Mult Mai Muloqoti Taite

US009993566B2MULT MAIMULOQOTI TAITE (12 ) United States Patent ( 10 ) Patent No. : US 9 , 993 ,566 B2 Liu et al. (45 ) Date of Patent : * Jun. 12 , 2018 (54 ) SEZ6 MODULATORS AND METHODS OF ( 56 ) References Cited USE U . S . PATENT DOCUMENTS @(71 ) Applicant: AbbVie Stemcentrx LLC , North 5 , 112 , 946 A 5 / 1992 Maione Chicago , IL (US ) 5 ,336 ,603 A 8/ 1994 Capon et al. 5 , 349 ,053 A 9 / 1994 Landolfi @(72 ) Inventors : David Liu , South San Francisco , CA 5 , 359 ,046 A 10 / 1994 Capon et al. (US ) ; Deepti Rokkam , Sunnyvale , CA 5 , 447 , 851 A 9 / 1995 Beutler et al. (US ) ; Sheila Bheddah , San Francisco , 5 , 530 , 101 A 6 / 1996 Queen et al. 5 ,622 , 929 A 4 / 1997 Willner et al . CA (US ) ; Javier Lopez -Molina , New 5 ,693 , 762 A 12 / 1997 Queen et al. York , NY (US ) ; Laura Saunders, San 6 ,180 , 370 B1 1 / 2001 Queen et al. Francisco , CA (US ) 6 , 214 , 345 B1 4 / 2001 Firestone et al. 6 , 362 , 331 B1 3 / 2002 Kamal et al. @ 7 , 049 , 311 B1 5 /2006 Thurston et al. ( 73 ) Assignee : AbbVie Stemcentrx LLC , North 7 ,087 ,409 B2 9 / 2006 Barbas, III et al . Chicago , IL (US ) 7 , 189 , 710 B2 3 / 2007 Kamal et al. 7 ,407 ,951 B2 8 / 2008 Thurston et al. ( * ) Notice : Subject to any disclaimer , the term of this 7 , 422 , 739 B2 9 / 2008 Anderson et al . patent is extended or adjusted under 35 7 , 429 , 658 B2 9 / 2008 Howard et al . U . S . C . 154 (b ) by 0 days. days . 7 , 521, 541 B2 4 / 2009 Eigenbrot et al. 7, 557 ,099 B2 7 / 2009 Howard et al . This patent is subject to a terminal dis 7 ,619 , 068 B2 11/ 2009 Pilkington et al. claimer . 7 , 632 , 678 B2 12 / 2009 Hansford et al. 7 , 741 , 319 B2 6 / 2010 Howard et al. 8 ,034 ,808 B2 10 / 2011 Delavault et al. (21 ) Appl. No. : 14 /915 , 563 8 , 163, 736 B2 . 4 /2012 Gauzy et al . 8 ,788 ,213 B2 7 / 2014 Bright ( 22 ) PCT Filed : Aug . 27, 2014 8 , 889 ,833 B2 11/ 2014 Yue et al. 9 ,676 ,850 B2 * 6 /2017 Saunders ...... C07K 16 / 28 ( 86 ) PCT No. : PCT/ US2014 / 053014 2003 /0211991 Al 11 /2003 Su $ 371 ( c ) ( 1 ), (Continued ) ( 2 ) Date : Feb . 29 , 2016 FOREIGN PATENT DOCUMENTS ( 87 ) PCT Pub . No .: W02015 /031541 CN 1343774 A 4 /2002 EP 0367166 5 / 1990 PCT Pub . Date : Mar. 5 , 2015 (Continued ) (65 ) Prior Publication Data US 2016 / 0287720 A1 Oct . 6 , 2016 OTHER PUBLICATIONS Gorlov et al. Seizure 6 - like (SEZÓL ) gene and risk for lung cancer . Related U . S . Application Data Cancer Research 67 ( 17 ) : 8406 -8411 , Sep . 1 , 2007. * ( 60 ) Provisional application No. 61 / 871, 289 , filed on Aug . Gussow et al . (Methods in Enzymology . 1991 ; 203: 99 - 121) . * George et al . (Circulation . 1998 ; 97 : 900 - 906 ). * 28 , 2013 NM _ 001098635 — Homo sapiens seizure related 6 homolog (51 ) Int . Ci. (SEZ6 ) , transcript variant 2 , mRNA . NM _ 021286 — Mus musculus seizure related gene 6 (Sezo ), tran CO7K 16 / 00 ( 2006 .01 ) script variant 1 , mRNA . A61K 4748 ( 2006 . 01) NM _ 178860 — Homo sapiens seizure related 6 homolog (SEZO ) , CO7K 16 / 28 ( 2006 .01 ) transcript variant 1 , mRNA . GOIN 33 / 574 ( 2006 .01 ) NP _ 001092105 — seizure protein 6 homolog isoform 2 precursor CO7K 16 / 18 ( 2006 .01 ) [Homo sapiens] . A61K 47/ 68 ( 2017 . 01 ) NP _ 001099224 — seizure protein 6 homolog precursor [Rattus A61K 39 / 00 ( 2006 . 01 ) norvegicus ] . (52 ) U .S . CI. (Continued ) CPC .. . . A61K 47 /48384 ( 2013 .01 ); A61K 47/ 6803 ( 2017 .08 ) ; A61K 47 /6843 ( 2017 .08 ); A61K 47 /6849 ( 2017 . 08 ) ; CO7K 16 / 18 ( 2013 .01 ) ; Primary Examiner — Brad Duffy CO7K 16 / 28 ( 2013 .01 ) ; GOIN 33 / 57407 (74 ) Attorney, Agent, or Firm — Womble Bond Dickinson (2013 .01 ) ; GOIN 33 /57423 ( 2013 .01 ) ; A61K (US ) LLP 2039 / 505 ( 2013 .01 ) ; CO7K 2317 /20 ( 2013 .01 ) ; CO7K 2317 / 24 ( 2013 .01 ) ; CO7K 2317 / 33 (57 ) ABSTRACT ( 2013 .01 ); CO7K 2317 / 34 ( 2013 .01 ) ; CO7K 2317 / 565 (2013 . 01 ); COZK 2317 /73 ( 2013 .01 ) ; Novel modulators , including antibodies and derivatives CO7K 2317/ 77 (2013 .01 ) thereof , and methods of using such modulators to treat (58 ) Field of Classification Search proliferative disorders are provided . None See application file for complete search history . 41 Claims, 72 Drawing Sheets US 9, 993 ,566 B2 Page 2

( 56 ) References Cited Bork et al ., “ The CUB domain . A widespread module in develop mentally regulated proteins, ” J Mol Biol . ( 1993 ) 231( 2 ): 539 - 45 . U . S . PATENT DOCUMENTS Capel et al . , " Heterogeneity of human IgG Fc receptors , " Immunomethods. (Feb . 1994 ) 4 ( 1 ): 25 - 34 . 2004 /0067490 A1 4 /2004 Zhong et al. Carrodus , N . L . , et al. , “ Seizure - Related Gene 6 : A Modulator of 2004 /0101920 A1 5 /2004 Radziejewski et al. Excitatory Synapse Development, ” Australian Neuroscience Soci 2005 / 0008625 AL 1 / 2005 Balint et al . 2005 / 0238649 Al 10 / 2005 Doronina et al . ety Annual Meeting , Auckland , (Jan . 31 - Feb . 3 , 2011 ) p . 87. 2007 / 0141066 A1 6 / 2007 Phillips et al . Chao et al. , “ Isolating and engineering human antibodies using 2007 /0292414 Al 12 / 2007 Duntsch et al . yeast surface display ,” Nature ( Sep . 6 , 2007 ) 2 :755 -768 with 2008 /0138313 Al 6 / 2008 Frankel erratum . 2008 /0175870 A1 7 / 2008 Mather et al . Chothia et al ., “ Canonical Structures for Hypervariable Regions of 2009 /0155255 Al 6 / 2009 Glaser et al. Immunoglobulins, ” J . Mol. Biol. ( 1987 ) 196 :901 -917 . 2010 /0162416 Al 6 / 2010 Krtolica et al. Chothia et al. , “ Conformations o fimmunoglobulin ,” Nature ( 1989 ) 2010 /0273160 A1 10 / 2010 Donahoe et al. 342 :877 -883 . 2010 /0275280 AL 10 / 2010 Clevers et al . Chumsae et al. , “ Identification and Localization of Unpaired Cys 2011/ 0020221 A1 1 / 2011 Berman et al . teine Residues in Monocolonal Antibodies by Fluorescence Label 2011/ 0033378 AL 2 / 2011 Dimasi et al . ing and Mass Spectrometry ,” Anal. Chem . (2009 ) 81: 6449 -6457 . 2011/ 0256157 Al 10 / 2011 Howard et al. Cochran et al . “ Domain - level antibody epitope mapping through 2011/ 0301334 AL 12 / 2011 Bhakta et al. 2012 /0078028 AL 3 / 2012 Satpayev et al. yeast surface display of epidermal growth factor receptor frag 2012 / 0244171 AL 9 / 2012 Li et al . ments , " J Immunol Methods . ( 2004 ) 287 ( 1 - 2 ) : 147 -58 . 2012 / 0328624 Al 12 / 2012 Yoshida et al. Cook , M . , et al. , " Notch in the development of thyroid C -cells and 2013 /0028919 Al 1 /2013 Howard et al. the treatment of medullary thyroid cancer, ” Am J . Transl Res. , 2013 / 0061340 Al 3 / 2013 Dylla et al. (2010 ) 2 : 119 - 25 PMID : 20182588 . 2013 /0061342 A1 3/ 2013 Dylla et al. Denardo et al . , “ Comparison of 1 , 4 , 7 , 10 -tetraazacyclododecane 2013 /0171170 A1 7 / 2013 Ebens et al . N ,N ', N ", N " - tetraacetic acid (DOTA )- peptide - ChL6 , a novel 2013 /0260385 Al 10 / 2013 Dylla et al . immunoconjugate with catabolizable linker, to 2 -iminothiolane -2 2015 / 0018531 Al 1 / 2015 Saunders et al. [ p - (bromoacetamido ) benzyl ] -DOTA - ChL6 in breast cancer 2015 / 0030636 A1 1 / 2015 Dylla et al . xenografts , ” Clin Cancer Res ( 1998 ) 4 : 2483 - 2490 . 2016 /0175460 AL 6 / 2016 Arathoon et al . Dubowchik et al. , “ Cathepsin B - labile dipeptide linkers for 2016 /0176964 A16 / 2016 Arathoon et al . lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and FOREIGN PATENT DOCUMENTS antigen - specific in vitro anticancer activity , " Bioconjug Chem . ( Jul. - Aug. 2002 ) 13 ( 4 ) :855 -69 . EP 0307434 9 / 1993 ?? 2617730 7 / 2013 Dylla et al. , “ Colorectal Cancer Stem Cells Are Enriched in WO WO 91/ 06570 5 / 1991 Xenogeneic Tumors Following Chemotherapy, " PLoS One, (2008 ) WO WO 96 / 04388 2 / 1996 3 ( 6 ) : e2428 . WO WO 98 / 52976 11 / 1998 Fuhrmann et al ., “ Poster Presentations— Immunomodulatory WO WO 00 / 34317 6 / 2000 Agents and Interventions Abstract 5625 : In vitro and in vivo WO WO 01/ 83552 A2 11 / 2001 pharmacology of MEDI- 565 (MT111 ) , a novel CEA/ CD3 -bispecific WO WO 03 / 075957 9 / 2003 singlechain BiTE antibody in development for the treatment of WO WO 2011 / 128650 10 / 2011 gastrointestinal adenocarcinomas , " Annual Meeting of AACR W0 WO 2011 / 130613 10 / 2011 Abstract Cancer Research (Apr . 15 , 2010 ) vol. 70 , Issue 8 , Supple WO WO 2011/ 130616 10 / 2011 ment 1 No . 5625 . WO WO 2012 /012801 1 / 2012 Galluzzo et al ., “ Notch signaling in lung cancer, " Expert Rev WO WO 2012 /031280 3 / 2012 WO WO 2013 /053873 4 / 2013 Anticancer Ther . ( Apr. 2011) 11 ( 4 ) :533 -40 PMID : 21504320 . WO WO 2013 / 119960 8 / 2013 Garnett , “ Targeted drug conjugates: principles and progress , " WO WO 2013 / 119964 8 / 2013 Advanced Drug Delivery Reviews 53 (2001 ) 171 - 216 . WO WO 2013 / 126810 A18 / 2013 Gene Cards (“ SEZ6 Gene” definition ; pp . 1 - 14 ( Jan . 15 , 2016 ) ) . WO WO 2015 / 031693 3 / 2015 Gunnersen et al. , “ Sez - 6 proteins affect dendritic arborization WO WO 2015 / 031698 3 /2015 patterns and excitability of cortical pyramidal neurons, " Neuron . WO WO 2016 / 064749 4 /2016 (Nov . 21 , 2007 ) 56 ( 4 ) :621 - 39 PMID : 18031681 . Gunnersen , Jenny M . , et al. , " Seizure -Related Gene 6 ( Sez - 6 ) in Amacrine Cells of the Rodent Rretina and the Consequence of Gene OTHER PUBLICATIONS Deletion ,” PLOS ONE (2009 ) 4 ( 8 ): e6546 . NP _ 001139913 — synaptojanin - 1 [Salmo salar) . Haddad , R . I . , “ How to incorporate new tyrosine kinase inhibitors in NP _ 001230261 — seizure 6 -like protein 2 isoform 5 precursor the treatment of patients with medullary thyroid cancer, " J Clin [Homo sapiens ] . Oncolo . ( 2013 ) 31 : 3618 - 20 PMID : 24002516 . NP _ 066938 — seizure 6 - like protein isoform 1 precursor [Homo Harris et al. , “ Targeting embryonic signaling pathways in cancer sapiens) therapy ," Expert Opin Ther Targets (Jan . 2012 ) 16 ( 1 ) : 131 -45 . NP _ 067261 — seizure protein 6 isoform 1 precursor [Mus Herbst et al. , " SEZ - 6 : promoter selectivity , genomic structure and musculus ) . localized expression in the brain , ” Brain Res Mol Brain Res . (Mar . NP _ 849191. 3 — seizure protein 6 homolog isoform 1 precursor 1997 ) 44 ( 2 ) : 309 - 22 PMID : 9073173 . [Homo sapiens ) Hochleitner et al ., " Characterization of a discontinuous epitope of XP _ 511368 — Predicted : seizure protein 6 homolog isoform X2 the human immunodeficiency virus (HIV ) core protein p24 by [Pan troglodytes ]. epitope excision and differential chemical modification followed by Ashkenazi et al ., “ Protection against endotoxic shock by a tumor mass spectrometric peptide mapping analysis , " Protein Sci . (Mar . necrosis factor receptor immunoadhesin ,” Proc Natl Acad Sci US 2000 ) 9 ( 3 ) :487 - 96 PMID : 10752610 . A (Dec . 1 , 1991 ) 88 (23 ): 10535 - 10539 . Hoey et al ., “ DLL4 blockade inhibits tumor growth and reduces Bjellqvist et al. , “ The focusing positions of polypeptides in immo tumor - initiating cell frequency, ” Cell Stem Cell . ( Aug . 7 , 2009 ) bilized pH gradients can be predicted from their amino acid 5 ( 2 ) : 168 - 77 PMID : 19664991 . sequences, ” Electrophoresis ( 1993 ) 14 : 1023 - 1031. Ishikawa et al. , “ Characterization of SEZ6L2 cell - surface protein as Boder et al. , Yeast surface display for screening combinatorial a novel prognostic marker for lung cancer, " Cancer Sci. (2006 ) polypeptide libraries, Nature ( Jun . 1997 ) 15 :553 - 557 . 97 (8 ) :737 -45 . US 9 , 993 ,566 B2 Page 3

( 56 ) References Cited Vermeer et al. , “ The thermal stability of immunoglobulin : unfolding and aggregation of a multi - domain protein , " Biophys J . ( Jan . 2000 ) OTHER PUBLICATIONS 78 ( 1 ) : 394 -404 . Vermeer et al. , “ The unfolding /denaturation of immunogam Jones et al. , “ Replacing the complementarity -determining regions in maglobulin of isotype 2b and its F ( ab ) and F ( c ) fragments, " a human antibody with those from a mouse, ” Nature ( 1986 ) Biophys. J . ( 2000 ) 79 ( 4 ) : 2150 -2154 PMID : 11023918 . 321: 522 - 525 — Abstract . Klimstra et al ., " The pathologic classification of neuroendocrine Vié et al ., “ Human fusion proteins between interleukin 2 and IgM tumors : a review of nomenclature, grading , and staging systems, " heavy chain are cytotoxic for cells expressing the interleukin 2 Pancreas. (Aug . 2010 ) 39 (6 ) : 707 - 12 PMID : 20664470 . receptor, ” Proc Nati Acad Sci USA (Dec . 1 , 1992 ) 89 (23 ) : 11337 Klöppel , “ Classification and pathology of gastroenteropancreatic 11341. neuroendocrine neoplasms, ” Endocr Relat Cancer . (Oct . 17 , 2011) Visvader et al. , “ Cancer stem cells in solid tumours: accumulating 18 Suppl 1 :S1 - 16 PMID : 22005112 . evidence and unresolved questions, ” Nat Rev Cancer . ( Oct. 2008 ) Maccallum et al. , “ Antibody -antigen interactions: contact analysis 8 ( 10 ) :755 -68 PMID : 18784658 . and binding site topography, ” J. Mol. Biol. ( 1996 ) 262 :732 - 745 . Waldmann et al. , “ Microarray analysis reveals differential expres Mulley et al . , “ The Role of Seizure -Related SEZ6 as a Susceptibility sion of benign and malignant pheochromocytoma, ” Endocr. Relat. Gene in Febrile Seizures, ” Neurol Res Int. (2011 ) 2011: 917565 Cancer ( 2010 ) 17 ( 3 ): 743 -56 . PMID : 21785725 . Xiong et al. , “ Development of tumor targeting anti -MUC - 1 NCBI protein database search (( “ human seizure related 6 multimer : effects of di- scFv unpaired cysteine location on homologue ” or “ SEZ6 " ) and (Homo sapiens )) (pp . 1 - 2 , Jun . 3 , PEGylation and tumor binding , ” Prot. Eng . , (2006 ) 19 ( 8 ) : 359 - 367 . 2016 ) . Yao , J . C . , et al. , " One hundred years after “ carcinoid” : epidemiol Osaki, George, et al. , “ The distribution of the seizure - related gene ogy of and prognostic factors for neuroendocrine tumors in 35 ,825 6 (Sez - 6 ) protein during postnatal development of the mouse cases in the United States, " J Clin Oncol. (2008 ) 26 : 3063 -72 . forebrain suggests multiple functions for this protein : An analysis PMID : 18565894 . using a new antibody, ” Brain Research (Feb . 10 , 2011) 1386 :58 -69 , Yu , Z . L . , et al . , “ Febrile seizures are associated with mutation of XP028186555 . Peterson et al. , “ Enzymatic Cleavage of Peptide - Linked Radiolabels seizure -related (SEZ ) 6 , a brain - specific gene, " J Neurosci Res. from Immunoconjugates, ” Bioconjugate Chem . ( 1999 ) ( 10 )4 :553 ( 2007 ) 85 : 166 -72 . PMID : 17086543 . 557 . Zheng et al. , “ Administration of noncytolytic IL - 10 / Fc in murine Ravetch et al. , “ Fc receptors ,” Annu Rev Immunol. ( 1991 ) 9 :457 -92 . models of lipopolysaccharide - induced septic shock and allogeneic Reineke , “ Antibody Epitope Mapping Using Arrays of Synthetic islet transplantation ,” J Immunol. (May 15 , 1995 ) 154 ( 10 ) : 5590 -600 Peptides ,” Methods Mol Biol . (2004 ) 2 48 : 443 -63 . PMID : 7730658 . Schuldes et al. , “ Loss of in vitro cytotoxicity of cisplatin after Zimmerman et al. , “ A triglycine linker improves tumor uptake and storage as stock solution in cell culture medium at various tem biodistributions of 67 - Cu - labeled anti - neuroblastoma MAb chCE7 peratures ,” Cancer . ( 1997 ) 79 ( 9 ) : 1723 - 8 PMID : 9128988 . F (ab ') 2 fragments ,” Nucl Med Biol. (1999 ) 26 (8 ): 943 -50 PMID : Schulenburg et al. , “ Neoplastic stem cells : current concepts and 10708309 . clinical perspectives, " Crit Rev Oncol Hematol. (Nov . 2010 ) IPRP dated Jun . 4 , 2014 , issued in PCT application (No . PCT/ 76 ( 2 ) :79 - 98 PMID : 20185329 . US2013/ 027476 ). Shao et al. , “ Expression of SEZ6 gene in human cancer cell ," International Search Report dated Apr. 16 , 2013 , issued in PCT Journal of Tongi University (Medical Science ) (2009 ) 30 ( 1 ) : 36 -39 application (No . PCT/US2013 /027476 ) . ( English translation ) . Written Opinion dated Apr. 16 , 2013 , issued in PCT application (No . Shimizu -Nishikawa K et al. , " Cloning and expression of SEZ - 6 , a PCT/ US2013 /027476 ) . brain - specific and seizure - related cDNA , ” Mol Brain Res . (Feb . Search report dated Dec . 12, 2014 , issued in PCT application (No . 1995 ) 28 ( 2 ) :201 - 10 PMID 7723619 . PCT/ US2014 /053014 ) . Shimizu -Nishikawa K et al. , “ Cloning and characterization of Written Opinion dated Dec . 12 , 2014 , issued in PCT application seizure -related gene , SEZ - 6 ," Biochem and Biophy Res Comm (No . PCT/ US2014 /053014 ) . (Nov . 2 , 1995 ) 216 ( 1 ) :382 - 389 . IPRP dated Mar. 1 , 2016 , issued in PCT application (No . PCT/ Tang et al. , Picoplatin overcomes resistance to cell toxicity in US2014 /053014 ) . small- cell lung cancer cells previouisly treated with cisplatin and Search report dated May 26 , 2017 , issued in European application carboplatin , Cancer Chemother Pharm (2011 ) 67 : 1389 - 1400 . (No . 14839298 .8 ). UniProtKB /Swiss -Prot database entry Q53EL9 — Seizure protein 6 homolog * cited by examiner U . S . Patent Jun . 12 , 2018 Sheet 1 of 72 US 9 , 993 , 566 B2

Homo sapiens seizure related 6 homolog ( SEZ6 ) , transcript variant 1 , MRNA > gi | 148839279 | ref | NM 178860 . 41 ( SEQ ID NO : 1 ) GATCCCCGGCGCCGTCGCCAGGCGCTGGCCGTGGTGCTGATTCTGTCAGGCGCTGGCGGCGGCAGCGGCGGTGACGGCTGCGG CCCCGCTCCCTCTACCCGGCCGGACCCGGCTCTGCCCCCGCGCCCAAGCCCCACCAAGCCCCCCGCCCTCCCGCCGCGGTCCC AGCCCAGGGCGCGGCCGCAACCAGCACCATGCGCCCGGTAGCCCTGCTGCTCCTGCCCTCGCTGCTGGCGCTCCTGGCTCACG GACTCTCTTTAGAGGCCCCAACCGTGGGGAAAGGACAAGCCCCAGGCATCGAGGAGACAGATGGCGAGCTGACAGCAGCCCCC ACACCTGAGCAGCCAGAACGAGGCGTCCACTTTGTCACAACAGCCCCCACCTTGAAGCTGCTCAACCACCACCCGCTGCTTGA GGAATTCCTACAAGAGGGGCTGGAAAAGGGAGATGAGGAGCTGAGGCCAGCACTGCCCTTCCAGCCTGACCCACCTGCACCCT TCACCCCAAGTCCCCTTCCCCGCCTGGCCAACCAGGACAGCCGC?CTGTCTTTACCAGCCCCACTCCAGCCATGGCTGCGGTA CCCACTCAGCCCCAGTCCAAGGAGGGACCCT' GGAGTCCGGAGTCAGAGTCCCCTAT GCTICGAATCACAGCT' CCCCTACCTCC AGGGCCCAGCATGGCAGTGCCCACCCTAGGCCCAGGGGAGATAGCCAGCACTACACCCCCCAGCAGAGCCTGGACACCAACCC AAGAGGGTCCTGGAGACATGGGAAGGCCGTGGGTTGCAGAGGTTGTGTCCCAGGGCGCAGGGATCGGGATCCAGGGGACCATC ACCTCCTCCACAGCTICAGGAGATGATGAGGAG??????????????????????????????????????????GTCCAGAC ACCAGGCCCTTGTAGCTGGAATTTCTCAGGCCCAGAGGGCTCTCTGGACTCCCCTACAGACCTCAGCTCCCCCACTGATGTTG GCCTGGACTGCTTCTTCTACATCTCTGTCTACCCTGGCTATGGCGTGGAAATCAAGGTCCAGAATATCAGCCTCCGGGAAGGG GAGACAGTGACTGTGGAAGGCCTGGGGGGGCCTGACCCACTGCCCCTGGCCAACCAGTCTTTCCTGCTGCGGGGCCAAGTCAT CCGCAGCCCCACCCACCAAGCGGCCCTGAGGTICCAGAGCCT' CCCGCCACCGGCT GGCCCTGGCACCTICCATITCCATTACC AAGCCTATCTCCTGAGCTGCCACTTTCCCCGTCGTCCAGCTTATGGAGATGTGACTGTCACCAGCCTCCACCCAGGGGGTAGT GCCCGCTTCCATTGT' GCCACTGGCTACCAGCTGAAGGGCGCCAGGCATCTCACCTGTCTCAATGCCACCCAGCCCTICTGGGA TTCAAAGGAGCCCGTCTGCATCGCTGCTTGCGGCGGAGTGATCCGCAATGCCACCACCGGCCGCATCGTCTCTCCAGGCTTCC CGGGCAACTACAGCAACAACCTCACCTGTCACTGGCTGCTTGAGGCTCCTGAGGGCCAGCGGCTACACCTGCACTTTGAGAAG GTTTCCCTGGCAGAGGATGATGACAGGCTCATCATTCGCAATGGGGACAACGTGGAGGCCCCACCAGTGTATGATTCCTATGA GGTGGAATACCTGCCCATTGAGGGCCTGCTCAGCTCTGGCAAACACTTCTTTGTTGAGCTCAGTACTGACAGCAGCGGGGCAG CTGCAGGCATGGCCCTGCGCTATGAGGCCTTCCAGCAGGGCCATTGCTATGAGCCCTTTGTCAAATACGGTAACTTCAGCAGC AGCACACCCACCTACCCTGTGGGTACCACTGTGGAGTTCAGCTGCGACCCTGGCTACACCCTGGAGCAGGGCTCCATCATCAT CGAGTGTGTTGACCCCCACGACCCCCAGTGGAATGAGACAGAGCCAGCCTGCCGAGCCGTGTGCAGCGGGGAGATCACAGACT CGGCTGGCGTGGTACTCTCTCCCAACTGGCCAGAGCCCTACGGTCGTGGGCAGGATTGTATCTGGGGTGTGCATGTGGAAGAG GACAAGCGCATCATGCTGGACATCCGAGTGCTGCGCATAGGCCCTGGTGATGTGCTTACCTTCTATGATGGGGATGACCTGAC GGCCCGGGTTCTGGGCCAGTACTCAGGGCCCCGTAGCCACTTCAAGCTCTTTACCTCCATGGCTGATGTCACCATTCAGTTCC AGTCGGACCCCGGGACCTCAGTGCTGGGCTACCAGCAGGGCTTCGTCATCCACTTCTTTGAGGTGCCCCGCAATGACACATGT CCGGAGCTGCCTGAGATCCCCAATGGCTGGAAGAGCCCATCGCAGCCTGAGCTAGTGCACGGCACCGTGGTCACTTACCAGTG CTACCCTGGCTACCAGGTAGTGGGATCCAGTGTCCTCATGTGCCAGTGGGACCTAACTTGGAGTGAGGACCTGCCCTCATGCC AGAGGGTGACTTCCTGCCACGATCCTGGAGATGTGGAGCACAGCCGACGCCTCATATCCAGCCCCAAGTTTCCCGTGGGGGCC ACCGTGCAATATATCTGTGACCAGGGTTTTGTGCTGATGGGCAGCTCCATCCTCACCTGCCATGATCGCCAGGCTGGCAGCCC CAAGTGGAGTGACCGGGCCCCTAAATGTCTCCTGGAACAGCTCAAGCCATGCCATGGTCTCAGTGCCCCTGAGAATGGTGCCC GAAGTCCTGAGAAGCAGCTACACCCAGCAGGGGCCACCATCCACTTCTCGTGTGCCCCTGGCTATGTGCTGAAGGGCCAGGCC AGCATCAAGTGTGTGCCTGGGCACCCCTCGCATTGGAGTGACCCCCCACCCATCTGTAGGGCTGCCTCTCTGGATGGGTT??? CAACAGTCGCAGCCTGGATGTTGCCAAGGCACCTGCTGCCTCCAGCACCCTGGATGCTGCCCACATTGCAGCTGCCATCTTCT TGCCACTGGTGGCGATGGTGTTGTTIGGTAGGAGGTGTATACT' TCTACTICTCCAGGCTCCAGGGAAAAAGCTCCCTGCAGCTG CCCCGCCCCCGCCCCCGCCCCTACAACCGCATTACCATAGAGTCAGCGTTTGACAATCCAACTTACGAGACTGGATCTCTTTC CTTTGCAGGAGACGAGAGAATATGAAGTCTCCATCTAGGTGGGGGCAGTCTAGGGAAGTCAACTCAGACTTGCACCACAGTCC AGCAGCAAGGCTCCTIGCTICCTGCTGTCCCTICCACCTCCT'GTATATACCACCTAGGAGGAGATGCCACCAAGCCCT CAAGAA GTTGT GCCCTICCCCGCCTGCGATGCCCACCATGGCCTATTITCTIGGTGTCATEGCCCACTIGGGGCCCTI??TIGGGCCCA TGTCAGGGGGCATCTACCTGTGGGAAGAACATAGCTGGAGCACAAGCATCAACAGCCAGCATCCTGAGCCTCCTCATGCCCTG GACCAGCCTGGAACACACTAGCAGAGCAGGAGTACCTTTCTCCACATGACCACCATCCCGCCCTGGCATGGCAACCTGCAGCA GGATTAACTTGACCATGGTGGGAACTGCACCAGGGTACTCCTCACAGCGCATCACCAATGGCCAAAACTCCTCTCAACGGTGA CCTCTGGGTAGTCCTGGCATGCCAACATCAGCCTCTTGGGAGGTCTCTAGTTCTCTAAAGTTCTGGACAGTTCTGCCTCCTGC CCTGTCCCAGTGGAGGCAGTAATTCTAGGAGATCCTAAGGGGTTCAGGGGGACCCTACCCCCACCTCAGGTTGGGCTTCCCTG GGCACTCATGCTCCACACCAAAGCAGGACACGCCATTTTCCACTGACCACCC?A?ACCCTGAGGAAAGGGAGACTTTCCTCCG AT' G 'ITTATTTAGCTGTTGCAAACATCITCACCCTAATAGTCCCTCCTCCAATTCCAGCCACTGICAGGCI??ICCTCITGACC ACTGTGTTATGGGATAAGGGGAGGGGGTGGGCATATTCTGGAGAGGAGCAGAGGTCCAAGGACCCAGGAATTTGGCATGGAAC AGGTGGTAGGAGAGCCCCAGGGAGACGCCCAGGAGCTGGCTGAAAGCCACTTTGTACATGTAATGTATTATATGGGGTCTGGG CTCCAGCCAGAGAACAATCTTTTATTTCTGTTGTTTCCTTATTAAAATGGTGTTTTTGGAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA FIG . 1A U . S . Patent Jun . 12, 2018 Sheet 2 of 72 US 9 , 993 , 566 B2

