADAM9 and the ADAM9 of a non- human primate (e.g. , a cynomolgus monkey).
  • the invention additionally pertains to all such ADAM9-binding molecules that comprise a Light Chain Variable (VL) Domain and/or a Heavy Chain Variable (VH) Domain that has been humanized and/or deimmunized so as to exhibit reduced immunogenicity upon administration of such ADAM9-binding molecule to a recipient subject.
  • the invention is also directed to pharmaceutical compositions that contain any of such ADAM9-binding molecules, and to methods involving the use of any of such ADAM9-binding molecules in the treatment of cancer and other diseases and conditions.
  • ADAM is a family of proteins involved in various physiologic and pathologic processes (Amendola, R.S. et al. (2015) "ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2 " J. Leukocyte Biol. 97(5):951-962; Edwars, D R. et al. (2008) “The ADAM Metalloproteases " Molec. Aspects Med. 29:258-289). At least 40 gene members of the family have been identified, and at least 21 of such members are believed to be functional in humans (Li, J. et al. (2016) "Overexpression of ADAM9 Promotes Colon Cancer Cells Invasion " J. Invest. Surg.
  • ADAM family members have a well-conserved structure with 8 domains, among which are a metalloprotease domain and an integrin-binding (disintegrin) domain (Duffy, M.J. et al. (2009) "The Role Of ADAMs In Disease Pathophysiology," Clin. Chim. Acta 403 :31-36).
  • the ADAM metalloprotease domain acts as a sheddase and has been reported to modulate a series of biologic processes by cleaving transmembrane proteins, which then can act as soluble ligands and regulate cellular signaling (Amendola, R.S. et al.
  • ADAM9 is a member of the ADAM family of molecule. It is synthesized as an inactive form which is proteolytically cleaved to generate an active enzyme. Processing at the upstream site is particularly important for activation of the proenzyme. ADAM9 is expressed in fibroblasts (Zigrino, P. et al. (2011) “The Disintegrin-Like And Cysteine-Rich Domains Of ADAM-9 Mediate Interactions Between Melanoma Cells And Fibroblasts " J. Biol. Chem. 286:6801-6807), activated vascular smooth muscle cells (Sun, C. et al.
  • ADAM9's metalloprotease activity participates in the degradation of matrix components, to thereby allow migration of tumor cells (Amendola, R.S. et al. (2015) "ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2 " J. Leukocyte Biol. 97(5):951-962). Its disintegrin domain, which is highly homologous to many snake-venom disintegrins, allows the interaction between ADAM9 and integrins, and enables ADAM9 to modulate, positively or negatively, cell adhesion events (Zigrino, P. et al.
  • ADAM9 has been found to be relevant to disease, especially cancer. ADAM9 has been found to cleave and release a number of molecules with important roles in tumorigenesis and angiogenesis, such as TEK, KDR, EPHB4, CD40, VCAM1 and CDH5. ADAM9 is expressed by many types of tumor cells, including tumor cells of breast cancers, colon cancers, gastric cancers, gliomas, liver cancers, non-small cell lung cancers, melanomas, myelomas, pancreatic cancers and prostate cancers (Yoshimasu, T. etal. (2004) "Overexpression Of ADAM9 In Non-Small Cell Lung Cancer Correlates With Brain Metastasis; Cancer Res.
  • ADAM9 expression has been found to correlate positively with tumor malignancy and metastatic potential (Amendola, R.S. et al. (2015) "ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Inte grins And CXCR2 " J. Leukocyte Biol. 97(5):951-962; Fan, X. et al. (2016) "ADAM9 Expression Is Associate with Glioma Tumor Grade and Histological Type, and Acts as a Prognostic Factor in Lower-Grade Gliomas " Int. J. Mol. Sci. 17: 1276: 1-11; Li, J. et al.
  • ADAM9 and its secreted soluble isoform seem to be crucial for cancer cells to disseminate (Amendola, R.S. et al. (2015) "ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2 " J. Leukocyte Biol. 97(5):951-962; Fry, J.L. et al. (2010) "Secreted And Membrane-Bound Isoforms Of Protease ADAM9 Have Opposing Effects On Breast Cancer Cell Migration " Cancer Res.
  • ADAM9 As a potential target for anticancer therapy (Peduto, L. (2009) "ADAM9 As A Potential Target Molecule In Cancer " Curr. Pharm. Des. 15:2282-2287; Duffy, M.J. et al. (2009) “Role Of ADAMs In Cancer Formation And Progression " Clin. Cancer Res. 15: 1140-1144; Duffy, M.J. et al. (2011) "The ADAMs Family Of Proteases: New Biomarkers And Therapeutic Targets For Cancer?" Clin. Proteomics 8:9: 1-13; Josson, S. et al.
  • Prostate 71(3):232-240 see also US Patent Publication Nos. 2016/0138113, 2016/0068909, 2016/0024582, 2015/0368352, 2015/0337356, 2015/0337048, 2015/0010575, 2014/0342946, 2012/0077694, 2011/0151536, 2011/0129450, 2010/0291063, 2010/0233079, 2010/0112713, 2009/0285840, 2009/0203051, 2004/0092466, 2003/0091568, and 2002/0068062, and PCT Publication Nos.
  • ADAM9 has also been found to be relevant to pulmonary disease and inflammation (see, e.g., US Patent Publication Nos. 2016/0068909; 2012/0149595; 2009/0233300; 2006/0270618; and 2009/0142301).
  • Antibodies that bind to ADAM9 are commercially available from Abeam, Thermofisher, Sigma-Aldrich, and other companies.
  • the present invention is directed to molecules, such as monospecific antibodies and bispecific, trispecific or multispecific binding molecules, including diabodies, BiTEs, and antibodies that are capable of specifically binding to "Disintegrin and Metalloproteinase Domain-containing Protein 9" ("ADAM9").
  • ADAM9 "Disintegrin and Metalloproteinase Domain-containing Protein 9"
  • the invention particularly concerns such binding molecules that are capable of exhibiting high affinity binding to human and non-human ADAM9.
  • the invention further particularly relates to such molecules that are thereby cross-reactive with human ADAM9 and the ADAM9 of a non- human primate (e.g. , a cynomolgus monkey).
  • the invention additionally pertains to all such ADAM9-binding molecules that comprise a Light Chain Variable (VL) Domain and/or a Heavy Chain Variable (VH) Domain that have been humanized and/or deimmunized so as to exhibit reduced immunogenicity upon administration of such ADAM9-binding molecule to a recipient subject.
  • the invention is also directed to pharmaceutical compositions that contain any of such ADAM9-binding molecules, and to methods involving the use of any of such ADAM9-binding molecules in the treatment of cancer and other diseases and conditions.
  • the invention provides an ADAM9-binding molecule that comprises an ADAM9-binding domain, wherein such ADAM9-binding domain comprises a Light Chain Variable (VL) Domain and a Heavy Chain Variable (VH) Domain, wherein such Heavy Chain Variable Domain comprises a CDRHI Domain, a CDRH2 Domain and a CDRH3 Domain, and such Light Chain Variable Domain comprises a CDRLI Domain, a CDRL2 Domain, and a CDRL3 Domain, wherein:
  • such CDRHI Domain, CDRH2 Domain and CDRH3 Domain have the amino acid sequence of the CDRHI Domain, CDRH2 Domain and CDRH3 Domain of a Heavy Chain Variable (VH) Domain of an optimized variant of MAB- A; and such CDRLI Domain, CDRL2 Domain, and CDRL3 Domain have the amino acid sequence of the CDRLI Domain, CDRL2 Domain, and CDRL3 Domain of the Light Chain Variable (VL) Domain of MAB-A; or
  • such CDRHI Domain, CDRH2 Domain and CDRH3 Domain have the amino acid sequence of the CDRHI Domain, CDRH2 Domain and CDRH3 Domain of the Heavy Chain Variable (VH) Domain of MAB-A; and such CDRLI Domain, CDRL2 Domain, and CDRL3 Domain have the amino acid sequence of the CDRLI Domain, CDRL2 Domain, and CDRL3 Domain of a Light Chain Variable (VL) Domain of an optimized variant of MAB-A; or
  • C such CDRHI Domain, CDRH2 Domain and CDRH3 Domain have the amino acid sequence of the CDRHI Domain, CDRH2 Domain and CDRH3 Domain of a Heavy Chain Variable (VH) Domain of an optimized variant of MAB- A; and such CDRLI Domain, CDRL2 Domain, and CDRL3 Domain have the amino acid sequence of the CDRLI Domain, CDRL2 Domain, and CDRL3 Domain of a Light Chain Variable (VL) Domain of an optimized variant of MAB-A
  • the invention particularly concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding domain possesses:
  • VH Heavy Chain Variable (VH) Domain of MAB-A
  • VL Light Chain Variable
  • VH Heavy Chain Variable
  • VL Chain Variable (VL) Domain of an optimized variant of MAB-A
  • VL Light Chain Variable
  • the invention additionally concerns the embodiment of all such ADAM9- binding molecules, wherein such Heavy Chain Variable (VH) Domain of such optimized variant of MAB-A comprises the amino acid sequence of SEQ ID NO: 15:
  • Xi is M or I
  • X2 is N or F
  • X 5 is S or G
  • X 6 is P, F, Y, W, I, L, V, T, G or D;
  • X 7 , Xs, X9, Xio, and Xn are selected such that:
  • X6 is F, Y or W;
  • X 7 is N or H;
  • Xs is S or K;
  • X9 is G or A;
  • Xio is T or V; and
  • Xn is M, L or K;
  • the invention additionally concerns the embodiment of all such ADAM9- binding molecules, wherein such CDRHI Domain, CDRH2 Domain and CDRH3 Domain of such Heavy Chain Variable (VH) Domain of such optimized variant of MAB-A respectively have the amino acid sequences of:
  • X 2 , X3 , 4, and X5 are independently selected, and wherein: X2 is N or F; X3 is K or R; X4 is K or Q; and X5 is S or G; and
  • X 6 is P, F, Y, W, I, L, V, T, G or D, and X 7 , X 8 , X9, X10, and Xn are selected such that:
  • X10 is W or F; and Xn is M, L or K;
  • X10 is T or V; and Xn is M, L or K;
  • X10 is V; and Xn is M, L or K;
  • X 7 is G
  • X 8 is K, M or N
  • X 9 is G
  • X10 is V or T; and Xn is L or M;
  • the invention additionally concerns the embodiment of all such ADAM9- binding molecules, wherein such Heavy Chain Variable (VH) Domain of such optimized variant of MAB-A is selected from the group consisting of:
  • the invention additionally concerns the embodiment of all such ADAM9- binding molecules, wherein such Light Chain Variable (VL) Domain comprises the amino acid sequence of SEQ ID NO:53:
  • X12, X13, Xi4, X15, Xi6, and Xi 7 are independently selected, and wherein: Xi 2 is K or R; X13 is D or S;
  • Xi4 is M or L; X15 is H or Y;
  • Xi6 is E or S; and X17 is D or T.
  • the invention additionally concerns the embodiment of all such ADAM9- binding molecules, wherein such CDRL I Domain, CDRL2 Domain and CDRL3 Domain of such Light Chain Variable (VL) Domain of such optimized variant of MAB-A respectively have the amino acid sequences of:
  • X15, Xi6, and X17 are independently selected, and wherein: X15 is H or Y; Xi6 is E or S; and X17 is D or T.
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such Light Chain Variable (VL) Domain of such optimized variant of MAB-A is selected from the group consisting of:
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein the ADAM9-binding domain comprises:
  • CDRL2 Domain that comprises the amino acid sequence AASDLES (SEQ ID NO: 13); or (3) a CDRL3 Domain that comprises the amino acid sequence QQSHEDPFT (SEQ ID NO:14);
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein the ADAM9-binding domain comprises the CDRHI Domain that comprises the amino acid sequence SYWMH (SEQ ID NO:8), the CDRH2 Domain that comprises the amino acid sequence E I I P I FGHTNYNEKFKS (SEQ ID NO:35), and the CDR H 3 Domain that comprises the amino acid sequence GGYYYYPRQGFLDY (SEQ ID NO:45)
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein the ADAM9-binding domain comprises the CDRLI Domain that comprises the amino acid sequence KASQSVDYSGDSYMN (SEQ ID NO:62), the CDR L 2 Domain that comprises the amino acid sequence AASDLES (SEQ ID NO: 13), and the CDRL3 Domain that cpomprises the amino acid sequence QQSHEDPFT (SEQ ID NO:14).
  • the ADAM9-binding domain comprises the CDRLI Domain that comprises the amino acid sequence KASQSVDYSGDSYMN (SEQ ID NO:62), the CDR L 2 Domain that comprises the amino acid sequence AASDLES (SEQ ID NO: 13), and the CDRL3 Domain that cpomprises the amino acid sequence QQSHEDPFT (SEQ ID NO:14).
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding domain comprises:
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding domain comprises a CDRHI domain, a CDRH2 domain, and a CDRH3 domain and a CDRL I domain, a CDRL2 domain, and a CDRL3 domain having the sequences selected from the group consisting of:
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) having sequences that are at least 90%, at least 95%, or at least 99% identical to sequences selected from the group consisting of:
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) having the sequences selected from the group consisting of:
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding domain has at least a 150-fold enhancement in binding affinity to cyno ADAM9 and retains high affinity binding to human ADAM9 as compared to MAB-A.