Homo sapiens seizure related 6 homolog ( SEZ6 ) , transcript variant 2 , MRNA > gi | 148839345 | ref | NM _ 001098635 . 11 ( SEQ ID NO : 2 ) GATCCCCGGCGCCGTCGCCAGGCGCTGGCCGTGGTGCTGATTCTGTCAGGCGCTGGCGGCGGCAGCGGCGGTGACGGCTGCGG CCCCGCTCCCTCTACCCGGCCGGACCCGGCTCTGCCCCCGCGCCCAAGCCCCACCAAGCCCCCCGCCCTCCCGCCGCGGTCCC AGCCCAGGGCGCGGCCGCAACCAGCACCATGCGCCCGGTAGCCCTGCTGCTCCTGCCCTCGCTGCTGGCGCTCCTGGCTCACG GACTCTCTTTAGAGGCCCCAACCGTGGGGAAAGGACAAGCCCCAGGCATCGAGGAGACAGATGGCGAGCTGACAGCAGCCCCC ACACCTGAGCAGCCAGAACGAGGCGTCCACTIT' GTCACAACAGCCCCCACCTIGAAGCT' GCTCAACCACCACCCGCT' GCTIGA GGAATTCCTACAAGAGGGGCTGGAAAAGGGAGATGAGGAGCTGAGGCCAGCACTGCCCTTCCAGCCTGACCCACCTGCACCCT TCACCCCAAGTCCCCTTCCCCGCCTGGCCAACCAGGACAGCCGCCCTGTCTTTACCAGCCCCACTCCAGCCATGGCTGCGGTA CCCACTCAGCCCCAGTCCAAGGAGGGACCCTGGAGTCCGGAGTCAGAGTCCCCTATGCTTCGAATCACAGCTCCCCTACCTCC AGGGCCCAGCATGGCAGTGCCCACCCTAGGCCCAGGGGAGATAGCCAGCACTACACCCCCCAGCAGAGCCTGGACACCAACCC AAGAGGGTCCTGGAGACATGGGAAGGCCGTGGGTTGCAGAGGTTGTGTCCCAGGGCGCAGGGATCGGGATCCAGGGGACCATC ACCTCCTCCACAGCTICAGGAGATGAT' GAGGAG??????????????????????T'CACCACCACCAT CACCACAGTCCAGAC ACCAGGCCCTTGTAGCTGGAATTTCTCAGGCCCAGAGGGCTCTCTGGACTCCCCTACAGACCTCAGCTCCCCCACTGATGTTG GCCTGGACTGCTTCTTCTACATCTCTGTCTACCCTGGCTATGGCGTGGAAATCAAGGTCCAGAATATCAGCCTCCGGGAAGGG GAGACAGTGACTGTGGAAGGCCTGGGGGGGCCTGACCCACTGCCCCTGGCCAACCAGTCTTTCCTGCTGCGGGGCCAAGTCAT CCGCAGCCCCACCCACCAAGCGGCCCTGAGGTTCCAGAGCCTCCCGCCACCGGCTGGCCCTGGCACCTTCCATTTCCATTACC AAGCCTATCTCCTGAGCTGCCACTTTCCCCGTCGTCCAGCTTATGGAGATGTGACTGTCACCAGCCTCCACCCAGGGGGTAGT GCCCGCTICCATTGTGCCACT' GGCTACCAGCT' GAAGGGCGCCAGGCATCTCACCT' GTCTCAATGCCACCCAGCCCTT ' CTGGGA TTCAAAGGAGCCCGTCTGCATCGCT' GCTTGCGGCGGAGT GATCCGCAATGCCACCACCGGCCGCATCGTCTCTCCAGGCTTCC CGGGCAACTACAGCAACAACCTCACCTGTCACTGGCTGCTTGAGGCTCCTGAGGGCCAGCGGCTACACCTGCACTTTGAGAAG GTTTCCCTGGCAGAGGATGATGACAGGCTCATCATTCGCAATGGGGACAACGTGGAGGCCCCACCAGTGTATGATTCCTATGA GGTGGAATACCTGCCCATTGAGGGCCTGCTCAGCTCTGGCAAACACTTCTTTGTTGAGCTCAGTACTGACAGCAGCGGGGCAG CTGCAGGCATGGCCCTGCGCTATGAGGCCTTCCAGCAGGGCCATTGCTATGAGCCCTTTGTCAAATACGGTAACTTCAGCAGC AGCACACCCACCTAC?CTGTGGGTACCACTGTGGAGTICAGCTGCGACCCTGGCTACACCCTGGAGCAGGGCTCCATCATCAT CGAGTGTGTTGACCCCCACGACCCCCAGTGGAATGAGACAGAGCCAGCCTGCCGAGCCGTGTGCAGCGGGGAGATCACAGACT CGGCTGGCGTGGTACTCTCTCCCAACTGGCCAGAGCCCTACGGTCGTGGGCAGGATTGTATCTGGGGTGTGCATGTGGAAGAG GACAAGCGCATCATGCTGGACATCCGAGTGCTGCGCATAGGCCCTGGTGATGTGCTTACCTTCTATGATGGGGATGACCTGAC GGCCCGGGTTCTGGGCCAGTACTCAGGGCCCCGTAGCCACTTCAAGCTCTTTACCTCCATGGCTGATGTCACCATTCAGTTCC AGTCGGACCCCGGGACCTCAGTGCTGGGCTACCAGCAGGGCTTCGTCATCCACTTCTTTGAGGTGCCCCGCAATGACACATGT CCGGAGCTGCCTGAGATCCCCAATGGCTGGAAGAGCCCATCGCAGCCTGAGCTAGTGCACGGCACCGTGGTCACTTACCAGTG C?ACCCT'GGCTACCAGGTAGTGGGATCCAGTGTCCTCATGTGCCAGTGGGACCTAACTIGGAGTGAGGACCTGCCCTCATGCC AGAGGGTGACTTCCTGCCACGATCCTGGAGATGTGGAGCACAGCCGACGCCTCATATCCAGCCCCAAGTTTCCCGTGGGGGCC ACCGTGCAATATATCTGTGACCAGGGTTTTGTGCTGATGGGCAGCTCCATCCTCACCTGCCATGATCGCCAGGCTGGCAGCCC CAAGTGGAGTGACCGGGCCCCTAAATGTCTCCTGGAACAGCTCAAGCCATGCCATGGTCTCAGTGCCCCTGAGAATGGTGCCC GAAGTCCTGAGAAGCAGCTACACCCAGCAGGGGCCACCATCCACTTCTCGTGTGCCCCTGGCTATGTGCTGAAGGGCCAGGCC AGCATCAAGIGTGTGCCTGGGCACCCCTCGCATTGGAGTGACCCCCCACCCATCTGTAGGGCTGCCTCTCTGGATGGGT???? CAACAGTCGCAGCCTGGATGTTGCCAAGGCACCTGCTGCCTCCAGCACCCTGGATGCTGCCCACATTGCAGCTGCCATCTTCT TGCCACTGGTGGCGATGGTGTTGTTGGTAGGAGGTGTATACTTCTACTTCTCCAGGCTCCAGGGAAAAAGCTCCCTGCAGCTG ????????????????GCC?CTACAACCGCATTACCAT?G?GTCAGCGTTTGACAATCCAACTTACGAGACTGGAGAGACGAG AGAATATGAAGTCTCCATCTAGGTGGGGGCAGTCTAGGGAAGTCAACTCAGACTTGCACCACAGTCCAGCAGCAAGGCTCCTT GCTTCCTGCTGTCCCTCCACCTCCT' GTATATACCACCTAGGAGGAGATGCCACCAAGCCCTCAAGAAGT' TGTGCCCTTCCCCG CCTGCGATGCCCACCATGGCCTATTITUTTGGTGTCATIGC?CACTIGGGGcccTTCATIGGGCCCATGTCAGGGGGCAT'c?? CCTGTGGGAAGAACATAGCTGGAGCACAAGCATCAACAGCCAGCATCCTGAGCCTCCTCATGCCCTGGACCAGCCTGGAACAC ACT' AGCAGAGCAGGAGTACCTITCTCCACATGACCACCATCCCGCCCIGGCAT' GGCAACCT' GCAGCAGGATTAACTTGACCAT GGTGGGAACTGCACCAGGGTACTCCTCACAGCGCCATCACCAATGGCCAAAACTCCTCTCAACGGTGACCTCTGGGTAGTCCT GGCATGCCAACATCAGCCTCTTGGGAGGTCTCTAGTTCTCTAAAGTTCTGGACAGTTCTGCCTCCTGCCCTGTCCCAGTGGAG GCAGTAATTCTAGGAGATCCTAAGGGGTTCAGGGGGACCCTACCCCCACCTCAGGTTGGGCTTCCCTGGGCACTCATGCTCCA CACCAAAGCAGGACACGCCATTTTCCACTGACCACCC?A?ACCCTGAGGAAAGGGAGACTTTCCTCCGATGITTATTTAGCTG TTGCAAACATCTICACCCTAATAGTCCCTccTCCAATTCCAGCCACTTGTCAGGCTCTCCTCTTGACCACTGTGTTATGGGAT AAGGGGAGGGGGTGGGCATATTCTGGAGAGGAGCAGAGGTCCAAGGACCCAGGAATTTGGCATGGAACAGGTGGTAGGAGAGC CCCAGGGAGACGCCCAGGAGCTGGCTGAAAGCCACTTTGTACATGTAATGTATTATATGGGGTCTGGGCTCCAGCCAGAGAAC AATCTTTTATTTCTGTTGTTTCCTTATTAAAATGGTGTTTTTGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA FIG . 1B U . S . Patent Jun . 12 , 2018 Sheet 3 of 72 US 9, 993, 566 B2

(SEQIDNO:3)

FIG.1C Homosapiensseizureprotein6homologisoform1precursor >gi|148839280refNP_849191.3 MRPVALLLLPSLLALLAHGLSLEAPTVGKGQAPGIEETDGELTAAPTPEQPERGVHFVTTAPTLKLLNHH PLLEEFLQEGLEKGDEELRPALPFQPDPPAPFTPSPLPRLANQDSRPVFTSPTPAMAAVPTQPQSKEGP WSPESESPMLRITAPLPPGPSMAVPTLGPGEIASTTPPSRAWTPTQEGPGDMGRPWVAEVVSQGAGI GIQGTITSSTASGDDEETTTTTTIITTTITTVQTPGPCSWNFSGPEGSLDSPTDLSSPTDVGLDCFFYISVYP GYGVEIKVQNISLREGETVTVEGLGGPDPLPLANQSFLLRGQVIRSPTHQAALRFQSLPPPAGPGTEHEH YQAYLLSCHEPRRPAYGDVTVTSLHPGGSARFHCATGYQLKGARHLTCLNATQPFWDSKEPVCIAACGG VIRNATTGRIVSPGFPGNYSNNLTCHWLLEAPEGQRLHLHFEKVSLAEDDDRLIIRNGDNVEAPPVYDSY EVEYLPIEGLLSSGKHFFVELSTDSSGAAAGMALRYEAFQQGHCYEPFVKYGNFSSSTPTYPVGTTVEFS CDPGYTLEQGSITIECVDPHDPQWNETEPACRAVCSGEITDSAGVVLSPNWPEPYGRGQDCIWGVHVE EDKRIMLDIRVLRIGPGDVLTFYDGDDLTARVLGQYSGPRSHEKLFTSMADVTIQFQSDPGTSVLGYQQ GFVIHEFEVPRNDTCPELPEIPNGWKSPSQPELVHGTVTYQCYPGYQWVGSSVLMCQWDLTWSEDL PSCQRVTSCHDPGDVEHSRRLISSPKFPVGATVQYICDQGFVLMGSSILTCHDRQAGSPKWSDRAPKCL LEQLKPCHGLSAPENGARSPEKQLHPAGATIHFSCAPGYVLKGQASIKCVPGHPSHWSDPPPICRAASL DGFYNSRSLDVAKAPAASSTLDAAHIAAAIFLPLVAMVLLVGGVYFYFSRLQGKSSLQLPRPRPRPYNRITI ESAFDNPTYETGSLSFAGDERI U . S . Patent Jun . 12, 2018 Sheet 4 of 72 US 9 ,993 , 566 B2

(SEQIDNO:4)

FIG.1D Homosapiensseizureprotein6homologisoform2precursor

>gi148839346|refNP_001092105.11 MRPVALLLLPSLLALLAHGLSLEAPTVGKGQAPGIEETDGELTAAPTPEQPERGVHFVTTAPT LKLLNHHPLLEEFLQEGLEKGDEELRPALPFQPDPPAPFTPSPLPRLANQDSRPVFTSPTPAM AAVPTQPQSKEGPWSPESESPMLRITAPLPPGPSMAVPTLGPGEIASTTPPSRAWTPTQEG PGDMGRPWVAEVVSQGAGIGIQGTITSSTASGDDEETTTTTTIITTTITTVQTPGPCSWNFS GPEGSLDSPTDLSSPTDVGLDCFFYISVYPGYGVEIKVONISLREGETVTVEGLGGPDPLPLA NQSFLLRGQVIRSPTHQAALRFQSLPPPAGPGTEHEHYQAYLLSCHEPRRPAYGDVTVTSLH PGGSARFHCATGYQLKGARHLTCLNATOPFWDSKEPVCIAACGGVIRNATTGRIVSPGFPG NYSNNLTCHWLLEAPEGQRLHLHFEKVSLAEDDDRLIIRNGDNVEAPPVYDSYEVEYLPIEG LLSSGKHFFVELSTDSSGAAAGMALRYEAFQQGHCYEPFVKYGNFSSSTPTYPVGTTVEFSC DPGYTLEQGSINTECVDPHDPQWNETEPACRAVCSGEITDSAGWVLSPNWPEPYGRGQDCI WGVHVEEDKRIMLDIRVLRIGPGDVLTFYDGDDLTARVLGQYSGPRSHFKLFTSMADVTIQ FQSDPGTSVLGYQQGFVIHEFEVPRNDTCPELPEIPNGWKSPSQPELVHGTVTYQCYPGY QVVGSSVLMCQWDLTWSEDLPSCQRVTSCHDPGDVEHSRRLISSPKFPVGATVQYICDQG FVLMGSSILTCHDRQAGSPKWSDRAPKCLLEQLKPCHGLSAPENGARSPEKQLHPAGATIH FSCAPGYVLKGQASIKCVPGHPSHWSDPPPICRAASLDGFYNSRSLDVAKAPAASSTLDAAHJAAAIFLPLVAMVLLVGGVYFYFSRLQGKSSLQLPRPRPRPYNRITIESAFDNPTYETGETREYE VSI U . S . Patent Jun . 12, 2018 Sheet 5 of 72 US 9 ,993 , 566 B2

80 160 240 320 400 480 560 640 720 800 880 960

994 (961)RPRPRPYNRITIESAFDNPTYETGSLSFAGDERISEQIDNO:3 (961)RPRPRPYNRITIESAFDNPTYETGETREYEVSI-SEQIDNO:4 AlignmentofSEZ6Isoforms (161)MAVPTLGPGEIASTTPPSRAWTPTQEGPGDMGRPWVAEVVSQGAGIGIQGTITSSTASGDDEETTTTTTIITTTITTVQT (241)PGPCSWNFSGPEGSLDSPTDLSSPTDVGLDCFFYISVYPGYGVEIKVONISLREGETVTVEGLGGPDPLPLANOSFLLRG FIG.1E (1)MRPVALLLLPSLLALLAHGLSLEAPTVGKGQAPGIEETDGELTAAPTPEQPERGVHFVTTAPTLKLLNHHPLLEEFLQEG (1)MRPVALLLLPSLLALLAHGLSLEAPTVGKGQAPGIEETDGELTAAPTPEQPERGVHFVTTAPTLKLLNHHPLLEEFLQEG 81 1)LEKGDEELRPALPFOPDPPAPFTPSPLPRLANODSRPVFTSPTPAMAAVPTOPOSKEGPWSPESESPMLRITAPLPPGPS (81)LEKGDEELRPALPFQPDPPAPFTPSPLPRLANQDSRPVETSPTPAMAAVPTQPQSKEGPWSPESESPMLRITAPLPPGPS 161 (161)MAVPTLGPGEIASTTPPSRAWTPTOEGPGDMGRPWVAEVVSQGAGIGIOGTITSSTASGDDEETTTTTTIITTTITTVOT 241 (241)PGPCSWNFSGPEGSLDSPTDLSSPTDVGLDCFFYISVYPGYGVEIKVONISLREGETVTVEGLGGPDPLPLANQSFLLRG321 (321)QVIRSPTHQAALRFQSLPPPAGPGTFHFHYQAYLLSCHFPRRPAYGDVTVTSLHPGGSARFHCATGYQLKGARHLTCLNA (321)OVIRSPTHQAALRFOSLPPPAGPGTFHFHYQAYLLSCHFPRRPAYGDVTVTSLHPGGSARFHCATGYOLKGARHLTCLNA 401 (401)TOPEWDSKEPVCIAACGGVIRNATTGRIVSPGFPGNYSNNLTCHWLLEAPEGORLHLHFEKVSLAEDDDRLIIRNGDNVE (401)TOPFWDSKEPVCIAACGGVIRNATTGRIVSPGFPGNYSNNLTCHWLLEAPEGQRLHLHFEKVSLAEDDDRLIIRNGDNVE 481 (481)APPVYDSYEVEYLPIEGLLSSGKHFFVELSTDSSGAAAGMALRYEAFQQGHCYEPFVKYGNFSSSTPTYPVGTTVEFSCD (481)APPVYDSYEVEYLPIEGLLSSGKHFFVELSTDSSGAAAGMALRYEAFOOGHCYEPFVKYGNFSSSTPTYPVGTTVEFSCD 561 (561)PGYTLEOGSIIIECVDPHDPOWNETEPACRAVCSGEITDSAGVVLSPNWPEPYGRGODCIWGVHVEEDKRIMLDIRVLRI (561)PGYTLEQGSIIIECVDPHDPQWNETEPACRAVCSGEITDSAGVVLSPNWPEPYGRGQDCIWGVHVEEDKRIMLDIRVLRI 641 (641)GPGDVLTFYDGDDLTARVLGQYSGPRSHFKLFTSMADVTIQFQSDPGTSVLGYQQGEVIHFFEVPRNDTCPELPEIPNGW (641)GPGDVLTFYDGDDLTARVLGQYSGPRSHFKLFTSMADVTIQFOSDPGTSVLGYQOGFVIHFFEVPRNDTCPELPEIPNGW 721 (721)KSPSOPELVHGTVVTYQCYPGYQVVGSSVLMCOWDLTWSEDLPSCORVTSCHDPGDVEHSRRLISSPKFPVGATVQYICD (721)KSPSQPELVHGTVVTYQCYPGYQVVGSSVLMCQWDLTWSEDLPSCQRVTSCHDPGDVEHSRRLISSPKFPVGATVQYICD 801 (801)QGFVLMGSSILTCHDRQAGSPKWSDRAPKCLLEQLKPCHGLSAPENGARSPEKQLHPAGATIHFSCAPGYVLKGQASIKC (801)QGFVLMGSSILTCHDRQAGSPKWSDRAPKCLLEOLKPCHGLSAPENGARSPEKQLHPAGATIHESCAPGYVLKGQASIKC 881 (881)VPGHPSHWSDPPPICRAASLDGFYNSRSLDVAKAPAASSTLDAAHIAAAIFLPLVAMVLLVGGVYFYFSRLQGKSSLQLP (881)VPGHPSHWSDPPPICRAASLDGFYNSRSLDVAKAPAASSTLDAAHIAAAIFLPLVAMVLLVGGVYFYFSRLQGKSSLQLP 961

hSEZ6v1(NP_849191) hSEZ6v1(NP849191) hSEZ6v1(NP849191) hSEZ6v1(NP849191) hSEZ6v1(NP849191) hSEZ6vi(NP849191) hSEZ6v1(NP_849191) hSEZ6v1(NP849191) hSEZ6v1(NP849191) hSEZ6v1(NP849191) hSEZ6v1(NP_849191) hSEZ6v1(NP849191) hSEZ6v1(NP849191) hSE26v2(NP_001092105) hSE26v2(NP001092105) hSEZ6v2(NP001092105) hSEZ6v2(NP001092105) hSEZ6v2(NP001092105) hSEZ6v2(NP_001092105) hSEZ6v2(NP001092105 hSE26V2(NP001092105) hSEZ6v2(NP001092105) hSEZ6v2(NP001092105) hSEZ6v2(NP001092105) hSEZ6v2(NP_001092105) hSEZ6V2(NP001092105) U . S . Patent Jun . 12 , 2018 Sheet 6 of 72 US 9, 993 , 566 B2

cell878|-8131817748175068516905721574-5081511397395169336|-771 SchematicRepresentationofSEZ6ProteinExtracellularDomain Pro-Rich|Sushi2CUB345Sushi1 FIG.1F

N-terminal domain U . S . Patent Jun . 12 , 2018 Sheet 7 of 72 US 9 , 993 , 566 B2

tel 90.4% 96.7%

192190an)660TDOAN( esnoushojoulousaisey 90.5% PercentIdentityBetweenFullLength MatureSEZ6ProteinsfromvariousSpecies walau)( 97.5% 93.0% FIG.2A

91.7% EOSOTELOOAX)

1???? -Cu2uunH (1616D8AN) Susau AN)197190( SEZ6 ISOLZGOLOGAN) TEOSOTILOOax U . S . Patent Jun . 12, 2018 Sheet 8 of 72 US 9, 993 , 566 B2

protein NP849191 NP001092105 NP_066938 NP001171702 NP001171703 NP_001701704 NP001171705 NP_001171706 NP_036542 NP963869 NP_001107571 NP001107572 NP001230261 NP001230262

HomoSapiensSEZ6,SEZÓLandSEZ6L2 SequenceAccessionNumbers-NCBI NM178860 NM001098635 NM021115 NM001184773 NM_001184774 NM_001184775 NM_001184776 NM_001184777 NM012410 NM_201575 NM001114099 NM001114100 NM_001243332 NM001243333 FIG.2B

2 Tam Hammo

SSSSSSSS SEZE SEZGL2 U . S . Patent Jun . 12, 2018 Sheet 9 of 72 US 9 ,993 , 566 B2

SEZOLIUS 42.9% 41.1% SEZGL245 INP001230261) 43.2% 40.5% Completeprotein INP_066938NP001230261) PercentIdentitybetweenVarious ProteinsandSEZ6L2SEZ6LSEZÓ,Human 42.5% SEZOLI INP066938) 42.4% FIG.2C

Homosapiens INP_849191) SEZGLYA (NP066938) Homosapiens SEZONA INP_849191) SEZGLI INP_066938) U . S . Patent Jun . 12 , 2018 Sheet 10 of 72 US 9 , 993 , 566 B2

(SEQIDNO:5)

FIG.3A >CDNASequenceofhumanSEZ6ORF CTGAGCCTGGAGGCCCCAACCGTGGGGAAAGGACAAGCCCCAGGCATCGAGGAGACAGATGGCGAGCTGACAGCAGCCCCCACACCTGAGCAGCCAGAAC GAGGCGTCCACTTTGTCACAACAGCCCCCACCTTGAAGCTGCTCAACCACCACCCGCTGCTTGAGGAATTCCTACAAGAGGGGCTGGAAAAGGGAGATGAGG AGTTGAGGCCAGCACTGCCCTTCCAGCCTGACCCACCTGCACCCTTCACCCCAAGTCCCCTTCCCCGCCTGGCCAACCAGGACAGCCGCCCTGTCTTTACCAGO CCCACTCCAGCCATGGCTGCGGTACCCACTCAGCCCCAGTCCAAGGAGGGACCCTGGAGTCCGGAGTCAGAGTCCCCTATGCTTCGAATCACAGCTCCCCTAC CTCCAGGGCCCAGCATGGCAGTGCCCACCCTAGGCCCAGGGGAGATAGCCAGCACTACACCCCCCAGCAGAGCCTGGACACCAACCCAAGAGGGTCCTGGA GACATGGGAAGGCCGTGGGTTGCAGAGGTTGTGTCCCAGGGCGCGGGGATCGGGATCCAGGGGACCATCACCTCCTCCACAGCTTCAGGAGATGATGAGG AGACCACCACTACCACCACCATCATCACCACCACCATCACCACAGTCCAGACACCAGGCCCTTGTAGCTGGAATTTCTCAGGCCCAGAGGGCTCTCTGGACTCC CCTACAGACCTCAGCTCCCCCACTGATGTTGGCCTGGACTGCTTCTTCTACATCTCTGTCTACCCTGGCTATGGCGTGGAAATCAAGGTCCAGAATATCAGCCT CCGGGAAGGGGAGACAGTGACTGTGGAAGGCCTGGGGGGGCCCGACCCACTGCCCCTGGCCAACCAGTCTTTCCTGCTGCGGGGCCAAGTCATCCGCAGCC CCACCCACCAAGCGGCCCTGAGGTTCCAGAGCCTC?CGCCACCGGCTGGCCCTGGCACCTICCATTTCCATTACCAAGCCTATCTCCTGAGCTGCCACTITcccc GTCGTCCAGCTTATGGAGATGTGACTGTCACCAGCCTCCACCCAGGGGGTAGTGCCCGCTTCCATTGTGCCACTGGCTACCAGCTGAAGGGCGCCAGGCATC TCACCTGTCTCAATGCCACCCAGCCCTTCTGGGATTCAAAGGAGCCCGTCTGCATCGCTGCTTGCGGCGGAGTGATCCGCAATGCCACCACCGGCCGCATCGT CTCTCCAGGCTTCCCGGGCAACTACAGCAACAACCTCACCTGTCACTGGCTGCTTGAGGCTCCTGAGGGCCAGCGGCTACACCTGCACTTTGAGAAGGTTTCC CTGGCAGAGGATGATGACAGGCTCATCATTCGCAATGGGGACAACGTGGAGGCCCCACCAGTGTATGATTCCTATGAGGTGGAATACCTGCCCATTGAGGG CCTGCTCAGCTCTGGCAAACACTTCTTTGTTGAGCTCAGTACTGACAGCAGCGGGGCAGCTGCAGGCATGGCCCTGCGCTATGAGGCCTTCCAGCAGGGCCA TTGCTATGAGCCCTTTGTCAAATACGGTAACTTCAGCAGCAGCACACCCACCTACCCTGTGGGTACCACTGTGGAGTTCAGCTGCGACCCTGGCTACACCCTG GAGCAGGGCTCCATCATCATCGAGTGTGTTGACCCCCACGACCCCCAGTGGAATGAGACAGAGCCAGCCTGCCGAGCCGTGTGCAGCGGGGAGATCACAGA CTCGGCTGGCGTGGTACTCTCTCCCAACTGGCCAGAGCCCTACGGTCGTGGGCAGGATTGTATCTGGGGTGTGCATGTGGAAGAGGACAAGCGCATCATGCT GGACATCCGAGTGCTGCGCATAGGCCCTGGTGATGTGCTTACCTTCTATGATGGGGATGACCTGACGGCCCGGGTTCTGGGCCAGTACTCAGGGCCCCGTAG CCACTTCAAGCTCTTTACCTCCATGGCTGATGTCACCATTCAGTTCCAGTCGGACCCCGGGACCTCAGTGCTGGGCTACCAGCAGGGCTTCGTCATCCACTTCT TTGAGGTGCCCCGCAATGACACATGTCCGGAGCTGCCTGAGATCCCCAATGGCTGGAAGAGCCCATCGCAGCCTGAGCTAGTGCACGGCACCGTGGTCACTT ACCAGTGCTACCCTGGCTACCAGGTAGTGGGATCCAGTGTCCTCATGTGCCAGTGGGACCTAACTTGGAGTGAGGACCTGCCCTCATGCCAGAGGGTGACTT CCTGCCACGATCCTGGAGATGTGGAGCACAGCCGACGCCTCATATCCAGCCCCAAGTTTCCCGTGGGGGCCACCGTGCAATATATCTGTGACCAGGGTTTTGT GCTGATGGGCAGCTCCATCCTCACCTGCCATGATCGCCAGGCTGGCAGCCCCAAGTGGAGTGACCGGGCCCCTAAATGTCTCCTGGAACAGCTCAAGCCATG CCATGGTCTCAGTGCCCCTGAGAATGGTGCCCGAAGTCCTGAGAAGCAGCTACACCCAGCAGGGGCCACCATCCACTTCTCGTGTGCCCCTGGCTATGTGCTG AAGGGCCAGGCCAGCATCAAGTGTGTGCCTGGGCACCCCTCGCATTGGAGTGACCCCCCACCCATCTGTAGGGCTGCCTCTCTGGATGGGTTCTACAACAGT CGCAGCCTGGATGTTGCCAAGGCACCTGCTGCCTCCAGCACCCTGGATGCTGCCCACATTGCAGCTGCCATCTTCTTGCCACTGGTGGCGATGGTGTTGTTGG TAGGAGGTGTATACTICTACTICTCCAGGCTCCAGGGAAAAAGCTcccTGCAGCTGCCCCGCCCCCGCCCCCGCCCCTACAACCGCATTACCAT?G?GTCAGC GTTTGACAATCCAACTTACGAGACTGGATCTCTTTCCTTTGCAGGAGACGAGAGAATA U . S . Patent Jun . 12, 2018 Sheet 11 of 72 US 9 , 993 , 566 B2

(SEQIDNO:6)

FIG.3B ORFSEZOhumanofTranslation> LSLEAPTVGKGQAPGIEETDGELTAAPTPEQPERGVHFVTTAPTLKLLNHHPLLEEFLQEGLEKGDE ELRPALPFQPDPPAPFTPSPLPRLANQDSRPVFTSPTPAMAAVPTQPQSKEGPWSPESESPMLRIT APLPPGPSMAVPTLGPGEIASTTPPSRAWTPTQEGPGDMGRPWVAEVVSQGAGIGIQGTITSSTASGDDEETTTTTTIITTTITTVQTPGPCSWNFSGPEGSLDSPTDLSSPTDVGLDCFFYISVYPGYGVE IKVQNISLREGETVTVEGLGGPDPLPLANQSFLLRGQVIRSPTHQAALRFQSLPPPAGPGTEHEHY QAYLLSCHEPRRPAYGDVTVTSLHPGGSARFHCATGYQLKGARHLTCLNATQPFWDSKEPVCIAA CGGVIRNATTGRIVSPGFPGNYSNNLTCHWLLEAPEGQRLHLHFEKVSLAEDDDRLIIRNGDNVE APPVYDSYEVEYLPIEGLLSSGKHFFVELSTDSSGAAAGMALRYEAFQQGHCYEPFVKYGNESSST PTYPVGTTVEFSCDPGYTLEQGSITIECVDPHDPQWNETEPACRAVCSGEITDSAGVVLSPNWPEP YGRGQDCIWGVHVEEDKRIMLDIRVLRIGPGDVLTFYDGDDLTARVLGQYSGPRSHEKLFTSMAD VTIQFQSDPGTSVLGYQQGFVIHFFEVPRNDTCPELPEIPNGWKSPSQPELVHGTVTYQCYPGY QWVGSSVLMCQWDLTWSEDLPSCQRVTSCHDPGDVEHSRRLISSPKEPVGATVQYICDQGFVL MGSSILTCHDRQAGSPKWSDRAPKCLLEQLKPCHGLSAPENGARSPEKQLHPAGATIHFSCAPGY VLKGQASIKCVPGHPSHWSDPPPICRAASLDGFYNSRSLDVAKAPAASSTLDAAHIAAAIFLPLVA MVLLVGGVYFYFSRLQGKSSLQLPRPRPRPYNRITIESAFDNPTYETGSLSFAGDERI U . S . Patent Jun . 12 , 2018 Sheet 12 of 72 US 9 ,993 ,566 B2

80 160 240 320 400 480 560 640 720 800 880 960

(SEQIDNO:7) (SEQIDNO:3)

AlignmentofSEZ6Variants 994 FIG.3C

hSE26BC146292(81)LEKGDEELRPALPFOPDPPAPFTPSPLPRLANQDSRPVFTSPTPAMAAVPTOPQSKEGPWSPESESPMLRITAPLPPGPS 161 481 721 hSE26BC146292(1)MRPVALLLLPSLLALLAHGLSLEAPTVGKGOAPGIEETDGELTAAPTPEOPERGVHFVTTAPTLKLLNHHPLLEEFLOEG hSEZ6NP849191(1)MRPVALLLLPSLLALLAHGLSLEAPTVGKGOAPGIEETDGELTAAPTPEOPERGVHFVTTAPTLKLLNHHPLLEEFLOEG 81 241 321 401 561 641 801 881 961 hSE26NP849191(81)LEKGDEELRPALPFOPDPPAPFTPSPLPRLANQDSRPVFTSPTPAMAAVPTOPQSKEGPWSPESESPMLRITAPLPPGPS hSEZ6BC146292(161)MAVPTLGPGEIASTTPPSRANTPTQEGPGDMGRPWVAEVVSOGAGIGINGTITSSTASGDDEETTTTTTIITTTITTVQT hSEZ6NP_849191(161)MAVPTLGPGEIASTTPPSRANTPTQEGPGDMGRPWVAEVVSQGAGIGIQGTITSSTASGDDEETTTTTTIITTTITTVOT hSE26BC146292(241)PGPCSWNFSGPEGSLDSPTDLSSPTDVGLDCFFYISVYPGYGVEIKVONISLREGETVTVEGLGGPDPLPLANQSFLLRG hSEZ6NP849191(241)PGPCSWNFSGPEGSLDSPTDLSSPTDVGLDCFFYISVYPGYGVEIKVONISLREGETVTVEGLGGPDPLPLANQSFLLRG hSEZ6BC146292(321)QVIRSPTHQAALRFQSLPPPAGPGTFHFHYQAYLLSCHFPRRPAYGDVTVTSLHPGGSARFHCATGYQLKGARHLTCLNA hSEZ6NP849191(321)OVIRSPTHOAALRFOSLPPPAGPGTFHFHYOAYLLSCHFPRRPAYGDVTVTSLHPGGSARFHCATGYOLKGARHLTCLNA hSEZ6BC146292(401)TOPEWDSKEPVCIGECPGVIRNATTGRIVSPGFPGNYSNNLTCHWLLEAPEGQRLHLHFEKVSLAEDDDRLIIRNGDNVE hSEZ6NP849191(401)TOPFWDSKEPVCIAACGGVIRNATTGRIVSPGFPGNYSNNLTCHWLLEAPEGORLHLHFEKVSLAEDDDRLIIRNGDNVE hSEZ6BC146292(481)APPVYDSYEVEYLPIEGLLSSGKHFFVELSTDSSGAAAGMALRYEAFOOGHCYEPFVKYGNFSSSTPTYPVGTTVEFSCD hSE26NP849191(481)APPVYDSYEVEYLPIEGLLSSGKHFFVELSTDSSGAAAGMALRYEAFQQGHCYEPFVKYGNFSSSTPTYPVGTTVEFSCD (561)PGYTLEQGSIIIECVDPHDPOWNETEPACRAVCSGEITDSAGVVLSPNWPEPYGRGODCIWGVHVEEDKRIMLDIRVLRIhSEZ6BC146292 hSEZ6NP849191(561)PGYTLEQGSIIIECVDPHDPOWNETEPACRAVCSGEITDSAGVVLSPNWPEPYGRGODCIWGVHVEEDKRIMLDIRVLRI hSE26BC146292(641)GPGDVLTFYDGDDLTARVLGQYSGPRSHEKLETSMADVTIQFQSDPGTSVLGYQQGEVIHFFEVPRNDTCPELPEIPNGW hSEZ6NP849191(641)GPGDVLTFYDGDDLTARVLGQYSGPRSHEKLFTSMADVTIQFQSDPGTSVLGYQQGFVIHFFEVPRNDTCPELPEIPNGW hSE26BC146292(721)KSPSQPELVHGTVVTYQCYPGYQVVGSSVLMCOWDLTWSEDLPSCORVTSCHDPGDVEHSRRLISSPKFPVGATVQYICD hSEZ6NP849191(721)KSPSQPELVHGTVVTYQCYPGYQVVGSSVLMCQWDLTWSEDLPSCQRVTSCHDPGDVEHSRRLISSPKFPVGATVQYICD hSE26BC146292(801)OGFVLMGSSILTCHDRQAGSPKWSDRAPKCLLEQLKPCHGLSAPENGARSPEKOLHPAGATIHFSCAPGYVLKGQASIKC HSEZ6NP849191(801)QGFVLMGSSILTCHDRQAGSPKWSDRAPKCLLEQLKPCHGLSAPENGARSPEKOLHPAGATIHFSCAPGYVLKGQASIKC hSE26BC146292(881)VPGHPSHWSDPPPICRAASLDGFYNSRSLDVAKAPAASSTLDAAHIAAAIFLPLVAMVLLVGGVYFYFSRLQGKSSLQLP hSEZ6NP849191(881)VPGHPSHWSDPPPICRAASLDGFYNSRSLDVAKAPAASSTLDAAHIAAAIFLPLVAMVLLVGGVYFYFSRLQGKSSLQLP hSE26BC146292(961)RPRPRPYNRITIESAFDNPTYETGSLSFAGDERIhSEZ6NP849191(961RPRPRPYNRITIESAFDNPTYETGSLSFAGDERI) U . S . Patent Jun . 12 , 2018 Sheet 13 of 72 US 9 ,993 , 566 B2

(SEQIDNO:8)

FIG.4A >humanSEZ6-FcORF ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCCGGGTCCACTGGTGACGGCGCGCCTGGATCCCTGAGCCTGGAGGCCCCAACCGTGGGGAAAGG ACAAGCCCCAGGCATCGAGGAGACAGATGGCGAGCTGACAGCAGCCCCCACACCTGAGCAGCCAGAACGAGGCGTCCACTTTGTCACAACAGCCCCCACCTTGAAGCTGCT CAACCACCACCCGCTGCTTGAGGAATTCCTACAAGAGGGGCTGGAAAAGGGAGATGAGGAGTTGAGGCCAGCACTGCCCTTCCAGCCTGACCCACCTGCACCCTTCACCCC AAGTCCCCTCCCCGCCTGGCCAACCAGGACAGCCGCCCTGTCTTTACCAGCCCCACTCCAGCCATGGCTGCGGTACCCACTCAGCCCCAGTCCAAGGAGGGACCCTGGAGT CCGGAGTCAGAGTCCCCTATGCTTCGAATCACAGCTCCCCTACCTCCAGGGCCCAGCATGGCAGTGCCCACCCTAGGCCCAGGGGAGATAGCCAGCACTACACCCCCCAGCA GAGCCTGGACACCAACCCAAGAGGGTCCTGGAGACATGGGAAGGCCGTGGGTTGCAGAGGTTGTGTCCCAGGGCGCGGGGATCGGGATCCAGGGGACCATCACCTCCTCC ACAGCTTCAGGAGATGATGAGGAGACCACCACTACCACCACCATCATCACCACCACCATCACCACAGTCCAGACACCAGGCCCTTGTAGCTGGAATTTCTCAGGCCCAGAGG GCTCTCTGGACTCCCCTACAGACCTCAGCTCCCCCACTGATGTTGGCCTGGACTGC?????????????cTGTCTAC?CTGGCTATGGCGTGGAAATCAAGGTCCAGAATATCA GCCTCCGGGAAGGGGAGACAGTGACTGTGGAAGGCCTGGGGGGGCCCGACCCACTGCCCCTGGCCAACCAGTCTTTCCTGCTGCGGGGCCAAGTCATCCGCAGCCCCACC CACCAAGCGGCCCTGAGGTTCCAGAGCCTCCCGCCACCGGCTGGCCCTGGCACCTTCCATTTCCATTACCAAGCCTATCTCCTGAGCTGCCACTTTCCCCGTCGTCCAGCTTAT GGAGATGTGACTGTCACCAGCCTCCACCCAGGGGGTAGTGCCCGCTTCCATTGTGCCACTGGCTACCAGCTGAAGGGCGCCAGGCATCTCACCTGTCTCAATGCCACCCAGC CCTTCTGGGATTCAAAGGAGCCCGTCTGCATCGCTGCTTGCGGCGGAGTGATCCGCAATGCCACCACCGGCCGCATCGTCTCTCCAGGCTTCCCGGGCAACTACAGCAACAA CCTCACCTGTCACTGGCTGCTTGAGGCTCCTGAGGGCCAGCGGCTACACCTGCACTTTGAGAAGGTTTCCCTGGCAGAGGATGATGACAGGCTCATCATTCGCAATGGGGAC AACGTGGAGGCCCCACCAGTGTATGATTCCTATGAGGTGGAATACCTGCCCATTGAGGGCCTGCTCAGCTCTGGCAAACACTTCTTTGTTGAGCTCAGTACTGACAGCAGCG GGGCAGCTGCAGGCATGGCCCTGCGCTATGAGGCCTTCCAGCAGGGCCATTGCTATGAGCCCTTTGTCAAATACGGTAACTTCAGCAGCAGCACACCCACCTACCCTGTGGG TACCACTGTGGAGTTCAGCTGCGACCCTGGCTACACCCTGGAGCAGGGCTCCATCATCATCGAGTGTGTTGACCCCCACGACCCCCAGTGGAATGAGACAGAGCCAGCCTGC CGAGCCGTGTGCAGCGGGGAGATCACAGACTCGGCTGGCGTGGTACTCTCTCCCAACTGGCCAGAGCCCTACGGTCGTGGGCAGGATTGTATCTGGGGTGTGCATGTGGA AGAGGACAAGCGCATCATGCTGGACATCCGAGTGCTGCGCATAGGCCCTGGTGATGTGCTTACCTTCTATGATGGGGATGACCTGACGGCCCGGGTTCTGGGCCAGTACTC AGGGCCCCGTAGCCACTTCAAGCTCTTTACCTCCATGGCTGATGTCACCATTCAGTTCCAGTCGGACCCCGGGACCTCAGTGCTGGGCTACCAGCAGGGCTTCGTCATCCACT TCTTTGAGGTGCCCCGCAATGACACATGTCCGGAGCTGCCTGAGATCCCCAATGGCTGGAAGAGCCCATCGCAGCCTGAGCTAGTGCACGGCACCGTGGTCACTTACCAGTGCTACCCTGGCTACCAGGTAGTGGGATCCAGTGTCCTCATGTGCCAGTGGGACCTAACTTGGAGTGAGGACCTGCCCTCATGCCAGAGGGTGACTTCCTGCCACGATCCTGG AGATGTGGAGCACAGCCGACGCCTCATATCCAGCCCCAAGTTTCCCGTGGGGGCCACCGTGCAATATATCTGTGACCAGGGTTTTGTGCTGATGGGCAGCTCCATCCTCACC TGCCATGATCGCCAGGCTGGCAGCCCCAAGTGGAGTGACCGGGCCCCTAAATGTCTCCTGGAACAGCTCAAGCCATGCCATGGTCTCAGTGCCCCTGAGAATGGTGCCCGA AGTCCTGAGAAGCAGCTACACCCAGCAGGGGCCACCATCCACTTCTCGTGTGCCCCTGGCTATGTGCTGAAGGGCCAGGCCAGCATCAAGTGTGTGCCTGGGCACCCCTCG CATTGGAGTGACCCCCCACCCATCTGTAGGGCTGCCTCTCTGGATGGGTTCTACAACAGTCGCAGCCTGGATGTTGCCAAGGCACCTGCTGCCTCCAGCACCCTGGATGCTG CCCACCTGGCCGGCCACAGATCTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGAccGTCAGTc??cc????cccccCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC ACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCT CCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCAGGGAGGAGATGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCATGCTGGACTCCGACG GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCTCCGGGTTGA U . S . Patent Jun . 12, 2018 Sheet 14 of 72 US 9 , 993, 566 B2

(SEQIDNO:9)

FIG.4B >humanSEZ6-Fcprotein METDTLLLWVLLLWVPGSTGDGAPGSLSLEAPTVGKGQAPGIEETDGELTAAPTPEQPERGVHFVTTAPTLKLLNHHPLLEEFLQEGLEKGDEELRPALPFQPDPPAPFTPSPLPRLANQDSRPVFTSPTPAMAAVPTQPQSKEGPWSPES ESPMLRITAPLPPGPSMAVPTLGPGEIASTTPPSRAWTPTQEGPGDMGRPWVAEVVSQGAGIGIQGTITSSTASG DDEETTTTTTUTTTITTVQTPGPCSWNFSGPEGSLDSPTDLSSPTDVGLDCFFYISVYPGYGVEIKVQNISLREGETV TVEGLGGPDPLPLANQSFLLRGQVIRSPTHQAALRFQSLPPPAGPGTFHFHYQAYLLSCHFPRRPAYGDVTVTSLH PGGSARFHCATGYQLKGARHLTCLNATQPFWDSKEPVCIAACGGVIRNATTGRIVSPGFPGNYSNNLTCHWLLEAPEGQRLHLHFEKVSLAEDDDRLIIRNGDNVEAPPVYDSYEVEYLPIEGLLSSGKHFFVELSTDSSGAAAGMALRYEA FQQGHCYEPFVKYGNFSSSTPTYPVGTTVEFSCDPGYTLEQGSIJIECVDPHDPQWNETEPACRAVCSGEITDSAG WLSPNWPEPYGRGQDCIWGVHVEEDKRIMLDIRVLRIGPGDVLTFYDGDDLTARVLGQYSGPRSHFKLFTSMA DVTIQFQSDPGTSVLGYQQGFVIHFFEVPRNDTCPELPEIPNGWKSPSQPELVHGTVVTYQCYPGYQVVGSSVLM CQWDLTWSEDLPSCQRVTSCHDPGDVEHSRRLISSPKFPVGATVQYICDQGFVLMGSSILTCHDRQAGSPKWSD RAPKCLLEQLKPCHGLSAPENGARSPEKQLHPAGATIHFSCAPGYVLKGQASIKCVPGHPSHWSDPPPICRAASLD GFYNSRSLDVAKAPAASSTLDAAHLAGHRSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWWVDVSHED PEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTOKSLSLSPG U . S . Patent Jun . 12 , 2018 Sheet 15 of 72 US 9 ,993 ,566 B2

(SEQIDNO:10) wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww

FIG.5A >cDNAsequenceencodingmaturemouseSEZ6protein CTCTCCTCAGAGGCTCCGATCACGGGGGAAGGTCATGCCACGGGCATCAGGGAGACGGATGGGGAGCTGACCGCAGCCCCTACACCTGAGCAGTCAGACCGAGGCGTCCACTTCGTCACCACAGCCCCTACCCTCAAGCTGCTCAACCACCACCCACTTCTGGAAGAATTTCTTCAAGAGGGGCTAGAAAGAGAGGAAGCGC CGCAGCCTGCACTGCCCTTCCAGCCGGACTCACCTACACACTTTACTCCAAGCCCCCTCCCCCGCCTCACCAACCAGGACAACCGCCCCGTCTTTACCAGTCCG ACTCCAGCCGTGGCTGCAGCACCCACCCAGCCCCACTCCAGGGAGAAACCTTGGAACCTAGAATCCAAACCCCCTGAGCTTTCTATCACATCGTCCCTTCCTCC AGGGCCGAGTATGGCAGTGCCCACACTGCTCCCAGAGGACAGACCCAGTACTACACCCCCTAGCCAAGCATGGACTCCAACTCAGGAGGGTCCTGGAGACA TGGACAGACCTTGGGTTCCAGAGGTCATGTCTAAGACCACAGGGCTTGGTGTCGAGGGAACCATTGCCACCTCCACAGCTTCAGGGGATGACGAAGAGACC ACTACCACCATCATTACCACTACTGTCACCACAGTTCAGCCACCAGGCCCCTGTAGCTGGAATTTCTCAGGCCCAGAGGGCTCTCTGGATTCCCCCACGGCCCC CAGCTCACCCTCTGATGTTGGCCTGGACTGTTTCTACTATATCTCTGTCTACCCTGGATATGGAGTAGAGATCAAGGTGGAGAACATCAGCCTTCAGGAAGGG GAGACCATCACCGTGGAGGGCCTGGGGGGCCCCGATCCACTGCCCTTGGCTAACCAGTCGTTCCTGCTGAGGGGCCAGGTCATCCGCAGCCCCACCCACCA AGCAGCCCTGAGGTTCCAGAGCCTCCCGCTACCCGCTGGGCCTGGCACTTTCCATTTCCGCTACCAAGCCTATCTCCTGAGCTGCCACTTTCCCCGACGTCCAG CGTATGGAGATGTGACTGTCACCAGTCTCCACCCAGGAGGCAGCGCCCACTTCCATTGTGCCACTGGCTACCAGCTCAAGGGTGCCAGGTTCCTCACCTGTCT CAATGCCACCCAGCCCTTTTGGGATTCCCAAGAGCCTGTTTGCATTGCTGCTTGTGGTGGAGTGATTCGGAATGCCACCACTGGCCGCATTGTCTCTCCTGGC TTCCCGGGGAACTACAGCAACAACCTCACCTGCCACTGGTTGCTAGAGGCTCCAGAGAGCCAGCGGCTGCACCTGCACTTTGAAAAGGTCTCCCTGGCAGAA GACGACGACAGGCTCATCATCCGCAATGGAAATAACGTGGAGGCCCCGCCGGTGTACGACTCCTATGAGGTGGAATACCTGCCCATTGAGGGCCTGCTCAG CTCTGGCAGACACTTCTTCGTGGAGTTCAGTACTGACAGCAGTGGGGCAGCTGCAGGCATGGCCCTGCGCTATGAGGCCTTCCAGCAAGGACATTGCTATGA GCCCTTTGTCAAATACGGCAACTTCAGCAGCAGTGCACCGTCCTACCCTGTGGGTACAACTGTGGAGTTCAGCTGTGACCCTGGCTACACCCTGGAGCAGGG CTCCATCATCATCGAATGCGTCGACCTCCACGACCCCCAGTGGAATGAGACAGAGCCAGCCTGCCGAGCCGTGTGCAGCGGGGAGATCACAGACTCTGCAG GCGTGGTGCTCTCTCCAAACTGGCCGGAGCCTTATGGCCGAGGGCAGGACTGCATCTGGGGTGTGCATGTGGAGGAGGACAAGCGCATCATGCTGGACAT CCGAGTGCTGCGCATAGGCTCTGGGGATGTACTGACCTTCTACGATGGGGATGACCTCACAGCCCGGGTCCTGGGCCAATACTCAGGGCCCCGTGGCCACTT CAAGCTCTTTACCTCCATGGCCGATGTCACCATCCAGTTCCAGTCAGACCCTGGGACCTCGGCGCTGGGTTACCAGCAAGGATTTGTCATCCACTTCTTTGAG GTTCCCCGCAACGACACATGTCCAGAGCTACCCGAGATCCCCAACGGCTGGAAGAACCCATCACAGCCTGAGCTGGTGCACGGCACGGTGGTCACCTATCAG TGCTACCCTGGTTACCAGGTGGTGGGATCCAGTATTCTCATGTGCCAGTGGGACCTAAGCTGGAGTGAGGACCTGCCTTCATGCCAGAGAGTGACATCTTGC CATGACCCAGGGGATGTGGAGCACAGCCGACGCCTCATATCCAGCCCCAAGTTTCCCGTGGGAGCAACTGTGCAATATGTCTGTGACCAGGGTTTTGTGCTG ACGGGGAGTGCCATTCTCACCTGCCATGATCGGCAAGCAGGCAGTCCCAAGTGGAGTGACAGGGCCCCCAAGTGTCTCTTGGAACAATTCAAGCCGTGCCA TGGCCTCAGCGCCCCGGAGAATGGTGCCCGCAGCCCTGAGAAGCGGCTTCACCCAGCAGGGGCCACCATCCACTTCTCCTGTGCCCCTGGTTATGTGCTGAA GGGCCAGGCCAGCATCAAATGCGTGCCTGGACACCCCTCGCATTGGAGTGACCCACCACCCATCTGTAGGGCTGCCTCTCTGGATGGGTTCTACAACGGCCG TAGCCTGGATGTTGCCAAGGCACCTGCCGCCTCCAGTGCCCTGGACGCTGCTCACCTGGCTGCTGCCATCTTCCTACCATTGGTGGCCATGGTGTTGCTGGTG GGAGGAGTGTACCTCTATTTTTCCAGATTCCAGGGGAAAAGTCCCCTGCAACTTCCCCGAACTCATCCTCGCCCCTATAACCGCATCACGGTAGAGTCAGCAT TTGACAATCCAACTTATGAGACTGGATCTCTTTCCTTTGCAGGAGACGAGAGAATATGA atent Jun . 12 , 2018 Sheet 16 of 72 US 9, 993, 566 B2

(SEQIDNO:11)

APAASSALDAAHLAAAIFLPLVAMVLLVGGVYLYFSRFQGKSPLQLPRTHPRPYNRITVESAFDNPTYETGSLSFAGDERI FIG.5B ORFSEZ6mouseofTranslation> LSSEAPITGEGHATGIRETDGELTAAPTPEQSDRGVHFVTTAPTLKLLNHHPLLEEFLQEGLEREEAPQPALPFQPDSPTHFT PSPLPRLTNQDNRPVFTSPTPAVAAAPTOPHSREKPWNLESKPPELSITSSLPPGPSMAVPTLLPEDRPSTTPPSQAWTPT QEGPGDMDRPWVPEVMSKTTGLGVEGTIATSTASGDDEETTTTIITTTVTTVQPPGPCSWNFSGPEGSLDSPTAPSSPSD VGLDCFYYISVYPGYGVEIKVENISLQEGETITVEGLGGPDPLPLANQSFLLRGQVIRSPTHQAALRFQSLPLPAGPGTEHER YQAYLLSCHEPRRPAYGDVTVTSLHPGGSAHFHCATGYQLKGARFLTCLNATQPFWDSQEPVCIAACGGVIRNATTGRIVS PGFPGNYSNNLTCHWLLEAPESQRLHLHFEKVSLAEDDDRLIIRNGNNVEAPPVYDSYEVEYLPIEGLLSSGRHFFVEFSTD SSGAAAGMALRYEAFQQGHCYEPFVKYGNFSSSAPSYPVGTTVEFSCDPGYTLEQGSIIIECVDLHDPQWNETEPACRAV CSGEITDSAGWLSPNWPEPYGRGQDCIWGVHVEEDKRIMLDIRVLRIGSGDVLTFYDGDDLTARVLGQYSGPRGHEKLF TSMADVTIQFQSDPGTSALGYQQGFVIHFFEVPRNDTCPELPEIPNGWKNPSQPELVHGTWTYQCYPGYQWGSSILMC QWDLSWSEDLPSCQRVTSCHDPGDVEHSRRLISSPKFPVGATVQYVCDQGFVLTGSAILTCHDRQAGSPKWSDRAPKCLL EQFKPCHGLSAPENGARSPEKRLHPAGATIHFSCAPGYVLKGQASIKCVPGHPSHWSDPPPICRAASLDGFYNGRSLDVAK atent Jun . 12 , 2018 Sheet 17 of 72 US 9 , 993, 566 B2

(SEQIDNO:12)

FIG.5C >cDNAsequenceofratSEZ6ORF ctctcctcagaggctccaatcacgggggaaggtcaagccacgggcatcagggagatggatggggagctgaccgcagcccctacacctgagcagtcagaccgaggcgtccacttcgtcaccac agcccctaccctcaagctactcaaccaccacccacttctggaggaatttcttcaagaggggctagaagggagagaggaagctccgaggccggcactgcccttccagccagactcacctacac cctttactccaagcccccttccccgcctcaccaaccaggacaaccgccctgtctttaccagtccgacgccagctgtagctgcggcacccacgcagccccactccagaaagaaaccctggaacc cagagtcagagcccccggagctttacatcacatctcccctccctccagggccgagtatggcagtgcccacactgcacccagaggacagacccagcactacaccccccagccaagcatggact ccaacccaggagggtcctggagacatgggcagaccttgggttccagagatcatgtctaagaccacagggcttggtatcgaggggaccattgccacctccacagcttcaggggatgacgaaga gaccaccaccaccaccatcattaccaccgtcaccacaattcagccaccaggcccctgtagctggaatttctcaggcccggagggctctctggattcccctgcggtccccagcgtcccctctgatg ttggcctggactgtctctactacatctctgtctaccctggatatggagtcgagatcaaggtgaagaacatcagccttcaggaaggagagaccataaccgtggagggcctgggggggcctgacc cactgcccttggctaaccagtctttcctgctgaggggccaggtcatccgcagccccacccaccaggcagccgtgaggttccaaagccttccacttcccgctggacctggtactttccatttccact accaagcctatctcctgagctgccactttcctcggcgtccagettatggagatgtgactgtcaccagcctccacccaggaggcagcgcccgcttccactgtgccactggctaccagctaaagggt gccaggttcctcacctgtctcaatgccacccagcccttttgggattcccaagagcctgtctgcattgctgcttgtggaggagtgattcggaatgccaccactggccgcattgtctctcctggctttcc cgggaactacagcaacaacctcacctgccactggctgctagaagcccccgagagccagcggctgcacctgcactttgaaaaggtctccctggcagaagatgacgacaggctcatcatccgta acgggaataacgtggaggccccgccagtgtatgactcctatgaggtggagtacctgcccattgagggcctgctcagttctggcagacacttcttcgtggagttcagtactgacagcagcggggc agccgcaggcatggcactgcgctatgaggccttccagcaaggacattgctatgagccctttgtcaaatacggtaacttcagcagcagcgcaccgtcctaccctgtgggtacgactgtggagttc agctgtgaccctggctacaccctggagcagggttccatcatcatcgaatgcgtcgacctccgtgacccccagtggaatgagacagaaccagcctgccgagccgtgtgcagcggggagatcac agactctgcaggcgtggtgctctctccaaactggccggagccttatggccgagggcaggactgcatctggggtgtgcatgtggaggaggacaagcgcatcatgctggacatccgagtgctgcg cataggctctggggatgtactgaccttctacgatggggatgacctgacagcccgggtcctgggccaatactcagggccccgtggccacttcaagctctttacctccatggctgatgtcaccattc agttccagtcagaccctgggacgtcggcgctgggttaccagcaaggatttgtcatccacttctttgaggtgccccgcaatgacacatgtccagagcttcccgagatccccaacggctggaagaa cccatcacagcctgagctggtgcatggcacggtggtcacctatcagtgctaccccggttaccaggtggtgggatccagtattctcatgtgccagtgggacctgagctggagtgaggacctgccct catgccagagagtgacatcctgccatgacccaggggatgtggagcacagccgacgcctcatatccagcctcaagtttcctgtgggagcaactgtgcagtatatctgtgaccagggttttgtgctc acgggtagcgccatccttacttgccatgatcgtcaagcgggcagtcccaagtggagtgacagggcccccaagtgtctcttggaacagttcaaaccatgtcatggcctcagtgcccctgagaatg gtgcccgcagccctgagaagaggctccacccagcaggggccaccattcacttctcctgtgcccctggttatgtgctgaagggccaggccagcatcaaatgcgtgcctggacacccctcacattg gagtgatcctccacccatctgtagggctgcttctctggatgggttctacaacggccgtagcctggatgttgccaaggcacctgccacctccagtgccctggatgctgcccacatggcagctgcca tctttctaccattggtggccatggtgttgctggtgggaggagtgtacctctatttctccagactccagggaaaaagtcctctgcagcttcccggaactcatcctcgcccctataaccgtatcacggt agagtcagcatttgacaatccaacttatgagaccogatctctttcctttgcaggagacgagagaata U . S . Patent Jun . 12, 2018 Sheet 18 of 72 US 9, 993, 566 B2

(SEQIDNO:13)

FIG.5D >TranslationofratSEZ6ORE LSSEAPITGEGQATGIREMDGELTAAPTPEQSDRGVHFVTTAPTLKLLNHHPLLEEFLQEGLEGREEAPRPALPFQPDSPTPFTPS PLPRLTNQDNRPVFTSPTPAVAAAPTQPHSRKKPWNPESEPPELYITSPLPPGPSMAVPTLHPEDRPSTTPPSQAWTPTQEGPG DMGRPWVPEIMSKTTGLGIEGTIATSTASGDDEETTTTTIITTYTTIQPPGPCSWNFSGPEGSLDSPAVPSVPSDVGLDCLYYISV YPGYGVEIKVKNISLQEGETITVEGLGGPDPLPLANQSFLLRGQVIRSPTHQAAVRFQSLPLPAGPGTEHEHYQAYLLSCHEPRRP AYGDVTVTSLHPGGSARFHCATGYQLKGARFLTCLNATQPFWDSQEPVCIAACGGVIRNATTGRIVSPGFPGNYSNNLTCHWL LEAPESQRLHLHFEKVSLAEDDDRLIIRNGNNVEAPPVYDSYEVEYLPIEGLLSSGRHFFVEFSTDSSGAAAGMALRYEAFQQGH CYEPFVKYGNFSSSAPSYPVGTTVEFSCDPGYTLEQGSITIECVDLRDPQWNETEPACRAVCSGEITDSAGWVLSPNWPEPYGRG QDCIWGVHVEEDKRIMLDIRVLRIGSGDVLTFYDGDDLTARVLGQYSGPRGHFKLFTSMADVTIQFQSDPGTSALGYQQGFVI HFFEVPRNDTCPELPEIPNGWKNPSQPELVHGTWTYQCYPGYQWGSSILMCQWDLSWSEDLPSCQRVTSCHDPGDVEHS RRLISSLKFPVGATVQYICDQGFVLTGSAILTCHDRQAGSPKWSDRAPKCLLEQFKPCHGLSAPENGARSPEKRLAPAGATIHFSC APGYVLKGQASIKCVPGHPSHWSDPPPICRAASLDGFYNGRSLDVAKAPATSSALDAAHMAAAIFLPLVAMVLLVGGVYLYFSR LQGKSPLQLPGTHPRPYNRITVESAFDNPTYETGSLSFAGDERI U . S . Patent Jun . 12, 2018 Sheet 19 of 72 US 9 , 993, 566 B2

(SEQIDNO:14)

FIG.5E >cDNAsequenceofcynomolgusSEZ6ORF atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacggcgcgccactcagcagcgaagctcccacaatgggcaagggacaggcccccggaattga agaaaccgatggcgaactcaccgctgcccctacccctgagcaacccgaaaggggagtgcactttgtgaccaccgctcccaccctgaagctgctcaatcaccaccccctcctggaggagtttct gcaggaaggcctggaaaaaggcgacgaggaactcagacctgccctgcccttccaacccgaccctcctaccccctttacacctagccctctccctagactggccaaccaagactccagacctgt gttcaccagccctacacctgctacagctgccgtccctacccaacctcaatccaaggagggaccttggagcctcgagagcgagcctcccgtgctgagaatçacagctcctctccctcctggccctt ccatggctgtccccacactcggacctggcgaaaggcccagcacaacacccccctccagagcctggacccctacacaagaaggccctggcgacatgggaaggccttgggtccctgaagtcgtg agccaaggcgccggcatcggaatccagggaaccatcgccagctccacagccagcggagacgatgaggaaacaaccaccacaaccaccatcatcaccaccacaatcacaacagtccagacc cccggcccttgcagctggaatttttccggccctgagggatccctggattcccccacagatctgtcctcccctcctgacgtgggcctcgactgtttcttctatatctccgtgtatcctggctacggcgt cgaaatcaaagtccagaacatctccctgagggagggcgaaacagtcaccgtggaaggactgggcggacccgctcctctgcctctcgccaaccaatccttcctcctcaggggccaagtgattag atcccccacacatcaagctgctctcaggttccaaagcctccctccccccgctggacccggaacctttcacttccactaccaagcctatctcctcagctgccatttcccccacaggcccgcttatgg agatgtcacagtcacctccctgcatcctggcggctccgctagattccactgegctaccggataccaactcaagggcgccaggcatctgacatgtctcaatgctacccagcccttctgggacagc aaggagcccgtctgcattgccgcttgcggaggcgtcatcagaaatgccaccaccggcagaatcgtgagccccggcttccctggcaactactccaacaacctgacatgccactggctgctggaa gctcctgagggccagagactgcatctgcacttcgagaaggtcagcctggccgaagatgacgacagactcatcatcaggaacggcgacaacgtggaggctccccccgtctatgattcctacgag gtcgagtacctccccatcgagggactgctgtcctccggcaagcattttttcgtggagctgtccacagattccagcggagctgccgccggaatggctctcaggtacgaggctttccaacagggcca ctgttacgagccctttgtgaagtacggcaacttctccagctccgctcctacctaccccgtcggcacaaccgtcgaatttagctgcgaccctggatacacactcgagcaaggctccatcatcatcg agtgtgtcgacccccacgacccccaatggaacgagacagagcccgcctgtagggccgtgtgtagcggagagattaccgactccgccggagtggtgctctcccctaattggcctgaaccctacg gcagaggacaagattgtatttggggcgtccatgtcgaggaggacaagaggattatgctcgacgtgagggtgctgaggattggacctggcgacgtgctcacattctatgacggcgacgatctca ccgccagagtcctgggacaatactccggccctcacagccacttcaagctgttcaccagcatggctgacgtgaccatccagttccagtccgatcctggaacatccgtgctgggataccagcaggg cttcgtcatccacttcttcgaggtccccaggaacgacacctgccccgaactgcccgagattcccaacggctggaaatccccctcccaacctgatctcgtgcacggcaccgtcgtcacctaccaat gctaccctggataccaagtcgtcggcagcagcgtgctgatgtgccaatgggacctcacctggagcgaggatctgccctcctgccagagagtcacctcctgccacgatcccggcgatgtggaac actccaggaggctgattagctcccccaagttccctgtcggagccaccgtgcaatacatctgcgaccagggctttgtgctgaccggaaccagcatcctcacatgccacgacaggcaagctggato ccccaagtggtecgatagggcccccaaatgcctcctggaacagctgaagccttgtcatggcctcagcgctcctgaaaacggcgctaggagccccgaaaagaggctccaccctgccggagcca ccatccacttttcctgtgcccccggatacgtgctgaagggccaggcctccattaagtgcgtgcccggacatccttcccactggtccgacccccctcccatctgtaaagccgcctccctggacggat tctataacagcagaagcctggacgtcgctaaggcccctgctgcttcctccaccctggatgctgctcacatcgctgctgccatctttctgcccctcgtcgccatggtgctgctggtgggaggcgtct acttctacttctccaggctgcagggaaagagctccctgcaactgcctaggacaagacccaggccctacaataggatcacagtcgagagcgccttcgacaaccccacatacgagacaggatcc ctgagctttgccggagacgagagaatt U . S . Patent Jun . 12 , 2018 Sheet 20 of 72 US 9 ,993 ,566 B2

(SEQIDNO:15)

FIG.5F >TranslationofcynomolgusSEZ6ORF METDTLLLWVLLLWVPGSTGDGAPLSSEAPTMGKGQAPGIEETDGELTAAPTPEQPER GVHFVTTAPTLKLLNHHPLLEEFLQEGLEKGDEELRPALPFQPDPPTPFTPSPLPRLANQ DSRPVFTSPTPATAAVPTQPQSKEGPWSLESEPPVLRITAPLPPGPSMAVPTLGPGERPS TTPPSRAWTPTQEGPGDMGRPWVPEVVSQGAGIGIQGTIASSTASGDDEETTTTTTIITT TITTVQTPGPCSWNFSGPEGSLDSPTDLSSPPDVGLDCFFYISVYPGYGVEIKVQNISLR EGETVTVEGLGGPAPLPLANQSFLLRGQVIRSPTHQAALRFQSLPPPAGPGTFHFHYQAY LLSCHEPHRPAYGDVTVTSLHPGGSARFHCATGYQLKGARHLTCLNATQPFWDSKEPVCI AACGGVIRNATTGRIVSPGFPGNYSNNLTCHWLLEAPEGQRLHLHFEKVSLAEDDDRLII RNGDNVEAPPVYDSYEVEYLPIEGLLSSGKHFFVELSTDSSGAAAGMALRYEAFQQGHCY EPFVKYGNFSSSAPTYPVGTTVEFSCDPGYTLEQGSITIECVDPHDPQWNETEPACRAVC SGEITDSAGWLSPNWPEPYGRGQDCIWGVHVEEDKRIMLDVRVLRIGPGDVLTFYDGDD LTARVLGQYSGPHSHEKLFTSMADVTIQFQSDPGTSVLGYQQGFVIHFFEVPRNDTCPEL PEIPNGWKSPSQPDLVHGTVVTYQCYPGYQWVGSSVLMCQWDLTWSEDLPSCQRVTSCHD PGDVEHSRRLISSPKFPVGATVQYICDQGFVLTGTSILTCHDRQAGSPKWSDRAPKCLLE QLKPCHGLSAPENGARSPEKRLHPAGATIHFSCAPGYVLKGQASIKCVPGHPSHWSDPPP ICKAASLDGFYNSRSLDVAKAPAASSTLDAAHIAAAIFLPLVAMVLLVGGVYFYFSRLQG KSSLQLPRTRPRPYNRITVESAFDNPTYETGSLSFAGDERI U . S . Patent Jun . 12, 2018 Sheet 21 of 72 US 9 , 993, 566 B2

(SEQIDNO:16)

FIG.5G >cDNAsequenceofhumanSEZOLECD ctcgagagggatgctctgcctgagggagatgcttcccctctcggaccttatctgctgcccagcggagctcctgagaggggatcccccggaaaggagcatcccgaagaaagagtggtcacagct ccccctagctccagccagagcgctgaggtgctgggagaactggtcctcgacggaacagccccttccgcccatcacgatattcctgccctcagccctctcctccccgaggaagctaggcctaaac acgccctcccccctaaaaagaagctgccttccctcaagcaggtcaattccgccaggaagcagctcagacccaaggccacctccgctgctacagtccaaagagctggatcccagcctgccagc cagggactcgatctgctcagcagctccacagaaaaacctggacctcctggcgatcctgaccctattgtggccagcgaagaagctagcgaagtccctctgtggctggacaggaaggagtccgct gtccccaccacacccgctcctctccagatcagccccttcacctcccagccttatgtcgctcatacactgcctcagaggcctgagcctggcgaacctggacctgacatggctcaggaggctcctca ggaggacaccagccctatggccctgatggataagggcgagaatgaactgaccggaagcgccagcgaggaaagccaggagaccaccaccagcacaatcatcaccaccaccgtcatcaccac cgaacaggcccccgctctgtgttccgtgtccttttccaaccccgagggctacattgacagcagcgattaccccctgctccctctcaacaacttcctcgagtgcacctacaatgtgaccgtgtacac cggctacggagtcgaactccaggtgaagtccgtgaacctctccgatggcgaactgctctccattaggggcgtcgatggccctacactcaccgtcctggctaaccaaaccctgctcgtcgaaggc caggtgattaggtcccccaccaacaccatctccgtctacttcaggacctttcaagacgacggactgggaaccttccaactgcattaccaggccttcatgctgtcctgtaatttccccaggagaccc gactccggagacgtcaccgtcatggatctgcactccggaggcgtggcccactttcattgtcacctcggctacgagctccagggcgccaagatgctgacatgcatcaacgccagcaaacctcact ggtccagccaggagcctatctgtagcgctccttgcggcggagccgtgcacaatgctacaattggcagagtgctcagcccttcctaccctgaaaacaccaacggctcccagttctgcatctggac aatcgaggcccccgaaggccaaaagctgcacctgcactttgagaggctcctgctccacgacaaagacaggatgaccgtgcactccggccagaccaataagtccgccctcctgtatgacagcc tgcagacagagtccgtcccttttgaaggcctgctgtccgagggcaataccatcaggattgagttcacatccgaccaagccagggctgctagcaccttcaacattaggtttgaggctttcgaaaag ggacactgctacgagccctatattcagaatggcaatttcacaacctccgaccccacctacaatatcggcacaattgtggagtttacctgcgaccctggacacagcctggagcagggacctgcca tcatcgaatgcatcaacgtcagggacccctactggaacgacacagaacctctgtgtagggctatgtgcggaggcgaactgagcgctgtggctggagtcgtgctctcccctaactggcccgaac cctatgtggagggcgaagattgcatctggaagatccacgtcggcgaggaaaaaaggatctttctggacatccagttcctgaatctctccaacagcgacatcctgaccatctacgacggagatga ggtcatgccccacattctgggccagtatctcggaaactccggcccccaaaagctctactcctccacccccgacctcacaatccaattccacagcgatcctgctggcctcatctttggaaagggac aaggctttatcatgaattacatcgaggtcagcagaaacgacagctgctccgacctgcctgagatccagaacggatggaagaccacctcccacaccgagctcgtcaggggagctaggatcaca taccagtgcgaccccggatacgacatcgtcggctccgataccctgacatgccagtgggatctgagctggagctccgacccccccttttgtgagaagatcatgtactgcaccgaccccggcgaag togatcatagcaccaggctcatcagcgatcctgtgctgctcgtcggcacaaccatccaatacacctgtaaccccggattcgtgctcgaaggatcctccctgctcacctgttacagcagggaaacc ggcacccccatttggacatccaggctgcctcactgcgtgtccgaagagagcctggcttgcgataatcccggcctgcctgagaacggataccagattctgtacaaaaggctgtacctccccggcg agtccctgaccttcatgtgctacgaaggattcgagctcatgggcgaagtcaccatcaggtgcatcctcggccagccctcccactggaacggacctctccccgtctgtaaggtcaatcaggattcc ttcgagcacgctctggaagtcgctgaggctgccgccgagacaagcctggaaggcggc U . S . Patent Jun . 12 , 2018 Sheet 22 of 72 US 9 ,993 ,566 B2

(SEQIDNO:17)

FIG.5H >humanSEZÓLECDprotein METDTLLLWVLLLWVPGSTGDHGAPLERDALPEGDASPLGPYLLPSGAPERGSPGKEHPEERVVT APPSSSQSAEVLGELVLDGTAPSAHHDIPALSPLLPEEARPKHALPPKKKLPSLKQVNSARKQLRPK ATSAATVQRAGSQPASQGLDLLSSSTEKPGPPGDPDPIVASEEASEVPLWLDRKESAVPTTPAPLQI SPFTSQPYVAHTLPQRPEPGEPGPDMAQEAPQEDTSPMALMDKGENELTGSASEESQETTTSTIL TTTVITTEQAPALCSVSFSNPEGYIDSSDYPLLPLNNFLECTYNVTVYTGYGVELQVKSVNLSDGELL SIRGVDGPTLTVLANQTLLVEGQVIRSPTNTISVYFRTFQDDGLGTFOLHYQAFMLSCNFPRRPDS GDVTVMDLHSGGVAHEHCHLGYELQGAKMLTCINASKPHWSSQEPICSAPCGGAVHNATIGRV LSPSYPENTNGSQFCIWTIEAPEGQKLHLHFERLLLHDKDRMTVHSGQTNKSALLYDSLQTESVPF EGLLSEGNTIRIEFTSDQARAASTENIRFEAFEKGHCYEPYIQNGNETTSDPTYNIGTIVEFTCDPGH SLEQGPATIECINVRDPYWNDTEPLCRAMCGGELSAVAGWLSPNWPEPYVEGEDCIWKIHVGEE KRIFLDIQFLNLSNSDILTIYDGDEVMPHILGQYLGNSGPQKLYSSTPDLTIQFHSDPAGLIFGKGQG FIMNYIEVSRNDSCSDLPEIQNGWKTTSHTELVRGARITYQCDPGYDIVGSDTLTCQWDLSWSSD PPFCEKIMYCTDPGEVDHSTRLISDPVLLVGTTIQYTCNPGFVLEGSSLLTCYSRETGTPIWTSRLPH CVSEESLACDNPGLPENGYQILYKRLYLPGESLTFMCYEGFELMGEVTIRCILGQPSHWNGPLPVC KVNQDSFEHALEVAEAAAETSLEGGLAGHHHHHHHHH U . S . Patent Jun . 12, 2018 Sheet 23 of 72 US 9 ,993 ,566 B2

(SEQIDNO:18)