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding domain comprises a CDRHI domain, a CDRH2 domain, and a CDRH3 domain and a CDRL I domain, a CDRL2 domain, and a CDRL3 domain having the sequences selected from the group consisting of:
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) having sequences that are at least 90%, at least 95%, or at least 99% identical to sequences selected from the group consisting of:
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) having the sequences selected from the group consisting of:
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the invention additionally concerns the embodiment of all such ADAM9- binding molecules, wherein such molecule is a monospecific ADAM9-binding antibody or an ADAM9-binding fragment thereof, or wherein such molecule is a bispecific antibody.
  • the invention additionally concerns the embodiment of all such ADAM9- binding molecules, wherein such molecule is a diabody, such diabody being a covalently bonded complex that comprises two, three, four or five polypeptide chains.
  • the invention additionally concerns the embodiment of all such ADAM9- binding molecules, wherein such molecule is a trivalent binding molecule, such trivalent binding molecule being a covalently bonded complex that comprises three, four, five, or more polypeptide chains.
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such molecule comprises an Albumin-Binding Domain (ABD).
  • ADAM9-binding molecules comprises an Albumin-Binding Domain (ABD).
  • ABSD Albumin-Binding Domain
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such molecule comprises an Fc Region, and particularly the embodiment wherein such Fc Region is a variant Fc Region that comprises:
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such one or more amino acid modification(s) that reduce(s) the affinity of the variant Fc Region for an FcyR comprise:
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such one or more amino acid modification(s) that that enhance(s) the serum half-life of such ADAM9-binding molecule comprise:
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such molecule is bispecific and comprises an epitope-binding site capable of immunospecific binding to an epitope of ADAM9 and an epitope-binding site capable of immunospecific binding to an epitope of a molecule present on the surface of an effector cell.
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such molecule comprises two epitope-binding sites capable of immunospecific binding to epitope(s) of ADAM9 and two epitope-binding sites capable of immunospecific binding to epitope(s) of a molecule present on the surface of an effector cell.
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such molecule is trispecific and comprises:
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such molecule is capable of simultaneously binding to ADAM9 and such molecule present on the surface of an effector cell.
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such molecule present on the surface of an effector cell is CD2, CD3, CD8, TCR, or KG2D.
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such effector cell is a cytotoxic T-cell or a Natural Killer (NK) cell.
  • NK Natural Killer
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such first molecule present on the surface of an effector cell is CD3 and such second molecule present on the surface of an effector cell is CD8
  • the invention additionally concerns the embodiment of such ADAM9-binding molecules, wherein such ADAM9-binding molecule mediates coordinated binding of a cell expressing ADAM9 and a cytotoxic T cell.
  • the invention additionally concerns a pharmaceutical composition that comprises an effective amount of any of the above-described ADAM9-binding molecules and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention additionally concerns the use of any of the above-described ADAM9-binding molecules, or the use of the above-described pharmaceutical composition in the treatment of a disease or condition associated with, or characterized by, the expression of ADAM9.
  • the invention particularly concerns such use wherein such disease or condition associated with, or characterized by, the expression of ADAM9 is cancer, and especially wherein such cancer is selected from the group consisting: bladder cancer, breast cancer, cervical cancer, colorectal cancer (especially an adenocarcinoma, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, primary colorectal lymphoma, leiomyosarcoma, melanoma, or squamous cell carcinoma), esophageal cancer, gastric cancer, head and neck cancer, liver cancer, non-small-cell lung cancer (especially a squamous cell carcinoma, adenocarcinoma, or large-cell undifferentiated carcinoma), myeloid cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, and uterine cancer.
  • cancer is selected from the group consisting: bladder cancer, breast cancer, cervical cancer, colorectal cancer (especially an adenocarcinoma, gastrointestinal car
  • the invention additionally concerns a method for treating a disease or condition associated with, or characterized by, the expression of ADAM9 in a subject comprising administering to such subject an effective amount of any of the above-described ADAM9-binding molecules, or any of the above-described pharmaceutical compositions.
  • the invention particularly concerns such method wherein such disease or condition associated with, or characterized by, the expression of ADAM9 is cancer, and especially wherein such cancer is selected from the group consisting: bladder cancer, breast cancer, cervical cancer, colorectal cancer (especially an adenocarcinoma, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, primary colorectal lymphoma, leiomyosarcoma, melanoma, or squamous cell carcinoma), esophageal cancer, gastric cancer, head and neck cancer, liver cancer, non-small-cell lung cancer (especially a squamous cell carcinoma, adenocarcinoma, or large-cell undifferentiated carcinoma), myeloid cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, and uterine cancer.
  • cancer is selected from the group consisting: bladder cancer, breast cancer, cervical cancer, colorectal cancer (especially an adenocarcinoma, gastrointestinal car
  • Figure 1 provides a schematic of a representative covalently bonded diabody having two epitope-binding sites composed of two polypeptide chains, each having an E- coil or K-coil Heterodimer-Promoting Domain (alternative Heterodimer-Promoting Domains are provided below).
  • a cysteine residue may be present in a linker and/or in the Heterodimer-Promoting Domain as shown in Figure 3B.
  • VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern.
  • Figure 2 provides a schematic of a representative covalently bonded diabody molecule having two epitope-binding sites composed of two polypeptide chains, each having a CH2 and CH3 Domain, such that the associated chains form all or part of an Fc Region. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern.
  • Figures 3A-3C provide schematics showing representative covalently bonded tetravalent diabodies having four epitope-binding sites composed of two pairs of polypeptide chains (i.e., four polypeptide chains in all).
  • One polypeptide of each pair possesses a CH2 and CH3 Domain, such that the associated chains form all or part of an Fc Region.
  • VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern.
  • the two pairs of polypeptide chains may be same.
  • the resulting molecule possesses four epitope-binding sites and is bispecific and bivalent with respect to each bound epitope.
  • the VL and VH Domains recognize the same epitope (e.g., the same VL Domain CDRs and the same VH Domain CDRs are used on both chains) the resulting molecule possesses four epitope-binding sites and is monospecific and tetravalent with respect to a single epitope.
  • the two pairs of polypeptides may be different.
  • FIG. 3A shows an Fc Region-containing diabody which contains a peptide Heterodimer-Promoting Domain comprising a cysteine residue.
  • Figure 3B shows an Fc Region-containing diabody, which contains E-coil and K-coil Heterodimer-Promoting Domains comprising a cysteine residue and a linker (with an optional cysteine residue).
  • Figure 3C shows an Fc-Region-Containing diabody, which contains antibody CHI and CL domains.
  • Figures 4A-4B provide schematics of a representative covalently bonded diabody molecule having two epitope-binding sites composed of three polypeptide chains. Two of the polypeptide chains possess a CH2 and CH3 Domain, such that the associated chains form all or part of an Fc Region.
  • the polypeptide chains comprising the VL and VH Domain further comprise a Heterodimer-Promoting Domain. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern.
  • Figure 5 provides the schematics of a representative covalently bonded diabody molecule having four epitope-binding sites composed of five 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.
  • the polypeptide chains comprising the linked VL and VH Domains further comprise a Heterodimer-Promoting Domain. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern.
  • Figures 6A-6F provide schematics of representative Fc Region-containing trivalent binding molecules having three epitope-binding sites.
  • Figures 6A and 6B respectively, illustrate schematically the domains of trivalent binding molecules comprising two diabody-type binding domains and a Fab-type binding domain having different domain orientations in which the diabody-type binding domains are N-terminal or C-terminal to an Fc Region.
  • the molecules in Figures 6A and 6B comprise four chains.
  • Figures 6C and 6D respectively, illustrate schematically the domains of trivalent binding molecules comprising two diabody-type binding domains N-terminal to an Fc Region, and a Fab-type binding domain in which the light chain and heavy chain are linked via a polypeptide spacer, or an scFv-type binding domain.
  • the trivalent binding molecules in Figures 6E and 6F respectively, illustrate schematically the domains of trivalent binding molecules comprising two diabody-type binding domains C-terminal to an Fc Region, and a Fab-type binding domain in which the light chain and heavy chain are linked via a polypeptide spacer, or an scFv-type binding domain.
  • the trivalent binding molecules in Figures 6C-6F comprise three chains. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern.
  • Figures 7A-7C present the results of an immunohistochemistry (IHC) studies and show the ability of MAB-A to specifically label a variety of non-small cell lung cancer types (Figure 7A, Panels 1-8), breast cancer cells, prostate cancer cells, gastric cancer cells ( Figure 7B, Panels 1-6), and colon cancer cells ( Figure 7C, Panels 1-8) while the isotype control failed to specifically label any of these cancer cell types ( Figures 7A-7C).
  • IHC immunohistochemistry
  • Figures 8A-8B present the results of cell staining studies and show that MAB- A binds to human ADAM9, and to a lesser extent, cynomolgus monkey ADAM9, transiently expressed on the surface of 293-FT and CHO-K cells ( Figure 8A and Figure 8B, respectively).
  • Figures 9A-9B depict the amino acid sequences of the murine anti-ADAM9- VH Domain aligned with several humanized/optimized variants of MAB-A ( Figure 9A, SEQ ID NOs:7, 16, 17, 18, 19, 21, 22, 23 and 28) and the murine anti-ADAM9-VL Domain aligned with several humanized/optimized variants of MAB-A ( Figure 9B, SEQ ID NOsrll, 51, 52, 53 and 54). Positions substituted within the CDRs during the initial optimization are underlined as follows: potential deamidation and isomeration sites are indicated with a single underline, lysine residues are indicated with double underline, additional labile residues are indicated with a double dashed underline.
  • Figures 10A-10B present the ELISA binding curves of the ten selected optimized hMAB-A clones comprising CDRH3 variants, the parental hMAB-A (2.2), and an isotype control antibody.
  • Figure 10A presents the binding curves for cynoADAM9 and
  • Figure 10B presents the binding curves for huADAM9. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is directed to molecules, such as monospecific antibodies and bispecific, trispecific or multispecific binding molecules, including diabodies, BiTEs, and antibodies that are capable of specifically binding to "Disintegrin and Metalloproteinase Domain-containing Protein 9" ("ADAM9").
  • ADAM9 "Disintegrin and Metalloproteinase Domain-containing Protein 9"
  • the invention particularly concerns such binding molecules that are capable of exhibiting high affinity binding to human and non-human ADAM9.
  • the invention further particularly relates to such molecules that are thereby cross-reactive with human ADAM9 and the ADAM9 of a non- human primate (e.g. , a cynomolgus monkey).
  • the invention additionally pertains to all such ADAM9-binding molecules that comprise a Light Chain Variable (VL) Domain and/or a Heavy Chain Variable (VH) Domain that has been humanized and/or deimmunized so as to exhibit reduced immunogenicity upon administration of such ADAM9-binding molecule to a recipient subject.
  • the invention is also directed to pharmaceutical compositions that contain any of such ADAM9-binding molecules, and to methods involving the use of any of such ADAM9-binding molecules in the treatment of cancer and other diseases and conditions.
  • 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.
  • scFv single-chain Fvs
  • Fab fragments F(ab') fragments
  • disulfide-linked bispecific Fvs sdFv
  • intrabodies and epitope-binding fragments of any of the above.
  • antibody includes immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an epitope-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.
  • class e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2
  • 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).
  • antibodies have been shown to be useful as therapeutic agents. Over 200 antibody -based drugs have been approved for use or are under development.
  • Antibodies are capable of "immunospecifically binding" to a polypeptide or protein or a non-protein molecule due to the presence on such molecule of a particular domain or moiety or conformation (an “epitope").
  • An epitope-containing molecule may have immunogenic activity, such that it elicits an antibody production response in an animal; such molecules are termed "antigens.”
  • 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 that viral epitope with greater affinity, avidity, more readily, and/or with greater duration than it immunospecifically binds to other viral epitopes or to non-viral epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that immunospecifically binds to a first target may or may not specifically or preferentially bind to a second target.
  • “immunospecific binding" to a particular epitope does not necessarily require (although it can include) exclusive binding to that epitope.
  • reference to binding means “immunospecific" 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.
  • the term "monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring or 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.
  • the term 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," IL AR 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," IL AR J. 37(3): 119-125).
  • Ribi see, e.g., Jennings, V.M. (1995) "Review of Selected Adjuvants Used in Antibody Production," IL AR J. 37(3): 119-125.
  • cells should be kept intact and preferably viable when used as immunogens
  • 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 monospecific or multispecific ⁇ e.g., bispecific, trispecific and tetraspecific) molecules of the invention as well as an affinity optimized, 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” Region (“H”) located between the CHI and CH2 Domains.
  • VL Variable Domain
  • CL Constant Domain
  • H Three Constant Domains
  • H Hinge” Region
  • the amino-terminal (“N-terminal”) portion of each chain includes a Variable Domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy- terminal (“C-terminal”) 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 n and c represent, respectively, the N-terminus and the C-terminus of the polypeptide).
  • the Variable Domains of an IgG molecule consist of 1, 2, and most commonly 3, complementarity determining regions ("CDR", i.e., CDR1, CDR2 and CDR3, respectively), which contain the residues in contact with epitope, and non-CDR segments, referred to as framework regions ("FR"), which in general maintain the structure and determine the positioning of the CDR regions so as to permit such contacting (although certain framework residues may also contact the epitope).
  • CDR complementarity determining regions
  • FR framework regions
  • the VL and VH Domains typically have the structure: n-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4-c (where "n” denotes the N-terminus and "c" denotes the C-terminus).
  • Polypeptides that are (or may serve as) the first, second, third, and fourth FR of the Light Chain of an antibody are herein respectively designated as: FR L I Domain, FR L 2 Domain, FR L 3 Domain, and FR L 4 Domain.