FIG.51

>cDNAsequenceofhumanSEZ6L2ECD ctgcctctcaaagaggaagagattctccccgagcccggatccgagacacccacagtggcttccgaagccctcgctgaactgctgcacggagccctcctgagaaggggacctgaaatgggcta tctccctggctccgacagagatcccacactcgccacacctcctgctggacagaccctcgctgtgccttccctgcccagagccacagaacccggaacaggccctctcacaacagctgtgacccct aacggcgtcagaggagctggacctacagcccctgagctgctgacacctcctcctggcacaaccgctcctcctcctccttcccctgctagccctggaccccctctcggacctgaaggaggcgagg aggagacaaccaccaccattattaccaccaccaccgtgacaaccacagtgaccagccctgtcctgtgcaacaacaacatcagcgaaggcgaaggctatgtggaatcccctgacctgggctcc cctgtgtccagaacactcggcctcctggattgcacatactccattcacgtgtaccccggctacggaatcgagattcaggtgcagaccctgaatctgtcccaggaggaggaactgctggtgctgg ctggcggaggaagccctggcctcgctcctagactcctcgctaactcctccatgctcggcgaaggccaggtcctcagatcccctaccaacaggctgctcctgcacttccagagccccagagtgcc tagaggaggcggcttcaggattcactaccaggcctatctcctgagctgtggattccctcccagacccgctcatggcgatgtctccgtcaccgacctccaccccggaggaacagccaccttccact gtgattccggataccagctgcaaggcgaggagaccctgatttgcctcaatggcaccaggcccagctggaacggagagacacctagctgcatggctagctgcggcggaaccatccataatgcc accctcggcaggatcgtcagccctgaacctggcggagctgtgggacctaacctcacatgcagatgggtgatcgaagctgctgaaggcaggagactccacctccacttcgagagggtgtccctg gacgaggacaacgacaggctcatggtcagaagcggcggaagccctctcagccctgtgatttacgacagcgacatggacgatgtgcctgagaggggcctcatctccgatgcccaaagcctgta cgtggaactcctctccgagacccccgctaaccccctcctcctgagcctcagattcgaggccttcgaggaggacagatgtttcgctccttttctggcccatggcaacgtgaccacaaccgaccccg agtacagacccggagctctggctaccttcagctgtctgcctggctacgccctcgaacctcccggacctcctaatgccatcgaatgtgtggatcccaccgaaccccattggaacgacaccgagcc cgcttgtaaggctatgtgcggcggagaactcagcgaacctgccggagtggtcctctcccctgattggccccagagctattcccccggacaagactgtgtctggggcgtgcacgtccaggagga aaagaggatcctcctccaggtggagattctgaacgtcagagagggagacatgctgaccctgttcgacggagacggaccttccgccagagtcctcgctcagctgagaggccctcagcccagaa ggagactgctcagctccggccccgatctgacactccagtttcaggccccccctggcccccctaatcctggcctgggacagggcttcgtgctccacttcaaggaggtccccaggaatgatacatg ccccgaactgcctcctcccgagtggggatggaggacagcttcccatggcgacctgatcaggggaaccgtgctgacatatcagtgtgaacccggctacgagctgctgggaagcgatatcctgac ctgtcagtgggatctctcctggagcgctgctccccctgcctgtcagaaaatcatgacctgcgctgaccctggagagatcgctaacggccacaggaccgcttccgacgctggatttcccgtgggct cccacgtgcaatacaggtgcctccccggatactccctcgaaggcgctgccatgctgacatgctacagcagggacaccggcacacccaagtggtccgacagggtgcccaaatgtgctctgaagt acgagccctgtctcaatcccggagtgcccgagaacggataccagaccctgtacaagcaccactatcaggccggegaatccctgagattcttctgctacgagggcttcgagctcatcggcgagg tgacaattacctgtgtgcccggccatccttcccagtggaccagccagccccctctctgtaaggtcgcctacgaagagctgctcgacaataggaagctggaggtcacccagaccaccgacccttc cagacaactggaaggeggc U . S . Patent Jun . 12 , 2018 Sheet 24 of 72 US 9, 993, 566 B2

(SEQIDNO:19)

WNGETPSCMASCGGTIHNATLGRIVSPEPGGAVGPNLTCRWVIEAAEGRRLHLHFERVSLDEDN FIG.5J >humanSEZ6L2ECDprotein METDTLLLWVLLLWVPGSTGDGAPLPLKEEEILPEPGSETPTVASEALAELLHGALLRRGPEMGYLPGSDRDPTLATPPAGQTLAVPSLPRATEPGTGPLTTAVTPNGVRGAGPTAPELLTPPPGTTAPPPPS PASPGPPLGPEGGEEETTTTIITTTTVTTTVTSPVLCNNNISEGEGYVESPDLGSPVSRTLGLLDCTY SIHVYPGYGIEIQVQTLNLSQEEELLVLAGGGSPGLAPRLLANSSMLGEGQVLRSPTNRLLLHFQSP RVPRGGGFRIHYQAYLLSCGFPPRPAHGDVSVTDLHPGGTATFHCDSGYQLQGEETLICLNGTRPS DRLMVRSGGSPLSPVIYDSDMDDVPERGLISDAQSLYVELLSETPANPLLLSLRFEAFEEDRCFAPF LAHGNVTTTDPEYRPGALATESCLPGYALEPPGPPNAIECVDPTEPHWNDTEPACKAMCGGELSE PAGWVLSPDWPQSYSPGQDCVWGVHVQEEKRILLQVEILNVREGDMLTLFDGDGPSARVLAQL RGPQPRRRLLSSGPDLTLQFQAPPGPPNPGLGQGFVLHEKEVPRNDTCPELPPPEWGWRTASH GDLIRGTVLTYQCEPGYELLGSDILTCQWDLSWSAAPPACQKIMTCADPGEIANGHRTASDAGFP VGSHVQYRCLPGYSLEGAAMLTCYSRDTGTPKWSDRVPKCALKYEPCLNPGVPENGYQTLYKHH YQAGESLRFFCYEGFELIGEVTITCVPGHPSQWTSQPPLCKVAYEELLDNRKLEVTQTTDPSRQLEG GLAGHHHHHHHHH. U . S . Patent Jun . 12, 2018 Sheet 25 of 72 US 9 ,993 . 566 B2

3. 11,4 C 14.1 HEY1 5.6 9.3 0.3 1.4 0. 2602.0 0633.9 285.29 ?.80. 1.51.? 2.10 55.1 72.6 35.1 4.3 ?. 2.4 1.9 8.1 0.7 HESE |0.2 ? ? NE

:: : :: : :: :

9.13 q 1.17 13.813 11.9 thyA 5.4 23.2 5 5.6?, 28.5 ?.19.4 37.0 423.204 1.3119 18.22k8 14.660401 ?. 0. ?. 0.30.3 ?. O> Q. 7.5s6,84SA?? 17.7 ?? 0.2 01.2?114 970. 23.0|3.4 0.46 94.026907282.70.7 4.0. L2888023.125 5 4410.215 11.4 3450.10.3 612550. 0. 0.0. ? 5.7578418S634 .0 80.8 4 ??? 0.114 0. 0.24. ??. 3.

3.0 0. 0.4 0. 0.2 ?.0. 0. 0. 0. 3.5

0 0.8ao256asks11.3 0.4158 * 0.3246.8* 5.0 ?.9 5.6 1.3 0.1 0.6 1.4 3.2

273.122485 27.504 0.4385.982114331 0.75.7 S854.0 8a500.94415. 14.5 316. 71.7 8.1 FIG,6A 37.956.8 0.20.7 RelativeExpressionValuesformRNATranscriptsAssociatedwith 881.540, .1195,80 20.01325?? 84.5532149 81.5 NeuroendocrineTumorsinNTXLinesusingWholeTranscriptomeSequencing DLL3DLL4NOTCH1NOTCH2NOTCH3NTCH4ASCL1NCAM1CHGAHES1 0.2 1768 27 .154 0.1 12?212.6 07.61042 be10.5 4.50114.478 616.04233. 24.0 8.2 5.6 7.6 0. 31.01998?. 1,6 11, 7. 65.4157?101.017,4 78.1 3.60.4 10.51

12.2119187 0. 0.1 0.1 4.3010.6 8 P 16.021.6 10.8343 0.5 70. ,93 16.0 65.4 0. 0. ?. 0.7 0. ?. 0.5 9.1 0, 0.1 1,6 Q. . 0. |0. 833 DL1. T6, 2.4 17.0 .36 10.9 0. 34.2 26.9 0.2 7,4 2.4 1.81.8 0.80.8 2.6 74. 2.8 Q.] 1.703 ?.3 ?. 0.3 7.1 0.3

LU137p0-LU_Ad LU146p0-LU_Ad LU153p0-LU_Ad LU49p4–0_scc ov7zMETpg-Oy ov91METp0-Ov LU37p3-LCNEC LU64p2-SCLC LU73p1-ScLC SCLCLU86p3- LU95p2-SCLC LU7Op4-LU_scC scCLULU76p5- oy26p3-Q\ oy100p0-Oy Oyoy45p3- Oy55p5-Qy NormalLung1 NormalLung2 NormalLung3 NormalLung4 NormalOvary U . S . Patent Jun . 12 , 2018 Sheet 26 of 72 US 9 ,993 ,566 B2

F 1XMelanocytes?3LugLung NomalNormal Normal > 2Liver Nomal 31LierKidney Normal Nomal 12Kidney Nomal > 2Heart Normal 1&Heartuolo Normal RelativeExpressionValuesForSEZ6TranscriptsinVarious 2Colon NomalEWION FIG.6B CSC1Colon LU95p2 Nomal SamplesasDeterminedbyWholeTranscriptomeSequencing NTG2LU95p main akinghowget min CSCLU86p3 ** NTGLUB?p3 ** CSCLU7301LU6402 Olenkannmanegen CSCNTGLU6402LU4904 NTGLU49p4 for op 3007 # transcript rpm NTGLU37p3 U . S . Patent Jun . 12 , 2018 Sheet 27 of 72 US 9 ,993 ,566 B2

wwwwwwwwwwwwwww swwwy W4XXXXXXXXXXXXXXXXXXXXXXXX

WWWWWWW

* * * * *

Www

wwwwwwwwwwwwwwwwwwwwwwwwwwwwww wwwww WwXrArr*rrrrrrrrrrrrrrrrrrrrrrrrrrrr wwwwwwwwwwwwww FIG.7A MicroarrayDataShowingClusters G SC 49 MARSHAWANA wwwwwwwwwwwwwwwwwwwwwwwww ??? WWWWWWWWWWWWWWWWWWWwwww Attloftot MS Cluster ofTumorswithNeuroendocrineFeatures * * * wwwwww ??????????????? WWW. www www BRO wwwwwwwwwwwwwwwwwwww WWWWW LIOOSOLO wwwwwwwwww -(1)(3) WATU CDE *2) LONGO 20 Cluster

* SCC LUTO2 wwwwwwwwwwwwwwwwwwww WWW LUSIONER 1 WARWAINWARNAWWWWWWWWWWWWWWWWWWWWWWWWWNA KOKKAKAKKAKAKAGUKATIKA wwwwwwwwwwwwwAll ???????? U . S . Patent Jun . 12 , 2018 Sheet 28 of 72 US 9 ,993 ,566 B2

) SCC( LU85 81 1 5239 34301 1249_23643224 134286 24722241 425106 1228251 213160 9028 9184011119 858 ClusterG (°°S )6007 con 94 ) SCLC ( LU100 45 14261 309135 712948 85334 7747 3612899 6884 9166624919 27311 ClusterE EM6 53 97264378 46561833 63941824 77894249 19923613

) SCLC( LU95 2477 1264 6 ) SCLC (qc - LU64 236513624.151 12583585-229 433 149842349297 261825193296 9749 665605204 907344622275391 ClusterD 339011 7024 35962 )SCLC ( LU86 | 7950 40434737 904 ) LCNEC (qc LU37 ) SCLC( LU102 82067408 5869 FIG.7B AverageNormalizedIntensityValues OV26 9664658982389382 2632937146133 ClusterC 75142759075065794 14325341147154725842757 34359784907356164166254 2216152152151614561242 82551875801999727 613733906875636633191515917 forCommonMarkersofNeuroendocrinePhenotype ) LCNEC( LU50 8352 191020541881.191115731262 1316 1831373508273451803315 KD66 5361670902

) samples 48( wo 9 2 67 Median 2441 415 NKX2-1 Gene Symbol ASCL1 CALCA CALCB CHGA CHGB DDC NCAM1 UCHL1 POMC SST SYP GDNFfamilyreceptoralpha1GFRA1 SomatostatinReceptor5SSTR5 KITCD117,Kitreceptor ENO2EnolaseNeural)Gamma( Achaete-scutecomplex homolog1 ChromograninA ChromograninB decaroxylaseDopa PGP9.5 Proopiomelanocortin Synaptophysin Calcitonin CGRP CD56 Somatostatin TTF1 U . S . Patent Jun . 12, 2018 Sheet 29 of 72 US 9 ,993 ,566 B2

) SCC( LU85 492 33 80 153 ClusterG 120 469 2024 678 438 322 1029 ) SCC (LU49 8 29 601 357 2665 28 3776 9131 159 4720. 37 1474 14 1226 )SCLC ( LU95 10298 514 5814 673 335 2502 311 1 136 69 5167 ClusterD ) SCLC ( LU64 3390 98 3137 198 137 2716 680 102 521 513 381 11 37 15 2678_ )SCIC (LU86 11 218 1686 763 5456 1660 2645 743 324 1039 105 14 4502

) LCNEC ( LU37 9664 179 5233 280 82 279 77 132 247 NA14 12 72 N 5 2278 FIG.7C AverageNormalizedIntensityValues forSelectedGenesintheNotchPathwayandASCL1 ) SCLC( LU102 12169 497 5884 301 92 416 103 148 95 420 17 16 2717 OV26 406 481 116 110 41 12 91 13 ClusterC 9382 6232 592 _398 1933 ) LCNEC ( LU50 8238 565 3985 445 129 101 132 114 529 23 NA 2255 KD66 6589 4584 601 128 196 86 157 159 335 34 6 13 1891

)samples 48( No 117 89 87 125 26 101 14 Median 350 614 670 666 1289

ASCL1 DLL1 DLL3 DLL4 HES1 HES6 HEY1 HEYL JAG1 JAG2 NOTCH1 NOTCH2 NOTCH3 NOTCH4 RBP) U . S . Patent Jun . 12 , 2018 Sheet 30 of 72 US 9 ,993 ,566 B2

. 4

. .

. -

- .AS10HN9d$:010708

* * * * * . . . : : : : : ...... IL NeuroendocrineTumorsDerivedbyqRT-PCR ! ! . . IONE $0 ExpressionofHES6mRNAin HES6_HS00936587g1 ucitopu FIG.7D

33N01017.PV:01AOXVW3 .

......

. .WONKOLULON

. .

03+ 5e 00 + 50 01 - 50 02- Se : GUSB . ACTB Norm( Expression Relative U . S . Patent Jun . 12 , 2018 Sheet 31 of 72 US 9 ,993 ,566 B2

o ) SCC( 2006 106 120 492 33 26 29 65 271 20 51 19 17 27 10 227 $ 801 9 3247 348

)SCC ( co 18 188 29 6032 665 28 34161 61 42 108 54 11 11 00 93 (15LU49 ) " Ea a - 4133430459069088372060 0795) 261 81 2899 04 54 ASO1619 201 9992 1880 279 LU100 30503724 2937SA ClusterDEG EM6 14 12 2013 361 146 758 118 1634121 301911 147 512 135 277527 697 ) SCLC( 416 116 981 229 5607 6528 3296 213 4621597 2014 333 514 5814 335 799 45791168 HA 4085 619 7824 5631 2940 ) SCLC( 25196126 6423 135 231 4359206 2257 5124 742 10613585 5555 L364 8 HORA ) SCLC( 26:18. 23 807WA 37 48 113390 548 2731 445 218 1686 551 1642 13131087 2317 2816 13:50 1258 LU86 2600 WM )LCNEC ( 16 91 25 86 LENZ 6643653 327 1409 1265 9 (575 ) 92 33 62 32 1902 75 42 ZOINT SSBA 416 11561409 1870 241 _3963872 11083958 tub222 18521336597 361 $1802734 FIG.7E 26 OV 36 3262. 481 29 35 52 |_7811123279 135 53 15 29 ClusterC _396 406 370 1069012 2468 423- LevelsofSelectedGenesLinkedToNeuralPhenotypes ) LCNEC( 658998238333239344035493987 oo875 730 2432 348 3983129 361 23 12 11 40 797 2620 299 1963 65.2 318 143 25 1667360 5641 10403 1237 3508 18 KD66 2081 18 44 49 941 43

9 62 36 82 123 51 350 670 117 48 21 59 66 145 77 23 26 118 60 113 13 10 26 18 163 48( samples) 5858 1464 5404 Median NGFRAP18484 2624KRT10 19604KRT19 1647SEMA4C Symbol NKX2-2 NKX2-1 Gene ASCL1 BEX1 BEX4 NCAM1 CLDN11 KRT17 KRT7 DLL1 DLL3 HES1 HES6 ISL1 LHX2 NEUROD1 NEUROD2 NRCAM LMO3 SCGN SCG2 SCG3 SEMA6C SEZ6 SEZOL SEZ6L2 SOX11 SOX2 SOX4 SNAP25 SYT4 ZIC2

SRY(sexdeterminingregionY)-box11 SRY(sexdeterminingregionY-box2 SRY(sexdeterminingregionY)-box4 Synaptosomal-associatedprotein,25kDa Achaete-scutecomplexhomolog1 Brain-expressedXlinkedprotein1 Brain-expressedXlinkedprotein3 Brain-expressedXlinkedprotein4 Hairyandenhancerofsplit1 Hairyandenhancerofsplit6 Neurogenicdifferentiation1 Neurogenicdifferentiation2 Neuronalcelladhesionmolecule Seizurerelated6homolog Seizurerelated6homologlike Seizurerelatedhomolog6like2 Cytokeratin10 Cytokeratin17 Cytokeratin19 Cytokeratin7 NK2homeobox2 Rhombotin-3 Secretogranin11 SecretograninIII Semaphorin4C Semaphorin6C SynaptotagminIV Claudin11 Delta-like1 Delta-like3 LIMhomeobox1 Zicfamilymember2 CD56 Islet1 Secretagogin TTF-1 Sin' fü?jd ung' ?ZT 8107 v??4S T€ JO TL SN 6 866 999 78

pancreasNormal Colon Normal D EM6 L- KDY66 D OV26 SCCD - LU120 SCC - LV104 SCC - LU92 SCC - LU85 SCC - LU76 D SCC - LU7O D- SCC - LU49 D- Ad - LU134 SEZO ADD LU97 FIG.7F LCNEC - LUSO AverageNormalizedIntensity LCNEC - LU37 ValuesForSEZ6ObtainedViaMicroarrayAnalysis SCLC - LU124 SCLC LUT17 SCLC - LU102 SCLC - LU100 SCLCD - LU95 SCLC - LWSG D - SCLC - LU80 SCLC - LU73 SCLC LU64

intensity Normalized U . S . Patent Jun . 12 , 2018 Sheet 33 of 72 US 9 ,993 ,566 B2

LU8503La

"LU122p0L??20 LU12480* NeuroendocrineNTXTumorLinesDeterminedbyqRT-PCR : LU12p0** * **** only these 100thLUTthe i LU102p1w ExpressionofSEZ6mRNATranscriptsinSelected LU100p1wat FIG.8A

LA44 W *** **** * )p& LU6 lung liverpancreas heartkidney ?colon Normalt Relativ U . S . Patent Jun . 12 , 2018 Sheet 34 of 72 US 9 ,993 ,566 B2

THY

(DOH

deID la • • • • SCLC-LU @ @D old oo SCC-LU DO LCC-LU NTXTumorLinesDeterminedbyqRT-PCR FIG.8B ExpressionofSEZ6mRNATranscriptsin CAR-LU ••• Ad-LU see to og * 46 : 0UD Norm

.. . 10-51 NormTox 10104 Expression Relative U . S . Patent Jun . 12 , 2018 Sheet 35 of 72 US 9 ,993 ,566 B2

W $ U * rund Buddet Plout Tostia Stomach # Postate e : * Par* i WOXD *-*- wy Aunt FIG.9B eweduwwwwwwwwwwwwwwwwwwwww DAT Kidney heudo3 tu uden * *- Calon * * Ercast * * * * 444 ExpressioninVariousWholeTumorSamplesUsingqRT-PCR Limon kashewwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwAdrenat ) ACTB SYBR norm( Expression Relative

AbsoluteandNormalizedSEZ6mRNA 06* $ uan . Badder * pro . 4 , . Tostis 0 ? Sto?? » . Prostata # ?? QOB 36 Paine * 1 Ovary

* * . what - un? * Livar V6'91: . Kidex w Esopa * etrium Erdon W af? illCervix o interne mwanan YAdemani A ) PCR -ORT ( Ct U . S . Patent Jun . 12 , 2018 Sheet 36 of 72 US 9 ,993 ,566 B2

FIG.10 ProteinSequencesofExemplaryMouse SEZ6ModulatorLightChainVariableRegions 10C–FulllengthLCandHCAASEQSofhs017.200200vl1 Sequencelisting-NASEQS(includingHumanized) 10A-LCAASEQS(incl.Humanized) 10B-HCAASEQS(incl.Humanized) U . S . Patent Jun . 12 , 2018 Sheet 37 of 72 US 9 ,993 ,566 B2

SEQIDNO 20 30 32 3434 36 38 40 42 44 46 48 50 www FR4 FGGGTKLK 1- FGGGTKLEIK QQYYSYPTFGGGTKLEIK LQYDDFPWTFGGGTKLEIK FQGSHVPYTFGGGTKLEIK FGAGTKLELK FGGGTKLK FGSGTKLEIK CDRL3 KQSYNLRT HQINDDPWT IIIII FR3 GVPSRFSGSGSGTQYSLKINSLQSEDFGNYYCQHFWGTPRT|WYQQKQGKSPQLLVYAATNLADFGGGTKLEIK WYQQKPRSSPTPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNSPITFGAGTKLELK WFQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQDRKVPWTFGGGTKLEIK WYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSDYPFTFGSGTKLVIK WYQQKPRSSPKPWIYLISNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNNPPTFGSGTKLELK WYOOKSGTSPKRWIYDTSKLPSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQOWSSTPPT WYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLELK CDRL2 AASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC ProteinSequencesofExemplaryMurineSEZ6ModulatorLightChainVariableRegions WFQQKPGKSPKTLIYRANRLIDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYC DWMTQTPLSLPVSLGDQASISCRSSQSLVHSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTLIPYTFGGGTKLDIK RSSOSIVHINRHTYLGWYLOKPGOSLKLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDMGVYYCFOGTHVPFT FIG.10A FR2FR2 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYFCQQYYNYPYT DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKSYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC SC17.10DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKTEDLALYYCQQYYWFPYTFGGGTKLEIK WYQQKPGQPPKLLIY CDRL1 SANSTVSFMY RASANINSNLV KASQDINSYLT RASSSVSSSYLH SASSSVNYMY SASSSVSYMH KASDHINNWLA -

RASESVEYYGTSLMQ - KPSOSVDYDGDSYMN

LICE 5C17.3DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC I- SC17.14DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC

- FR1 SC17.16DIQMTQSPASLSVSVGETVTITC| QIVLTQSPALMSASPGEKVSLTC DIKMTQSPSSMYASLGERVTITC SC17.11ENVLTQSPAIMSASPGEKVTMTC| $217.15QIVLTQSPALMSASPGEKVTMTC| SC17.17QIVLTOSPAIMSASPGEKVTMTC| SC17.18DIOMTOSSSYLSVSLGGRVTITC SC17.22OWLTQTPLSLPVSLGDQASISC SC17.1 5217.2 SC17,9 SC17.38DIVVTOSPASLAVSLGQRATISC SC17.4 SC17.8 SC17.19DIVLTOSPASLAVSLGORAAISC Name Y-YYYYY U . S . Patent Jun . 12 , 2018 Sheet 38 of 72 US 9 ,993 ,566 B2

SEQIDNO 52 54 56 58 60 62 64 66 68 70 74 80 82

FR4 -

- - - FGGGTKLEIK

- KQSYNLRTFGGGTKLEIK - QQSNSWPLTFGAGTKLELK QQYSGYPLTFGAGTKLELK - FGAGTKLELKQQWNTNPPT QOWSSTPPTFGGGTKLEIK

CORL3 -

- - LQYDEFPPT

FR3 WYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTOFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK WYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLOPEDFGSYYCQHFWSTPPTFGGGTKLEIK WYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSAYPFTFGSGTKLEIK WYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK WYQQKSSTSPKLWIYDTSKLTSGVPGRFSGSGSGNSYSLTISNMEAEDVATYYC|FOGSGYPLTFGSGTKLEIK WYOOKSSTSPKLWIYDTSKLTSGVPGRFSGSGSGNSYSLTISNMEAEDVATYYCFOGSGYPLTFGSGTKLEIK WYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPRTFGGGTKLEIK WYQOKSGTSPKRWIYOTSKLASGVPARFSGSGSGSSYSLTISSMEAEDAATYYC ProteinSequencesofExemplaryMurineSEZ6ModulatorLightChainVariableRegions CDR2 WYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLRINSLESEDIADYYC GVPARFSGSASGTSYSLTISSVEAEDAATYYCWYQQKSGASPKLWIYSTSNLAS| WFOOKPGKSPKTLIYRANRLVDGVPSRFSGSGSGODYSLTISSLEYEDMGIYYC GVPTRFSGSGSGTSYSLTISSMGAEDAATYYCWYQQKPRSSPKPWIYLTSNLAS FIG.10ACont FR2 5C17.40DVVMTQTPLSRPVTLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVENRESGVPDRFSGSGSGTOFTLKISRVEAEDLGVYFCSQNTHVPFTFGSGTKLEIK SC17.27|DWMTOTPLTLSVTIGOPASISCKSSOSLLESDGKTYLNWLLORPGOSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLOVYYCWOGIOHPRTFGGGTKLEIK SC17.39DVLMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYFHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTYVPWTFGGGTKLEIK CDRL1 SC17.24DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKSYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC RASQSIGTSIH KASQDVGTDVA RASSSVISSYLH RASGNIHNYLV KASODINSYLS RASSSMSSSYLH SASSSVSYMY SASSSVSYMY SASSSVNYMY SASSSVNYMY KASQSVNNDVA SASSSVSYMH

FR1

- - SC17.46SFVMTQTPKFLLVSAGDRVTITC| - QIVLTQSPALMSASPGEKVTMTC SC17.41QIVLTQSPALMSASPGEKVTMTC SC17.45ENVLTOSPAIMSASPGEKVTMTC 5C17.47QIVLTOSPAIMSASPGEKVSMTC Name 5C17.28DILLTQSPAILSVSPGEGVSFSC SC17.29DIVMTQSHKEMSTSVGDRVSITC SC17.30ENVLTQSPAIVSASPGEKVTMTC SC17.32DIOMTQSPASLSASVGETVTMTC SC17.34DIKMTQSPSSMYASLGERVTITC SC17.35ENVLTOSPAIMSASPGEKVTLTC SC17.36 5C17.42ENVLTQSPAIMSASPGEKVTMTC U . S . Patent Jun . 12 , 2018 Sheet 39 of 72 US 9 , 993 , 566 B2

TW SEQIDNO 84 86 88 90 92 94 96 98 100 102 104 106 108 110 112 114

. FR4 FGGGTKLEIK FGGGTKLEIK |FGGGTKLEIK FGGGTKLEIK QOGYSSPFT|FGGGTKLEIK FGGGTKLEIK QHFWSTPPTFGGGTKVEIK CDRL3 WWW QQYSKLPYT

FR3 WYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPPT|FGAGTKLELK WYQQKPGQPPKLLIKYASNLES|GVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPWTFGGGTKLEIK WYQQKPGHPPKLLIRYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIK KSSQSLLYSDGKWLLQRPGQSPKRLIYLVSKLDSTYLNGVPDRFTGSGSGTDFTLKISRVEAEDLGLYYCWQGTHFPWT|FGGGTKLEIK WW WYQQKPGSSPKLWIYSTSNLASGVPPRFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPPTFGGGTKLEIK WOOKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQOYHSYPWT GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC WYQQROGKSPQLLVYNAKTLAVGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWNTPPTFGGGTKLEIK WYQQKQGKSPQLLVYNAKALADGVPSRESGSGSGTQYSLKINSLOPEDFGSYYCQHFWSIPPT| CDRL2 FIG.10ACont ProteinSequencesofExemplaryMurineSEZ6ModulatorLightChainVariableRegions ???????????????? WYQOKOGKSPOLLVYNAKTLADGVPSRFSGSGSGTOYSLKINSLOPEDFGTYFCQHFWSIPPT YASNRYTWYQQKPGQSPTLLIS WYOOKPDGTVKLLIYYTSSLHSGVPSKFSGSGSGTDYSLTISNLEPEDIATYYC WYQQKQGKSPQLWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLRINSLOPEDFGSYYCLVYNAKTI FR2 SC17.76DIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVEYPRT|FGGGTKLEIK SC17.49DWMTOTPLTLSVTIGOPASISCKSSOSLLESDGKTYLNWLLORPGOSPKRLIY|LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWOGIQHPRT SC17.56DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYOQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTOFTLTISSVKAEDLAVYFCQQYYNYPYTFGGGTKLEIK SC17.81DIVMSQSPSSLTVSVGEKVTLSCKSSQSLLYSTNOKIYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLAISNVKAEDLAVYYCQQYYSYPYTFGGGTKLEIK CDRL1 TASSSVSSSYLH RASGNLHNYLA SASSSVSYIY KASQSVSNDVA RASGNIHNYLT SASQGISNYLN KASDHINNWLA RASGNIHNYLA RASGNIHNYLARASGNIHNYLA

FR1 DIVLTQSPASLAASLGQRATISCRASQSVSTSSYSYMH SC17.50DIVLTQSPASLAASLGQRATISCRASQSVSTSSYSYMH SC17.54DWLTQTPLTLSVTIGQPASISC 5C17.57QIVLTOSPAIMSASLGERVTMTC SC17.59DIQMTQSPASLSASVGETVTITC| SC17.61QIVLTOSPAIMSASPGEKVTISC SC17.63SIVMTQTPKFLLVSAGDRVAITC TVTIACDIQMTQSPASLSASVGESC17.71 SC17.72DIOMTOTTSSLSASLGDRVTISC SC17.74DIQMTOSSSYLSVSLGGRVTITC SC17.77DIOMTOSPASLSASVGETVTITC SC17.79DIQMTQSPASLSASVGETVTITC Name SC17.53 ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? atent Jun . 12 , 2018 Sheet 40 of 72 US 9 ,993 ,566 B2

SEQIDNO 116 118 120 122 124 126 128 130 132 134 136 138 140 142 144 146 FR4FR4 FGSGTKLEIK AQNLEHPTFGGGTKLEIK CDRL3 HQWSSFT

FR3 WYQQKPGQPPKVLIYRASNLESGIPARFSGSGSRTDFTLTINPVEDEDVATYYCQQSNEDPYT|FGGGTKLEIK WYQOKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMETEDAATYYCQOWSNTPPTFGSVTKLEIK WYQQKSGTSPKRWIY|DTSKLASGVPARFSGSGSGTSYSLTISNMEAEDAATYYCQQWSSTPPTFGGGTKLEIK WYQQKPDGTVTLLIYYTSSLHSGVPSRESGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPWTFGGGTKLEIK WYQQKSGTSPKRWIYDTSKLASGVPPRFSGRGSGTSYSLTISSMEAEDAATYYCQHWSSNPPTFGAGTKLEMK ProteinSequencesofExemplaryMurineSEZ6ModulatorLightChainVariableRegions CDRL2 WYQQKSGTSPKLLIYSTSNLASGYPSRFSGSGSGTFYSLTISSVEAEDAADYYC FIG.10ACont FR2 SC17.87DWMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLISKVSNRFSGVPDRFSGSGSGTOFTLKISRVEAEDLGVYFCSQSTHVPPMFGGGTRLEIK SC17.89DVLMTOTPLSLPVSLGDQASISCRSSOSIVHSNGNTYLEWYLOKPGOSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC|FOGSHVPFTFGSGTKLEIK SC17.115DIVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYFCSQSTHLPRTFGGGTKLEIK| SC17.90DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRKSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCHQYYSYPLTFAAGTKLELK 5217.91DVVMTOTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLLWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFALKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK SC17.93DIVMSQSPSSLAVSVGEKVTMTC|KSSOSLLYSSNOKNYLAWYOQKPGOSPKLLIYWASTRESIGVPDRFIGSGSGTDFTLTISSVKAEDLAIYYCQQYYRYPLTFGAGTKLELK 5217.97DWMTQTPLSLPVSLGDQASISCRSSQSLVHNNGNTYLHWYLQKPGQSPNLLIY|KVSNRFSGVPDRFSGSGSGTDFTLKISIVEAEDLGLYFCSQSTHVPRTFGGGTKLEIK SC17.99DIVMSQSPSSLAVSVGEKVTMNCESSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRDSGVPDRFTGSGSGTOFTLTISSVRAEDPAVYYCQQYYSYPLTFGAGTKLELR 5C17.114DWMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSSRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPFTFGSGTKLEIK CDRL1 SASSSVSYMH SASSSISYMH SASSSVSYMH SASSSVSYMM SC17.85DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPERFSSSGSGSDFTLRISRVEAEDVGVYYC www.wwwwwwwwwwwwwww SASQGINNYLN

FR1 SC17.121QIVLTOSPAIMSASPGEKVTMTC SC17.82QIVLTOSPAIMSASLGEEITLTC QIVLTQSPAIMSASPGEKVTMTC|SC17.84 $217.102QIVLTQSPAIMSASPGEKVTMTC SC17.120DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMH Name SC17.95DIQMTQTTSSLSASLGDRVTISC U . S . Patent Jun . 12 , 2018 Sheet 41 of 72 US 9 ,993 ,566 B2

SEQIDNO 148 150 152 154 156 158 160 162 164 166 168 FR4 FGGGTKLEIK QOWSSTPPTFGSGTKLEIK CDRL3 QQLVEYPRT EIPARFSGSGSGTDFTLTINPVEADDVATYYCQQSHEDPYTFGGGTKMEIK FR3 WYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISNMEAEDAATYYCQQWSSTPPTFGGGTKLEIK WFQQKPGKSPETLIYRANRLIDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPPTFGGGTKLEIK WYQOKSGTSPKRWIYDTSKLASGVPARFTGSGSGTSYSLTISSMEAEDAATYYCQOWSSSPPTFGAGTKLELK WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDATTYYCQHSRELPYTFGGGTKLEIK WFQOKPGOPPKLLIYAASNVOSGIPARFSGSGSGTDFTLNIHPVEEEDAATFYC1QOSIEDPPTFGGGTKLEIK CORL2 RASESVDSYGNSFMHWYQQKPGQPPKPLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIK FIG.10ACont ProteinSequencesofExemplaryMurineSEZ6ModulatorLightChainVariableRegions WYOOKSGTSPKRWIY|DTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC RASNLESWFQQKPGQPPKLLIY FR2FR2 SC17.156DVVMTQSPLSLPVSLGDQASISC|RSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSGSGTDFTLKISRVEAEDLGVYYCFOGSHVPPTFGGGTKLEIK SC17.161DIVMSOSPSSLAVSVGEKVTMNCESSOSLLYNSNOKNYLAWYQOKPGOSPKLLIYWASTRDSGVPDRFTGSGSGTDFTLTISSVRADDPAVYYCQOYFNYPLTFGAGTKLELK CDRL1 SC17.122DIVITQDDLSNPVTSGESVSISC|RSSKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYC SASSSVSYMH RASESVDSYGNSFMH SASSSISYMH KASQDINSYLS SASSSVSYMH FR1 SC17,193DLVLTQSPASLAVSLGQRATISC|RASESVSTSGYSYM 5C17.200DIVLTQSPASLAVSLGQRATIFC|RASQSVDYNGISYMH SC17.166QIVLTQSPAIMSASPGEKVTMTC 5C17.187DIKMTQSPSSMYASLGERVTLTC Name QIVLTOSPAIMSASPGEKVTMTCs017.140 SC17.151DIVLTQFPASLAVSLGQRATIPC SC17.191QIVLTOSPAIMSASPGEKVTMTC SC17,199DIVLTQSPASLAVSLGQRATISC U . S . Patent Jun . 12 , 2018 Sheet 42 of 72 US 9 , 993 , 566 B2

SEQIDNO 170 172 174 176 178 180 182 www 184 186 188 190 192

ww-wwwiv FR4 FGGGTKVEIK w CDRL3 KOSYNLRT WWW

-11

11 WFQQKPGKAPKSLIYRANRLVDGVPSRFSGSGSGTDYTLTISSLOPEDFATYYCLQYDEFPPT|FGQGTKLEIK 11- FR3 11- WYQQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTOFTLTISSLOPEDFATYYCQHFWGTPRT|FGGGTKLEIK WYQQKPGQAPRLLIYRASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSHEDPYT|FGQGTKLEIK -11 WYQQKPGQAPRLLIYAASNVQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSIEDPPT|FGGGTKVEIK 11

-11 RASQSVDYDGISYMHWYQQKPGQAPRLLIYAASNVQSGIPARFSGSGSGTOFTLTISSLEPEDFAVYYCQQSIEDPPT|FGGGTKVEIK WYOOKPGOAPRLLIYDTSKLPSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQOWSSTPPT|FGQGTKLEIK WYOQKPDQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNSWPLT|FGQGTKLEIK WYOOKPGKAPKLLIYYASNRYTGVPSRFSGSGSGTDFTLTISSLOPEDFATYFCOODYSSPRT|FGOGTKLEIK 11

11-

CDRL? .

. . FIG.10ACont . DIVMTQSPDSLAVSLGERATINCESSQSLLYNSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYFNYPLTFGQGTKLEIK ProteinSequencesofExemplaryHumanizedSEZ6ModulatorLightChainVariableRegions . DIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFOGSHVPPTFGGGTKLEIK FR2 DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNOKSYLAWYOQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTOFTLTISSLQAEDVAVYYC GVPDRFSGSGSGTOFTLTISSLQAEDVAVYYCFGQGTKLEIKHQYYSYPLT|DIVMTQSPDSLAVSLGERATINCWYQQKPGQPPKLLIYWASTRKSKSSQSLLYSSNQKNYLAh5C17.155 wwwwwwwwwwwww CDRL1 RASANINSNLV SASSSVSYMM RASQSIGTSIH KASODINSYLS KASOSVNNDVA RASESVDSYGNSFMH RASQSVDYNGISYMH FR1FR1 DIQMTOSPSSLSASVGDRVTITC EIVLTOSPATLSLSPGERATLSC EIVLTQSPDFQSVTPKEKVTITC AIQMTOSPSSLSASVGDRVTITC| EIVLTOSPATLSLSPGERATLSC EIVLTOSPATLSLSPGERATLSC Name hSc17.16 hSC17.17 hs017.24 hs017.28 hs017.34 hs017.46 hs017.151 WILIMALIIMIMII- n5C17.156 hs017.161 hs017.200 H5C17.2001L1EIVLTOSPATLSLSPGERATLSC U . S . Patent Jun . 12 , 2018 Sheet 43 of 72 US 9 ,993 ,566 B2

SEQIDNO 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 FR4FR4 WGQGTTLTVSS |WGQGTTLTVSS WGQGTLVTVSA WGQGTTLTVSS WGQGTSVTVSS WGQGTSVTVSS WGQGTLVTVSA WGQGTLVTVSA WGQGTLVTVSA WGQGTTLTVSS WGQGTLVTVSA WGQGTLVTVSA | RHRYDGFRYAIDYWGQGTSVTVSS| | RDYYGSSYVMFAYWGQGTLVTVSA| | |KGRTARATRGFAYWGHGTLVTVSA GYYGSSYDALDYIWGQGTSVTVSS CDRH3 TNWDYFDY SYSNYFDY RGTPGKPLW YDKGFDY IRQYYYAMDY GGTGYTMDY NKGWFAY GPAAY GNVY WGTTWGAN TYSYYSYEFAY SRYYYDAYGFAY FR3 RISITRDTSKNOFFLQLNSVTTEDTATYYCAT KATLTADKSSSTAYMQLNSLTSEDSAVYFCAR KATLTVDTSSSSAYMQLSSLTSEDSAVYFCAR KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR RLTISKDTSNNQVELKIASWTADTATYFCAR |KATLTVDKSSSTAYMQLSSLTSEDSAVYYCVR RFAFSLETSASTAYLOINNLKNEDTATYFCVK KATITADTSSNIAYIQLSSLTSEDSAVYYCAG |KATITADTSSNTAYLQLSSLTSEDTAVYYCVR KATLTVDKSSSTAYMELRSLTSEDSAIYYCAR KATLTVDKSSSTAYMELNSLTSEDSAVYYCTR RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR KATLTVHKSSSTAYMELRSLTSEDSAVYYCAR RLTISKOTSSNQVFLKITSVDTADTATYYCAR RISITROTSKNOFFLQLNSVTTEDTATYYCAR RFAFSLETSASTAYLQINNLKNEDMATYFCTR ProteinSequencesofExemplaryMurineSEZ6ModulatorHeavyChainVariableRegions CDRH2 NIHNSGGTNYNPSLKS YIYPRDGSTKYNEEFKG AIDPSDSYTSYNPKFKG EINPNNGGTAYNQKFRG PIWWNGDKYYNPALKS VINPSNGRTNYNEKEKN WINTETGEPTYSEDFKG EILPGSGNTNNNEKFKG RIDPANVNTKYDPKFQG EVIPYNDETFYNRKFKD VINPYNGNTRYNQMEKG TITSGGGNTYYPDSVKG EINPNIGGTGYNQKFKG DIWWDDSKYYNPSLKS YIHYSGSTNYNPSLRS WINTYTGEPTYADDFKG FIG.10B FR2 WVKQSHGQSLEWIG WMKQRPEQGLEWIG DYWMNWKQRPGOGLEWIG CVKQRPGQGLEWIG WVKQAPGKGLKWLG SYWIEWIKORPGHGLEWIG DTYMHWVKQRPEQGLEWIG DYNMNWVKQSHGKSLEWIG SYAMSWVRQTPEKRLEWVA WVKONOGKSLEWIG WVKQAPGKGLKWMG CDRH1 DVQLQDSGPGLVKPSQSLSVTCTVTGYSITWGYYWNWIRQFPGNKLEWMG DHTIH QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGMSVGWVRQPSGRGLEWLA SYWIH DYSMH DYNMH NYGMN FR1 SC17.16EVQLQQSGPELMKPGASVKMSCKASGYTFTDYNMYWYKONQGKSLEWIG QVQLQQPGAEIVBPGASVKLSCKASGYTFT SC17.11EVQLQQSGPELVKPGALVMMSCKASGYTFTDYYMH SC17.18QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSTMGVGWIRQPSGKGLEWLA 5C17.19DVQLQESGPGLVKPSQSLSVTCTVTGYSIT5SYTWNWIRQFPGNKLEWMG

SC17.1 SC17.2QVQLQQSDAELVKPGASVKISCKVSGYTFT SC17.9 QVQLQQPGAELVKPGASVKLSCKASGYTFPSC17.3 SC17.4 SC17.8 SC17.38 QIQLVQSGPELKKPGETVKISCKASGYTFT QVHLQQSGTEVMKPGASVKISCKATGYTFS EVQLQQSGAELVKPGASVKLSCTASGFNIKSC17.10 5C17.14EVQLQQSGPVLVKPGASVKMSCKASGYTIT SC17.15DVKLVESGGGLVKLGGSLKLSCAASGFTFS SC17.17EVOLQOSGPEVMKPGASVKMSCKASGYTFT SC17.22QIOMMOSGPELKKPGETVKISCKASGYSFT Name U . S . Patent Jun . 12 , 2018 Sheet 44 of 72 US 9 ,993 ,566 B2

SEQIDNO 5353 55 57 59 63 65 67 69 79 81 83

FR4 WGQGTLVTVS ruri WGQGTTLTVSS WGPGTLVTVSA WGAGTTVTVSS WGQGTTLTVSS WGQGTSVTVSS WGQGTLVTVSA WGQGTSVTVSS WGQGTLVTVSA WGQGTTLTVSS WGOGTPVTVSV uri-ruri WGOGTTLTVSS WGQGTTLTVSS WGQGTSVTV WGOGTTLTVSS CDRH3 WFSY GYPAFDY RGWYLTOYAMDY PSNWVFDY GGRGFGY GNH GHYDY SYSNYFDY GGVRYFDV HYYYAMDY SITTAWFAY TSYYNKFLPFAY RDYYGTSYVMFAY -"rurir EYYDGTYDAMDY GGYFLYYFDY RFIISRDNAKNILYLQMSSLKSEDTATYYCARDDYYGSSPSYWYFDVWGAGTTVTVSS TIR FR3 KATLTADKSSSTAYMOLNSLTSEDSAVYFCAR |KATLTADKSSSTAYMELRSLTSEDSAVYFCTR KASLTADNPSSTAYMHLSSLTSEDSAIYFCAR KATLTVDK55STSYMELRSLTSEDSAVYYCAG KATLTVDKSSSTAYMELRSLTSEDSAVYYCSR KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR RISITROTSKNQYYLQLNSVTSEDTATYYCAR KATLTVDTSFSSAYMQLSSLTSEDSAVYFCAS RFTISRDNAKNTLYLOMSSLKSEDTAMYYCAR IIIIIII RFTISRDNAKNTLFLOMSSLKSEDTAMYYCTN KATLTVDTSSSTAYMQLSSLTSDDSAVYYCAR KATLTVDKSSSTAYMOLKSLTSEDSAVYYCAR FIG.10BCont RegionsVariableHeavyModulatorChainMurineExemplarySEZ6ProteinSequencesof CDRH2 YISSNDGTIYYADTVRGRETISRDNAKNTLFLQMTSLRSEDTAMYYCAR EIHPHSGSTNYNEKFKGKATLTVDTSSSTAYVDLSSLTSEDSAVYYCVG TVV YIYPRDGSTKYNEEFKG GIDPETGGTAYNQKFKG YISSGSGGTTYNQKFKG EINPHNGGTGYNQKFKG WVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVFLQMNNLRTEDTGIYYCTR YIYPDNGGAGYNQKFKG EINPYNGETFYNOKFKG YINYSGSTYYNPSLKS TIDPSDSYTRYNQKFKG TISSGGGNTYYPDSVKG TINSGGSNTYYPDSVKG NIFPDTTTTNYNEKFKS EINPSTGGTTYNQNFKA FR2 DYYMAWVRQVPEKGLEWANINYDGSSTYYLDSLKS WMKORPEOGLEWIG WVKQTPVHGLEWIG WVKQSHGKSLEWIG WVRQTPEKRLEWVA var WVKQSPGQGLEWIG WVKORPGQGLEWIG CDRH1 DHTIH RSYIHWVKQRPGQGLEWIG NYWMS DYNMDWVKOSHGKRLEWIG DYYIH SDYWNWIRKFPGKKVEYMG SYGMHWVRQAPEKGLEWVA SYAMS RYTMSWVRQTPEKRLEWAA SYWMH SYWIN QVQLQQPGAEIVRPGASVKLSCKASGYTFTDYWMNWVKQRPGQGLEWIG Iruru FR1 5C17.29EVQLQQSGPELMKPGASVKMSCKASGYTFTDYNMHWVKQNQGKSLEWIG YIRRI SC17.47EVQLQQSGPELVKPGASVKISCKASGYSFTDYYMRWKQSPEKSLEWIG

SC17.27QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMH -I

I-

- - 5017.39EVOLVESGGGLVQPGGSRKLSCAASGETES SC17.45QVQLQQPGSVLVRPGDSVKLSCKASGYTFT SC17.28QVHLPQSRPELVKPGASVKISCKASGYGFT SC17.36EVQLQESGPSLVKPSQSQSLTCSVTGDSIT SC17.40 SC17.24QVOLOOSDAELVKPGASVKISCKVSGYTFT 5C17.30EVKLVESEGGLVQPGSSMKLSCTASGFTFS SC17.34EVOLQOSGPELVKPGSSVKISCKASGYTFT SC17.35EVQLQQSGPELVKPGALVKMSCKASGYTFT SC17.41 SC17.46QVQLQQPGAELVKPGASMKLSCKASGYTFT - SC17.42DVKLVESGGGLVRPGGSLKLSCAASGFTFS EVKLEESGGGLVQPGGSMKLSCVASGFTFSSC17.32 DVKLVESGGGLVKLGGSLKLSCAASGFTFS Name - [Typetext] U . S . Patent Jun . 12 , 2018 Sheet 45 of 72 US 9 ,993 ,566 B2

SEQIDNO 85 87 89 91 93 95 97 101 103 105 107 109 111 113 115 www FR4 WGQGTLVTVSA| WGPGTLVTVSA WGTGTTVTVSS WGQGTLVTVRA WGQGTTLTVS WGQGTTLTV WGQGTSVTVSS WGQGTTLTV WGQGTSVTVSS WGQGTLVTVSA WGQGTLVTVSA WGQGTSVTVSS WGQGTSVTVSS watoto WGQGTLVTVSA CDRH3 WFSY VYYGSTYGYFDV SYDYDTWFGY y IGDSSPSDY GRYYGHDYAMDYWGQGTSVTVSS LWDFAMDY MITPYYFDY HYYYAMDY DDGYYVFFAY GDYDGSLWFAY HGWG HYDYAMDY HYYYALDY terte GNAY RLTISKDTSKNQVFLKIANVDTADTATYYCARIGYYSGSSRCWYFDVWGTGSTVTVSS foto FR3 RFTISRDNAKNTLFLQMTSLRSEDTAMYYCAR KATLTADKSSSTAYMELRSLTSEDSAVYFCTR KATLTVDNSSNTTYMELRSLTSEDSAVYYCAR RFAFSLETSASTASLQIINLKNEDTATYFCAR RFAFSLETSASTAFLQIYNLKNEDSATYFCAR KATLTADKSSSTAYMQLNSLISEDSWYFCAR RLTISRDNGKNTLYLQMSSLRSEDTALYYCVR RLSITKDNSKSQVFLKMNSLQTDDTAMYYCAS RFTVSRDNAKNTLYLQMSSLKSEDTAMYYCSR towwwwwfotortortortortorowotworowowowowowowowwwwwwwwwwwwwwwwwwwwwwww KATLTADTSSNTAYLQLSSLTSEDTAVYYCGR FIG.10BCont ProteinSequencesofExemplaryMurineSEZ6ModulatorHeavyChainVariableRegions CDRH2 GIDPETGGTAYNQKFKG YIHPYNGGSGYNQKFKR WVROSPEKGLEWVAEIRMKSNNYATHYAESVKGRFTISRDDSKSCWLOMNNLRPEDTGIYYCTR WINTETGEPTVAEDFKG YIYPGDSNTKYNEKFKG NYGMNWVKCAPGKGLKWMAWINTYTGEPTYADDFKG NYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTR QIWWDDYKYYNPALKS WVRQSPEKGLEWVAEIRLKSNNYSTHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTR AINSNGGSTYYPDTVKG VIWGGGSTNYNSALKS TISSGGTFTYYPDSVKG WVROSPEKGLEWAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRVEDTAIYYCTR RIDPANVNTKYDPKFQG FR2 DYGMHWVRQAPEKGLEWAYISSGSRTIYYADTVKG WVROSPEKGLELVAEIRLISNNYATHYAESVKGRFTISRDDSKSSVYLOMNNLRAEDTGIYYCTR WVKQTPVHGLEWIG WVKQSHGKRLEWIG WVRQSPGKGLKWMG WVKQRPEQGLEWIG ?????????????????? wwwwwwwwwwwwwww WLKQRPEQGLEWIG SYGMSWRQTPEKRLEWVA SYGVDWVRQSPGKGLEWLG SYGMSWVRQTPDKRLEWVA ww CDRHI DYEMH NYWIN DFSIH OVTLKESGPGILOPSOTLSLTCSFSGFSLSTFGMGVGWIROPSGKGLEWLA NYWMN NYWMN DYWMN DTYYH FR1 SC17.53EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMH SC17.59EVKLEESGGGLVQPGGSMKLSCVASGFTFS SC17.63QVQLQQSDAELVKPGASVKISCKAAGYTFTDLTIH QVQLKOSGPGLVAPSQSLSITCTVSGFSLT SC17.56 SC17.77EVKLEESGGGLVOPGGSMKLSCVASGFTFS 5C17.74 SC17.76EVOLVESGGDLVKPGGSLKLSCVASGFTFS SC17.49QVQLQQSGAELVRPGASVTLSCKASGYTFT SC17.50EVOLVESGGGLVKPGGSLKLSCAASGFTFS SC17.54 5C17.57QIQLVOSGPELKKPGETVKISCKASDYTFT SC17.71EVKLEESGGGLVOPGGSMKLSCVASGIIFS SC17.72EVOLVESGGGLVKPGGSLKLSCAASGFTFS SC17.81EVQLQQSGAELVKPGASVKLSCTASGENIN EVKLEESGGGLVOPGGSMKLSCVASGFTFS QIQLVOSGPELKKPGETVKISCKASGYTFT SC17.61 SC17.79EVKLEESGGGLVQPGGSMKLSCVASGFTFS Name [Typetext] U . S . Patent Jun . 12 , 2018 Sheet 46 of 72 US 9 ,993 ,566 B2

SEQIDNO 117 119 121 123 125 127 129 131 133 135 137 139 - 141 143 145 147

FR4FR4 WGQGSTLTVSS WGQGTSVTVSS WGQGTTLTVSS WGQGTQVTVSA WGQGTLVTVST WGQGTLVTVSA WGQGTLVTVSA WGAGTTVTVSS WGQGTSVTVSS AGLGNYVWAMDY|WGOGASVTVSS WGQGTLVTVSA WGAGTTVTVSS WGOGTLVTVSA WGQGTTLTVSS WGQGTLVTVSA WGQGTTLTVSS CDRH3 SGDLYYYAMDY DGNYCFDY QSYSDYVSFAY NTPFAY RDGYFFPWFAY WTLFTY PENWYFDV EDYDGGDYAMDY SYGSSPFDY TGTSY GRWYFDV PAWFAY GNYDY SGSGRFAY GLYFFDY FR3 KATLTVDKSLSTAYMRLNSLTSEDSAVYYCSR KATLTVDKSSSTAYMDLRSLTSEDSAVYYCAR RFTISRDNAKNTLYLQMSSLKSEDTAMYYCVG KASITADTSSNTAYLQLSSLTSEDTAVYYCTT |KATLTVDTSSSTAYVDLSSLTSEDSAVYYCAR RFTISRDNAKNTLFLQMTSLRSEDTAMYYCAR KATLTVDKSSSTAYMQLSSPTSEDSAVYYCAR KATLTVOKSSSTAYMELRSLKSEDSAVYYCAR KATLTADKSSSTAYMQLGSLTSEDSAVYYCAR KATLTVDKSSSTAYMELRSLTSEDSAVYCAM KATLTVDKSSSTAYMELNSLTSEDSAVYYCVI KATLTVDTSSSTAYVDLSSLTSEDSAVYYCAG KAKLTAVTSASTAYMELSSLTNEDSAVYYCSR KATMTVDKSSSTAYMELRSLTSEDSAVYYCVR WVRQSPEKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSSAYLQMNSLRAEDTGIYYCVS FIG.10BCont ProteinSequencesofExemplaryMurineSEZ6ModulatorHeavyChainVariableRegions CDRH2 RVNPNNGGASYNHKFKG EVNPNTGGIGYNQKFKG TISTGGTYTYYPDSVKG RIDPESDNTLYDPKFQG VINPGSGGTNYNEKFKVKATLTADKSSSTAYMQLTSLTSDDSAVYFCTR EIHPNNGSTNYNEKFKG YISRGSSTIHYADTVKG VINPRNGRNNYNEKFKT GINPNNGITSYNOKFKG YINPSTDYTEYNQKFKD EVNPNIGGIGYNQKFKG RVNTNNGGTSYDQKFEG EIHPNSGNTNYNEKFKG AFYPGNSGTYYNQKFKD EINPNTGGTGYNQKFQG FR2 |WVKOSHGKSLEWIG WVKQNQGKSLEWIG WVYQRPEQGLEWIG WVKQRPGQGLEWIG WVKQRPGQGLEWIG WVRQAPEKGLEWVA WVKQRPGQGLEWIG WVKLSHGKSLEWIG WVKQNQGKSLEWIG WVKQRPGQGLEWIG WVKORPGOGLEWIG WVKQHQGKSLEWIG CDRH1 DSYMN DYNMH DYYIH SYWMH DYGMH SYWVH EYTMO SYWMHWVKQRPGQGLEWIG DAWMD DYNIH SYWMH SYWMH DHNIH

HWAHVAHVAR

W wu-www FR1 SC17.85EVOLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLEWVA SC17.89QVQLQQSGAELVRPGTSVKVSCKTSGYAFTNYLIE SC17.114EVQLQQSGPEMVKPGASVKISCKASGYTFTDYYMHWVKQSHGKSLEWIG SC17.82|EVOLOOSGPELVKPGASVKMSCKASGYTFT SC17.120|EVOLEOSGTVLARPGASVKMSCKASGYTFT 5C17.87EVQLQQSGAELLKPGASVKLSCTASGLNIK SC17.90QVQLQQPGSVLVRPGASVKLSCKASGYTFT SC17.91EVOLVESGGGLVKPGGSRKLSCAASGFTFS SC17.93QVQLQQPGAELVKPGASVMLSCKASGYTFT SC17.95EVELQOSGPELVKPGASVKISCKTSGNTYT SC17.97QVQLPQSGAELAKPGASVKISCKASGFTFT 5C17.99EVKLEESGGGLVOPGGSMKLSCAASGFTFS Name $C17.84EVQLQQSGPELMKPGASVKMSCKASGYIFT SC17.102EVOLQOSGPELMKPGASVKMSCKASGDTFT SC17.115QVQLQQSGSVLVRPGASVKLSCKASGYTFT SC17.121EVQLQQSGPELMKPGASVKMSCKASGYTFT [Typetext] U . S . Patent Jun . 12 , 2018 Sheet 47 of 72 US 9 ,993 ,566 B2

NOIDSEQ 149 151 153 155 157 159 161 163 165 167 169

FR4 WGQGTLVTVS WGQGTLVTVSA WGAGTTVTVSS WGQGTLVTVSA WGQGTTLTVSS - WGQGTTLTVSS WGOGTLVTVSA WGQGTALTVSS WGQGTLVTVSA WGQGTTLTVSS - WGQGTLVTVSA

- CDRH3 HGWG SGKGYFAY FYGLYFAY TGTSY DGNYCFDY - SGTGWFAY GLVMDY - SITAAWFAY IPSLRRYYFDY FR3 RFTISRDNAKNTLYLOMSSLKSEDTAMYYCSR KATLTVDKFSSTAFIELRSLTSEDSAIYYCTR KAKLTAVTSASTLYMELSSLTNEDSAVYYCTR RLTISKATSSNQVFLILTSVDTADTATYYCAT KATLTVDKSSSTAYMDLRSLTSEDSAVYYCAR KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR KAKLTAVTSASTAYMELSSLTNEDSAVYYCTR KATLTADKSSTTAYMQLSSLTSVDSAVYFCTR RLTISKOTSNNOVFLKIANVDTADTATYYCARMVYYDYDGGFAY FIG.10BCont ProteinSequencesofExemplaryMurineSEZ6ModulatorHeavyChainVariableRegions CDRH2 EINPNTGGTGYNQKFKG AIYPGKNDTTYNQKFKG HIFWDDDKWYNPSLKS WVRQSPEKGLEWVAEIRSKPNNHATYYAESVKGRFTISRDDSKSSAYLOMNSLRAEDTGIYYCVS EVNPNTGGIGYNQKFKG NIYPNNGGAGYNQNEKD EINPNTGGTGYNQKFKD HIWWNDNKYYNTALKS AIYPGNSDTSYNHKFKG RIYPGEGDTNYSGNFEG FR2 |TISSGGTYTYYPDSVKGSYGMSWVRQTPDKRLEWA WVKQRPGQGLEWIG WVKQNQGKSLEWIG WVKQSHGKRLEWIG WVKORPGQGLEWIG CDRH1 SYWMH DAWMD DYNMH DYNMD SYWMHWVKQRPGQGLEWIG SSWIN

FR1 $C17.140EVQLQQSGPELMKPGASVKMSCKASGYTFTDYNMHWVKQNQGKSLEWIG SC17.156QVTLKESGPGILOPSQTLSLTCSFSGFSLSTSGMGVSWIRKTSGKGLEWLA SC17.191EVQLQQSGPELMKPGASVKMSCKASGYTFTDYNMHWVKQNQGKSLEWIG SC17.193OVTLKESGPGILOPSOTLSLTCSFSGFSLITYGIGVGWIROPSGKGLEWLA

Nane SC17.122EVHLVESGGDLVKPGGSLKLSCAASGFTFS SC17.151EVQLQQSGTVLARPGASVKMSCKASGYTFT SC17.161EVKLEESGGGLVQPGGSMKLSCAASGFTFS 5C17.166EVQLQQSGPELMKPGASVKMSCKASGYIFT SC17.187EVHLQOSGPELVNPGSSVKISCKAAGYTFT SC17.199EVQLQQSGTVLARPGASVRMSCKASGYTFT SC17.200QVQLQQSGPELVKPGASVKISCKASGYAFS [Typetext] U . S . Patent Jun . 12 , 2018 Sheet 48 of 72 US 9 ,993 ,566 B2

- SEQIDNO 171 173 175 177 179 - 181 183 185 187 189 191 193 194 195 196

- FR4 wwwwwwwwwwwwwwwwwwwww WGQGTTVTVSS WGQGTTVTVSS WGQGTTVTVSS WGOGTLVTVSS WGQGTLVTVSS WGQGTLVTV WGOGTLVTVSS WGQGTLVTVSS WGQGTLVTVSS ttttttt WGQGTLVTV WGQGTLVTV WGQGTLVTV WGQGTLVTV CDRH3 YDKGFDY SYSNYFDY GGVRYFDV SITTAWFAY SGKGYFAY WTLFTY FYGLYFAY TGTSY GLVMDY WTLFTY WTLFTY WTLFTY WTLFTY RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR|TYSYYSYEFAYWGQGTLVTVSS RVTLTRDTSISTAYMELSRLRSDDTAVYYCAREYYDGTYDAMDYWGOGTLVTVSS RVTMTROTSTSTVYMELSSLRSEDTAVYYCAR| FR3

- -

- RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR

- HIFWDDDKWYNPSLKS|RLTISKDTSKSOWLTMTNMDPVDTATYYCAT -

- KVTMTRDTSISTAYMELSRLRSDDTAVYYCAR RVTITADTSTDTAYMELSSLRSEDTAVYYCAR RVTSTRDTSISTAYMELSRLRSDDTAVYYCAR RVTITVDTSASTAYMELSSLRSEDTAVYYCSR RVTMTROTSTSTVYMELSSLRSEDTAVYYCAR RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR VTISADKSISTAYLQWSSLKASDTAMYYCTR to RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR |RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR t ProteinSequencesofExemplaryHumanizedSEZ6ModulatorHeavyChainVariableRegions CDRH2 betyderdetfor FIG.10BCont

EINPNNGGTAYNQKFRG EINPNIGGTGYNQKFKG YIYPRDGSTKYNEEFKG YISSGSGGTTYNQKFKG YIYPDNGGAGYNQKFKG - NIFPDTTTTNYNEKFKG EIHPNNGSTNYNEKEKG RIYPGEGDTNYSGNFEG EIHPNNGSTNYNEKFKG EIHPNNGSTNYNEKFKG EIHPNNGSTNYNEKFKG EIHPNDGSTNYNEKFKG

- - DAWMDWRQAPGQRLEWMGEIRSKPNNHATYYAESVKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR ebelievethat FR2 - WVRQAPGQGLEWMGAIYPGKSDTTYNQKFKG -

- WVRQAPGQGLEWMGDYNMY WVRQAPGKGLEWIG WVRQAPGQGLEWMG WROAPGQRLEWIG WVRQAPGQGLEWIG SYWMHWRQAPGQGLEWMG WVROMPGKGLEWMG W WVRQAPGQGLEWMG WVRQAPGQGLEWMG YYWMHWVRQAPGQGLEWMG SYWMHWVRQAPGQGLEWMG CDRH1 QVQLVOSGAEVKKPGASVKVSCKASGYTFTDYNMHWRQAPGQGLEWMG DHTIH wwwwwwwwwwww DYNMD SYWIN SYWMH QVTLKESGPVLVKPTETLTLTCTVSGFSLSTSGMGVSWIRQPPGKALEWLA SSWIN SYWMH FR1 QVQLVOSGAEVKKPGASVKVSCKASGYTFTRSYIH QVQLVOSGAEVKKPGASVKVSCKASGYTFT EVOLVOSGAEVKKPGATVKISCKVSGYTFT OVQLVOSGAEVKKPGASVKVSCKASGYTFT OVQLVOSGAEVKKPGASVKVSCKASGYTFT QVQLVQSGAEVKKPGASVKVSCKASGYTFT VOLVOSGAEVKKPGASVKVSCKASGYTEN QVQLVQSGAEVKKPGASVKVSCKASGFTFS EVQLVOSGAEVKKPGESLKISCKGSGYSFT wwwwwwwwwwwwwwwwwwwwww HSC17.155VH1|QVQLVQSGAEVKKPGASVKVSCKASGYTFD hs017.155VH2QVQLVOSGAEVKKPGASVKVSCKASGYTFTSYWMH H5C17.16 hs017.17 hSc17.24 H5C17.28 ASC17.34 - h5C17.46 - h5C17.151 HSC17.155 hs017.156 hSC17.161 HSC17.200 h5C17.155VH3QVQLVQSGAEVKKPGASVKVSCKASGYTEN Name - HSC17.155VH4QVOLVQSGAEVKKPGASVKVSCKASGYTFN - U . S . Patent Jun . 12 , 2018 Sheet 49 of 72 US 9 ,993 ,566 B2

SEQIDNO 197 198 199 FR4 WGQGTLVTV WGQGTLVTV WGOGTLVTVSS CDRH3 WTLFTY WTLFTY TGTSY FR3 LSSLRSEDTAVYYCARRVTMTRDTSTSTVYME RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR RegionsVariableHeavyModulatorChainExemplaryHumanizedSEZ6ProteinSequencesof CDRH2 EIHPNSGSTNYNEKFKG FIG.10BCont. FR2 WVRQAPGQGLEWMG WVROAPGKGLEWVGEIRSKPNNHATYYAESVKGRFTISRODSKNSLYLOMNSLKTEDTAVYYCAR CDRH1 SYWMH DAWMD

FR1

Name hSC17.155VH5 HSC17.155VH6QVQLVQSGAEVKKPGASVKVSCKASGYTEN hs017.161VH1EVOLVESGGGLVOPGGSLRLSCAASGFTFS U . S . Patent Jun . 12 , 2018 Sheet 50 of 72 US 9 ,993 ,566 B2

SEQID NO 400 401 402

AminoAcidSequencesofExemplaryFullLengthHumanizedAnti-SEZ6AntibodyLightand FullSequence HeavyChains EVHNAKTKPREEQYNSTYRVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FIG.10C LightEIVLTQSPATLSLSPGERATLSCRASQSVDYNGISYMHWYQQKPGQAPRLLIYAASNVQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSIEDPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL FSCSVMHEALHNHYTOKSLSLSPG h5C17.2001L1LightEIVLTQSPATLSLSPGERATLSCRASQSVDYDGISYMHWYQQKPGQAPRILIYAASNVQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSIEDPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Chain GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVVDVSHEDPEVKENWYVDGV Name hs017.200 NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC hs017.200HeavyEVOLVQSGAEVKKPGESLKISCKGSGYSFTSSWINWVROMPGKGLEWMGRIYPGEGDTNYSGNFEGQVTISADKSISTAYLQWSSLKASDTAMYYCTRGLVMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL U . S . Patent Jun . 12 , 2018 Sheet 51 of 72 US 9 , 993 , 566 B2

SEZ6L2XR NO Yes ??? Yes Yes Yes No No No No

SEZOLXR No NO Yes ?? No NO No NO No No No ON No

N.D N.D ND ON Yes N.D Yes ND Yes No ??? No

|RatXRCynoXR Yes(79,5). )1Yes( Yes(5.0) Yes(17.4) Yes(31) 0)3.Yes( (Kd,nM) No ??. No Yes No No No FIG.11A SEZ6ModulatorCharacteristics Mouse XR No Yes Yes Yes Yes ON NO Yes No3 ON3 3

N.D N.D 22.1 17.4 HuAffinity (Kd,nM) ND ND 5.3 62,0

DE A

24SC17. Clone SC173SC173 SC174 SC176 SC17.7 SC1717 SC17.19 SC17.26 S61728 SC17.34 $67.36 SC17.42 SC17.45SC17245 SC17.46 SC1749 U . S . Patent Jun . 12 , 2018 Sheet 52 of 72 US 9 ,993 ,566 B2

SD4**** SD3 SD2CUB2 CorrelationBetween Domain-LevelMappingandInVitroEfficacy .- CUB1 FIG.11B N3 ***

# * * * * * * * * * * * * * S 8 50pm at ) % ( Cells SEZ6- 293 Live U . S . Patent Jun . 12 , 2018 Sheet 53 of 72 US 9 ,993 ,566 B2

21 Cells293THEK-EngineeredonExpression blulu FMOControl(grayfilled) Anti-SEZ6Antibody(blackline) FIG.12A DetectionofSEZ6Surface lauldumneavoastingaanbanlieu SEZ6(SC17.33)? BAVANNAVAALANN YANNYANNYANNYANNYANNYANNALANNYAANNYA U . S . Patent Jun . 12 , 2018 Sheet 54 of 72 US 9 ,993 ,566 B2

€ Skin Prostate 0 0 6Pancreas 88 Ovarian SCLC-Lung SquamousLCNEC -Lung Carcinoid-Lung FIG.12B isUpregulatedinCertainNTXTumors @00000LiverAdeno - Lung SEZ6ProteinExpression 00 Kidney Breast 0.001@ 117 Normal protein total mg per SEZ6 ng U . S . Patent Jun . 12, 2018 Sheet 55 of 72 US 9 ,993 ,566 B2

Tumor (MFI) 199 810 300 1400 237 192 AntiSEZ6Antibody(blackline) XXX * 289 112 22.6 Line(MFI) LU3729.4 LU86|95.1 *37.3LU50 FMO(grayfilled) *

FMOTumor X XX LU100 LU73 KDY66 44654444445444 wwwwwwwwwww wwwwww LU50 KDY66 SEZ6(SC17.10) ????????????????????????????????????????????????????????????????????????????????????????????????????????????? debebandinumándoteldardnombre inimestekvinnen DetectSurfaceExpressionofSEZ6inNTXTumors wwwwwwwwwwwwwwwwwwwwwwww wwwwwwwwwww SEZ6(SC17.28)?SEZ6SC176 10% ?????????????????????????? FIG.13A SelectedSEZ6Modulators LU86 LU73 Baustoiminnallisestikokon10% 10% kallisessaquestapara SEZ6(SC17.6)

rekisteritiseeritzt wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww 1 LU37 LU100 10* asionnement SEZ6(SC17.6)? SEZ6(SC17.42) connaissan inngangssystem10* and winderile mimbwiniowdwww mihimiwa inhimin U . S . Patent Jun . 12 , 2018 Sheet 56 of 72 US 9 ,993 ,566 B2

+ + + + + + + +

FFFFFRIRA w

time FW.FR SC17.42? rekisteriMediationin 6-87*ZIOS .A .FRP

the .A forOTZIOS .RFR mengangg p ng ••••••••••••••••••••••••»hogy a ä y * * * * * * * * * * * * * * * * 727. 1990 .S

. boru-bWer

Wir ! SC17.42?