  • polypeptides that are (or may serve as) the first, second, third and fourth FR of the Heavy Chain of an antibody are herein respectively designated as: FRHI Domain, FRH2 Domain, FRH3 Domain and FRH4 Domain.
  • Polypeptides that are (or may serve as) the first, second and third CDR of the Light Chain of an antibody are herein respectively designated as: CDRLI Domain, CDRL2 Domain, and CDRL3 Domain.
  • polypeptides that are (or may serve as) the first, second and third CDR of the Heavy Chain of an antibody are herein respectively designated as: CDRHI Domain, CDRH2 Domain, and CDR H 3 Domain.
  • CDRL I 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 a specific epitope regardless of whether such protein is an antibody having light and heavy chains or is a diabody or a single-chain binding molecule (e.g., an scFv, a BiTe, etc.), or is another type of protein.
  • epitope-binding fragment means a fragment of an antibody capable of immunospecifically binding to an epitope
  • epitope-binding site refers to a portion of a molecule comprising an epitope-binding fragment.
  • An epitope-binding fragment may contain any 1, 2, 3, 4, or 5 the CDR Domains of an antibody, or may contain all 6 of the CDR Domains of an antibody and, although capable of immunospecifically binding to such epitope, may exhibit an immunospecificity, affinity or selectivity toward such epitope that differs from that of such antibody.
  • an epitope-binding fragment will contain all 6 of the CDR Domains of such antibody.
  • An epitope-binding fragment of an antibody may be a single polypeptide chain (e.g., an scFv), or may comprise two or more polypeptide chains, each having an amino terminus and a carboxy terminus (e.g., a diabody, a Fab fragment, an Fab 2 fragment, etc.).
  • a polypeptide chain e.g., an scFv
  • two or more polypeptide chains each having an amino terminus and a carboxy terminus
  • a diabody, a Fab fragment, an Fab 2 fragment, etc. Unless specifically noted, the order of domains of the protein molecules described herein is in the "N-terminal to C-terminal" direction.
  • the invention particularly encompasses single-chain Variable Domain fragments ("scFv") comprising an anti-ADAM9-VL and/or VH Domain of the invention as well as multispecific binding molecules comprising such anti-ADAM9-VL and/or VH Domains.
  • Single-chain Variable Domain fragments comprise VL and VH Domains that are linked together using a short "Linker" peptide.
  • Linkers can be modified to provide additional functions, such as to permit the attachment of a drug or to permit attachment to a solid support.
  • the single-chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used.
  • 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.
  • 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 the CDRHI , CDRH2, CDRH3, CDRLI , CDRL2, and CDRL3 Domains of humanized variants of the anti-ADAM9 antibodies of the invention, as well as VL Domains that contain any 1, 2, or 3 of such CDRLS and VH Domains that contain any 1, 2, or 3 of such CDRHS, as well as multispecific-binding molecules comprising the same.
  • the term "humanized” antibody refers to a chimeric molecule having an epitope-binding site of an immunoglobulin from a non-human species and a remaining immunoglobulin structure that is based upon the structure and /or sequence of a human immunoglobulin. Humanized antibodies are generally prepared using recombinant techniques.
  • the anti-ADAM9 antibodies of the present invention include humanized, chimeric or caninized variants of an antibody that is designated herein as "MAB-A.”
  • the polynucleotide sequences that encode the Variable Domains of MAB-A may be used for genetic manipulation to generate MAB-A derivatives possessing improved or altered characteristics (e.g., affinity, cross-reactivity, specificity, etc.).
  • the general principle in humanizing an antibody involves retaining the basic sequence of the epitope- binding portion of the antibody, while swapping the non-human remainder of the antibody with human antibody sequences. There are four general steps to humanize a monoclonal antibody.
  • the term “optimized” antibody refers to an antibody having at least one amino acid which is different from the parent antibody in at least one complementarity determining region (CDR) in the light or heavy chain variable region, which confers a higher binding affinity, (e.g., a 2-fold or more fold) higher binding affinity, to human ADAM9 and/or cynomolgus monkey ADAM9 as compared to the parental antibody.
  • CDR complementarity determining region
  • the epitope-binding site may comprise either a complete Variable Domain fused to one or more Constant Domains or only the CDRs of such Variable Domain grafted to appropriate framework regions.
  • Epitope-binding sites may be wild-type or may be modified by one or more amino acid substitutions, insertions or deletions. Such action partially or completely eliminates the ability of the Constant Region to serve as an immunogen in recipients (e.g., human individuals), however, 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.
  • 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.
  • Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy” Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity " Science 239: 1534-1536; Kettleborough, C. A. et al.
  • humanized antibodies preserve all CDR sequences (for example, a humanized murine antibody which contains all six of the CDRs present in the murine antibody).
  • humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that differ in sequence relative to the CDRs of the original antibody.
  • a number of humanized antibody molecules comprising an epitope-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent or modified rodent Variable Domain 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 etal. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224; Shaw et al.
  • CDRs complementarity determining regions
  • Fey Receptors Fey Receptors
  • Fc Region is a domain that is recognized by cellular “Fc Receptors,” including but not limited to Fc gamma Receptors ("FcyRs").
  • Fc Region is used to define the C-terminal region of an IgG Heavy Chain that comprises the CH2 and CH3 Domains of that chain.
  • An Fc Region is said to be of a particular IgG isotype, class or subclass if its amino acid sequence is most homologous to that isotype, relative to other IgG isotypes.
  • 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 IgG2 is (SEQ ID NO:2):
  • amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG4 is (SEQ ID NO:4):
  • the numbering of the residues in the constant region of 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, NHl, MD (1991) ("Kabat”), expressly incorporated herein by reference.
  • the term "the EU index as set forth in Kabat” refers to the numbering of the Constant Domains of human IgGl EU antibody provided in Kabat. 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, and the CDRs are identified as defined by Kabat (it will be understood that CDRHI as defined by Chothia, C. & Lesk, A. M. ((1987) "Canonical structures for the hypervariable regions of immunoglobulins " J. Mol. Biol. 196:901-917) begins five residues earlier).
  • 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
  • the antibodies of the present invention may 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.
  • the C- terminal residue of the CH3 Domain is an optional amino acid residue in the ADAM9- binding molecules of the invention.
  • ADAM9-binding molecules lacking the C-terminal residue of the CH3 Domain are also specifically encompassed by the instant invention are such constructs comprising the C- terminal lysine residue of the CH3 Domain.
  • the Fc Region of natural IgG antibodies is capable of binding to cellular Fc gamma Receptors (FcyRs). Such binding results in the transduction of activating or inhibitory signals to the immune system.
  • FcyRs Fc gamma Receptors
  • the ability of such binding to result in diametrically opposing functions reflects structural differences among the different FcyRs, and in particular reflects whether the bound FcyR possesses an Immunoreceptor Tyrosine-Based Activation Motif ("ITAM”) or an Immunoreceptor Tyrosine-Based Inhibitory Motif ("ITIM").
  • ITAM Immunoreceptor Tyrosine-Based Activation Motif
  • ITIM Immunoreceptor Tyrosine-Based Inhibitory Motif
  • ITAM-containing FcyRs include FcyRI, FcyRIIA, FcyRIIIA, and activate the immune system when bound to Fc Regions (e.g., aggregated Fc Regions present in an immune complex).
  • FcyRIIB is the only currently known natural ITFM-containing FcyR; it acts to dampen or inhibit the immune system when bound to aggregated Fc Regions.
  • Human neutrophils express the FcyRIIA gene.
  • FcyRIIA clustering via immune complexes or specific antibody cross- linking serves to aggregate ITAMs with receptor-associated kinases which facilitate ITAM phosphorylation.
  • ITAM phosphorylation serves as a docking site for Syk kinase, the activation of which results in the activation of downstream substrates (e.g., PI3K).
  • downstream substrates e.g., PI3K.
  • Cellular activation leads to release of pro-inflammatory 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.
  • the ITIM in FcyRIIB becomes phosphorylated and attracts the SH2 domain of the inositol polyphosphate 5 '-phosphatase (SHIP), which hydrolyzes phosphoinositol messengers released as a consequence of ITAM-containing FcyR- mediated tyrosine kinase activation, consequently preventing the influx of intracellular Ca ++ .
  • SHIP inositol polyphosphate 5 '-phosphatase
  • cross-linking of FcyRIIB dampens the activating response to FcyR ligation and inhibits cellular responsiveness. B- cell activation, B-cell proliferation and antibody secretion is thus aborted.
  • 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's Light Chain and Heavy Chain and, in particular, interaction of its VL and VH Domains forms one of the two epitope-binding sites of a natural antibody, such as an IgG. Natural antibodies are capable of binding to only one epitope species (i.e., they are monospecific), although they can bind multiple copies of that epitope species (i.e., exhibiting bivalency or multivalency).
  • 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.
  • bispecific antibody formats In order to provide molecules having greater capability than natural antibodies, a wide variety of recombinant bispecific antibody formats have been developed (see, e.g., PCT Publication Nos. WO 2008/003116, WO 2009/132876, WO 2008/003103, WO 2007/146968, WO 2009/018386, WO 2012/009544, WO 2013/070565), most of which use linker peptides either to fuse a further epitope-binding fragment (e.g., an scFv, VL, VH, etc.) to, or within the antibody core (IgA, IgD, IgE, IgG or IgM), or to fuse multiple epitope- binding fragments (e.g., two Fab fragments or scFvs).
  • linker peptides either to fuse a further epitope-binding fragment (e.g., an scFv, VL, VH, etc.) to, or within the
  • Linker peptides to fuse an epitope-binding fragment (e.g., an scFv, VL, VH, etc.) to a dimenzation domain such as the CH2-CH3 Domain or alternative polypeptides (see, e.g., PCT Publication Nos. WO 2005/070966, WO 2006/107786A WO 2006/107617A, WO 2007/046893).
  • PCT Publication Nos. WO 2013/174873, WO 2011/133886 and WO 2010/136172 disclose 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 (see, e.g., PCT Publication Nos.
  • WO 2008/027236; WO 2010/108127 to allow them to bind to more than one antigen.
  • PCT Publication Nos. WO 2013/163427 and WO 2013/1 19903 disclose modifying the CH2 Domain to contain a fusion protein adduct comprising a binding domain.
  • PCT Publication Nos. WO 2010/028797, WO 2010/028796 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 Publication Nos. WO 2003/025018 and WO 2003/012069 disclose recombinant diabodies whose individual chains contain scFv Domains.
  • WO 2013/006544 discloses multivalent Fab molecules that are synthesized as a single polypeptide chain and then subjected to proteolysis to yield heterodimeric structures.
  • PCT Publication Nos. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2008/024188, WO 2007/024715, WO 2007/075270, WO 1998/002463, WO 1992/022583 and WO 1991/003493 disclose adding additional binding domains or functional groups to an antibody or an antibody portion (e.g., adding a diabody to the antibody's light chain, or adding additional VL and VH Domains to the antibody's light and heavy chains, or adding a heterologous fusion protein or chaining multiple Fab Domains to one another).
  • 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 using a short linking peptide.
  • Bird, R.E. et al. (1988) Single-Chain Antigen-Binding Proteins;' Science 242:423-426) describes examples 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.
  • bispecific binding molecules ⁇ e.g., non-monospecific diabodies
  • a "trans" binding capability sufficient to co-ligate and/or co-localize different cells that express different epitopes
  • a "cis" binding capability sufficient to co-ligate and/or co- localize different molecules expressed by the same cell.
  • Bispecific binding molecules ⁇ e.g., non-monospecific diabodies
  • Bispecific binding molecules 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. 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: 1221-1225).
  • bispecific (or tri- or multispecific) diabodies can be used (in “cis”) to co-ligate molecules, such as receptors, etc., that are present on the surface of the same cell. Co-ligation of different cells and/or receptors is useful to modulate effector functions and/or immune cell signaling.
  • Multispecific molecules comprising epitope-binding sites may be directed to a surface determinant of any immune cell such as CD2, CD3, CD8, CD 16, T-Cell Receptor (TCR), NKG2D, etc., which are expressed on T lymphocytes, Natural Killer (NK) cells, Antigen-Presenting Cells or other mononuclear cells.
  • TCR T-Cell Receptor
  • NK Natural Killer
  • Antigen-Presenting Cells or other mononuclear cells are useful in the generation of multispecific binding molecules capable of mediating redirected cell killing.
  • 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 non-functional 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® diabodies stable, covalently bonded heterodimeric non-monospecific diabodies, termed DART® diabodies; see, e.g., US Patent Nos. 9,296,816 and 9,284,375 and US Patent Publication Nos. 2015/0175697; 2014/0255407; 2014/0099318; 2013/0295121; WO 2012/018687; WO 2012/162068; 2010/0174053; WO 2010/080538; 2009/0060910; 2007-0004909; European Patent Publication Nos. EP 2714079; EP 2601216; EP 2376109; EP 2158221; EP 1868650; and PCT Publication Nos.
  • 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 one or more pairs of such polypeptide chains to one another.
  • 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 involved polypeptide chains, stabilizing the resulting diabody without interfering with the diabody's binding characteristics.
  • Such molecules can be made to be bispecific (or multispecific) and thus may be made to co-ligate two or more molecules. Such co-ligation permits one to provide an enhanced immunotherapy. Additionally, because the individual polypeptide chains of such molecules form a covalently bonded complex, the molecules exhibit far greater stability than diabodies involving non-covalently bonded polypeptide chains.
  • WO 1999/057150, WO 2003/025018, and WO 2013/013700 which are formed by the homo-dimerization of two identical polypeptide chains, each possessing a VH1, VL2, VH2, and VL2 Domain.