SC17.28 * SEZ6ProteinisaBiomarkerofCSCsasDeterminedbyFACS an FIG.13B 58.% SC17.10 's? *$! 1247 VTT NA * * * wwwwwwwww homeentertain willy N

m 42

0.347 -W nouo . 80

-+ wirriwaywverirrt The ini+*-

MOPC? * MOPC? ** K i .7%98 dow 558 *+ Honeywnythingpa Now Fb780 CD324 CD324 LU86 LU117 LU64 U . S . Patent Jun . 12, 2018 Sheet 57 of 72 US 9 ,993 ,566 B2

100 (375)

(0/5)

50 DaysPost-Transplant FIG.14B CD324+SEZ6 -CD324+SEZ6 EnhancedTumorigenicityinaLungTumor(LU37) 1000 o TumorCellsExpressingSEZ6Exhibit )mm3 ( Voume Tumor

- *

- MC

SortedFACS -

* * * * * * * * * * * Pripyretiyermeria

- - - eparatorprogryzargram FIG.14A Parent S *7 47 PAKOW SEZ6 wbs. . www 199 CD324 U . S . Patent Jun . 12 , 2018 Sheet 58 of 72 US 9 ,993 ,566 B2

103.0 95.1 114.5 101.2 113.7 ND ND ND ND ND ND ND ND ND ND 10PM ND ND ND ND ND ND ND1 ND ND ND ND ...... lalalalalalal2| 2| 2 | 2| 2 2 2 2 22

101,3 96.4 105.1 108.3 99.6 62.8 97.7 95.4 107.8 111.7 100.9 97,8 17.0 105.4 104.0 107.4 106.3 113.0 53.5 18.8 97.0 71ND ND |111.9 ttttttttttttt 102.3ND |ND111.4 110.8 114.0 wwwwwwwwwww YYYYY 1000M50PM 111. ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Clone SC17.57 SC17.58 SC17.59 SC17,60 SC17.61 SC17,72 SC17,73 SC17.74 SC17,75 SC17.76 SC17.81 SC17.82 SC17.83 SC17.84 SC17.86 SC17.87 SC17.88 SC17.89 SC17,90 SC17.91 SC17.92 SC17.93 SC17.95 SC17,96 SC17.97 SC17,99 SC17.100

w 103.3 50.0 122.9 17.6 16.7 12.9 104.8 121.8 11.3 98.1 91.7 36.8 86.3 12.6 39.2 39,3 109.9 102.1 99.9 28.9 16.5 10PM 87.4 wwwwwwwwww 41.8 92.2 23.6 8.6 99.6

WIN CytotoxicAgentstoSEZ6-ExpressingHEK293TCells 500M ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 16.3 ND ND ND ND ND ND ND ND FIG.15A 112.4 104.7 15.9 9.8 104.9 121.7 11.1 12.7 21.3 102.7 105.4 111.6 11.0 12.4 SEZ6ModulatorsFacilitateDeliveryof 100PM 22.6 47.0 10.4 8. 78.4 9.6 60.3 58.5 21.5 47.3 15.0 8.2 77.0 Clone SC17.29 SC17.30 SC17.31 SC17.32 SC1733 SC17.34 SC17,35 SC17.36 SC17.37 SC17,38 SC17.39 SC17.40 SC17.41 SC17.42 SC17,43 SC17.44 SC17.45 SC17,46 SC17,47 SC17,49 SC17.50 SC17,51 SC17,52 SC17.53 SC17.54 SC17,55 SC17,56

10PM 94.3 94.7 96.9 70.1 38.5 wwwwwwwwwwwwwwwwwwwwwwwwww 34,7 14.3 49.4 22.8 24.4 92.2 64.5 1117 103.4 71.8 110.6 21.4 89.6 14.4 62.7 9.68 38.0 77.5 88.8 15.6 33.6 17.9

12.8 ND11 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 102.3LND 97.3IgG2a1004 26.3 16.9 18.7 8. 23.9 15.2 13.8 56.6 23.8 112.4 107.0 34.9 95.1 11.3 59,3 9.2 36.4 46.9 13.3 49.5 58.7 13.4 21.7 12.8 est Clone|100PM50PM IgG1|103. IgG2b102.3

W IgG2h SC17.1 SC17.3 SC17.4 SC17.6 SC17.7 SC17.8 SC17.9 SC17.10 SC17.11 SC17.12 SC17.13 SC17.14 SC17.15 SC17,17 SC17,18 SC17,19 SC1721 SC17.22 SC17,23 SC17.24 SC17.25 SC17.26 SC17.27 SC17.28 U . S . Patent Jun . 12 , 2018 Sheet 59 of 72 US 9 ,993 ,566 B2

ND ND ND ND ND ND ND ND ND ND ND ND 10PM ND ND ND ND ND etetett ND ND ND NDND ND ND ND

102.5 102.1 21.7 101.2 10.9 105.1 13.5 21.2 98.8 17,3 103.6 21.1 38.0 106.8 7.0 108.0 21.0 9. 40.1 96,2 114.1 70.2 63.2 91.3 0 50pM 21.+ ww.

w 100PM ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Clone SC17.177 SC17.178 SC17.179 SC17.180 SC17.181 SC17.182 SC17.184 SC17.186 SC17.187 SC17.188 SC17.189 SC17.190 SC17.191 SC17.192 SC17.193 SC17.195 SC17.196 SC17,197 SC17.198 SC17.199 SC17.200 SC17.201 SC17,203 SC17204

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 10PM ND ND . www 91ND 106.5ND 19.2ND 103.4ND 109.7ND ND108,9 68.9ND 107.7 101.4 100.8 108.9 30.1 182.8ND SEZ6ModulatorsFacilitateDeliveryof CytotoxicAgentstoSEZ6-ExpressingHEK293TCells 50pM 92.9 15.3 22.5 97.0 96.7 45.9 8.1 102.0 15.3 19.5 18.3 46.0 15. FIG.15A(Cont) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

|Clone1000M SC17.136ND SC17.138ND SC17.140ND SC17.142ND SC17.143ND SC17.145ND SC17.146ND SC17.147ND SC17.176) SC17.137 SC17.149 SC17.151 SC17.154 SC17.155 SC17.156 SC17.161 SC17.162 SC17.163 SC17.166 SC17.167 SC17.168 SC17.169 SC17.170 SC17.173 SC17.175

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND |10PM 11ND 16.2ND 109.9ND 116.6 104.7 108.3 102.7 102.9 108.2 50PM 50.7 13.6 111.4 39.4 115.2 36.0 11.4 10.7 95.4 4107. 111.0 9.1 107. 98,7 80.4 37.3 99.0

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 100PM wwwww Clone SC17.101 SC17.102 SC17.103 SC17.104 SC17.105 SC17.106 SC17.107 SC17.108 SC17.109 SC17.112 SC17.114 SC17.115 SC17.116 SC17.117 SC17.119 SC17.120 SC17.121 SC17.122 SC17.124 SC17.125 SC17.129 SC17.130 SC17.133 SC17.134 SC17.135 U . S . Patent Jun . 12 , 2018 Sheet 60 of 72 US 9 ,993 ,566 B2

IgG2b

4 .

+ . 33:SC17 - LUBO .

120,entre100PM X 6.¢17 $ wwwwwwwwwwwww wwwwwwwwww W pszyjewou SelectedSEZ6Modulators MediateKillingofNSCLCNTXTumorCells LU100p3 FIG.15B 500PM 50PM gG20 6 § qñ LU37 33.SC17 ) %( RLU normalized mething1000M

) % ( RLU normalized U . S . Patent Jun 12 , 2018 Sheet 61 of 72 US 9 , 993, 566 B2

??????????????????? ???????????????????

??? ???????????-????

? .? 1095 1986 ??????

?????????

?????????? LU80

????? LLLLLLLLLLLLLLLLL

????????? LU73 1064

???????????????

- LU195

IHCMeasurementsofSEZ6ExpressiononSCLCNTX ?? LL181 FIG.16A

- CellularH-ScoreForMembraneStaining =??????????????? LU150 149

* *. 1U148 |"'-?????? LU126 ????? 10124 ???? ????????????????????????????????????????????????????????????????????????. ???????????? ????????????? ??????????????? LU102

????????? LU100 ???????????.

300300 150 100 5 U . S . Patent Jun . 12 , 2018 Sheet 62 of 72 US 9 ,993 ,566 B2

LungNormal

CellSpindle /SCLC IHCMeasurementsofSEZ6ExpressioninLungCancer TissueMicroarrays FIG.16B Adenocarcinoma/SCLC SCLC 3007 200 ) Staining Membrane( Score - H Cellular U . S . Patent Jun . 12, 2018 Sheet 63 of 72 US 9 ,993 ,566 B2

+,10% +,40% H+,80% +,80% SC17.140 +/,90% -/+,10% +,90% +,75% +,95%

MouseIgG1 ND IHCMeasurementofSEZ6ExpressioninMedullaryThyroid Tumors FIG.16C MouseIgG2a

Patient1 Patient2 Patient3 Patient4 Patient5 Patient6 Patient8 Patient9 Patient10 (Thy1pOMET) U . S . Patent Jun . 12 , 2018 Sheet 64 of 72 US 9 ,993 ,566 B2

1000 100

$0 ngimiAntibody 0.1 SC1728-A0C2 •MsigG1ADC2 #901724-ADC) SC17.34-ADC2 Hitta 0.00101

) % ( RLU Normalized SCLCandOVNTXTumorGrowthInVitro FIG.17A MurineSEZ6ADCModulatorsSuppress 1000 deiprecise 100 enmanos en erting tuntunen nereti LU64 ngimiAntibody commentareconciudadesconsisteattentesquewas MsIgG1.ADC2 ÁSC1724-A0C2 SC17.28-A0C2 SC17.34-ADC2 0.00101110

) % ( RLU Normalized U . S . Patent Jun . 12, 2018 Sheet 65 of 72 US 9 ,993 ,566 B2

60 n=5/group XA 1020304050 44 ?????????

??? DaysfromRandomization MsigG1-ADC1 SC17.3-ADC1 SC17.17-ADC1 SC17.24-ADC1 OSC17.34-ADC1 VSC17.46-ADC1

Saw ????????????????????????????????????????????• • ??. ???? 09WU * * * 0 #

van it Writiwite * TAUX * ** ** ** * * ** WinW 0

1,790 1,500 1.250 1.000 2901 ( um ) Funto A Jouni LU50 SCLCandNSCLCNTXTumorGrowthInVivo FIG.17B

MurineSEZ6ADCModulatorsSuppress . 25 ???????????? n=4/group * * * * * * * * ? www WY

in SAXY * * … ennek i Am S 255075100 ?????????? C? was DaysfromKandomization MslgG1-ADC1 sc17.3-ADC1 SC17.24-ADC1 SC17.26-ADC1 Vsc17.28-ADC1 SC17.34-ADC1 * *

** * * * * * * ** * * * * * * * * * * * * * ** * * * * * * * ** * * * * * *

. ? ? ,???? . ???? ?????? ,????? ? ???, ??? . ,??????? ? ??? . ???????????? ,??? ? ????? ? ??, ?????????????? ? ?

1,400 +LU86 ) max( Volume Tumor U . S . Patent Jun . 12 , 2018 Sheet 66 of 72 US 9 ,993 ,566 B2

hSC17.200-ADC1 ADC1hulgG1-v 10000

VitroCellsInCancerThyroidGrowthofSuppress 100 HumanizedSEZ6ADCModulators -I FIG.18A ADCPM 1

0.01

z i g f ? 0+0.0001 ) % ( RLU Normalized U . S . Patent Jun . 12 , 2018 Sheet 67 of 72 US 9 ,993 ,566 B2

VSC17.200-ADC1

HumanizedSEZ6ADCModulatorsSuppressGrowthof ThyroidTumorsInVivo FIG.18B 102030 Daysfromrandomization

2007 100 50 - ) mm( Volume Tumor U . S . Patent Jun . 12 , 2018 Sheet 68 of 72 US 9 ,993 ,566 B2

80 . <

. n=5/group

XXK heregetto * * * * 2040 DaysfroniRandomization HulgG1-ADC1 hSC17.17-ADC1 hs017.34-ADC1 SC17.46-ADC1

wrmWNA UMYWKAve NWASAMA W US * * ** * * *Userstwo Wasiwrnwy r . ric vr4416wi? * * ?* ????????werswewwwwww??? 0 V

wwwwwwwwwwwwwwww 1,200 +60 800 2033 6 LUNI) Ownjon jouni SuppressSCLCNTXTumorGrowthInVivo LU64 HumanizedSEZ6ADCModulators FIG.18C 50

* * * * * n=5/group

. 10203040 DaysfromRandomization HulgG1-ADC1 hSC17.17-ADC1 hSC17.24-ADC1 hSC17.34-ADC1 Vhs017.46-ADC1

Musisivi

* * * * * * * * * * * * *14 + 19r * * 0

> » ??????????? ?????????????? * * * * * * * * * * *

1,200 LU80 ( KALI DIO A Joun k . U . S . Patent Jun . 12 , 2018 Sheet 69 of 72 US 9 ,993 ,566 B2

???????.????+133????? n=5/group . wees 4060 ** * Na . . HulgG1-ADC1 hSC17.17-ADC1 hSC17.34-ADC1 Vhs017.46-ADC1 20 DaysfromRandomization

VASAR " * * W * * * NUMEURS L A * V* * * * * * ** * * * * * * * * * * * * * ** * * * * * * * * ** * ** * * * * * * * ** * * * ** * ** * * * ** ** * * * * * * * * * ** * * * ???? * * * * 12576UNLARI ( nity * i Pik * * * * * VISION 0

. 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . * . * . * . . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . 1 . 1 . . . . . * . * . . . * . * ...... * .* . * . . 4 . 4. 4 . 4. 1

001) 1.000 000 3 ( UNU ) UNO A JOUUNI ShowDifferentialEfficacyonSCLCNTXTumorGrowthInVivo ITINI HumanizedSEZ6ADCModulators FIG.18D

* * * * 1444 m 100 * * * * * * * * * * n=4/group Irrer

95 ,14

5075

. . .

. - - - - ak - hSC17.17-ADC1 hSC17.24-ADC1 hSC17.34-ADC1 . HulgG1-ADC1 VhSC17.46-ADC1 SW 2002 B RandomizationDaysfrom ???? ?????????????? : 25 * * * ??????????????????????????????????????????????????????????????? * * * * * ** * * * * * * * * * * * * * * * * * * ** > V I X X * * WM VWwWowowworks VAWATAN * * * * * ** * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * 0

XY XUNUN w 1,200 se o LU1171,000 ) mm( Volume Tumor atent Jun . 12 , 2018 Sheet 70 of 72 US 9 ,993 ,566 B2

1000 mmm.LU124p2 -LU1240XAHIp2 -LU1240XAHIp3 1011100 cellline FIG.19 Www UMOxaliplatin Invitrosensitivityparentalofandoxaliplatin-resistanttumor WITHIN 1111111111111111011 primullaperucapanpropoTTTT- itust 0.01 M 11:11

HOT etafiti

1409 120 0- 0.001 RLU control % atent Jun . 12 , 2018 Sheet 71 of 72 US 9 ,993 ,566 B2

LU124p1 LU124p3 SEZ6mRNAexpressiondeterminedbymicroarray FIG.20 LU1240XAHIp3 6000 2000 Value Intensity Normalized U . S . Patent Jun . 12 , 2018 Sheet 72 of 72 US 9 ,993 ,566 B2