  • a representative human ADAM9 polypeptide (NCBI Sequence NP_003807, including a 28 amino acid residue signal sequence, shown underlined) has the amino acid sequence (SEQ ID NO:5):
  • residues 1-28 are a signal sequence
  • residues 29-697 are the Extracellular Domain
  • residues 698-718 are the Transmembrane Domain
  • residues 719-819 are the Intracellular Domain.
  • Three structural domains are located within the Extracellular Domain: a Reprolysin (M12B) Family Zinc Metalloprotease Domain (at approximately residues 212-406); a Disintegrin Domain (at approximately residues 423-497); and an EGF-like Domain (at approximately residues 644-697).
  • a number of post-translational modifications and isoforms have been identified and the protein is proteolytically cleaved in the trans-Golgi network before it reaches the plasma membrane to generate a mature protein.
  • the removal of the pro-domain occurs via cleavage at two different sites. Processed most likely by a pro-protein convertase such as furin, at the boundary between the pro-domain and the catalytic domain (Arg- 205/Ala-206).
  • An additional upstream cleavage pro-protein convertase site (Arg-56/Glu-57) has an important role in the activation of ADAM9.
  • a representative cynomolgus monkey ADAM9 polypeptide (NCBI Sequence XM_005563126.2, including a possible 28 amino acid residue signal sequence, shown underlined) has the amino acid sequence (SEQ ID NO:6):
  • the Reprolysin (M12B) Family Zinc Metalloprotease Domain of the protein is at approximately residues 212-406); the Disintegrin Domain of the protein is at approximately residues 423-497.
  • ADAM9-binding molecules of the invention are characterized by any one, two, three, four, five, six, seven, or eight of the following criteria:
  • ADAM9 e.g. , ADAM9 of cynomolgus monkey
  • At least 100-fold enhancement e.g., at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550- fold, or at least 600-fold enhancement
  • binding affinity e.g. , as measured by BIACORE® analysis
  • human ADAM9 e.g. , as measured by BIACORE® analysis
  • the binding constants of an ADAM9-binding molecule may be determined using surface plasmon resonance e.g., via a BIACORE® analysis.
  • Surface plasmon resonance data may be fitted to a 1 : 1 Langmuir binding model (simultaneous ka kd) and an equilibrium binding constant KD calculated from the ratio of rate constants kd/ka.
  • binding constants may be determined for a monovalent ADAM9- binding molecule (i.e., a molecule comprising a single ADAM9 epitope-binding site), a bivalent ADAM9-binding molecule (i.e., a molecule comprising two ADAM9 epitope- binding sites), or ADAM9-binding molecules having higher valency (e.g., a molecule comprising three, four, or more ADAM9 epitope-binding sites).
  • a monovalent ADAM9- binding molecule i.e., a molecule comprising a single ADAM9 epitope-binding site
  • a bivalent ADAM9-binding molecule i.e., a molecule comprising two ADAM9 epitope- binding sites
  • ADAM9-binding molecules having higher valency e.g., a molecule comprising three, four, or more ADAM9 epitope-binding sites.
  • the present invention particularly encompasses ADAM9-binding molecules (e.g., antibodies, diabodies, trivalent binding molecules, etc.) comprising anti-ADAM9 Light Chain Variable (VL) Domain(s) and anti-ADAM9 Heavy Chain Variable (VH) Domain(s) that immunospecifically bind to an epitope of a human ADAM9 polypeptide.
  • ADAM9-binding molecules e.g., antibodies, diabodies, trivalent binding molecules, etc.
  • ADAM9-binding molecules e.g., antibodies, diabodies, trivalent binding molecules, etc.
  • ADAM9-binding molecules e.g., antibodies, diabodies, trivalent binding molecules, etc.
  • a murine anti-ADAM9 antibody that blocks the target protein processing activity of ADAM9, is internalized and having anti -tumor activity was identified (see, e.g., US Patent No. 8,361,475).
  • This antibody designated in US Patent Nos. 7,674,619 and 8,361,475 as an "anti-KID24" antibody produced by hybridoma clone ATCC PTA-5174, is designated herein as "MAB-A.”
  • MAB-A exhibits strong preferential binding to tumors over normal tissues (see, Figures 7A-7C).
  • MAB-A exhibited little or no staining across a large panel of normal cell types (Table 1).
  • Tissue MAB-A (1.25 ⁇ g/mL)
  • Interstitial cells (possibly Leydig cells) 2-3+ (gr c) ⁇ 5% and l+ (gr c) 10%
  • MAB-A binds human ADAM9 with high affinity, but binds non-human primate (e.g., cynomolgus monkey) ADAM9 to a lesser extent.
  • non-human primate e.g., cynomolgus monkey
  • VH and VL Domains of MAB-A are provided below.
  • the VH and VL Domains of MAB-A were humanized and the CDRs optimized to improve affinity and/or to remove potential amino acid liabilities.
  • the CDRH3 was further optimized to enhance binding to non-human primate ADAM9 while maintaining its high affinity for human ADAM9.
  • the preferred anti-human ADAM9-binding molecules of the present invention possess the 1, 2 or all 3 of the CDRHS of a VH Domain and/or 1, 2 or all 3 of the CDRLS of the VL Domain of an optimized variant of MAB-A, and preferably further possess the humanized framework regions ("FRs") of the VH and/or VL Domains of humanized MAB- A.
  • Other preferred anti-human ADAM9-binding molecules of the present invention possess the entire VH and/or VL Domains of a humanized/optimized variant of MAB-A.
  • Such preferred anti-human ADAM9-binding molecules include antibodies, bispecific (or multispecific) antibodies, chimeric or humanized antibodies, BiTes, diabodies, etc., as well as such binding molecules that additionally comprise a naturally occurring or a variant Fc Region.
  • the invention particularly relates to ADAM9-binding molecules comprising an ADAM9 binding domain that possess:
  • amino acid sequence of the VH Domain of the murine anti-ADAM9 antibody MAB-A is SEQ ID NO:7 (the CDRH residues are shown underlined):
  • amino acid sequence of the CDR H 1 Domain of MAB-A is (SEQ ID NO:8): SYWMH.
  • the amino acid sequence of the CDR H 2 Domain of MAB-A is (SEQ ID NO:9): E I I P INGHTNYNEKFKS .
  • amino acid sequence of the CDR H 3 Domain of MAB-A is (SEQ ID NO:
  • amino acid sequence of the CDR L 1 Domain of MAB-A is (SEQ ID NO:
  • amino acid sequence of the CDR L 2 Domain of MAB-A is (SEQ ID NO:
  • amino acid sequence of the CDR L 3 Domain of MAB-A is (SEQ ID NO:
  • amino acid sequences of certain preferred humanized/optimized anti- ADAM9-VH Domains of MAB-A are variants of the ADAM9-VH Domain of MAB-A (SEQ ID NO:7) and are represented by SEQ ID NO: 15 (CDRH residues are shown underlined):
  • Xi is M or I
  • X2 is N or F
  • X 5 is S or G
  • X 6 is P, F, Y, W, I, L, V, T, G or D
  • X 7 , Xs, X9, X10, andXn are selected such that:
  • X 7 is K or R; X 7 is N or H;
  • Xs is F or M; Xs is S or K;
  • X10 is W or F; and X10 is T or V; and
  • X 11 is M, L or K; X 11 is M, L or K; when X6 is I, L or V: (D) when X6 is T:
  • X 8 is K; Xs is K, M or N;
  • X10 is V; and X10 is V or T; and
  • X11 is M, L or K; X11 is L or M; when X6 is G: and (F) when X6 is D:
  • X 7 is G; X 7 is S;
  • X 8 is S; Xs is N;
  • X11 is L; X11 is L.
  • hMAB-A VH(2) (SEQ ID NO: 17) hMAB-A VH(2D) (SEQ ID NO:23) hMAB-A VH(3) (SEQ ID NO: 18) hMAB-A VH(2E) (SEQ ID NO:24) hMAB-A VH(4) (SEQ ID NO: 19) hMAB-A VH(2F) (SEQ ID NO:25) hMAB-A VH(2A) (SEQ ID NO:20) hMAB-A VH(2G) (SEQ ID NO:26) hMAB-A VH(2B) (SEQ ID NO:21) hMAB-A VH(2H) (SEQ ID NO:27) hMAB-A VH(2C) (SEQ ID NO:22) hMAB-A VH(2I) (SEQ ID NO:28) and hMAB-A VH(2J) (SEQ ID NO:29)
  • Suitable human amino acid sequences for the FRs of a humanized and/or optimized anti-ADAM9-VH Domain of MAB-A are:
  • FRHI Domain (SEQ ID NO:30): EVQLVESGGGLVKPGGSLRLSCAASGFTFS FR H 2 Domain (SEQ ID NO:31): WVRQAPGKGLEWVG
  • Suitable alternative amino acid sequences for the CDRHI Domain of an anti- ADAM9-VH Domain of MAB-A include:
  • Suitable alternative amino acid sequences for the CDRH2 Domain of an anti- ADAM9-VH Domain of MAB-A include:
  • Suitable alternative amino acid sequences for the CDRH3 Domain of an anti- ADAM9-VH Domain of MAB-A include:
  • SEQ ID NO:40 GGYYYYTGKGVLDY
  • SEQ ID NO:45 GGYYYYPRQGFLDY
  • ADAM9 binding molecules having a VH domain comprising:
  • X2 is N or F
  • X3 is K or R
  • X4 is K or Q; and X5 is S or G.
  • Xe is P, F, Y, W, I, L, V, T, G or D
  • X 7 , Xs, X9, Xio, and Xn are selected such that:
  • X 7 is K or R; X 7 is N or H;
  • Xs is F or M; Xs is S or K;
  • Xio is W or F; and Xio is T or V; and X 11 is M, L or K; X 11 is M, L or K; when X6 is I, L or V: (D) when Xe is T:
  • Xs is K; Xs is K, M or N;
  • Xio is V; and Xio is V or T; and X11 is M, L or K; X11 is L or M; when X6 is G: and (F) when Xe is D:
  • X 7 is G; X 7 is S;
  • Xs is S; Xs is N;
  • Xio is V; and Xio is V; and
  • X11 is L; X11 is L.
  • a first exemplary humanized/optimized IgGl Heavy Chain of a derivative/variant of MAB-A contains the hMAB-A VH (2) Domain (SEQ ID NO: 17), and has the amino acid sequence (SEQ ID NO:50): EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYWMHWVRQA PGKGLEWVGE
  • X is a lysine (K) or is absent.
  • a second exemplary humanized/optimized IgGl Heavy Chain of a derivative/variant of MAB-A contains the hMAB-A VH (2C) Domain (SEQ ID NO:22), and has the amino acid sequence (SEQ ID NO:51):
  • X is a lysine (K) or is absent.
  • a third exemplary humanized/optimized IgGl Heavy Chain of a derivative/variant of MAB-A contains the hMAB-A VH (21) Domain (SEQ ID NO:28), and has the amino acid sequence (SEQ ID NO:52):
  • the CH2-CH3 Domains of the Fc Region may be engineered for example, to reduce effector function.
  • the CH2-CH3 Domains of the exemplary humanized/optimized IgGl Heavy Chains of the invention comprise one or more subustitutions selected from: L234A and L235A.
  • a fourth exemplary humanized/optimized IgGl Heavy Chain of a derivative/variant of MAB-A contains the hMAB-A VH (21) Domain (SEQ ID NO:28), and further comprises the substitutions L234A, and L235A in the CH2-CH3 Domains of the Fc Region (SEQ ID NO: 106), underlined below) and has the amino acid sequence (SEQ ID NO:202):
  • X is a lysine (K) or is absent.
  • VL Domains of MAB-A are variants of the ADAM9-VL Domain of MAB-A (SEQ ID NO: 1
  • X12 is K or R
  • X13 is D or S
  • Xi4 is M or L; X15 is H or Y;
  • Xi6 is E or S; and X17 is D or T.
  • suitable human amino acid sequences for the FRs of a humanized and/or optimized anti-ADAM9-VL Domain of MAB-A are:
  • FRLI Domain (SEQ ID NO:58): DIVMTQSPDSLAVSLGERATISC
  • Suitable alternative amino acid sequences for the CDRLI Domain of an anti- ADAM9-VL Domain include:
  • SEQ ID NO:62 KASQSVDYSGDSYMN
  • Suitable alternative amino acid sequences for the CDRL3 Domain of an anti- ADAM9-VL Domain include:
  • the present invention encompasses anti-ADAM9 antibody VL Domain comprising:
  • An exemplary humanized/optimized IgGl Light Chain of a derivative/variant of MAB-A contains the hMAB-A VL (2) Domain (SEQ ID NO:55), and has the amino acid sequence (SEQ ID NO:68):
  • ADAM9-binding molecules e.g., antibodies, diabodies, trivalent binding molecules, etc.
  • ADAM9-binding molecules that immunospecifically bind to an epitope of a human ADAM9 polypeptide, and that comprise any of the above-provided MAB-A CDRHI , CDRH2, CDRH3, CDRLI , CDRL2, or CDR L 3, and particularly contemplates such ADAM9-binding molecules that comprise one of the above-provided MAB-A CDRHI, one of the above-provided MAB-A CDRH2, one of the above-provided MAB-A CDRH3, one of the above-provided MAB-A CDRLI, one of the above-provided MAB-A CDRL2, and one of the above-provided MAB-A CDRL3.
  • ADAM9-binding molecules that comprise one of the above-provided MAB-A CDRHI, one of the above-provided MAB-A CDRH2, one of the above-provided MAB-A C
  • the invention further contemplates such ADAM9-binding molecules that further comprise any of the above-provided humanized MAB-A FRH I , FRH2, FRH3, or FRH4, FRL I , FRL2, FRL3, or FRL4, and particularly contemplates such ADAM9-binding molecules that comprise FRHI , FRH2, FRH3, and FRH4, and/or that comprise FRL I , FRL2,
  • the ADAM9-binding molecules include a CDRHI domain, a CDRH2 domain, and a CDRH3 domain and a CDRL I domain, a CDRL2 domain, and a CDRL3 domain having the sequences selected from the group consisting of:
  • the ADAM9-binding molecules include a CDRHI domain, a CDRH2 domain, and a CDRH3 domain and a CDRLI domain, a CDRL2 domain, and a CDRL3 domain having the sequences of SEQ ID NOs:8, 35 and 45 and SEQ ID NOs:62, 13 and 14, respectively.
  • the ADAM9-binding molecules of the invention include a heavy chain variable domain (VH) and a light chain variable domain (VL) having sequences that are at least 90%, at least 95%, at least 99%, or are 100% identical to the sequences as follows:
  • substantially identical or “identical” is meant a polypeptide exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein.
  • such a sequence is at least 60%>, more preferably at least 80%> or at least 85%>, and more preferably at least 90%, at least 95% at least 99%), or even 100%> identical at the amino acid level to the polypeptide sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center
  • the ADAM9-binding molecules of the invention include a heavy chain variable domain (VH) and a light chain variable domain (VL) having sequences that are at least 90%, at least 95%, at least 99%, or are 100% identical to the sequences of SEQ ID NO: 28 and SEQ ID NO: 55, respectively.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the ADAM9-binding molecules of the invention comprise a heavy chain and a light chain sequence as follows:
  • the present invention also expressly contemplates ADAM9-binding molecules (e.g., antibodies, diabodies, trivalent binding molecules, etc.) that immunospecifically bind to an epitope of a human ADAM9 polypeptide, and that comprise any of the above-provided humanized/optimized anti-ADAM9 MAB-A VL or VH Domains.
  • ADAM9-binding molecules e.g., antibodies, diabodies, trivalent binding molecules, etc.
  • the present invention particularly contemplates such ADAM9-binding molecules that comprise any of the following combinations of humanized anti-ADAM9 VL or VH Domains:
  • the present invention specifically encompasses ADAM9-binding molecules comprising (i) a humanized/optimized anti-ADAM9-VL and/or VH Domain as provided above, and (ii) an Fc Region.
  • the ADAM9-binding molecules of the present invention are monoclonal antibodies comprising (i) a humanized/optimized anti-ADAM9-VL and/or VH Domain as provided above, and (ii) an Fc Region.
  • the ADAM9-binding molecules of the present invention are selected from the group consisting of: monoclonal antibodies, multispecific antibodies, synthetic antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFv), BiTEs, diabodies, and trivalent binding molecules.
  • the ADAM9-binding molecules of the present invention may be monospecific single-chain molecules, such as anti-ADAM9 single-chain variable fragments ("anti- ADAM9-scFvs”) or anti-ADAM9 Chimeric Antigen Receptors ("anti-ADAM9-CARs").
  • anti-ADAM9-scFvs anti-ADAM9 single-chain variable fragments
  • anti-ADAM9-CARs anti-ADAM9 Chimeric Antigen Receptors
  • scFvs are made by linking Light and Heavy Chain Variable Domains together via a short linking peptide.
  • First-generation Chimeric Antigen Receptors (“CARs") typically comprise the intracellular domain from the CD3 ⁇ - chain, which is the primary transmitter of signals from endogenous T-cell Receptors ("TCRs").
  • Second- generation CARs possess additional intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 4 IBB, ICOS, etc.) fused 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 CD3 ⁇ -CD28-41BB or CD3 ⁇ -CD28-OX40, in order to further augment their potency (Tettamanti, S. et al. (2013) "Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123- Specific Chimeric Antigen Receptor ,” Br. J. Haematol.
  • the anti-ADAM9-CARs of the present invention comprise an anti-ADAM9- scFv fused to an intracellular domain of a receptor.
  • the Light Chain Variable (VL) Domain and the Heavy Chain Variable (VH) Domain of the anti-ADAM9-scFv are selected from any of the humanized anti-ADAM9-VL and anti-ADAM9-VH Domains disclosed herein.
  • the VH Domain is selected from the group consisting of: hMAB-A VH(1) (SEQ ID NO: 16), hMAB-A VH(2) (SEQ ID NO: 17), hMAB-A VH(3) (SEQ ID NO: 18), hMAB-A VH(4) (SEQ ID NO: 19), hMAB-A VH(2A) (SEQ ID NO:20), hMAB-A VH(2B) (SEQ ID NO:21), hMAB-A VH(2C) (SEQ ID NO:22), hMAB-A VH(2D) (SEQ ID NO:23), hMAB-A VH(2E) (SEQ ID NO:24), hMAB-A VH(2F) (SEQ ID NO:25), hMAB-A VH(2G) (SEQ ID NO:26), hMAB-A VH(2H) (SEQ ID NO:27), hMAB-A VH(2A) (
  • the intracellular domain of the anti-ADAM9-CARs of the present invention is preferably selected from the intracellular domain of any of: 41BB-CD3 ⁇ b2c-CD3 ⁇ CD28, CD28-4-lBB-CD3C, CD28-CD3C, CD28-Fc8RIy, CD28mut-CD3C, CD28-OX40- CD3C, CD28-OX40-CD3C, CD3C, CD4-CD3C, CD4-Fc8RIy, CD8-CD3C, FcsRIy, FcsRIyCAIX, Heregulin-CD3C, IL-13-CD3C, or Ly49H-CD3C (Tettamanti, S. et al.
  • the present invention is also directed to multispecific (e.g., bispecific, trispecific, etc.) ADAM9-binding molecules comprising an epitope-binding site (preferably comprising 1, 2 or all 3 of the CDRHS of an anti-ADAM9-VFI Domain of the invention and/or 1, 2 or all 3 of the CDRLS of an anti-ADAM9-VL Domain of the invention, or such anti-ADAM9-VFI Domain and/or such anti-ADAM9-VL Domain) and further comprising a second epitope-binding site that immunospecifically binds to a second epitope, where such second epitope is (i) a different epitope of ADAM9, or (ii) an epitope of a molecule that is not ADAM9.
  • an epitope-binding site preferably comprising 1, 2 or all 3 of the CDRHS of an anti-ADAM9-VFI Domain of the invention and/or 1, 2 or all 3 of the CDRLS of an anti-ADAM9-VL Domain
  • Such multispecific ADAM9-binding molecules preferably comprise a combination of epitope-binding sites that recognize a set of antigens unique to target cells or tissue type.
  • the present invention relates to multispecific ADAM9-binding molecules that are capable of binding to an epitope of ADAM9 and an epitope of a molecule present on the surface of an effector cell, especially a T lymphocyte, a natural killer ( K) cell or other mononuclear cell.
  • such ADAM9-binding molecules of the present invention may be constructed to comprise an epitope-binding site that immunospecifically binds CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), or KG2D.
  • One embodiment of the present invention relates to bispecific ADAM9- binding molecules that are capable of binding to a "first epitope” and a "second epitope,” such epitopes not being identical to one another.
  • Such bispecific molecules comprise "VL1" / " VH1” domains that are capable of binding to the first epitope, and " VL2" / "VH2" domains that are capable of binding to the second epitope.
  • the notation “VL1" and “VH1” denote respectively, the Light Chain Variable Domain and Heavy Chain Variable Domain that bind the "first" epitope of such bispecific molecules.
  • VL2 and VH2 denote respectively, the Light Chain Variable Domain and Heavy Chain Variable Domain that bind the "second" epitope of such bispecific molecules. It is irrelevant whether a particular epitope is designated as the first vs. the second epitope; such notation having relevance only with respect to the presence and orientation of domains of the polypeptide chains of the binding molecules of the present invention.
  • one of such epitopes is an epitope of human ADAM9 and the other is a different epitope of ADAM9, or is an epitope of a molecule that is not ADAM9.
  • one of such epitopes is an epitope of human ADAM9 and the other is an epitope of a molecule (e.g., CD2, CD3, CD8, CD 16, T-Cell Receptor (TCR), KG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • a bispecific molecule comprises more than two epitope-binding sites.
  • Such bispecific molecules will bind at least one epitope of ADAM9 and at least one epitope of a molecule that is not ADAM9, and may further bind additional epitopes of ADAM9 and/or additional epitopes of a molecule that is not ADAM9.
  • the present invention particularly relates to bispecific, trispecific and multispecific ADAM9-binding molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) that possess epitope-binding fragments of antibodies (e.g., VL and VH Domains) that enable them to be able to coordinately bind to at least one epitope of ADAM9 and at least one epitope of a second molecule that is not ADAM9.
  • bispecific, trispecific and multispecific ADAM9-binding molecules e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.
  • epitope-binding fragments of antibodies e.g., VL and VH Domains
  • VL and VH Domains of the polypeptide domains of such molecules is coordinated so that the polypeptides chains that make up such multispecific ADAM9-binding molecules assemble to form at least one functional epitope-binding site that is specific for at least one epitope of ADAM9 and at least one functional epitope-binding site that is specific for at least one epitope of a molecule that is not ADAM9.
  • the multispecific ADAM9- binding molecules comprise 1, 2 or all 3 of the CDRHS of an anti-ADAM9-VH Domain of the invention and/or 1, 2 or all 3 of the CDRLS of an anti-ADAM9-VL Domain of the invention, or such anti-ADAM9-VH Domain and/or such anti-ADAM9-VL Domain, as provided herein.
  • the present invention encompasses bispecific antibodies capable of simultaneously binding to an epitope of ADAM9 and an epitope of a molecule that is not ADAM9.
  • the bispecific antibody capable of simultaneously binding to ADAM9 and a second molecule that is not ADAM9 is produced using any of the methods described in PCT Publication Nos.
  • One embodiment of the present invention relates to bispecific diabodies that are capable of binding to a first epitope and a second epitope, wherein the first epitope is an epitope of human ADAM9 and the second is an epitope of a molecule that is not ADAM9, preferably a molecule (e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), KG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • a molecule e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), KG2D, etc.
  • 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 diabody that is capable of simultaneously binding to an epitope of ADAM9 and the second epitope.
  • the first polypeptide chain of such an embodiment of bispecific 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 VLanti-ADAM9-vL or VLEpitope 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 VL a nti-ADAM9-vL) or ADAM9 (if such first polypeptide chain contains VLEpitope 2), a second intervening spacer peptide (Linker 2) optionally containing a cysteine residue, a Heterodimer-Promoting Domain and a C-terminus ( Figure 1).
  • the second polypeptide chain of this embodiment of bispecific diabodies comprises, in the N-terminal to C-terminal direction: an N-terminus, a VL Domain of a monoclonal antibody capable of binding to either the first or second epitope (i.e., either VLanti-ADAM9-vL or VLEpitope 2, and being the VL Domain not selected for inclusion in the first polypeptide chain of the diabody), an intervening spacer peptide (Linker 1), a VH Domain of a monoclonal antibody capable of binding to either the second epitope (if such second polypeptide chain contains VLanti-ADAM9-vL) or to ADAM9 (if such second polypeptide chain contains VLEpitope 2), a second intervening spacer peptide (Linker 2) optionally containing a cysteine residue, a Heterodimer-Promoting Domain, and a C-terminus ( Figure 1).
  • Linker 1 a VH Domain of a monoclonal antibody capable
  • the VL Domain of the first polypeptide chain interacts with the VH Domain of the second polypeptide chain to form a first functional epitope-binding site that is specific for a first antigen (i.e., either ADAM9 or a molecule that contains the second epitope).
  • a first antigen i.e., either ADAM9 or a molecule 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 epitope-binding site that is specific for a second antigen (i.e., either the molecule that comprises the second epitope or ADAM9).
  • 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 ADAM9 and to the second epitope (i.e., they collectively comprise VL a nti-ADAM9- VH a nti-
  • the length of the intervening spacer peptide is selected to substantially or completely prevent the VL and VH Domains of the polypeptide chain from binding to one another (for example consisting of from 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 intervening linker amino acid residues).
  • 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 (Linker 1) has the sequence (SEQ ID NO:69): GGGS GGGG.
  • the length and composition of the second intervening spacer peptide (“Linker 2") is selected based on the choice of one or more polypeptide domains that promote such dimerization (i.e., a "Heterodimer-Promoting Domain").
  • the second intervening spacer peptide (Linker 2) will comprise 3-20 amino acid residues.
  • a cysteine-containing second intervening spacer peptide (Linker 2) is utilized.
  • a cysteine-containing second intervening spacer peptide (Linker 2) will contain 1, 2, 3 or more cysteines.
  • a preferred cysteine-containing spacer peptide has the sequence GGCGGG (SEQ ID NO:70).
  • Linker 2 does not comprise a cysteine (e.g., GGG , GGGS (SEQ ID NO:71), LGGGS G (SEQ ID NO:72), GGGS GGGS GGG (SEQ ID NO:73), AS TKG (SEQ ID NO:74), LE PKS S (SEQ ID NO:75), APS S S (SEQ ID NO:76), etc.) 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:77) or VE PKS C ( SEQ ID NO:78) or AE PKS C (SEQ ID NO:79) on one polypeptide chain and GFNRGEC (SEQ ID NO:80) or FNRGEC (SEQ ID NO:81) on the other polypeptide chain (see, US Patent No. 9,296,816).