LU124p3

X OLU1240XAH1p3

SEZ6proteinexpressioninoxaliplatin-resistanttumorcellline SEZ6 FIG.21

? q? protein total mg per target ng US 9 , 993 ,566 B2 SEZ6 MODULATORS AND METHODS OF are non - responsive or minimally responsive to such treat USE ments . For example , in some patients tumors exhibit gene mutations that render them non -responsive despite the gen CROSS REFERENCED APPLICATIONS eral effectiveness of selected therapies . Moreover, depend 5 ing on the type of cancer and what form it takes some This application claims priority from U . S . Provisional available treatments , such as surgery , may not be viable Patent Application Ser . No . 61/ 871 , 289 filed on Aug. 28 , alternatives. Limitations inherent in current standard of care 2013 , which is incorporated herein by reference in its therapeutics are particularly evident when attempting to treat entirety . patients who have undergone previous treatments and have 10 subsequently relapsed . In such cases the failed therapeutic SEQUENCE LISTING regimens and resulting patient deterioration may contribute to refractory tumors which often manifest themselves as a The instant application contains a sequence listing which relatively aggressive disease that ultimately proves to be has been submitted in ASCII format via EFS - Web and is incurable . Although there have been great improvements in hereby incorporated by reference in its entirety . Said ASCII 15 the diagnosis and treatment of cancer over the years, overall copy , created on Aug . 27 , 2014 , is named “ S69697 1130WO survival rates for many solid tumors have remained largely SEQL 082714 for filing ” and is 462 ,073 bytes in size . unchanged due to the failure of existing therapies to prevent relapse , tumor recurrence and metastases . Thus , it remains a FIELD OF THE INVENTION challenge to develop more targeted and potent therapies for 20 proliferative disorders . This application generally relates to novel compounds, compositions and methods of their use in diagnosing , pre SUMMARY OF THE INVENTION venting , treating or ameliorating proliferative disorders and any expansion , recurrence , relapse or metastasis thereof. In These and other objectives are provided for by the present a broad aspect, the present invention relates to the use of 25 invention which , in a broad sense , is directed to methods, seizure related 6 homolog (SEZ6 ) modulators , including compounds , compositions and articles of manufacture that anti - SEZ6 antibodies and fusion constructs , for the treat - may be used in the treatment of SEZ6 associated disorders ment, diagnosis or prophylaxis of neoplastic disorders . ( e . g ., proliferative disorders or neoplastic disorders ). To that Selected embodiments of the present invention provide for end , the present invention provides novel seizure related 6 the use of such SEZ6 modulators , including antibody drug 30 homolog ( or SEZ6 ) modulators that effectively target tumor conjugates , for the immunotherapeutic treatment of malig - cells and / or cancer stem cells and may be used to treat nancies preferably comprising a reduction in tumor initiating patients suffering from a wide variety of malignancies . As cell frequency . Particularly preferred embodiments of the will be discussed in more detail herein , there are at least two invention comprise modulators that associate with specific naturally occurring SEZ6 isoforms or variants and the epitopes and use of the disclosed modulators to treat certain 35 disclosed modulators may comprise or associate selectively patient populations such as those suffering from medullary with one isoform or the other or with both . Moreover, in thyroid cancer , small cell lung cancer and patients that are certain embodiments the disclosed SEZ6 modulators may resistant to standard of care platinum based agents . further react with one or more SEZ family members ( e . g . , SEZOL or SEZ6L2 ) or, in other embodiments , may be BACKGROUND OF THE INVENTION 40 generated and selected for so as to exclusively associate or react with SEZ6 isoform ( s ) . In preferred embodiments the Stem and progenitor cell differentiation and cell prolif - invention is more particularly directed to isolated SEZO eration are normal ongoing processes that act in concert to modulators comprising antibodies ( i. e . , antibodies that support tissue growth during organogenesis and cell replace immunopreferentially bind , react with or associate with at ment and repair of most tissues during the lifetime of all 45 least one isoform of SEZ6 ) that, in particularly preferred living organisms. In the normal course of events cellular embodiments , are associated or conjugated to one or more differentiation and proliferation is controlled by numerous cytotoxic agents or therapeutic moieties , for example , factors and signals that are generally balanced to maintain auristatins, amanitins and pyrrolobenzodiazepines . More cell fate decisions and tissue architecture . Thus , to a large over, as discussed extensively below , such modulators may extent it is this controlled microenvironment that regulates 50 be used to provide pharmaceutical compositions useful for cell division and tissue maturation where signals are prop - the prophylaxis , diagnosis or treatment of proliferative dis erly generated based on the needs of the organism . In this orders . regard cell proliferation and differentiation normally occur in selected embodiments of the invention , SEZ6 modu only as necessary for the replacement of damaged or dying lators may comprise a SEZ6 polypeptide or fragments cells or for growth . Unfortunately , disruption of cell prolif - 55 thereof, either in an isolated form or fused or associated with eration and / or differentiation can result from a myriad of other moieties ( e . g ., FC -SEZÓ , PEG - SEZ6 or SEZ6 associ factors including , for example , the under- or overabundance ated with a targeting moiety ) . In other selected embodiments of various signaling chemicals , the presence of altered SEZ6 modulators may comprise SEZ6 antagonists which , microenvironments , genetic mutations or some combination for the purposes of the instant application , shall be held to thereof. When normal cellular proliferation and /or differen - 60 mean any construct or compound that recognizes, competes, tiation is disturbed or somehow disrupted it can lead to interacts , binds or associates with SEZ6 and neutralizes, various diseases or disorders including proliferative disor - eliminates , reduces , sensitizes , reprograms, inhibits or con ders such as cancer. trols the growth of neoplastic cells including tumor initiating Conventional treatments for cancer include chemo - cells . In preferred embodiments the SEZ6 modulators of the therapy , radiotherapy, surgery , immunotherapy ( e . g ., bio - 65 instant invention comprise anti -SEZ6 antibodies , or frag logical response modifiers , vaccines or targeted therapeu - ments or derivatives thereof, that have unexpectedly been tics ) or combinations thereof. Unfortunately , certain cancers found to silence , neutralize , reduce, decrease , deplete , mod US 9 ,993 ,566 B2 erate , diminish , reprogram , eliminate , or otherwise inhibit 158 , SEQ ID NO : 160 , SEQ ID NO : 162, SEQ ID NO : 164 , the ability of tumor initiating cells to propagate , maintain , SEQ ID NO : 166 and SEQ ID NO : 168 and wherein said expand , proliferate or otherwise facilitate the survival, recur - heavy chain variable region comprises an amino acid rence , regeneration and / or metastasis of neoplastic cells . In sequence having at least 60 % identity to an amino acid particularly preferred embodiments the antibodies or immu- 5 sequence selected from the group consisting of amino acid noreactive fragments may be associated with or conjugated sequences as set forth in SEQ ID NO : 21, SEQ ID NO : 23 , to one or more anti - cancer agents ( e . g ., a cytotoxic agent) . With regard to such modulators it will be appreciated that SEQ ID NO : 25 , SEQ ID NO : 27 , SEQ ID NO : 29 , SEQ ID compatible antibodies may take on any one of a number of NO : 31, SEQ ID NO : 33, SEQ ID NO : 35 , SEQ ID NO : 37 , forms including , for example , polyclonal and monoclonal 10 SEQ ID NO : 39 , SEQ ID NO : 41, SEQ ID NO : 43 , SEQ ID antibodies , chimeric , CDR grafted , humanized and human NO : 45 , SEQ ID NO : 47 , SEQ ID NO : 49, SEQ ID NO : 51 , antibodies and immunoreactive fragments and /or variants of SEQ ID NO : 53, SEQ ID NO : 55 , SEQ ID NO : 57 , SEQ ID each of the foregoing . Preferred embodiments will comprise NO : 59, SEQ ID NO : 61 , SEQ ID NO : 63 , SEQ ID NO : 65 , antibodies that are relatively non -immunogenic such as SEQ ID NO : 67, SEQ ID NO : 69 , SEQ ID NO : 71 , SEQ ID humanized or fully human constructs . Of course , in view of 1515 NON : 73 , SEQ ID NO : 75 , SEQ ID NO : 77 , SEQ ID NO : 79 , the instant disclosure those skilled in the art could readily SEQ ID NO : 81, SEQ ID NO : 83 , SEQ ID NO : 85 , SEQ ID identify one or more complementarity determining regions NO : 87 , SEO ID NO : 89 , SEO ID NO : 91 , SEO ID NO : 93 , (CDRs ) associated with heavy and light chain variable SEQ ID NO : 95 , SEQ ID NO : 97 , SEQ ID NO : 99 , SEQ ID regions of SEZ6 antibody modulators and use those CDRs NO : 101 , SEO ID NO : 103 , SEQ ID NO : 105 , SEQ ID NO : to engineer or fabricate chimeric , humanized or CDR grafted 20 107 , SEQ ID NO : 109 , SEQ ID NO : 111 , SEQ ID NO : 113 , antibodies without undue experimentation . Accordingly , in SEQ ID NO : 115 , SEQ ID NO : 117 , SEQ ID NO : 119 , SEO certain preferred embodiments the SEZ6 modulator com - ID NO : 121 , SEQ ID NO : 123 , SEQ ID NO : 125 , SEQ ID prises an antibody that incorporates one or more CDRS NO : 127 , SEQ ID NO : 129 , SEQ ID NO : 131, SEQ ID NO : derived from the light (FIG . 10A ) or heavy (FIG . 10B ) 133 , SEQ ID NO : 135, SEQ ID NO : 137 , SEQ ID NO : 139 , contiguous chain murine variable regions (SEQ ID NOS : 25 SEQ ID NO : 141, SEQ ID NO : 143 , SEQ ID NO : 145 , SEO 20 - 169) set forth therein . Such CDR grafted variable regions ID NO : 147 . SEO ID NO : 149 . SEO ID NO : 151. SEO ID having a human framework and variants thereof are also NO : 153 . SEO ID NO : 155 . SEO ID NO : 157. SEO ID NO : shown in FIG . 10 comprising SEQ ID NOS: 170 - 199 . In preferred embodiments such antibodies will comprise 159, SEQ ID NO : 161 , SEQ ID NO : 163, SEQ ID NO : 165 , monoclonal antibodies and , in even more preferred embodi - 30 SEQ ID NO : 167 and SEQ ID NO : 169. In other preferred ments , will comprise chimeric , CDR grafted or humanized embodiments the selected modulators will comprise heavy antibodies . and light chain variable regions that comprise 65, 70 , 75 or Exemplary nucleic acid sequences encoding each of the 80 % identity to the aforementioned murine sequences . In amino acid sequences set forth in FIGS . 10A and 10B are set still other embodiments the modulators will comprise heavy forth in the appended sequence listing and comprise SEQ ID 35 aand light chain variable regions that comprise 85 , 90 or even NOS : 220 to 399 . In this respect it will be appreciated that 95 % identity to the disclosed murine sequences. the invention further comprises nucleic acid molecules ( and Of course, in view of the instant disclosure those skilled associated constructs , vectors and host cells ) encoding dis - in the art could readily identify CDRs associated with each closed antibody variable region amino acid sequences of the aforementioned heavy and light chain variable regions including those set forth in the sequence listing . 40 and use those CDRs to engineer or fabricate chimeric , More particularly , in selected embodiments compatible humanized or CDR grafted antibodies without undue experi SEZ6 modulators may comprise an antibody having a light mentation . In this regard several website databases are chain variable region and a heavy chain variable region available that automatically designate CDRs and framework wherein said light chain variable region comprises an amino regions (as per any of the commonly used numbering acid sequence having at least 60 % identity to an amino acid 45 systems) upon entry of the subject heavy or light chain sequence selected from the group consisting of amino acid variable region nucleic acid or amino acid sequence . As sequences as set forth in SEQ ID NO : 20 , SEQ ID NO : 22 , such , in selected embodiments the present invention is SEQ ID NO : 24 , SEQ ID NO : 26 , SEQ ID NO : 28 , SEQ ID directed to anti -SEZ6 antibodies comprising one or more NO : 30 , SEQ ID NO : 32 , SEQ ID NO : 34 , SEQ ID NO : 36 , CDRs derived from a variable region sequence set forth in SEO ID NO : 38 , SEO ID NO : 40 , SEO ID NO : 42 , SEQ ID 50 FIG . 10A or FIG . 10B . In preferred embodiments such NO : 44 , SEQ ID NO : 46 , SEQ ID NO : 48 , SEQ ID NO : 50 , antibodies will comprise monoclonal antibodies and , in even SEO ID NO : 52 , SEQ ID NO : 54 , SEQ ID NO : 56 , SEQ ID more preferred embodiments will comprise chimeric , CDR NO : 58 , SEQ ID NO : 60 , SEQ ID NO : 62, SEQ ID NO : 64 , grafted or humanized antibodies . As discussed in more detail SEQ ID NO : 66 , SEQ ID NO : 68 , SEQ ID NO : 70 , SEQ ID below still other embodiments will comprise such antibodies NO : 72 , SEQ ID NO : 74 , SEQ ID NO : 76 , SEQ ID NO : 78 55 conjugated or associated with one or more cytotoxic agents . SEQ ID NO : 80 , SEQ ID NO : 82 , SEQ ID NO : 84 , SEQ ID Another aspect of the invention comprises modulators NO : 86 , SEQ ID NO : 88 , SEQ ID NO : 90 , SEQ ID NO : 92 , obtained or derived from SC17 . 1 , SC17 . 2 , SC17 . 3 , SC17 . 4 , SEQ ID NO : 94 , SEQ ID NO : 96 , SEQ ID NO : 98 , SEQ ID SC17 . 8 , SC17 . 9 , SC17 . 10 , SC17 . 11 , SC17 . 14 , SC17 . 15 , NO : 100 , SEQ ID NO : 102 , SEQ ID NO : 104 , SEQ ID NO : SC17 . 16 , SC17 . 17 , SC17 . 18 , SC17 . 19 , SC17 . 22 , SC17 . 24 . 106 , SEQ ID NO : 108 , SEQ ID NO : 110 , SEQ ID NO : 112 , 60 SC17 .27 , SC17 . 28 , SC17 .29 , SC17 . 30 , SC17 . 32 , SC17 .34 , SEQ ID NO : 114 , SEQ ID NO : 116 , SEQ ID NO : 118 , SEQ S C17 . 35 , SC17 . 36 , SC17 . 38 , SC17 . 39 , SC17 .40 , SC17 . 41 , ID NO : 120 , SEO ID NO : 122 , SEO ID NO : 124 , SEO ID SC17 . 42 , SC17 . 45 , SC17 . 46 , SC17 . 47 , SC17 .49 , SC17 . 50 , NO : 126 , SEQ ID NO : 128 , SEQ ID NO : 130 , SEQ ID NO : SC17 .53 , SC17 .54 , SC17 . 56 , SC17 . 57 , SC17 .59 , SC17 .61 , 132 , SEQ ID NO : 134 , SEQ ID NO : 136 , SEQ ID NO : 138 , SC17 .63 , SC17 .71 , SC17 .72 , SC17 .74 , SC17 .76 , SC17 .77 , SEQ ID NO : 140 , SEQ ID NO : 142 , SEQ ID NO : 144 , SEQ 65 SC17 .79 , SC17 .81 , SC17 . 82 , SC17 . 84 , SC17 . 85 , SC17 .87 , ID NO : 146 , SEQ ID NO : 148 , SEQ ID NO : 150 , SEQ ID SC17 . 89 , SC17 . 90 , SC17 .91 , SC17 . 93 , SC17 . 95 , SC17 .97 , NO : 152 , SEQ ID NO : 154 , SEQ ID NO : 156 , SEQ ID NO : SC17. 99, SC17 .102 , SC17 .114 , SC17 .115 , SC17 .120 , US 9 , 993 ,566 B2 SC17121 , SC17. 122 , SC17 .140 , SC17. 151, SC17. 156 , disclosed modulator or target binding moiety , L is an SC17 . 161, SC17 . 166 , SC17 . 187 , SC17 . 191 , SC17 . 193 , optional linker or linker unit , D is a compatible drug or SC17 . 199 and SC17 . 200 . prodrug and n is an integer from about 1 to about 20 . It will In yet other compatible embodiments the instant invention be appreciated that, unless otherwise dictated by context, the will comprise the CDR grafted or humanized SEZ6 modu - 5 terms " antibody -drug conjugate ” or “ ADC ” or the formula lators hSC17 . 16 , hsC17 . 17 , hSC17 . 24 , hSC17 .28 , SC17 . 34 , M - IL - D ] n shall be held to encompass conjugates comprising hSC17 .46 , SC17 . 151, SC17 . 155 , SC17 . 156 , SC17 . 161 and both therapeutic and diagnostic moieties . In such embodi SC17 . 200 . Still other embodiments are directed to a SEZ6 ments antibody -drug conjugate compounds will typically modulator comprising a humanized antibody wherein said comprise anti -SEZ6 as the modulator unit ( M ) , a therapeutic humanized antibody comprises a light chain variable region 10 or diagnostic moiety ( D ) , and optionally a linker ( L ) that and a heavy chain variable region wherein said light chain joins the drug and the antigen binding agent. Modulators can variable region comprises an amino acid sequence having at be directly or indirectly conjugated to a therapeutic or least 60 % identity to an amino acid sequence selected from diagnostic moiety . Modulators that are joined to the thera the group consisting of amino acid sequences as set forth in peutic or diagnostic moiety with a linker are referred to as SEQ ID NO : 170 , SEQ ID NO : 172 , SEQ ID NO : 174 , SEQ 15 being " indirectly conjugated ” whereas antibodies that are ID NO : 176 , SEQ ID NO : 178 , SEQ ID NO : 180 , SEQ ID joined to the therapeutic or diagnostic moiety in the absence NO : 182 , SEQ ID NO : 184 , SEQ ID NO : 186 , SEQ ID NO : of a linker are referred to as being “ directly conjugated ” . In 188 and SEQ ID NO : 190 and wherein said heavy chain a preferred embodiment, the antibody is a SEZ6 mAb variable region comprises an amino acid sequence having at comprising at least one CDR from the heavy and light chain least 60 % identity to an amino acid sequence selected from 20 variable regions as described above . the group consisting of amino acid sequences as set forth in As previously indicated one aspect of the invention may SEQ ID NO : 171, SEQ ID NO : 173, SEQ ID NO : 175, SEQ comprise the unexpected association of SEZ6 polypeptides ID NO : 177, SEQ ID NO : 179 and SEQ ID NO : 181, SEQ with cancer stem cells . Thus, in certain other embodiments ID NO : 183 , SEQ ID NO : 185 , SEQ ID NO : 187 , SEQ ID the invention will comprise a SEZ6 modulator that reduces NO : 189 and SEQ ID NO : 191. Additionally , certain human - 25 the frequency of tumor initiating cells upon administration ized variants of light (SEQ ID NO : 192 ) and heavy (SEQ ID to a subject. Preferably the reduction in frequency will be NO : 193 , SEO ID NO : 194 , SEQ ID NO : 195 , SEQ ID NO : determined using in vitro or in vivo limiting dilution analy 196 , SEQ ID NO : 197 , SEQ ID NO : 198 and SEQ ID NO : sis . In particularly preferred embodiments such analysis may 199 ) chain variable regions are provided in accordance with be conducted using in vivo limiting dilution analysis com the teachings herein . Moreover , as described immediately 30 prising transplant of live human tumor cells into immuno above nucleic acid sequences encoding the exemplified compromised mice . Alternatively , the limiting dilution humanized heavy and light chain variable regions are set analysis may be conducted using in vitro limiting dilution forth in the appended sequence listing as SEQ ID NOS : analysis comprising limiting dilution deposition of live 370 -399 . human tumor cells into in vitro colony supporting condi Besides the aforementioned aspects , other preferred 35 tions . In either case , the analysis , calculation or quantifica embodiments of the instant invention will comprise SEZ6 tion of the reduction in frequency will preferably comprise modulators associated or conjugated to one ormore drugs or the use of Poisson distribution statistics to provide an therapeutic moieties ( e . g . auristatins , amanitins and pyr - accurate accounting . It will be appreciated that , while such rolobenzodiazepines ) to provide modulator conjugates that quantification methods are preferred , other , less labor inten may be particularly effective in treating proliferative disor- 40 sive methodology such as flow cytometry or immunohisto ders (alone or in combination with other pharmaceutically chemistry may also be used to provide the desired values active agents ). More generally , once the modulators of the and , accordingly , are expressly contemplated as being within invention have been fabricated and selected they may be the scope of the instant invention . In such cases the reduc linked with , fused to , conjugated to ( e . g . , covalently or tion in frequency may be determined using flow cytometric non - covalently ) or otherwise associated with pharmaceuti- 45 analysis or immunohistochemical detection of tumor cell cally active or diagnostic moieties or biocompatible modi- surface markers known to enrich for tumor initiating cells . fiers. As used herein the term " conjugate " or " modulator As such , another preferred embodiment of the instant conjugate ” or “ antibody conjugate ” will be used broadly and invention comprises a method of treating a SEZ6 associated held to mean any biologically active or detectable molecule disorder comprising administering a therapeutically effec or drug associated with the disclosed modulators regardless 50 tive amount of a SEZ6 modulator to a subject in need thereof of the method of association . In this respect it will be whereby the frequency of tumor initiating cells is reduced . understood that such conjugates may, in addition to the Preferably the SEZ6 associated disorder comprises a neo disclosed modulators , comprise peptides , polypeptides , pro - plastic disorder. Again , the reduction in the tumor initiating teins , prodrugs which are metabolized to an active agent in cell frequency will preferably be determined using in vitro vivo , polymers , nucleic acid molecules, small molecules, 55 or in vivo limiting dilution analysis . binding agents , mimetic agents , synthetic drugs, inorganic In this regard it will be appreciated that the present molecules , organic molecules and radioisotopes. Moreover, invention is based , at least in part , upon the discovery that as indicated above the selected drug or therapeutic moiety SEZ6 immunogens are associated with tumor perpetuating may be covalently or non - covalently associated with , or cells ( i . e ., cancer stem cells ) that are involved in the etiology linked to , the modulator and exhibit various stoichiometric 60 of various neoplasia . More specifically , the instant applica molar ratios depending , at least in part , on the method used tion unexpectedly demonstrates that the administration of to effect the conjugation . various exemplary SEZ6 modulators can mediate , reduce , Particularly preferred aspects of the instant invention will deplete , inhibit or eliminate tumorigenic signaling by tumor comprise antibody modulator conjugates or antibody - drug initiating cells ( i. e ., reduce the frequency of tumor initiating conjugates that may be used for the diagnosis and /or treat- 65 cells ). This reduced signaling , whether by depletion , neu ment of proliferative disorders . Such conjugates may be tralization , reduction , elimination , reprogramming or silenc represented by the formula M - [L - D ] n where M stands for a ing of the tumor initiating cells or by modifying tumor cell US 9 ,993 ,566 B2 morphology ( e . g ., induced differentiation , niche disruption ) , Other embodiments comprise a method of reducing metas in turn allows for the more effective treatment of SEZO tasis or tumor recurrence following treatment comprising associated disorders by inhibiting tumorigenesis , tumor administering a SEZ6 modulator to a subject in need thereof. maintenance , expansion and/ or metastasis and recurrence . In a particularly preferred aspect of the invention the SEZ6 Besides the aforementioned association with cancer stem 5 modulator will specifically result in a reduction of tumor cells , there is evidence that SEZ6 isoforms may be impli - initiating cell frequency as determined using in vitro or in cated in the growth , recurrence or metastatic potential of vivo limiting dilution analysis tumors comprising neuroendocrine features. For the pur - More generally preferred embodiments of the invention poses of the instant invention such tumors will comprise comprise a method of treating a SEZ6 associated disorder in neuroendocrine tumors ( e . g . small cell lung cancer and 10 a subject in need thereof comprising the step of administer medullary thyroid tumors ) and pseudo neuroendocrine ing a SEZ6 modulator to the subject. In particularly pre tumors. Intervention in the proliferation of such tumorigenic ferred embodiments the SEZ6 modulator will be associated cells using the novel SEZ6 modulators described herein , ( e . g . , conjugated ) with an anti - cancer agent. In yet other may thereby ameliorate or treat a disorder by more than one embodiments the SEZ6 modulator will internalize following mechanism ( i . e . , tumor initiating cell reduction and disrup - 15 association or binding with SEZ6 on or near the surface of tion of oncogenic pathway signaling ) to provide additive or the cell . Moreover the beneficial aspects of the instant synergistic effects . Still other preferred embodiments may invention , including any disruption of signaling pathways take advantage of the cellular internalization of cell surface and collateral benefits , may be achieved whether the subject SEZ6 to deliver a modulator mediated anti - cancer agent. In tumor tissue exhibits elevated levels of SEZ6 or reduced or this regard it will be appreciated that the present invention 20 depressed levels of SEZ6 as compared with normal adjacent is not limited by any particular mechanism of action but tissue . Particularly preferred embodiments will comprise the rather encompasses the broad use of the disclosed modula treatment of disorders exhibiting elevated levels of SEZ6 on tors to treat SEZ6 associated disorders ( including various tumorigenic cells as compared to normal tissue or non neoplasia ). tumorigenic cells . Thus, in other embodiments the present invention will 25 In yet another aspect the present invention will comprise comprise the use of the disclosed modulators to treat tumors a method of treating a subject suffering from a neoplastic comprising neuroendocrine features, e . g . small cell lung disorder comprising the step of administering a therapeuti cancer and medullary thyroid tumors in a subject in need cally effective amount of at least one internalizing SEZO thereof. Of course the same modulators may be used for the modulator. Preferred embodiments will comprise the admin prophylaxis , prognosis , diagnosis, theragnosis , inhibition or 30 istration of internalizing antibody modulators wherein , in maintenance therapy of these same tumors . other selected embodiments , the internalizing antibody It has further been discovered that the disclosed modula modulators are conjugated or associated with a cytotoxic tors are effective in treating patients that are suffering from agent. small cell lung cancer or medullary thyroid cancer . More - Other embodiments are directed to a method of treating a over, as discussed in more detail below and set forth in the 35 subject suffering from a SEZ6 associated disorder compris Examples the anti -SEZ6 antibodies and antibody drug con - ing the step of administering a therapeutically effective jugates of the instant invention are particularly effective in amount of at least one depleting SEZ6 modulator. treating certain patient populations such as those suffering In yet another embodiment the present invention provides from forms of small cell lung cancer that are resistant to methods of maintenance therapy wherein the disclosed standard of care platinum based agents ( e . g . , carboplatin , 40 effectors or modulators are administered over a period of cisplatin or oxaliplatin ) . Accordingly, in selected embodi- time following an initial procedure (e . g ., chemotherapeutic , ments the present invention comprises a method of treating radiation or surgery ) designed to remove at least a portion of a patient suffering from platinum resistant small cell lung the tumor mass . Such therapeutic regimens may be admin cancer comprising the step of administering a SEZ6 modu istered over a period of weeks, a period of months or even lator. 45 a period of years wherein the SEZ6 modulators may act Other facets of the instant invention exploit the ability of prophylactically to inhibit metastasis and /or tumor recur the disclosed modulators to potentially disrupt oncogenic rence . In yet other embodiments the disclosed modulators pathways while simultaneously silencing tumor initiating may be administrated in concert with known debulking cells . Such multi -active SEZ6 modulators (e . g. , SEZO regimens to prevent or retard metastasis , tumor maintenance antagonists ) may prove to be particularly effective when 50 or recurrence . used in combination with standard of care anti - cancer agents It will further be appreciated that the SEZ6 modulators of or debulking agents . Accordingly preferred embodiments of the instant invention may be generated and selected to react the instant invention comprise using the disclosed modula - with known isoform (s ) of SEZ6 or a single isoform of the tors as anti -metastatic agents for maintenance therapy fol protein or, conversely , may comprise a pan - SEZ6 modulator lowing initial treatments . In addition , two or more SEZ6 55 that reacts or associates with at least one additional SEZO antagonists ( e . g . antibodies that specifically bind to two family member ( e . g . , SEZOL or SEZÓL2 and isoforms discrete epitopes on SEZ6 ) may be used in combination in thereof) in addition to SEZ6 . More specifically , as disclosed accordance with the present teachings . Moreover , as dis - herein preferred modulators such as antibodies may be cussed in some detail below , the SEZ6 modulators of the generated and selected so that they react with domains ( or present invention may be used in a conjugated or unconju - 60 epitopes therein ) that are exhibited by SEZ6 only or with gated state and , optionally , as a sensitizing agent in combi- domains that are at least somewhat conserved across two or nation with a variety of chemical or biological anti - cancer more of the SEZ6 family members . agents . In yet other preferred embodiments the modulators will Accordingly another preferred embodiment of the instant associate or bind to a specific epitope, portion , motif or invention comprises a method of sensitizing a tumor in a 65 domain of SEZ6 . As will be discussed in some detail below subject for treatment with an anticancer agent comprising both SEZ6 isoforms incorporate an identical extracellular the step of administering a SEZ6 modulator to said subject . region (see FIG . 1E ) comprising at least an N - terminal US 9 ,993 ,566 B2 10 domain , two alternating Sushi and CUB domains , and three contain a number of antibody modulators . For the purposes additional tandem Sushi domain repeats . In addition the ofthe instant disclosure the seven bins were termed bins A - F SEZ6 protein comprises a transmembrane domain and a and bin U . Bins A - F are unique bins and the antibodies cytoplasmic domain . Accordingly, in certain embodiments contained in each of these bins compete with each other for the modulators will bind or associate with the N - terminal 5 binding to the SEZ6 protein . Bin U contains antibodies that domain of SEZ6 (i . e . amino acids 1 -335 in the mature do not compete with antibodies in Bins A - F , but may protein ) or to an epitope therein . Other aspects of the instant compete for binding with each other . Thus , in selected invention comprise modulators that associate or bind to a embodiments the present invention will comprise a modu specific epitope located in a particular Sushi domain of lator residing in a bin selected from the group consisting of SEZ6 . In this regard the particular modulator may associate 10 bin A , bin B , bin C , bin D , bin E , bin F , and bin U . In other or bind to an epitope located in Sushi Domain 1 (amino acids embodiments the present invention comprises a modulator 336 - 395 ) , Sushi Domain 2 ( amino acids 511 - 572 ) , Sushi residing in a bin defined by a reference antibody selected Domain 3 ( amino acids 690 - 748 ) , Sushi Domain 4 (amino from the group consisting of SC17 .1 , SC17 .2 , SC17. 3 , acids 750 -813 ) or Sushi Domain 5 (amino acids 817 - 878 ) . SC17 . 4 , SC17 . 8 , SC17 . 9 , SC17 . 10 , SC17 . 11, SC17 . 14 , Other aspects of the instant invention comprise modulators 15 SC17 . 15 , SC17 . 16 , SC17 . 17 , SC17 . 18 , SC17 . 19 , SC17 .22 , that associate or bind to a specific epitope located in a SC17 . 24 , SC17 . 27 , SC17 . 28 , SC17 . 29 , SC17 . 30 , SC17 .32 , particular CUB -like domain of SEZ6 . In this regard the SC17 . 34 , SC17 . 35 , SC17 . 36 , SC17 . 38 , SC17 .39 , SC17 .40 , particular modulator may associate or bind to an epitope SC17 .41 , SC17 .42 , SC17 . 45 , SC17 . 46 , SC17 . 47 , SC17 .49 , located in CUB Domain 1 ( amino acids 397 -508 ) or CUB SC17 . 50 , SC17 .53 , SC17 .54 , SC17 . 56 , SC17 .57 , SC17 .59 , Domain 2 (amino acids 574 -685 ) . In a further embodiment 20 SC17 .61 , SC17 .63 , SC17 .71 , SC17 . 72 , SC17 . 74 , SC17 . 76 , the antibodies of the invention may bind to certain epitopes SC17 .77 , SC17 .79 , SC17 .81 , SC17 .82 , SC17 .84 , SC17 .85 , on SEZ6 . SC17 . 87 , SC17 .89 , SC17 . 90 , SC17 . 91 , SC17 . 93 , SC17 . 95 , In one embodiment, the invention provides for an isolated SC17 .97 , SC17 . 99 , SC17 . 102 , SC17 . 114 , SC17 . 115 , antibody that specifically binds to an epitope on a SEZ6 SC17 . 120 , SC17121 , SC17 . 122 , SC17 . 140 , SC17 . 151 , protein , wherein the epitope comprises amino acid residues 25 SC17 . 156 , SC17 . 161 , SC17 .166 , SC17 . 187 , SC17 .191 , selected from the group consisting of (i ) residues R762 , SC17 . 193 , SC17 . 199 and SC17 . 200 . In still other embodi L764 , 0777 , 1779 , D781 and Q782 ; ( ii ) residues R342 and ments the invention will comprise modulators from bin A , K389 and ( iii ) residues T352 , S353 and H375 . modulators from bin B , modulators from bin C , modulators In another embodiment the invention provides for an from bin D , modulators from bin E , modulators from bin F antibody drug conjugate comprising an antibody conjugated 30 or modulators from bin U . Yet other preferred embodiments directly or indirectly to a therapeutic moiety , wherein the will comprise a reference antibody modulator and any antibody specifically binds to an epitope on a SEZ6 protein , antibody that competes with the reference antibody. wherein the epitope comprises amino acid residues selected The term “ compete ” or “ competing antibody ” when used from the group consisting of (i ) residues R762, L764 , Q777 , in the context of the disclosed modulators means binding 1779 , D781 and Q782 ; (ii ) residues R342 and K389 and ( iii ) 35 competition between antibodies as determined by an assay residues T352 , S353 and H375 . in which a reference antibody or immunologically functional Of course it will be appreciated that each of the afore - fragment substantially prevents or inhibits ( e . g . , greater than mentioned domains may comprise more than one epitope 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % or and may be associated with more than one bin . With regard 90 % . ) specific binding of a test antibody to a common to modulator or antibody “ bins” it will be appreciated that 40 antigen . Compatible methods for determining such compe the SEZ6 antigen may be analyzed or mapped through tition comprise art known techniques such as , for example , competitive antibody binding using art recognized tech - bio - layer interferometry , surface plasmon resonance , flow niques to define specific bins located along the protein . cytometry , competitive ELISA , etc . While discussed in more detail herein and shown in In a selected embodiment the invention comprises a Examples 9 and 10 below , two antibodies ( one of which may 45 pan -SEZ6 modulator that associates with SEZ6 and at least be termed a “ reference antibody, ” “ bin delineating antibody ” one other SEZ6 family member ( e . g ., SEZÓL or SEZÓL2 ) . or “ delineating antibody ” ) may be considered to be in the In other selected embodiments the invention comprises a same bin if they substantially compete with each other for SEZ6 modulator that immunospecifically associates with binding to the target antigen . In such cases the subject one or more isoform of SEZ6 but does not immunospecifi antibody epitopes may be identical, substantially identical or 50 cally associate with any other SEZ6 family member. In yet close enough (either in a linear sense where they are other embodiments the present invention comprises a separated by a few amino acids or conformationally ) so that method of treating a subject in need thereof comprising both antibodies are sterically or electrostatically inhibited or administering a therapeutically effective amount of a pan precluded from binding to the antigen . Such defined bins SEZ6 modulator. Still other embodiments comprise a may be generally associated with certain SEZ6 domains 55 method of treating a subject in need thereof comprising ( e . g . the reference antibody will bind with an epitope administering a therapeutically effective amount of a SEZ6 contained in a specific domain ) though the correlation is not modulator that immunospecifically associates with one or always precise ( e . g . , there may be more than one bin in a more isoforms of SEZ6 but does not immunospecifically domain or the bin may be defined conformationally and associate with any other SEZ6 family member . comprise more than one domain ) . It will be appreciated that 60 In one embodiment the invention is directed to a method those skilled in the art can readily determine the relationship of treating a subject suffering from cancer comprising between the SEZ6 domains and empirically determined administering a therapeutically effective amount of an anti bins. body drug conjugate comprising an antibody conjugated With regard to the present invention competitive binding directly or indirectly to a therapeutic moiety , wherein the analysis using art -recognized techniques ( e . g . , ELISA , sur- 65 antibody specifically binds to an epitope on a SEZ6 protein , face plasmon resonance or bio -layer interferometry ) defined wherein the epitope comprises amino acid residues selected at least seven distinct bins, each of which was found to from the group consisting of (i ) residues R762 , L764 , Q777 , US 9 ,993 ,566 B2 11 12 1779 , D781 and Q782 ; ( ii ) residues R342 and K389 and ( iii ) some cases the reporter associated with the tumor sample residues T352 , S353 and H375. In preferred embodiments will be detected in vitro , for example , using immunohisto the therapeutic moiety will comprise auristatins, amanitins chemistry . In some cases the tumor sample will be exposed and pyrrolobenzodiazepines . In some cases the subject suf to an anti -SEZ6 antibody that binds to an epitope on a SEZO fering from cancer may have previously been treated with a 5 protein , wherein the epitope comprises amino acid residues platinum based agent. R762, L764 , Q777 , 1779 , D781 and Q782 . In another embodiment the invention is directed to a Such methods may be easily discerned in conjunction method of treating a subject suffering from medullary thy - with the instant application and may be readily performed roid cancer comprising administering a therapeutically using generally available commercial technology such as effective amount of an antibody drug conjugate comprising 10 automatic plate readers, dedicated reporter systems, etc . In an antibody conjugated directly or indirectly to a therapeutic selected embodiments the SEZ6 modulator will be associ moiety . In one embodiment the antibody drug conjugate a ted with tumor perpetuating cells present in the sample . In used to treat a subject suffering from medullary thyroid other preferred embodiments the detecting or quantifying cancer may comprise an antibody that specifically binds to step will comprise a reduction of tumor initiating cell an epitope on a SEZ6 protein , wherein the epitope comprises 15 frequency and detection thereof. Moreover , limiting dilution amino acid residues selected from the group consisting of ( i ) analysis may be conducted as previously alluded to above residues R762 , 1764 , Q777 , 1779 , D781 and Q782 ; ( ii ) and will preferably employ the use of Poisson distribution residues R342 and K389 and ( iii ) residues T352 , S353 and statistics to provide an accurate accounting as to the reduc H375 . In a preferred embodiment the therapeutic moiety tion of frequency . may comprise auristatins, amanitins and pyrrolobenzodiaz - 20 In a similar vein the present invention also provides kits epines . or devices and associated methods that are useful in the In a further embodiment the invention is directed to a diagnosis and monitoring of SEZ6 associated disorders such method of treating a subject suffering from platinum resis - as cancer . To this end the present invention preferably tant small cell lung cancer comprising administering a provides an article ofmanufacture useful for diagnosing or therapeutically effective amount of an antibody drug conju - 25 treating SEZ6 associated disorders comprising a receptacle gate comprising an antibody conjugated directly or indi- comprising a SEZ6 modulator and instructionalmaterials for rectly to a therapeutic moiety, wherein the antibody specifi - using said SEZ6 modulator to treat or diagnose the SEZ6 cally binds to an epitope on a SEZ6 protein , wherein the associated disorder. In selected embodiments the devices epitope comprises amino acid residues selected from the and associated methods will comprise the step of contacting group consisting of (i ) residues R762 , L764, Q777 , 1779 , 30 at least one circulating tumor cell . D781 and 2782 ; ( ii ) residues R342 and K389 and ( iii ) Other preferred embodiments of the invention also exploit residues T352 , S353 and H375 . In preferred embodiments the properties of the disclosed modulators as an instrument the patient suffering from platinum resistant small cell lung useful for identifying , characterizing , isolating , sectioning or cancer have previously been treated with a platinum based enriching populations or subpopulations of tumor initiating agent. In another preferred embodiments the therapeutic 35 cells through methods such as flow cytometric analysis moiety will comprise auristatins , amanitins and pyrroloben - including fluorescence activated cell sorting ( FACS ) or laser zodiazepines . mediated sectioning . Beyond the therapeutic uses discussed above it will also As such , another preferred embodiment of the instant be appreciated that the modulators of the instant invention invention is directed to a method of identifying , isolating , may be used to detect, diagnose or classify SEZ6 related 40 sectioning or enriching a population of tumor initiating cells disorders and , in particular , proliferative disorders . In some comprising the step of contacting said tumor initiating cells embodiments the modulator may be administered to the with a SEZ6 modulator. subject and detected or monitored in vivo . Those of skill in The foregoing is a summary and thus contains, by neces the art will appreciate that such modulators may be labeled sity , simplifications , generalizations , and omissions of or associated with markers or reporters as disclosed below 45 detail ; consequently, those skilled in the art will appreciate and detected using any one of a number of standard tech - that the summary is illustrative only and is not intended to niques ( e. g ., MRI, CAT scan PET scan , etc .) . be in any way limiting . Other aspects , features, and advan Thus, in some embodiments the invention will comprise tages of the methods , compositions and / or devices and / or a method of diagnosing, detecting or monitoring a SEZ6 other subject matter described herein will become apparent associated disorder in vivo in a subject in need thereof 50 in the teachings set forth herein . The summary is provided comprising the step of administering a SEZ6 modulator. to introduce a selection of concepts in a simplified form that In other instances the modulators may be used in an in are further described below in the Detailed Description . This vitro diagnostic setting using art - recognized procedures . As summary is not intended to identify key features or essential such , a preferred embodiment comprises a method of diag - features of the claimed subject matter , nor is it intended to nosing cancer ( e . g . medullary thyroid cancer or platinum 55 be used as an aid in determining the scope of the claimed resistant small cell lung cancer ) in a subject comprising the subject matter . steps of: (a ) providing a tumor sample from a subject; (b ) exposing the tumor sample to an anti -SEZ6 antibody labeled BRIEF DESCRIPTION OF THE FIGURES with a reporter wherein said anti - SEZ6 antibody associates with the tumor sample (e . g . a medullary thyroid tumor 60 FIGS. 1A - 1E are various representations of SEZ6 includ sample , a small cell lung cancer tumor sample or a platinum ing nucleic acid or amino acid sequences pertaining to the resistant small cell lung cancer tumor sample ); and ( c ) SEZ6 modulators described herein . FIGS . 1A and 1B (SEO detecting the reporter associated with the tumor sample . In ID NOS : 1 and 2 ) depict the full length mRNA sequence some embodiments, the step of providing the tumor sample containing the open reading frames (ORFs ) ( underlined ) may be performed separately from the step of exposing the 65 encoding the SEZ6 variants 1 and 2 , respectively . FIGS. 1C tumor sample to an anti- SEZ6 antibody or the step of and 1D (SEQ ID NOS: 3 and 4 ) provide the corresponding detecting the reporter associated with the tumor sample . In amino acid sequences of the ORFs denoted in FIGS. 1A and US 9 , 993 ,566 B2 13 14 1B , respectively , with the amino acid residues indicating the FIG . 7A -7F depict mRNA expression levels analyzed predicted transmembrane spanning domain for each protein using microarray . FIG . 7A is a graphical representation of isoform underlined and the amino acid residues ( residues unsupervised clustering ofmicroarray profiles for 46 tumor 1 - 19 ) indicating the signal peptide in bold and underlined ; lines and two normal tissues; FIGS. 7B and 7C are tabular FIG . 1E depicts the alignment of the two protein isoforms 5 representations of normalized intensity values correspond ( SEQ ID NOS : 3 and 4 ) to illustrate the sequence differences ing to relative expression levels of selected genes related to in the cytoplasmic termini of each isoform , with the under - neuroendocrine phenotypes (FIG . 7B ) or the Notch signal lined residues indicating the differences between the two ing pathway ( FIG . 7C ) wherein unshaded cells and rela sequences ; and FIG . 1F provides a schematic representation tively low numbers indicate little to no expression and of the extracellular region of the SEZ6 protein illustrating 10 darker cells and relatively higher numbers indicate higher the positions of the various domains. expression levels; FIG . 7D is a graphical representation FIGS . 2A - 2C provide a tabular representation of the showing relative expression levels of HES6 mRNA in percent identity at the protein level between the closest various tumors and control tissues as measured using qRT human isoform of SEZ6 and rhesus , cynomolgus , mouse or PCR ; FIG . 7E is a tabular representation of normalized rat SEZ6 proteins (FIG . 2A ) ; a tabular listing of various 15 intensity values corresponding to relative expression levels cDNA or protein sequence accessions for each of the of selected genes indicative of neurogenesis , neural com reported isoforms of the SEZ6 family of genes (FIG . 2B ) ; mitment, or differentiation towards neural fates , with un and the percent identity at the protein level between the shaded cells indicating little to no expression and darker longest isoforms of human SEZO , SEZÓL , and SEZ6L2 cells indicating higher expression levels ; and FIG . 7F is a proteins (FIG . 2C ). 20 graphical representation of normalized intensity values cor FIGS. 3A - 3C provide various representations of nucleic responding to relative expression of SEZ6 in various NTX acid or amino acid sequences related to the production of the tumor lines . immunogens or cell lines used to generate or characterize FIGS. 8A and 8B are graphical representations showing the SEZ6 modulators described herein . For human SEZ6 a relative expression levels of SEZ6 mRNA transcripts as specific cDNA clone ( FIG . 3A ; SEQ ID NO : 5 ) encoding the 25 measured by RT- PCR in a variety of RNA samples isolated complete mature human SEZ6 protein ( FIG . 3B ; SEQ ID from normal tissues or bulk neuroendocrine NTX tumors NO : 6 ) was constructed from a commercial cDNA clone ( FIG . 8A ) and a variety of other NTX tumors ( FIG . 8B ) . (BC146292 ; SEQ ID NO : 7 ) with known differences ( FIG . FIGS . 9A and 9B are graphical representations showing 3C ) from a database reference sequence , NP _ 849191 ( SEO the absolute (FIG . 9A ) or normalized (FIG . 9B ) mRNA ID NO : 3 ) , for the SEZ6 protein . 30 expression levels of human SEZ6 as measured by RT- PCR FIGS. 4A and 4B provide a cDNA (FIG . 4A ; SEQ ID NO : in whole tumor specimens (grey dot) or matched normal 8 ) used to express an Fc - SEZ6 construct in CHO - S cells and adjacent tissue (NAT ; white dot ) from patients with one of yield a protein immunogen ( FIG . 4B ; SEQ ID NO : 9 ) , eighteen different solid tumor types . comprising the ECD of human SEZO fused to a human IgG2 FIGS. 10A - 10B provide the continuous amino acid Fc domain , in which the underlined sequences correspond to 35 sequences of light ( FIG . 10A ) and heavy chain (FIG . 10B ) the human IgG2 Fc domain , the double underlined variable regions of a number of murine and humanized sequences correspond to the IgK signal peptide , and the exemplary SEZ6 modulators isolated , cloned and engi amino acids in bold font correspond to residues contributed neered as described in the Examples herein . The correspond by the restriction sites used to clone the hSEZ6 fragment. ing nucleic acid sequences are set forth in the appended FIGS. 5A -5J provide various representations of nucleic 40 sequence listing. FIG . 10C sets out the full length amino acid acid or amino acid sequences related to the production of the sequences of the light and heavy chains of the humanized immunogens or cell lines used to generate or characterize antibodies SC17 . 200 and SC17 .200vL1 . the SEZ6 modulators described herein , wherein the under - FIG . 11 sets forth various characteristics of exemplary lined sequences denote the ECD of protein for the specific modulators of the invention . FIG . 11A shows the biochemi SEZ6 or SEZ6 family member being illustrated , and the 45 cal and immunological properties of exemplary SEZ6 modu figures comprise the cDNA sequences for the constructs lators as represented in a tabular format; and FIG . 11B encoding mature murine SEZ6 (FIG . 5A , SEQ ID NO : 10 ) , provides a correlation between the domain to which an mature rat SEZO (FIG . 5C , SEQ ID NO : 12 ), mature antibody binds and the antibody 's efficacy in an in vitro cynomolgus SEZO ( FIG . 