  • the Heterodimer-Promoting Domains will comprise tandemly repeated coil domains of opposing charge for example, "E-coil” helical domains (SEQ ID NO:82): EVAALEK-EVAALEK-EVAALEK-EVAALEK), whose glutamate residues will form a negative charge at pH 7, and "K-coil” domains (SEQ ID NO:83): KVAALKE -KVAALKE -KVAALKE -KVAALKE), whose lysine residues will form a positive charge at pH 7.
  • E-coil helical domains
  • Heterodimer-Promoting Domains that comprise modifications of the above-described E- coil and K-coil sequences so as to include one or more cysteine residues may be utilized.
  • the presence of such cysteine residues permits the coil present on one polypeptide chain to become covalently bonded to a complementary coil present on another polypeptide chain, thereby covalently bonding the polypeptide chains to one another and increasing the stability of the diabody.
  • Heterodimer-Promoting Domains include a Modified E-Coil having the amino acid sequence EVAACE_K- EVAALEK-EVAALEK-EVAALEK (SEQ ID NO:84), and a modified K-coil having the amino acid sequence KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:85).
  • 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 a polypeptide chain 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. etal. (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: 86): LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.
  • deimmunized variants of SEQ ID NO:86 have the ability to attenuate or eliminate MHC class II binding. Based on combinational mutation results, the following combinations of substitutions are considered to be preferred substitutions for forming such a deimmunized ABD: 66D/70S +71A; 66S/70S +71A; 66S/70S +79A; 64A/65A/71A;
  • 64A/65A/71A+66S 64A/65A/71A+66D; 64A/65A/71A+66E; 64A/65A/79A+66S;
  • Variant deimmunized ABD having the amino acid sequence:
  • the first polypeptide chain of such a diabody having an ABD 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 ABD (which is preferably a deimmunized ABD).
  • Linker 3 is GGGS (SEQ ID NO:71).
  • One embodiment of the present invention relates to multispecific diabodies capable of simultaneously binding to an epitope of ADAM9 and a second epitope (i.e., a different epitope of ADAM9 or an epitope of a molecule that is not ADAM9) that comprise an Fc Region.
  • a second epitope i.e., a different epitope of ADAM9 or an epitope of a molecule that is not ADAM9
  • the addition of an IgG CH2-CH3 Domain 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 the diabody.
  • Such diabodies comprise, two or more polypeptide chains whose sequences permit the polypeptide chains to covalently bind to each other to form a covalently associated diabody that is capable of simultaneously binding to an epitope of ADAM9 and the second epitope.
  • Incorporating an 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 3C shows a representative four-chain diabody 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 (see, e.g., Figures 3A and 3B, US Patent Publication Nos. 2013-0295121; 2010- 0174053 and 2009-0060910; European Patent Publication Nos. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publication Nos.
  • a peptide comprising tandem coil domains of opposing charge such as the "E-coil” helical domains (SEQ ID NO:82): E VAALE K - E VAALE K - EVAALEK-EVAALEK or SEQ ID NO:84): E VAA£EK-E VAALE K-E VAALE K- E VAALE K); and the "K-coil” domains (SEQ ID NO:83): KVAALKE -KVAALKE - KVAALKE -KVAALKE or SEQ ID NO:85): KVAACKE -KVAALKE -KVAALKE - KVAALKE).
  • a representative coil domain containing four-chain diabody is shown in Figure 3B
  • the Fc Region-containing molecules of the present invention may include additional intervening spacer peptides (Linkers), generally such Linkers will be incorporated between a Heterodimer-Promoting Domain (e.g., an E-coil or K-coil) and a CH2-CH3 Domain and/or between a CH2-CH3 Domain and a Variable Domain (i.e., VH or VL).
  • the additional Linkers will comprise 3-20 amino acid residues and may optionally contain all or a portion of an IgG Hinge Region (preferably a cysteine-containing portion of an IgG Hinge Region).
  • Linkers that may be employed in the bispecific Fc Region- containing diabody molecules of the present invention include: GGGS (SEQ ID NO:71), LGGGS G (SEQ ID NO:72), GGGS GGGS GGG (SEQ ID NO:73), AS TKG (SEQ ID NO:74), LE PKS S (SEQ ID NO:75), APS S S (SEQ ID NO:76), APS S S PME (SEQ ID NO:90), VE PKSADKTHTCPPCP (SEQ ID NO:91), LE PKSADKTHTCPPC ( SEQ ID NO:92), DKTHTCPPCP (SEQ ID NO:93), GGC, and GGG.
  • LE PKS S (SEQ ID NO:75) may be used in lieu of GGG or GGC for ease of cloning. Additionally, the amino acids GGG, or LE PKS S (SEQ ID NO:75) may be immediately followed by DKTHTCPPCP ( SEQ ID NO:93) to form the alternate linkers: GGGDKTHTCPPCP (SEQ ID NO:94); and LE PKS S DKTHTCPPCP (SEQ ID NO:95).
  • Bispecific Fc Region-containing molecules of the present invention may incorporate an IgG Hinge Region in addition to or in place of a linker.
  • Exemplary Hinge Regions include: E PKS CDKTHTCPPCP (SEQ ID NO:96) from IgGl, ERKCCVECPPCP (SEQ ID NO:97) from IgG2, ELKT PLGDT T HTCPRCPE PK SCDTPPPCPR CPEPKSCDTP PPCPRCPEPK SCDTPPPCPR CP (SEQ ID NO:206) from IgG3, ESKYGPPCPSCP (SEQ ID NO:98) from IgG4, and ESKYGPPCPPCP (SEQ ID NO:99), which is an IgG4 hinge variant comprising a stabilizing S228P substitution (underlined) (as numbered by the EU index as set forth in Kabat) to reduce strand exchange.
  • E PKS CDKTHTCPPCP SEQ ID NO:96
  • ERKCCVECPPCP SEQ ID NO:97
  • Fc Region-containing diabodies of the invention may comprise four chains.
  • the first and third polypeptide chains of such a diabody contain three domains: (i) a VLl -containing Domain, (ii) a VH2-containing Domain, (iii) a Heterodimer-Promoting Domain, and (iv) a Domain containing a CH2-CH3 sequence.
  • the second and fourth polypeptide chains contain: (i) a VL2-containing Domain, (ii) a VH1 -containing Domain, and (iii) a Heterodimer-Promoting Domain, where the Heterodimer-Promoting Domains promote the dimerization of the first/third polypeptide chains with the second/fourth polypeptide chains.
  • the VL and/or VH Domains of the third and fourth polypeptide chains, and VL and/or VH Domains of the first and second polypeptide chains may be the same or different so as to permit tetravalent binding that is either monospecific, bispecific or tetraspecific.
  • VL3 and VH3 denote respectively, the Light Chain Variable Domain and Heavy Chain Variable Domain that bind a "third" epitope of such diabody.
  • VL4 and VH4 denote respectively, the Light Chain Variable Domain and Heavy Chain Variable Domain that bind a "fourth” epitope of such diabody.
  • Table 2 The general structure of the polypeptide chains of a representative four-chain bispecific Fc Region-containing diabodies of invention is provided in Table 2:
  • diabodies of the present invention are bispecific, tetravalent (i.e., possess four epitope-binding sites), Fc-containing diabodies that are composed of four total polypeptide chains ( Figures 3A-3C).
  • the bispecific, tetravalent, Fc-containing diabodies of the invention comprise two epitope-binding sites immunospecific for ADAM9 (which may be capable of binding to the same epitope of ADAM9 or to different epitopes of ADAM9), and two epitope-binding sites immunospecific for a second molecule (which may be capable of binding to the same epitope of the second molecule or to different epitopes of the second molecule).
  • the second molecule is a molecule (e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), KG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • an effector cell such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • the Fc Region-containing diabodies of the present invention 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 a diabody 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 a diabody comprises a CH2-CH3 sequence.
  • the first and second polypeptide chains of such a diabody associate together to form a VL1/VH1 epitope-binding site that is capable of binding to a first antigen (i.e., either ADAM9 or a molecule that comprises a second epitope), as well as a VL2/VH2 epitope-binding site that is capable of binding to a second antigen (i.e., either the molecule that contains the second epitope or ADAM9).
  • 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 bispecific diabodies have enhanced potency.
  • Figures 4A and 4B illustrate the structures of such diabodies.
  • Such Fc-Region-containing diabodies may have either of two orientations (Table 3): Table 3
  • diabodies of the present invention are bispecific, bivalent (i.e., possess two epitope-binding sites), Fc-containing diabodies that are composed of three total polypeptide chains ( Figures 4A-4B).
  • the bispecific, bivalent Fc-containing diabodies of the invention comprise one epitope-binding site immunospecific for ADAM9, and one epitope-binding site immunospecific for a second molecule.
  • the second molecule is a molecule (e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), KG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • an effector cell such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • the Fc Region-containing diabodies may comprise a total of five polypeptide chains.
  • two of said five polypeptide chains have the same amino acid sequence.
  • the first polypeptide chain of such a diabody contains: (i) a VHl -containing domain, (ii) a CHI -containing domain, and (iii) a Domain containing a CH2-CH3 sequence.
  • the first polypeptide chain may be the heavy chain of an antibody that contains a VHl and a heavy chain constant region.
  • the second and fifth polypeptide chains of such a diabody contain: (i) a VLl -containing domain, and (ii) a CL- containing domain.
  • the second and/or fifth polypeptide chains of such a diabody may be light chains of an antibody that contains a VLl complementary to the VHl of the first/third polypeptide chain.
  • the first, second and/or fifth polypeptide chains may be isolated from a naturally occurring antibody. Alternatively, they may be constructed recombinantly.
  • the third polypeptide chain of such a diabody contains: (i) a VHl -containing domain, (ii) a CHI- containing domain, (iii) a Domain containing a CH2-CH3 sequence, (iv) a VL2-containing Domain, (v) a VH3 -containing Domain and (vi) a Heterodimer-Promoting Domain, where the Heterodimer-Promoting Domains promote the dimerization of the third chain with the fourth chain.
  • the fourth polypeptide of such diabodies contains: (i) a VL3 -containing Domain, (ii) a VH2-containing Domain and (iii) a Domain that promotes heterodimerization and covalent bonding with the diabody's third polypeptide chain.
  • the first and second, and the third and fifth, polypeptide chains of such diabodies associate together to form two VLl/VHl epitope-binding sites capable of binding a first epitope.
  • the third and fourth polypeptide chains of such diabodies associate together to form a VL2/VH2 epitope-binding site that is capable of binding to a second epitope, as well as a VL3/VH3 binding site that is capable of binding to a third epitope.
  • the first and third polypeptides are bonded to one another through a disulfide bond involving cysteine residues in their respective constant regions.
  • the first and third polypeptide chains complex with one another to form an Fc Region.
  • VLl/VHl, VL2/VH2, and VL3/VH3 Domains may be the same or different so as to permit binding that is monospecific, bispecific or trispecific. As provided herein, these domains are preferably selected so as to bind an epitope of ADAM9, an epitope of second molecule and optionally an epitope of a third molecule.
  • VL and VH Domains of the polypeptide chains are selected so as to form VL/VH binding sites specific for a desired epitope.
  • the VL/VH binding sites formed by the association of the polypeptide chains may be the same or different so as to permit tetravalent binding that is monospecific, bispecific, trispecific or tetraspecific.
  • VL and VH Domains may be selected such that a multivalent diabody may comprise two binding sites for a first epitope and two binding sites for a second epitope, or three binding sites for a first epitope and one binding site for a second epitope, or two binding sites for a first epitope, one binding site for a second epitope and one binding site for a third epitope (as depicted in Figure 5).
  • Table 4 Table 4
  • diabodies of the present invention are bispecific, tetravalent (i.e., possess four epitope-binding sites), Fc-containing diabodies that are composed of five total polypeptide chains having two epitope-binding sites immunospecific for ADAM9 (which may be capable of binding to the same epitope of ADAM9 or to different epitopes of ADAM9), and two epitope-binding sites specific for a second molecule (which may be capable of binding to the same epitope of the second molecule or to different epitopes of the second molecule).
  • the bispecific, tetravalent, Fc- containing diabodies of the invention comprise three epitope-binding sites immunospecific for ADAM9 (which may be capable of binding to the same epitope of ADAM9 or to two or three different epitopes of ADAM9), and one epitope-binding site specific for a second molecule.
  • the bispecific, tetravalent, Fc-containing diabodies of the invention comprise one epitope-binding site immunospecific for ADAM9, and three epitope-binding sites specific for a second molecule (which may be capable of binding to the same epitope of the second molecule or to two or three different epitopes of the second molecule).
  • the VL and VH domains may be selected to permit trispecific binding. Accordingly, the invention also encompasses trispecific, tetravalent, Fc- containing diabodies.
  • the trispecific, tetravalent, Fc-containing diabodies of the invention comprise two epitope-binding sites immunospecific for ADAM9, one epitope-binding site immunospecific for a second molecule, and one epitope-binding site immunospecific for a third molecule.
  • the second molecule is a molecule (e.g., CD2, CD3, CD8, CD 16, T-Cell Receptor (TCR), KG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • an effector cell such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • the second molecule is CD3 and the third molecule is CD8.
  • a further embodiment of the present invention relates to trivalent binding molecules comprising an Fc Region capable of simultaneously binding a first epitope, a second epitope and a third epitope, wherein at least one of such epitopes is not identical to another.
  • Such trivalent binding molecules comprise three epitope-binding sites, two of which are Diabody-Type Binding Domains, which provide binding Site A and binding Site B, and one of which is a Fab-Type Binding Domain, or an scFv-Type Binding Domain, which provides binding Site C (see, e.g., Figures 6A-6F, and PCT Publication Nos: WO 2015/184207; and WO 2015/184203).
  • Such trivalent binding molecules thus comprise "VL1" / "VH1" domains that are capable of binding to the first epitope and "VL2" / “VH2” domains that are capable of binding to the second epitope and "VL3" and “VH3” domains that are capable of binding to the "third" epitope of such trivalent binding molecule.
  • a “Diabody-Type Binding Domain” is the type of epitope-binding site present in a diabody, and especially, a DART® diabody, as described above.
  • Fab-Type Binding Domains are epitope-binding sites that are formed by the interaction of the VL Domain of an immunoglobulin light chain and a complementing VH Domain of an immunoglobulin heavy chain.
  • Fab-Type Binding Domains differ from Diabody-Type Binding Domains in that the two polypeptide chains that form a Fab-Type Binding Domain comprise only a single epitope-binding site, whereas the two polypeptide chains that form a Diabody-Type Binding Domain comprise at least two epitope-binding sites.
  • scFv-Type Binding Domains also differ from Diabody-Type Binding Domains in that they comprise only a single epitope-binding site.
  • Fab- Type, and scFv-Type Binding Domains are distinct from Diabody-Type Binding Domains.
  • the trivalent binding molecules of the present invention will comprise four different polypeptide chains (see Figures 6A-6B), however, the molecules may comprise fewer or greater numbers of polypeptide chains, for example by fusing such polypeptide chains to one another (e.g., via a peptide bond) or by dividing such polypeptide chains to form additional polypeptide chains, or by associating fewer or additional polypeptide chains via disulfide bonds.
  • Figures 6C-6F illustrate this aspect of the present invention by schematically depicting such molecules having three polypeptide chains.
  • the trivalent binding molecules of the present invention may have alternative orientations in which the Diabody-Type Binding Domains are N-terminal ( Figures 6A, 6C and 6D) or C-terminal ( Figures 6B, 6E and 6F) to an Fc Region.
  • the first polypeptide chain of such trivalent binding molecules of the present invention contains: (i) a VL1 -containing Domain, (ii) a VH2- containing Domain, (iii) a Heterodimer-Promoting Domain, and (iv) a Domain containing a CH2-CH3 sequence.
  • the VL1 and VL2 Domains are located N-terminal or C-terminal to the CH2-CH3 -containing domain as presented in Table 4 (also see, Figures 6 A and 6B).
  • the second polypeptide chain of such embodiments contains: (i) a VL2-containing Domain, (ii) a VH1 -containing Domain, and (iii) a Heterodimer-Promoting Domain.
  • the third polypeptide chain of such embodiments contains: (i) a VH3 -containing Domain, (ii) a CHI- containing Domain and (iii) a Domain containing a CH2-CH3 sequence.
  • the third polypeptide chain may be the heavy chain of an antibody that contains a VH3 and a heavy chain constant region, or a polypeptide that contains such domains.
  • the fourth polypeptide of such embodiments contains: (i) a VL3 -containing Domain and (ii) a CL-containing Domain.
  • the fourth polypeptide chains may be a light chain of an antibody that contains a VL3 complementary to the VH3 of the third polypeptide chain, or a polypeptide that contains such domains.
  • the third or fourth polypeptide chains may be isolated from naturally occurring antibodies. Alternatively, they may be constructed recombinantly, synthetically or by other means.
  • the Light Chain Variable Domain of the first and second polypeptide chains are separated from the Heavy Chain Variable Domains of such polypeptide chains by an intervening spacer peptide having a length that is too short to permit their VL1/VH2 (or their VL2/VH1) domains to associate together to form epitope-binding site capable of binding to either the first or second epitope.
  • a preferred intervening spacer peptide (Linker 1) for this purpose has the sequence (SEQ ID NO:69): GGGSGGGG.
  • Other Domains of the trivalent binding molecules may be separated by one or more intervening spacer peptides (Linkers), optionally comprising a cysteine residue.
  • Linkers will typically be incorporated between Variable Domains (i.e., VH or VL) and peptide Heterodimer-Promoting Domains (e.g., an E-coil or K-coil) and between such peptide Heterodimer-Promoting Domains (e.g., an E-coil or K-coil) and CH2-CH3 Domains.
  • VH or VL Variable Domains
  • peptide Heterodimer-Promoting Domains e.g., an E-coil or K-coil
  • Exemplary linkers useful for the generation of trivalent binding molecules are provided above and are also provided in PCT Application Nos: PCT/US 15/33081; and PCT/US15/33076.
  • the first and second polypeptide chains of such trivalent binding molecules associate together to form a VLl/VHl binding site capable of binding a first epitope, as well as a VL2/VH2 binding site that is capable of binding to a second epitope.
  • the third and fourth polypeptide chains of such trivalent binding molecules associate together to form a VL3/VH3 binding site that is capable of binding to a third epitope.
  • the trivalent binding molecules of the present invention may comprise three polypeptides. Trivalent binding molecules comprising three polypeptide chains may be obtained by linking the domains of the fourth polypeptide N- terminal to the VH3 -containing Domain of the third polypeptide (e.g., using an intervening spacer peptide (Linker 4)).
  • a third polypeptide chain of a trivalent binding molecule of the invention containing the following domains is utilized: (i) a VL3 -containing Domain, (ii) a VH3 -containing Domain, and (iii) a Domain containing a CH2-CH3 sequence, wherein the VL3 and VH3 are spaced apart from one another by an intervening spacer peptide that is sufficiently long (at least 9 or more amino acid residues) so as to allow the association of these domains to form an epitope-binding site.
  • an intervening spacer peptide for this purpose has the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 100).
  • VLl/VHl, VL2/VH2, and VL3/VH3 Domains of such trivalent binding molecules may be different so as to permit binding that is monospecific, bispecific or trispecific.
  • the VL and VH Domains may be selected such that a trivalent binding molecule comprises two binding sites for a first epitope and one binding sites for a second epitope, or one binding site for a first epitope and two binding sites for a second epitope, or one binding site for a first epitope, one binding site for a second epitope and one binding site for a third epitope.
  • these domains are preferably selected so as to bind an epitope of ADAM9, an epitope of second molecule, and an epitope of a third molecule.
  • the second molecule is a molecule (e.g., CD2, CD3, CD8, CD 16, T-Cell Receptor (TCR), KG2D, etc) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • the third molecule is CD8.
  • One embodiment of the present invention relates to trivalent binding molecules that comprise two epitope-binding sites for ADAM9 and one epitope-binding site for a second molecule.
  • the two epitope-binding sites for ADAM9 may bind the same epitope or different epitopes.
  • Another embodiment of the present invention relates to trivalent binding molecules that comprise, one epitope-binding site for ADAM9 and two epitope-binding sites for a second molecule.
  • the two epitope-binding sites for the second molecule may bind the same epitope or different epitopes of the second molecule.
  • a further embodiment of the present invention relates to trispecific trivalent binding molecules that comprise, one epitope-binding site for ADAM9, one epitope-binding site for a second molecule, and one epitope-binding site for a third molecule.
  • the second molecule is a molecule (e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), KG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • the second molecule is CD3 and the third molecule is CD8.
  • such trivalent binding molecules may comprise three, four, five, or more polypeptide chains.]]
  • ADAM9-binding molecules e.g., antibodies, diabodies, trivalent binding molecules, etc.
  • a preferred CL Domain is a human IgG CL Kappa Domain.
  • the amino acid sequence of an exemplary human CL Kappa Domain is (SEQ ID NO:101):
  • an exemplary CL Domain is a human IgG CL Lambda Domain.
  • the amino acid sequence of an exemplary human CL Lambda Domain is (SEQ ID NO: 102):
  • the ADAM9-binding molecules of the invention may comprise an Fc Region.
  • the Fc Region of such molecules of the invention may be of any isotype (e.g., IgGl, IgG2, IgG3, or IgG4).
  • the ADAM9-binding molecules of the invention may further comprise a CHI Domain and/or a Hinge Region.
  • the CHI Domain and/or Hinge Region may be of any isotype (e.g., IgGl, IgG2, IgG3, or IgG4), and is preferably of the same isotype as the desired Fc Region.
  • An exemplary CHI Domain is a human IgGl CHI Domain.
  • the amino acid sequence of an exemplary human IgGl CHI Domain is (SEQ ID NO:103):
  • An exemplary CHI Domain is a human IgG2 CHI Domain.
  • the amino acid sequence of an exemplary human IgG2 CHI Domain is (SEQ ID NO:104):
  • An exemplary CHI Domain is a human IgG3 CHI Domain.
  • the amino acid sequence of an exemplary human IgG3 CHI Domain is (SEQ ID NO:207):
  • An exemplary CHI Domain is a human IgG4 CHI Domain.
  • the amino acid sequence of an exemplary human IgG4 CHI Domain is (SEQ ID NO:105):
  • One exemplary Hinge Region is a human IgGl Hinge Region.
  • the amino acid sequence of an exemplary human IgGl Hinge Region is (SEQ ID NO:96):
  • EPKSCDKTHTCPPCP EPKSCDKTHTCPPCP .
  • Another exemplary Hinge Region is a human IgG2 Hinge Region.
  • the amino acid sequence of an exemplary human IgG2 Hinge Region is (SEQ ID NO:97):
  • Another exemplary Hinge Region is a human IgG4 Hinge Region.
  • the amino acid sequence of an exemplary human IgG4 Hinge Region is (SEQ ID NO:98): ESKYGPPCPSCP.
  • an IgG4 Hinge Region may comprise a stabilizing mutation, such as the S228P substitution.
  • the amino acid sequence of an exemplary stabilized IgG4 Hinge Region is (SEQ ID NO:99): ESKYGPPCPPCP.
  • the Fc Region of the Fc Region-containing molecules (e.g., antibodies, diabodies, trivalent binding molecules, etc.) of the present invention may be either a complete Fc Region (e.g., a complete IgG Fc Region) or only a fragment of an Fc Region.
  • the Fc Region of the Fc Region-containing molecules of the present invention lacks the C-terminal lysine amino acid residue.
  • FcyRI CD64
  • FcyRIIA CD32A
  • FcyRIII CD16
  • FcyRIIB CD32B
  • FcRn neonatal Fc Receptor
  • Modification of the Fc Region may lead to an altered phenotype, for example altered serum half-life, altered stability, altered susceptibility to cellular enzymes or altered effector function. It may therefore be desirable to modify an Fc Region-containing ADAM9-binding molecule of the present invention with respect to effector function, for example, so as to enhance the effectiveness of such molecule in treating cancer. 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- Hodgkin's lymphoma, CLL, and Burkitt's lymphoma).
  • Molecules of the invention possessing such conferred or altered effector function activity are useful for the treatment and/or prevention of a disease, disorder or infection in which an enhanced efficacy of effector function activity is desired.
  • the Fc Region of the Fc Region- containing molecules of the present invention may be an engineered variant Fc Region.
  • the Fc Region of the bispecific Fc Region-containing molecules of the present invention may possess the ability to bind to one or more Fc receptors (e.g., FcyR(s)), more preferably such variant Fc Region have 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), e.g., will have enhanced binding to an activating receptor and/or will have substantially reduced or no ability to bind to inhibitory receptor(s).
  • the Fc Region of the Fc Region-containing molecules 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. etal. (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).
  • Table 6 lists exemplary single, double, triple, quadruple and quintuple substitutions (numbering is that of the EU index as in Kabat, and substitutions are relative to the amino acid sequence of SEQ ID NO:l) of exemplary modification that increase binding to activating receptors and/or reduce binding to inhibitory receptors.
  • Exemplary variants of human IgGl Fc Regions with reduced binding to CD32B and/or increased binding to CD16A contain F243L, R292P, Y300L, V305I or P396L substitutions, wherein the numbering is that of the EU index as in Kabat. These amino acid substitutions may be present in a human IgGl Fc Region in any combination.
  • the variant human IgGl Fc Region contains a F243L, R292P and Y300L substitution.
  • the variant human IgGl Fc Region contains a F243L, R292P Y300L, V305I and P396L substitution.
  • the Fc Regions of ADAM9-binding molecules of the present invention it is preferred for the Fc Regions of ADAM9-binding molecules 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).
  • the ADAM9-binding molecules of the present invention comprise an IgG Fc Region that exhibits reduced ADCC effector function.
  • the CH2-CH3 Domains of such ADAM9-binding molecules include any 1, 2, 3, or 4 of the substitutions: L234A, L235A, D265A, N297Q, and N297G, wherein the numbering is that of the EU index as in Kabat.
  • the CH2-CH3 Domains contain an N297Q substitution, an N297G substitution, L234A and L235A substitutions or a D265A substitution, as these mutations abolish FcR binding.
  • the ADAM9- binding molecules of the present invention comprise an IgG2 Fc Region (SEQ ID NO:2) or an IgG4 Fc Region (SEQ ID:NO:4).
  • the instant invention also encompasses the introduction of a stabilizing mutation, such as the Hinge Region S228P substitution described above (see, e.g., SEQ ID NO:99). Since the N297G, N297Q, L234A, L235A and D265A substitutions abolish effector function, in circumstances in which effector function is desired, these substitutions would preferably not be employed.
  • a stabilizing mutation such as the Hinge Region S228P substitution described above (see, e.g., SEQ ID NO:99). Since the N297G, N297Q, L234A, L235A and D265A substitutions abolish effector function, in circumstances in which effector function is desired, these substitutions would preferably not be employed.
  • a preferred IgGl sequence for the CH2 and CH3 Domains of the Fc Region- containing molecules of the present invention having reduced or abolished effector function will comprise the substitutions L234A/L235A (shown underlined) (SEQ ID NO:106):
  • X is a lysine (K) or is absent.
  • Region-containing molecules of the present invention comprises an S442C substitution
  • X is a lysine (K) or is absent.
  • a third preferred IgGl sequence for the CH2 and CH3 Domains of the Fc Region-containing molecules of the present invention comprises the L234A/L235A substitutions (shown underlined) that reduce or abolish effector function and the S442C substitution (shown underlined) that permits two CH3 domains to be covalently bonded to one another via a disulfide bond or conjugation of a drug moiety.
  • the amino acid sequence of such molecule is (SEQ ID NO: 108):
  • X is a lysine (K) or is absent.
  • the serum half-life of proteins comprising Fc Regions may be increased by increasing the binding affinity of the Fc Region for FcRn.
  • the term "half-life" as used herein means a pharmacokinetic property of a molecule that is a measure of the mean survival time of the molecules following their administration.
  • Half-life can be expressed as the time required to eliminate fifty percent (50%) of a known quantity of the molecule from a subject's (e.g., a human patient or other mammal) body or a specific compartment thereof, for example, as measured in serum, i.e., circulating half-life, or in other tissues.
  • an increase in half-life results in an increase in mean residence time (MRT) in circulation for the administered molecule.
  • MRT mean residence time
  • the ADAM9-binding molecules of the present invention comprise a variant Fc Region that comprises at least one amino acid modification relative to a wild-type Fc Region, such that said molecule has an increased half-life (relative to a molecule comprising a wild-type Fc Region).
  • the ADAM9- binding molecules of the present invention comprise a variant IgG Fc Region, wherein said variant Fc Region comprises a half-live extending amino acid substitution at one or more positions selected from the group consisting of 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, 433, 434, 435, and 436, wherein the numbering is that of the EU index as in Kabat.
  • Numerous mutations capable of increasing the half-life of an Fc Region-containing molecule are known in the art and include, for example M252Y, S254T, T256E, and combinations thereof. For example, see the mutations described in U.S. Patent Nos. 6,277,375, 7,083,784; 7,217,797, 8,088,376; U.S. Publication Nos. 2002/0147311; 2007/0148164; and PCT Publication Nos. WO 98/23289; WO 2009/058492; and WO 2010/033279, which are herein incorporated by reference in their entireties.
  • ADAM9- binding molecules with enhanced half-life also include those possessing variant Fc Regions comprising substitutions at two or more of Fc Region residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378, 428, 433, 434, 435 and 436.
  • two or more substitutions selected from: T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K, and Y436I, wherein the numbering is that of the EU index as in Kabat.
  • an ADAM9-binding molecule of the present invention possesses a variant IgG Fc Region comprising the substitutions:
  • an ADAM9-binding molecule of the present invention possesses a variant IgG Fc Region comprising any 1, 2, or 3 of the substitutions: M252Y, S254T and T256E.
  • the invention further encompasses ADAM9-binding molecules possessing variant Fc Regions comprising:
  • Region-containing molecules of the present invention comprises the M252Y, S254T and
  • Region-containing molecules of the present invention comprises the L234A/L235A substitutions (shown underlined) that reduce or abolish effector function and the M252Y,
  • amino acid sequence of such molecule is (SEQ ID NO:201):
  • X is a lysine (K) or is absent.
  • Region-containing molecules of the present invention comprises the L234A/L235A substitutions (shown underlined) that reduce or abolish effector function and the M252Y,
  • X is a lysine (K) or is absent.
  • an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a "knob", e.g., tryptophan) can be introduced into the CH2 or CH3 Domain such that 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).
  • the hole e.g., a substitution with glycine
  • a preferred knob is created by modifying an IgG Fc Region to contain the modification T366W.
  • a preferred hole is created by modifying an IgG Fc Region to contain the modification T366S, L368A and Y407V.
  • the protein A binding site of the hole-bearing CH2 and CH3 Domains of the third polypeptide chain is preferably mutated by amino acid substitution at position 435 (H435R).
  • the hole-bearing third polypeptide chain homodimer will not bind to protein A, whereas the bispecific heterodimer will retain its ability to bind protein A via the protein A binding site on the first polypeptide chain.
  • the hole-bearing third polypeptide chain may incorporate amino acid substitutions at positions 434 and 435 (N434A/N435K).
  • a preferred IgG amino acid sequence for the CH2 and CH3 Domains of the first polypeptide chain of an Fc Region-containing molecule of the present invention will have the "knob-bearing" sequence (SEQ ID NO:109):
  • a preferred IgG amino acid sequence for the CH2 and CH3 Domains of the second polypeptide chain of an Fc Region-containing molecule of the present invention having two polypeptide chains (or the third polypeptide chain of an Fc Region-containing molecule having three, four, or five polypeptide chains) will have the "hole-bearing" sequence (SEQ ID NO: 110):
  • X is a lysine (K) or is absent.
  • NO: 110 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:l).
  • the invention also encompasses such CH2-CH3 Domains, which comprise the wild-type alanine residues, alternative and/or additional substitutions which modify effector function and/or FyR binding activity of the Fc region.
  • the invention also encompasses such CH2-CH3 Domains, which further comprise one or more half-live extending amino acid substitutions.
  • the invention encompasses such hole-bearing and such knob-bearing CH2-CH3 Domains which further comprise the M252Y/S254T/T256E substitutions.
  • X is a lysine (K) or is absent.
  • the first polypeptide chain will have a "knob-bearing" CH2- CH3 sequence, such as that of SEQ ID NO: 109.
  • a "hole- bearing" CH2-CH3 Domain e.g., SEQ ID NO: 110 could be employed in the first polypeptide chain, in which case, a "knob-bearing" CH2-CH3 Domain (e.g., SEQ ID NO: 109) would be employed in the second polypeptide chain of an Fc Region-containing molecule of the present invention having two polypeptide chains (or in the third polypeptide chain of an Fc Region-containing molecule having three, four, or five polypeptide chains).
  • the invention encompasses ADAM9-binding molecules comprising CH2 and/or CH3 Domains that have been engineered to favor heterodimerization over homodimerization using mutations known in the art, such as those disclosed in PCT Publication Nos. WO 2007/1 10205; WO 201 1/143545; WO 2012/058768; and WO 2013/06867, all of which are incorporated herein by reference in their entirety.
  • the ADAM9-binding molecules of the invention can be engineered to comprise a combination of epitope-binding sites that recognize a set of antigens unique to a target cell or tissue type.
  • the present invention relates to multispecific ADAM9-binding molecules that are capable of binding to an epitope of ADAM9 and an epitope of a molecule present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • an effector cell such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.
  • the ADAM9-binding molecules of the present invention may be construction to comprise an epitope-binding site that immunospecifically binds CD2, CD3, CD8, CD 16, T-Cell Receptor (TCR), or KG2D.
  • the invention also relates to trispecific ADAM9-binding molecules that are capable of binding to an epitope of CD3 and an epitope of CD8 (see, e.g., PCT Publication No. WO 2015/026894).
  • the bispecific, trispecific or multispecific ADAM9- binding molecules of the invention are capable of binding to an epitope of ADAM9 and an epitope of CD2.
  • CD2 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 K cell nanotube formation (Mace, E.M. et al. (2014) "Cell Biological Steps and Checkpoints in Accessing NK Cell Cytotoxicity " Immunol. Cell. Biol. 92(3):245-255; Comerci, C.J. et al. (2012) "CD 2 Promotes Human Natural Killer Cell Membrane Nanotube Formation " PLoS One 7(10):e47664: l-12).
  • Molecules that specifically bind CD2 include the anti-CD2 antibody "Lo-CD2a.”
  • the bispecific, trispecific or multispecific ADAM9- binding molecules of the invention are capable of binding to an epitope of ADAM9 and 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
  • Molecules that specifically binds CD3 include the anti-CD3 antibodies "CD3 mAb-1" and "OKT3.”
  • the anti-CD3 antibody CD3 mAb-1 is capable of binding non-human primates (e.g., cynomolgus monkey).
  • the VH Domain of CD3 mAb-1 VH(1) may be used with the VL Domain of CD3 mAb-1 (SEQ ID NO:) to form a functional CD3-binding molecule in accordance with the present invention.
  • the VH Domain of CD3 mAb-1 VH(2) may be used with the VL Domain of CD3 mAb-1 (SEQ ID NO:) to form a functional CD3 -binding molecule in accordance with the present invention.
  • CD3 mAb-1 (D65G), comprises the VL Domain of CD3 mAb-1 (SEQ ID NO: 115) and a VH CD3 mAb-1 Domain having a D65G substitution (Kabat position 65, corresponding to residue 68 of SEQ ID NO: 113)
  • an affinity variant of CD3 mAb-1 may be incorporated.
  • Variants include a low affinity variant designated "CD3 mAb-1 Low” and a variant having a faster off rate designated "CD3 mAb-1 Fast.”
  • the VL Domain of CD mAbl (SEQ ID NO:115) is common to CD3 mAb-1 Low and CD3 mAbl Fast and is provided above.
  • the amino acid sequences of the VH Domains of each of CD3 mAb-1 Low and CD3 mAb-1 Fast are provided below.
  • Another anti-CD3 antibody which may be utilized is antibody Muromonab- CD3 "OKT3" (Xu et al. (2000) "In Vitro Characterization Of Five Humanized OKT3 Effector Function Variant Antibodies, " Cell. Immunol. 200: 16-26; Norman, D.J. (1995) "Mechanisms Of Action And Overview Of OKT3 " Ther. Drug Monit. 17(6):615-620; Canafax, D.M. et al. (1987) "Monoclonal Anti lymphocyte Antibody (OKT3) Treatment Of Acute Renal Allograft Rejection," Pharmacotherapy 7(4): 121-124; Swinnen, L.J. et al.
  • Additional anti-CD3 antibodies that may be utilized include but are not limited to those described in PCT Publication Nos. WO 2008/119566; and WO 2005/118635.
  • the bispecific, trispecific or multispecific ADAM9- binding molecules of the invention are capable of binding to an epitope of ADAM9 and an epitope of CD8.
  • CD8 is a T-cell co-receptor composed of two distinct chains (Leahy, D.J., (1995) "A Structural View of CD 4 and CD8 " FASEB J., 9: 17-25) that is expressed on Cytotoxic T-cells.
  • CD8 + T-cells The activation of CD8 + T-cells has been found to be mediated through co-stimulatory interactions between an antigemmajor histocompability class I (MHC I) molecule complex that is arrayed on the surface of a target cell and a complex of CD8 and the T-cell Receptor, that are arrayed on surface of the CD8 + T-cell (Gao, G., and Jakobsen, B., (2000). "Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-Cell Receptor" . Immunol Today 21 : 630-636). Unlike MHC II molecules, which are expressed by only certain immune system cells, MHC I molecules are very widely expressed.
  • MHC I an antigemmajor histocompability class I
  • cytotoxic T-cells are capable of binding to a wide variety of cell types. Activated cytotoxic T-cells mediate cell killing through their release of the cytotoxins perforin, granzymes, and granulysin.
  • Antibodies that specifically bind CD 8 include the anti-CD8 antibodies "OKT8" and "TRX2.”
  • multispecific ADAM9-binding molecules of the invention are capable of binding to an epitope of ADAM9 and an epitope of CD16.
  • CD16 is the FcyRIIIA receptor.
  • CD 16 is expressed by neutrophils, eosinophils, natural killer (NK) cells, and tissue macrophages that bind aggregated but not monomelic human IgG (Peltz, G. A. et al. (1989) "Human Fc Gamma RIII: Cloning, Expression, And Identification Of The Chromosomal Locus Of Two Fc Receptors For IgG," Proc. Natl. Acad. Sci.
  • Molecules that specifically bind CD 16 include the anti-CD 16 antibodies "3G8" and "A9.” Humanized A9 antibodies are described in PCT Publication No. WO 03/101485.
  • Additional anti-CD 16 antibodies that may be utilized include but are not limited to those described in PCT Publication Nos. WO 03/101485; and WO 2006/125668.
  • the bispecific, trispecific or multispecific ADAM9- binding molecules of the invention are capable of binding to an epitope of ADAM9 and 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.
  • Molecules 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.
  • multispecific ADAM9-binding molecules of the invention are capable of binding to an epitope of ADAM9 and an epitope of the KG2D receptor.
  • the KG2D receptor is expressed on all human (and other mammalian) Natural Killer cells (Bauer, S. et al. (1999) "Activation OfNK 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
  • Molecules that specifically bind to the NKG2D Receptor include the anti- NKG2D antibodies “KYK-1.0” and “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).

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CN114173778A (zh) * 2019-04-26 2022-03-11 洪明奇 Adam9抑制剂作为免疫调节剂的用途
EP3960171A4 (en) * 2019-04-26 2023-05-24 China Medical University USE OF AN ADAM9 INHIBITOR AS AN IMMUNOMODULATOR
CN114173778B (zh) * 2019-04-26 2024-12-27 洪明奇 Adam9抑制剂在制备用于制备免疫调节剂的药物中的应用
WO2022192134A1 (en) 2021-03-08 2022-09-15 Immunogen, Inc. Methods for increasing efficacy of immunoconjugates targeting adam9 for the treatment of cancer
WO2024199111A1 (zh) * 2023-03-24 2024-10-03 映恩生物制药（苏州）有限公司 靶向adam9的人源化抗体、其抗体药物偶联物及其应用

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