5E , SEQ ID NO : 14 ) , mature ECD killing assay . of the human SEZOL protein ( FIG . 5G , SEQ ID NO : 16 ), or 50 FIGS. 12A and 12B show detection of expression of SEZ6 the mature ECD of the human SEZÓL2 protein (FIG . 51, protein measured using an electrochemiluminescent assay . SEQ ID NO : 18 ) , or the corresponding proteins encoded by FIG . 12A shows SEZ6 expression in HEK - 293T cells engi these cDNA constructs , namely mature murine SEZ6 (FIG . neered to over - express human SEZ6 protein (h293T 5B , SEQ ID NO : 11 ) , mature rat SEZ6 (FIG . 5D , SEQ ID HUSEZ6 ) using the anti -SEZ6 antibody SC17 . 33 ; FIG . 12B NO : 13 ) , mature cynomolgus SEZO ( FIG . 5F , SEQ ID NO : 55 shows the relative protein expression of human SEZ6 in 15 ) , the mature ECD of the human SEZOL protein (FIG . 5H , various NTX tumor and normal tissue lysates . SEO ID NO : 17 ) , or the mature ECD of the human SEZÓL2 FIGS . 13A and 13B show detection by flow cytometry of protein (FIG . 5J, SEQ ID NO : 19 ). SEZ6 protein expression on NTX tumor cells using various FIGS . 6A and 6B are depictions of mRNA expression anti - SEZ6 antibodies ( FIG . 13A ) ; whereas FIG . 13B shows levels of various genes as measured using whole transcrip - 60 enhanced expression of SEZ6 protein in CSCs compared to tome (SOLID ) sequencing of mRNA derived from tumor NTG subpopulations using various anti - SEZ6 antibodies cell subpopulations or normal tissues. FIG . 6A is a tabular (FIG . 13B ) . representation of genes associated with tumors having neu FIGS . 14A and 14B show that CSCs expressing SEZO roendocrine features ; and FIG . 6B is a graphical represen exhibit enhanced tumorigenicity compared to CSCs that do tation of SEZ6 mRNA expression in normal tissues and 65 not express SEZ6 . FIG . 14A is a contour plot showing cell several non -traditional xenograft (NTX ) tumors derived sorting by FACS of the cells in a lung tumor (LU37 ) on the from lung cancers . basis of expression of CD324 ( a marker of CSCs ) and SEZ6 ; US 9 ,993 ,566 B2 15 16 FIG . 14B is a graphical representation of the growth of markers which may be exploited in the treatment of related tumor cells that are either CD324 + SEZ6 + ( black circles ) or diseases . Moreover, as shown in the instant application it has CD324 + SEZO (white circles ) after implantation into unexpectedly been found that SEZ6 markers or determinants immunocompromised mice . Tumor cells expressing both such as cell surface SEZ6 protein are associated with cancer CD324 and SEZ6 exhibit enhanced tumorigenicity . 5 stem cells (also known as tumor perpetuating cells ) and may FIGS . 15A and 15B provide , respectively, a tabular and be effectively exploited to eliminate or silence the same. The graphical representation illustrating that the disclosed modu - ability to selectively reduce or eliminate cancer stem cells lators may effectively be used as targeting moieties to direct ( e . g ., through the use of conjugated SEZ6 modulators ) is cytotoxic payloads to cells engineered to express SEZ6 particularly surprising in that such cells are known to ( FIG . 15A ) and NTX lung tumors (LU80 , LU37 and LU100 ) 10 generally be resistant to many conventional treatments . That grown in vitro ( FIG . 15B ) where the decrease in normalized is , the effectiveness of traditional, as well as more recent relative luminescence units (RLU ) is indicative of cell targeted treatment methods , is often limited by the existence killing through internalization of the saporin toxin . and / or emergence of resistant cancer stem cells that are FIG . 16 shows the quantification of SEZ6 expression in capable of perpetuating the cancer even in the face of these various SCLC and medullary thyroid tumors determined 15 diverse treatment methods. Further, determinants associated using immunohistochemistry (IHC ), where FIG . 16A shows with cancer stem cells often make poor therapeutic targets SEZ6 expression in NTX SCLC tumors , FIG . 16B shows due to low or inconsistent expression , failure to remain SEZ6 expression in SCLC tumor microarrays and FIG . 16C associated with the tumorigenic cell or failure to present at shows SEZ6 expression in primary medullary thyroid the cell surface . In sharp contrast to the teachings ofthe prior tumors . 20 art , the instantly disclosed compounds and methods effec FIGS. 17A - 17B depict the ability of conjugated anti - tively overcome this inherent resistance to specifically elimi SEZ6 mouse antibodies to retard in vitro and in vivo growth nate , deplete, silence or promote the differentiation of such of NTX tumor cells . FIG . 17A shows the results of an in cancer stem cells thereby negating their ability to sustain or vitro killing assay using anti - SEZ6 ADCs on SCLC ( LU64 ) re - induce the underlying tumor growth . and OV ( OV26 ) NTX tumor cell lines; whereas FIG . 17B 25 More specifically , it has been discovered that SEZ6 modu shows the effect of anti- SEZ6 ADCs on growth of SCLC lators such as those disclosed herein may advantageously be (LU86 ) and LCNEC (LU50 ) tumors in vivo . used in the prognosis , diagnosis , theragnosis , treatment or FIGS. 18A -18D depict the ability of conjugated human - prevention of proliferative disorders ( e. g. neoplastic disor ized anti- SEZ6 antibodies to retard growth of a thyroid cell ders ) in subjects in need thereof. Accordingly , while pre line in vitro ( FIG . 18A ) and in vivo (FIG . 18B ) ; and to retard 30 ferred embodiments of the invention will be discussed growth of four SCLC tumors in vivo ( LU80 , LU64 , LU111 extensively below , particularly in terms of particular and LU117 ) and achieve durable remission in immunodefi domains, regions or epitopes or in the context of cancer stem cient mice (FIGS . 18C and 18D ) . cells or tumors comprising neuroendocrine features and their FIG . 19 shows dose response curves of oxalaplatin in the interactions with the disclosed modulators , those skilled in parental small cell lung cancer (SCLC ) patient -derived 35 the art will appreciate that the scope of the instant invention xenograft ( PDX ) line , LU124p2 compared to the oxalapla - is not limited by such exemplary embodiments . Rather, the tin -resistant cell line LU1240XAHIp2 and most expansive embodiments of the present invention and LU1240XAHIp3 . the appended claims are broadly and expressly directed to FIG . 20 shows mRNA expression of SEZ6 in parental SEZ6 modulators (including conjugated modulators ) and SCLC PDX lines LU124p1 and LU124p3 , and in oxalapla - 40 their use in the prognosis , diagnosis , theragnosis , treatment tin -resistant cell line LU1240XAHIp3 . or prevention of a variety of SEZ6 associated or mediated FIG . 21 shows protein expression of SEZ6 in in parental disorders , including neoplastic or proliferative disorders, SCLC PDX line LU124p3 and in oxalaplatin -resistant cell regardless of any particular mechanism of action or specifi line LU124OXAHIp3. cally targeted tumor, cellular or molecular component. To that end , and as demonstrated in the instant applica DETAILED DESCRIPTION OF THE tion , it has unexpectedly been found that the disclosed SEZ6 INVENTION modulators can effectively be used to target and eliminate or otherwise incapacitate proliferative or tumorigenic cells and I. Introduction treat SEZ6 associated disorders ( e . g . , neoplasia ) . As used 50 herein a " SEZ6 associated disorder ” shall be held to mean While the present invention may be embodied in many any disorder or disease ( including proliferative disorders ) different forms, disclosed herein are specific illustrative that is marked , diagnosed , detected or identified by a phe embodiments thereof that exemplify the principles of the notypic or genotypic aberration of SEZ6 genetic compo invention . It should be emphasized that the present invention n ents or expression during the course or etiology of the is not limited to the specific embodiments illustrated . More - 55 disease or disorder . In this regard a SEZ6 phenotypic aber over , any section headings used herein are for organizational ration or determinant may , for example , comprise elevated purposes only and are not to be construed as limiting the or depressed levels of SEZ6 protein expression , abnormal subject matter described . Finally , for the purposes of the SEZ6 protein expression on certain definable cell popula instant disclosure all identifying sequence Accession num - tions or abnormal SEZ6 protein expression at an inappro bers may be found in the NCBI Reference Sequence (Ref - 60 priate phase or stage of a cell lifecycle . Of course , it will be Seq ) database and/ or the NCBI GenBank® archival appreciated that similar expression patterns of genotypic sequence database unless otherwise noted . determinants ( e . g ., mRNA transcription levels ) of SEZ6 may As previously alluded to , it has surprisingly been found also be used to classify or detect SEZ6 associated disorders . that the expression of SEZ6 is associated with neoplastic As used herein the term “ determinant” or “ SEZ6 deter growth and proliferative disorders , particularly in the 65 minant" shall mean any detectable trait , property , marker or instance of tumors with neuroendocrine features, and that factor that is identifiably associated with , or specifically SEZ6 and variants or isoforms thereof provide useful tumor found in or on a particular cell, cell population or tissue US 9 ,993 ,566 B2 17 18 including those identified in or on a tissue , cell or cell such “ isolated ” preparations may be intentionally formu population affected by a SEZ6 associated disease or disor lated or combined with inert or active ingredients as desired der . In selected preferred embodiments the SEZ6 modulators to improve the commercial , manufacturing or therapeutic may associate , bind or react directly with the SEZ6 deter - aspects of the finished product and provide pharmaceutical minant ( e . g ., cell surface SEZ6 protein or SEZ6 mRNA ) and 5 compositions. In a broader sense the same general consid thereby ameliorate the disorder . More generally determi erations may be applied to an “ isolated ” SEZ6 isoform or nants may be morphological , functional or biochemical in variant or an “ isolated ” nucleic acid encoding the same. nature and may be genotypic or phenotypic . In other pre ferred embodiments the determinant is a cell surface antigen Further, it has surprisingly been found that modulators or genetic component that is differentially or preferentially 10 interacting , associating or binding to particular SEZ6 expressed (or is not) by specific cell types ( e . g . , cancer stem domains ,motifs or epitopes are especially effective in elimi cells ) or by cells under certain conditions ( e . g . , during nating tumorigenic cells and /or silencing or attenuating specific points of the cell cycle or cells in a particular niche ). cancer stem cell effects on tumor growth or propagation . In still other preferred embodiments the determinant may That is , while modulators that react or associate with comprise a gene or genetic entity that is differently regulated 15 domains that are proximal to the cell surface ( e . g . one of the ( up or down ) in a specific cell or discrete cell population , a Sushi or CUB - like domains ) are effective in depleting or gene that is differentially modified with regard to its physical neutralizing tumorigenic cells it has unexpectedly been structure and chemical composition or a protein or collection discovered that modulators associating or binding to of proteins physically associated with a gene that show domains , motifs or regions that are relatively more distal to differential chemical modifications. Determinants contem - 20 the cell surface are also effective in eliminating , neutraliz plated herein are specifically held to be positive or negative ing , depleting or silencing tumorigenic cells . This is espe and may denote a cell , cell subpopulation or tissue ( e . g ., cially true of conjugated modulators such as , for example , tumors ) by its presence (positive ) or absence (negative ). anti- SEZ6 antibody drug conjugates comprising a cytotoxic In a similar vein " SEZ6 modulators” of the invention agent . broadly comprise any compound that recognizes, reacts, 25 While the present invention expressly contemplates the competes , antagonizes , interacts , binds, agonizes , or asso - use of any SEZ6 modulator in the treatment of any SEZO ciates with a SEZ6 variant or isoform (or specific domains, disorder , including any type of neoplasia , in particularly regions or epitopes thereof) or its genetic component. By preferred embodiments the disclosed modulators may be these interactions , the SEZ6 modulators may advanta used to prevent, treat or diagnose tumors comprising neu geously eliminate , reduce or moderate the frequency , activ - 30 ity , recurrence, metastasis or mobility of tumorigenic cells roendocrine features (genotypic or phenotypic ) including ( e. g. , tumor perpetuating cells or cancer stem cells ). Exem neuroendocrine tumors . True or " canonical neuroendocrine plary modulators disclosed herein comprise nucleotides , tumors ” (NETs ) arise from the dispersed endocrine system oligonucleotides , polynucleotides, peptides or polypeptides . and are typically highly aggressive . Neuroendocrine tumors In certain preferred embodiments the selected modulators 355 occurOcc in the kidney , genitourinary tract (bladder , prostate , will comprise antibodies to a SEZ6 protein isoform or ovary, cervix , and endometrium ) , gastrointestinal tract immunoreactive fragments or derivatives thereof. Such anti - ( stomach , colon ), thyroid (medullary thyroid cancer ), and bodies may be antagonistic or agonistic in nature and may lung ( small cell lung carcinoma and large cell neuroendo optionally be conjugated or associated with a therapeutic or crine carcinoma ) . Moreover , the disclosed modulators may diagnostic agent. Moreover , such antibodies or antibody 40 advantageously be used to treat, prevent or diagnose pseudo fragments may comprise depleting, neutralizing or internal neuroendocrine tumors (pNETs ) that genotypically or phe izing antibodies . In other embodiments , modulators within notypically mimic , comprise, resemble or exhibit common the instant invention will constitute a SEZ6 construct com - traits with canonical neuroendocrine tumors . “ Pseudo neu prising a SEZ6 isoform or a reactive fragment thereof. It will roendocrine tumors” are tumors that arise from cells of the be appreciated that such constructs may comprise fusion 45 diffuse neuroendocrine system or from cells in which a proteins and can include reactive domains from other poly - neuroendocrine differentiation cascade has been aberrantly peptides such as immunoglobulins or biological response reactivated during the oncogenic process . Such PNETs com modifiers . In still other aspects , the SEZ6 modulator will m only share certain genotypic , phenotypic or biochemical comprise a nucleic acid moiety ( e . g . miRNA , siRNA , characteristics with traditionally defined neuroendocrine shRNA , antisense constructs, etc . ) that exerts the desired 50 tumors , including the ability to produce subsets of biologi effects at a genomic level. Still other modulators compatible cally active amines , neurotransmitters , and peptide hor with the instant teachings will be discussed in detail below . mones . Accordingly , for the purposes of the instant inven More generally SEZ6 modulators of the present invention tion the phrases “ tumors comprising neuroendocrine broadly comprise any compound that recognizes , reacts , features” or “ tumors exhibiting neuroendocrine features” competes , antagonizes, interacts , binds, agonizes, or asso - 55 shall be held to comprise both neuroendocrine tumors and ciates with a SEZ6 determinant ( genotypic or phenotypic ) pseudo neuroendocrine tumors unless otherwise dictated by including cell surface SEZ6 protein . Whichever form of context. modulator is ultimately selected it will preferably be in an In preferred embodiments the disclosed modulators will isolated and purified state prior to introduction into a subject. be used to treat small cell lung cancer and , in particularly In this regard the term “ isolated SEZ6 modulator” or “ iso - 60 preferred embodiments , will be used to treat platinum resis lated SEZ6 antibody ” shall be construed in a broad sense and tant small cell lung cancer in a subject in need thereof . As in accordance with standard pharmaceutical practice to used herein and known in the art, the term “ platinum mean any preparation or composition comprising the modu - resistant small cell lung cancer " means the presence of lator in a state substantially free of unwanted contaminants tumors or tumorigenic small cell lung cancer cells in a (biological or otherwise ) . Moreover these preparations may 65 subject that are resistant or refractory to treatment with be purified and formulated as desired using various art standard of care platinum based agents such as carboplatin , recognized techniques . Of course , it will be appreciated that cisplatin and /or oxalaplatin . Such conditions are readily US 9 ,993 ,566 B2 19 20 diagnosed using standard clinical procedures and their rec members of this family of SEZ6 - like proteins in humans are ognition is well with the purview of a clinician of ordinary shown in FIG . 2C . Taken together SEZO , SEZÓL and skill in the art. SEZÓL2, including their various isoforms, will be termed Besides the association with tumors generally discussed the SEZ6 family for the purposes of the instant application . above , there are also indications of phenotypic or genotypic 5 SEZ6 modulators of the invention comprise modulators that association between selected tumor initiating cells ( TIC ) and are specific for each of SEZÓ , SEZÓL or SEZÓL2 . Alterna SEZ6 determinants . In this regard selected TICs ( e . g ., cancer tively , the modulators of the invention may cross react with stem cells ) may express elevated levels of SEZ6 proteins SEZ6 and one or both of SEZÓL and / or SEZ6L2 . when compared to normal tissue and non - tumorigenic cells The mature SEZ6 protein is composed of a series of (NTG ) , which together typically comprise much of a solid 10 structural domains : a cytoplasmic domain , a transmembrane tumor. Thus, SEZ6 determinants may comprise a tumor domain and an extracellular domain comprising a unique associated marker ( or antigen or immunogen ) and the dis N - terminal domain , followed by two alternating Sushi and closed modulators may provide effective agents for the CUB - like domains, and three additional tandem Sushi detection and suppression of TIC and associated neoplasia domain repeats . Two isoforms of the SEZ6 antigen exist, and due to altered levels of the proteins on cell surfaces or in the 15 differ only on the extreme carboxy terminal, cytoplasmic tumor microenvironment. Accordingly , SEZ6 modulators, domain . including immunoreactive antagonists and antibodies that FIG . 1F provides a schematic diagram of the extracellular associate , bind or react with the proteins, may effectively region of the SEZ6 protein , illustrating the general juxtapo reduce the frequency of tumor initiating cells and could be sition of the Sushi and CUB domains, and the N -terminal useful in eliminating , depleting , incapacitating , reducing , 20 domain . Generally, the domains are recognized as occurring promoting the differentiation of, or otherwise precluding or at about amino acid residues 336 -395 (Sushi Domain 1 ) , limiting the ability of these tumor- initiating cells to lie 397 - 508 ( CUB Domain 1 ) , 511 -572 (Sushi Domain 2 ) , dormant and /or continue to fuel tumor growth , metastasis or 574 -685 (CUB Domain 2 ) , 690 -748 (Sushi Domain 3 ), recurrence in a patient. In this regard those skilled in the art 750 - 813 (Sushi Domain 4 ), 817 - 878 (Sushi Domain 5 ) , with will appreciate that the present invention further provides 25 the N terminal domain at about amino acid residues 1 - 335 , SEZ6 modulators and their use in reducing the frequency of and a compositional bias of proline -rich residues at about tumor initiating cells . amino acid residues 71 - 169 . The Sushi repeats are similar to the short consensus II. SEZ6 Physiology repeats found in the other human complement regulatory 30 proteins ( i . e . , complement C3b /C4b binding sites ) . The SEZ6 (also known as seizure related 6 homolog ) is a type CUB - like domains are similar to CUB domains found in I transmembrane protein originally cloned from mouse other mammalian complement binding proteins which are cerebrum cortex -derived cells treated with the convulsant associated with a wide range of proteins that participate in pentylentetrazole (Shimizu -Nishikawa , 1995 ; PMID : numerous biological processes other than complement acti 7723619 ) . Representative SEZ6 protein orthologs include , 35 vation , including but not limited to patterning , axon guid but are not limited to , human (NP _ 849191 ; ance , inflammation , and tumor suppression (Bork and Beck NP _ 001092105 ) , chimpanzee (XP _ 511368, man , 1993 , PMID : 8510165 ) . Both the Sushi and CUB NP _ 001139913 ), mouse (NP _ 067261 ), and rat domains imply a function for SEZ6 involving binding of (NP 001099224 ) . In humans , the SEZ6 gene consists of 17 other proteins extracellularly . Proteins containing CUB exons spanning 51. 1 kBp located on chromosome 17q11 . 2 . 40 domains also have been linked to cell signaling pathways, Alternate splice acceptor sites only 16 base pairs apart and consistent with this function , the SEZ6 C -terminal within the last exon gives rise to two processed transcripts , cytoplasmic domains contain the Asn -Pro - Thr - Tyr motif one of approximately 4210 bases (NM 178860 ; FIG . 1A ) (SEQ ID NO : 200 ) , which is a potential target for phospho and one of approximately 4194 bases (NM _ 001098635 , rylation by Src tyrosine kinase family members. If true , this FIG . 1B ) . The former transcript encodes a 994 amino acid 45 would link SEZ6 to a cellular signal transduction pathway protein (NP _ 849191; FIG . 1C ) , whereas the latter encodes a leading to the activation of Ras, suggesting that SEZ6 may 993 amino acid protein (NP _ 001092105 ; FIG . 1D ) . These be a neurotrophic receptor. two protein isoforms of SEZ6 share overall 100 % identity Note that, the terms “ mature protein ” or “ mature poly across their extracellular domains and their transmembrane peptide ” as used herein refers to the form ( s ) of the SEZO domains , differing only in the final ten amino acid residues 50 protein produced without the signal peptide of 19 amino ( FIG . 1E ) . A third splice variant has been reported to acids that may be cleaved prior to cell surface expression . generate a secreted from of SEZ6 (Shimizu -Nishikawa , Unless otherwise indicated SEZ6 amino acid numbering ( for 1995 ; PMID : 7723619 ) , however it has not been included in domains , regions, epitopes , etc . ) will be in the context of a the RefSeqs associated within the NCBI database Gene page mature protein without the leader. entry . The modulators of the invention may bind to any of 55 SEZ6 is detectable by RT- PCR at low levels in kidney, the splice variants . liver, heart , lung and thymus of rodents , although strong The biological relevance of the isoforms is unclear, protein expression was seen only in brain , with a significant although one study has suggested opposing actions for the level expressed in testis (Herbst and Nicklin , 1997 , PMID : membrane versus soluble proteins when their expression is 9073173 ) . Using polyclonal sera to SEZ6 , protein expres restored in neurons from murine SEZ6 knockout mice 60 sion was detected in day 13 of developing mouse forebrain . (Gunnersen et al. 2007 , PMID : 18031681 ) . Cross species Strong staining was detected in the post -mitotic , maturing protein sequence identity for the SEZ6 proteins are listed in neurons of the developing cortical plate and sub - plate . This FIG . 2A . In the human genome, there are two closely related staining is diminished in the adult brain where the SEZO genes - seizure related 6 homolog - like (SEZÓL ) and seizure expression can be detected in other brain regions associated related 6 homolog like - 2 (SEZ6L2 ) , each of which has 65 with ongoing morphological plasticity , such as the hip multiple splice variants encoding numerous isoforms (FIG . pocampus , cerebellum , and olfactory bulb and in neurons of 2B ) . Percent identities for the longest protein of each of the the retina and spinal cord (Gunnersen et al. , 2007 , PMID : US 9 , 993 ,566 B2 18031681 ). The densest signals are found in regions with PMID : 18565894 ), in which various hormone and endocrine greatest concentration of neuronal cell bodies . In spite of markers are expressed and/ or secreted , and various neural widespread retinal expression of SEZ6 , retinal function in markers indicative of neurogenesis , neural commitment, or the absence of SEZ6 was not affected (Gunnersen et al. , differentiation towards neural fates are expressed . Tumors 2009 , PMID : 19662096 ) . The SEZ6 staining pattern is 5 with neuroendocrine features arise infrequently in a wide closely tied with the emergence of the neocortical layers and range of primary sites, and while their exhaustive classifi hippocampus, and implies a forebrain -specific role for this cation remains problematic (Yao ; PMID : 18565894 ; Klim gene during development. In human and mice SEZ6 was stra 2010; PMID : 20664470 ; Klöppel , 2011 ; PMID : found to be differentially expressed in highly specific 22005112 ) , they may be classified into four major types : low regions of the neocortex (Gunnersen et al ., 2007 , supra ) . 10 grade benign carcinoids, low - grade well -differentiated neu Mutations in the human SEZ6 gene have been linked to roendocrine tumors with malignant behavior, tumors with febrile seizures ( FS ), a convulsion associated with a rise in mixed neuroendocrine and epithelial features , and high body temperature and the most common type of seizure in grade poorly differentiated neuroendocrine carcinomas. Of childhood ( Yu et al. , 2007 , PMID : 17086543 ) . FS may be these classifications , the poorly differentiated neuroendo classified as simple or complex , depending upon duration , 15 crine carcinomas , which include small cell lung cancer recurrence , and extent of the body affected by the seizure. In (SCLC ) and subsets of non -small cell lung cancer (NSCLC ) , a Chinese cohort, no mutations in SEZ6 were found in 15 are cancer types with dismal prognoses. It has been postu healthy controls , but mutations were found in 21 of 60 lated that SCLC is bronchogenic in origin , arising in part patients with FS , with the most common type ofmutation from pulmonary neuroendocrine cells (Galluzzo and Boc being a heterozygous , cytosine insertion ( frame shift muta - 20 chetta , 2011 ; PMID : 21504320 ) . Whatever the cellular tion ) at position 1435 of the cDNA . The mutation incidence source of origin for these tumors , it is clear that they show was significantly higher in patients with complex FS and in a poorly differentiated endocrine phenotype , often are highly patients with a positive family history . As there is an 80 % proliferative and aggressive, and frequently over -express chance that children with complex FS will have seizures neural proteins. Similarly , medullary thyroid cancers later in life , the authors suggest that screening for mutations 25 (MTC ) , a special neuroendocrine tumor type that arises from in SEZ6 may be valuable in predicting FS recurrence or the the calcitonin -secreting parafollicular C cells of the thyroid , development of epilepsy ( Yu et al. , 2007, supra ). Later show neuroendocrine phenotypes consistent with both their studies have questioned the incidence , relevance , and ability mature endocrine function and their derivation from neural of this study to have adequate power to imply causality , but crest tissue (Cook et al. , 2010 ; PMID : 20182588 ) . While do support that SEZ6 may be one gene among many that 30 representing only about 3 - 5 % of thyroid cancers , MTC may play a role in seizure disorders (Mulley et al. , 2011, results in up to 14 % of all thyroid cancer deaths, is not very PMID : 21785725 ). responsive to standard chemotherapy or radiation treat The specific molecular functions of SEZ6 remain unclear. ments , and even with newer molecularly targeted tyrosine As discussed above, analysis of the structuralmodules of the kinase inhibitors such as cabozantinib and vandetanib , protein identified by homology and sequence analysis sug - 35 responds poorly to monotherapies (Haddad , 2013 ; PMID : gest a possible role in signaling , cell - cell communication , 24002516 ) . Given these examples of tumors with dismal and neural development. The neuronal dendritic branching prognoses , the resultant elevation of neural expression and connectivity that form the signaling networks that markers in these tumors that otherwise may be primarily constitute the brain ' s circuitry arise and are specified both by restricted to the nervous system or show limited expression intrinsic molecular programs in the neural cell as well as 40 during development, of which SEZ6 may be an exemplar, extrinsic signals . The process of dendritic growth in pyra - may therefore offer a unique therapeutic target for tumors midal neurons, the principal neuron in the mammalian with the neuroendocrine phenotype . forebrain , yields neurons with distinctive morphologies — a pyramidal cell body , and two distinct, complex dendritic III. Cancer Stem Cells trees : one emerging from the apex and the other from the 45 base of the cell body. Gunnersen et al. ( 2007 , supra ) have As alluded to above it has surprisingly been discovered shown that SEZ6 null mice exhibit an excess of short that aberrant SEZ6 expression ( genotypic and /or pheno dendrites in the dendritic trees of these neurons , yet display typic ) is associated with various tumorigenic cell subpopu no increase in the overall dendritic field , the range of lations . In this respect the present invention provides SEZ6 neurons with which a given neuron connects . Restoring the 50 modulators that may be particularly useful for targeting such expression of the membrane bound SEZ6 isoforms in the cells, and especially tumor perpetuating cells, thereby facili knockout neurons results in an anti - branching effect. In tating the treatment, management or prevention of neoplas behavioral tests the SEZ6 null mice display specific explor - tic disorders . Thus , in preferred embodiments modulators of atory , motor , and cognitive deficits . These data suggest that SEZ6 determinants ( phenotypic or genotypic ) may be SEZ6 is important for the achievement of the necessary 55 advantageously be used to reduce tumor initiating cell balance between dendrite elongation and branching during frequency in accordance with the present teachings and the elaboration of a complex dendritic arbor during devel - thereby facilitate the treatment or management of prolifera opment. tive disorders . Together, the studies above strongly suggest that the For the purposes of the instant application the term SEZ6 protein is important in the context of neural develop - 60 " tumor initiating cell ” ( TIC ) encompasses both “ tumor ment, and is likely to have some role in cell - cell commu- perpetuating cells” ( TPC ; i . e . , cancer stem cells or CSC ) and nication and signaling . Inappropriate reactivation of devel- highly proliferative " tumor progenitor cells ” ( termed opmental signaling pathways or disregulation of normal TProg ) , which together generally comprise a unique sub signaling pathways are commonly observed in tumors (Har - population ( i . e . 0 . 1 -40 % ) of a bulk tumor or mass . For the ris et al. , 2012 ) . One collection of tumors sharing features 65 purposes of the instant disclosure the terms “ tumor perpetu indicative of partial reactivation of developmental programs ating cells ” and “ cancer stem cells ” or “ neoplastic stem are tumors with neuroendocrine phenotypes ( Yao 2008 ; cells ” are equivalent and may be used interchangeably US 9 ,993 ,566 B2 23 24 herein . TPC differ from TProg in that TPC can completely reducing the frequency or altering the chemosensitivity of recapitulate the composition of tumor cells existing within a such SEZ6 positive tumor initiating cells regardless of the tumor and have unlimited self -renewal capacity as demon particular embodiment or mix represented in a tumor. strated by serial transplantation ( two or more passages In the context of the instant invention , TPC are more through mice ) of low numbers of isolated cells , whereas 5 tumorigenic , relatively more quiescent and often more TProg will not display unlimited self - renewal capacity . chemoresistant than the TProg ( both ETP and LTP ) , NTG Those skilled in the art will appreciate that fluorescence cells and the tumor - infiltrating non - TPC derived cells ( e . g . , activated cell sorting (FACS ) using appropriate cell surface markers is a reliable method to isolate highly enriched fibroblasts / stroma, endothelial & hematopoietic cells ) that cancer stem cell subpopulations ( e . g . , > 99. 5 % purity ) due , at 10 comprise the bulk of a tumor. Given that conventional least in part , to its ability to discriminate between single cells therapies and regimens have, in large part , been designed to and clumps of cells ( i . e . doublets , etc . ). Using such tech both debulk tumors and attack rapidly proliferating cells , niques it has been shown that when low cell numbers of TPC are likely to be more resistant to conventional therapies highly purified TProg cells are transplanted into immuno and regimens than the faster proliferating TProg and other compromised mice they can fuel tumor growth in a primary 15 bulkbull tumor cell populations. Further , TPC often express transplant. However , unlike purified TPC subpopulations the other characteristics that make them relatively chemoresis TProg generated tumors do not completely reflect the paren tant to conventional therapies , such as increased expression tal tumor in phenotypic cell heterogeneity and are demon of multi -drug resistance transporters, enhanced DNA repair strably inefficient at reinitiating serial tumorigenesis in sub - mechanisms and anti -apoptotic proteins . These properties , sequent transplants . In contrast , TPC subpopulations 20 each of which contribute to drug tolerance by TPC , consti completely reconstitute the cellular heterogeneity of paren - tute a key reason for the failure of standard oncology tal tumors and can efficiently initiate tumors when serially treatment regimens to ensure long - term benefit for most isolated and transplanted . Thus , those skilled in the art will patients with advanced stage neoplasia ; i. e . the failure to recognize that a definitive difference between TPC and adequately target and eradicate those cells that fuel contin TProg , though both may be tumor generating in primary 25 ued tumor growth and recurrence ( i . e . TPC or CSC ) . transplants, is the unique ability of TPC to perpetually fuel Unlike many prior art treatments, the novel compositions heterogeneous tumor growth upon serial transplantation at of the present invention preferably reduce the frequency of low cell numbers . Other common approaches to characterize tumor initiating cells upon administration to a subject TPC involve morphology and examination of cell surface regardless of the form or specific target ( e . g . , genetic mate markers , transcriptional profile , and drug response although 30 rial, SEZ . antibody or ligand fusion construct ) of the marker expression may change with culture conditions and selected modulator. As noted above , the reduction in tumor with cell line passage in vitro . initiating cell frequency may occur as a result of a ) elimi Accordingly , for the purposes of the instant invention nation , depletion , sensitization , silencing or inhibition of tumor perpetuating cells, like normal stem cells that support tumor initiating cells ; b ) controlling the growth , expansion cellular hierarchies in normal tissue , are preferably defined 35 or recurrence of tumor initiating cells ; c ) interrupting the by their ability to self -renew indefinitely while maintaining initiation , propagation , maintenance , or proliferation of the capacity for multilineage differentiation . Tumor perpetu tumor initiating cells ; or d ) by otherwise hindering the ating cells are thus capable of generating both tumorigenic survival, regeneration and / or metastasis of the tumorigenic progeny ( i. e ., tumor initiating cells : TPC and TProg ) and cells . In some embodiments , the reduction in the frequency non - tumorigenic (NTG ) progeny. As used herein a " non - 40 of tumor initiating cells occurs as a result of a change in one tumorigenic cell ” (NTG ) refers to a tumor cell that arises or more physiological pathways . The change in the pathway , from tumor initiating cells , but does not itself have the whether by reduction or elimination of the tumor initiating capacity to self - renew or generate the heterogeneous lin - cells or by modifying their potential ( e . g . , induced differ eages of tumor cells that comprise a tumor. Experimentally , entiation , niche disruption ) or otherwise interfering with NTG cells are incapable of reproducibly forming tumors in 45 their ability to influence the tumor environment or other mice , even when transplanted in excess cell numbers . cells , in turn allows for the more effective treatment of SEZ6 As indicated , TProg are also categorized as tumor initi - associated disorders by inhibiting tumorigenesis , tumor ating cells (or TIC ) due to their limited ability to generate maintenance and / or metastasis and recurrence . tumors in mice . TProg are progeny of TPC and are typically Among art - recognized methods that can be used to assess capable of a finite number of non - self- renewing cell divi - 50 such a reduction in the frequency of tumor initiating cells is sions . Moreover, TProg cells may further be divided into limiting dilution analysis either in vitro or in vivo , preferably early tumor progenitor cells (ETP ) and late tumor progenitor followed by enumeration using Poisson distribution statis cells (LTP ) , each of which may be distinguished by pheno - tics or assessing the frequency of predefined definitive type ( e . g . , cell surface markers) and different capacities to events such as the ability to generate tumors in vivo or not. recapitulate tumor cell architecture . In spite of such techni- 55 While such limiting dilution analysis comprise preferred cal differences , both ETP and LTP differ functionally from methods of calculating reduction of tumor initiating cell TPC in that they are generally less capable of serially frequency other, less demanding methods, may also be used reconstituting tumors when transplanted at low cell numbers to effectively determine the desired values, albeit slightly and typically do not reflect the heterogeneity of the parental less accurately , and are entirely compatible with the teach tumor. Notwithstanding the foregoing distinctions , it has 60 ings herein . Thus, as will be appreciated by those skilled in also been shown that various TProg populations can , on rare the art , it is also possible to determine reduction of fre occasion , gain self -renewal capabilities normally attributed quency values through well -known flow cytometric or to stem cells and themselves become TPC ( or CSC ) . In any immunohistochemical means. As to all the aforementioned event both types of tumor - initiating cells are likely repre - methods see , for example , Dylla et al . 2008 , PMID : sented in the typical tumor mass of a single patient and are 65 18560594 & Hoey et al. 2009 , PMID : 19664991 ; each of subject to treatment with the modulators as disclosed herein . which is incorporated herein by reference in its entirety and , That is , the disclosed compositions are generally effective in in particular , for the disclosed methods . US 9 , 993 ,566 B2 25 26 With respect to limiting dilution analysis , in vitro enu B - catenin , CD2 , CD3 , CD9 , CD14 , CD31, CD38 , CD44 , meration of tumor initiating cell frequency may be accom CD45 , CD74 , CD90 , CXCR4 , decorin , EGFR , CD105 , plished by depositing either fractionated or unfractionated CD64 , CD16 , CD16a, CD16b , GLI1, GLI2 , CD49b , and human tumor cells ( e . g . from treated and untreated tumors , CD49f. See , for example, Schulenburg et al. , 2010 , PMID : respectively ) into in vitro growth conditions that foster 5 20185329 , U . S . Pat. No. 7 ,632 ,678 and U . S . P .Ns . 2007 / colony formation . In this manner, colony forming cells 0292414 , 2008 /0175870 , 2010 /0275280 , 2010 /0162416 and might be enumerated by simple counting and characteriza 2011/ 0020221 each of which is incorporated herein by tion of colonies , or by analysis consisting of, for example , reference . It will further be appreciated that each of the the deposition of human tumor cells into plates in serial aforementioned markers may also be used as a secondary dilutions and scoring each well as either positive or negative 10 target antigen in the context of the bispecific or multispecific for colony formation at least 10 days after plating . In vivo antibodies of the instant invention . limiting dilution experiments or analyses , which are gener Similarly , non - limiting examples of cell surface pheno ally more accurate in their ability to determine tumor types associated with cancer stem cells of certain tumor initiating cell frequency encompass the transplantation of types include CD44 " iCD24low , ALDH * , CD133 + , CD1237, human tumor cells, from either untreated control or treated 15 CD34 +CD38 - , CD44 + CD24 - , CD46CD324 + CD66c - , populations, for example , into immunocompromised mice in CD133 +CD34 + CD10 - CD19 + , CD138 -CD34 -CD19 ++ , serial dilutions and subsequently scoring each mouse as CD133 +RC2 + , CD44 + a , B NiCD133 + , CD44 +CD24 + ESA +" , either positive or negative for tumor formation at least 60 CD271 " , ABCB5 + as well as other cancer stem cell surface days after transplant. The derivation of cell frequency values phenotypes that are known in the art. See , for example , by limiting dilution analysis in vitro or in vivo is preferably 20 Schulenburg et al. , 2010 , supra , Visvader et al. , 2008 , PMID : done by applying Poisson distribution statistics to the known 18784658 and U . S .P .N . 2008 /0138313 , each of which is frequency of positive and negative events , thereby providing incorporated herein in its entirety by reference. Those skilled a frequency for events fulfilling the definition of a positive in the art will appreciate that marker phenotypes such as event; in this case , colony or tumor formation , respectively. those exemplified immediately above may be used in con As to other methods compatible with the instant invention 25 junction with standard flow cytometric analysis and cell that may be used to calculate tumor initiating cell frequency, sorting techniques to characterize , isolate , purify or enrich the most common comprise quantifiable flow cytometric TIC and / or TPC cells or cell populations for further analysis . techniques and immunohistochemical staining procedures . Of interest with regard to the instant invention CD46 , Though not as precise as the limiting dilution analysis CD324 and , optionally , CD66c are either highly or hetero techniques described immediately above , these procedures 30 geneously expressed on the surface of many human colorec are much less labor intensive and provide reasonable values tal (“ CR ” ) , breast (“ BR ” ) , non -small cell lung (NSCLC ) , in a relatively short time frame. Thus , it will be appreciated small cell lung ( SCLC ) , pancreatic ( " PA ” ) , melanoma that a skilled artisan may use flow cytometric cell surface (“ Mel ” ) , ovarian (" OV ” ) , and head and neck cancer (“ HN ” ) marker profile determination employing one or more anti - tumor cells , regardless of whether the tumor specimens bodies or reagents that bind art- recognized cell surface 35 being analyzed were primary patient tumor specimens or proteins known to enrich for tumor initiating cells ( e . g . , patient -derived NTX tumors. potentially compatible markers as are set forth in PCT Using any of the above- referenced methods and selected application 2012 / 031280 which is incorporated herein in its markers as known in the art it is then possible to quantify the entirety ) and thereby measure TIC levels from various reduction in frequency of TIC (or the TPC therein ) provided samples . In still another compatible method one skilled in 40 by the disclosed SEZ6 modulators ( including those conju the art might enumerate TIC frequency in situ ( e . g ., in a gated to cytotoxic agents ) in accordance with the teachings tissue section ) by immunohistochemistry using one or more herein . In some instances , the compounds of the instant antibodies or reagents that are able to bind cell surface invention may reduce the frequency of TIC or TPC (by a proteins thought to demarcate these cells . variety of mechanisms noted above , including elimination , Those skilled in the art will recognize that numerous 45 induced differentiation , niche disruption , silencing , etc . ) by markers ( or their absence ) have been associated with various 10 % . 15 % . 20 % . 25 % . 30 % or even bv 35 % . In other populations of cancer stem cells and used to isolate or embodiments , the reduction in frequency of TIC or TPC characterize tumor cell subpopulations. In this respect exem may be on the order of 40 % , 45 % , 50 % , 55 % , 60 % or 65 % . plary cancer stem cell markers comprise OCT4 , Nanog , In certain embodiments , the disclosed compounds my STAT3 , EPCAM , CD24 , CD34 , NB84 , TrkA , GD2, CD133 , 50 reduce the frequency of TIC or TPC by 70 % , 75 % , 80 % , CD20 , CD56 , CD29, B7H3, CD46 , transferrin receptor, 85 % , 90 % or even 95 % . Of course it will be appreciated that JAM3, carboxypeptidase M , ADAM9, oncostatin M , Lgr5, any reduction of the frequency of the TIC or TPC likely Lgr6 , CD324 , CD325 , nestin , Sox1 , Bmi- 1 , eed , easyhl , results in a corresponding reduction in the tumorigenicity , easyh2 , mf2 , yyl , smarcA3, smarckA5 , smarcD3 , smarcEi , persistence , recurrence and aggressiveness of the neoplasia . mllt3 , FZD1, FZD2, FZD3, FZD4 , FZD6 , FZD7, FZD8, 55 FZDS , FZD10 , WNT2 , WNT2B , WNT3 , WNT5A , IV . SEZ6 Modulators WNT10B , WNT16 , AXIN1 , BCL9, MYC , ( TCF4 ) SLC7A8 , ILIRAP , TEM8, TMPRSS4 , MUC16 , GPRC5B , In any event, the present invention is directed to the use SLC6A14 , SLC4A11 , PPAP2C , CAV1, CAV2 , PTPN3 , of SEZ6 modulators, including SEZ6 antagonists , for the EPHA1, EPHA2, SLC1A1, CX3CL1, ADORA2A , MPZL1, 60 diagnosis , theragnosis , treatment and /or prophylaxis of vari FLJ10052 , C4. 4A , EDG3 , RARRES1, TMEPAI, PTS , ous disorders including any one of a number of SEZ6 CEACAM6 , NID2, STEAP, ABCA3 , CRIMI, ILIR1, associated malignancies. The disclosed modulators may be OPN3, DAF, MUC1, MCP, CPD , NMA, ADAM9 , GJA1, used alone or in conjunction with a wide variety of anti SLC19A2, ABCA1, PCDH7, ADCY9 , SLC39A1, NPC1, cancer compounds such as chemotherapeutic or immuno ENPP1, N33, GPNMB , LY6E , CELSR1, LRP3 , C20orf52 , 65 therapeutic agents (e .g ., therapeutic antibodies ) or biological TMEPAI, FLVCR , PCDHA10 , GPR54 , TGFBR3 , response modifiers . In other selected embodiments , two or SEMA4B , PCDHB2, ABCG2, CD166 , AFP, BMP - 4 , more discrete SEZ6 modulators may be used in combination US 9 ,993 ,566 B2 27 28 to provide enhanced anti - neoplastic effects or may be used substrates. In this respect it will be appreciated that SEZ6 to fabricate multispecific constructs . antagonists of the instant invention may comprise any In certain embodiments , the SEZ6 modulators of the ligand , polypeptide , peptide , fusion protein , antibody or present invention will comprise nucleotides , oligonucle - immunologically active fragment or derivative thereof that otides , polynucleotides, peptides or polypeptides . More par- 5 recognizes, reacts , binds, combines, competes, associates or ticularly , exemplary modulators of the invention may com otherwise interacts with the SEZ6 protein or fragment prise antibodies and antigen - binding fragments or thereof and eliminates, silences , reduces , inhibits , hinders , derivatives thereof, proteins , peptides, glycoproteins, glyco restrains or controls the growth of tumor initiating cells or peptides , glycolipids, polysaccharides , oligosaccharides , other neoplastic cells including bulk tumor or NTG cell . nucleic acids, antisense constructs , siRNA , miRNA , bioor - 10 Compatible antagonists may further include small molecule ganic molecules , peptidomimetics , pharmacological agents inhibitors , aptamers , antisense constructs , siRNA , miRNA and their metabolites, transcriptional and translation control and the like , receptor or ligand molecules and derivatives sequences , and the like . In certain embodiments the modu thereof which recognize or associate with a SEZ6 genotypic variantlators will derivative comprise orsoluble fragment SEZO thereof ( sSEZO including ) or a formfor , 15 or phenotypic determinant thereby altering expression pat example , SEZ6 fusion constructs ( e . g ., SEZ6 - Fc , SEZ6 terns or sequestering its binding or interaction with a sub targetingmoiety , etc .) or SEZ6 - conjugates ( e . g . , SEZ6 -PEG , strate , receptor or ligand . SEZ6 - cytotoxic agent, SEZ6 - brm , etc . ) . It will also be As used herein an antagonist refers to a molecule capable appreciated that, in other embodiments , the SEZ6 modula ofneutralizing , blocking , inhibiting, abrogating , reducing or tors comprise antibodies or immunoreactive fragments or 20 interfering with the activities of a particular or specified derivatives thereof. In particularly preferred embodiments protein , including the binding of receptors to ligands or the the modulators of the instant invention will comprise neu interactions of enzymes with substrates . More generally tralizing antibodies or derivatives or fragments thereof. In antagonists of the invention may comprise antibodies and other embodiments the SEZ6 modulators may comprise antigen -binding fragments or derivatives thereof, proteins , internalizing antibodies or fragments thereof. In still other 25 peptides , glycoproteins , glycopeptides , glycolipids, polysac embodiments the SEZ6 modulators may comprise depleting charides , oligosaccharides , nucleic acids, antisense con antibodies or fragments thereof. Moreover , as with the strestructs , siRNA , miRNA , bioorganic molecules, peptidomi aforementioned fusion constructs , these antibody modula - metics , pharmacological agents and their metabolites , tors may be conjugated , linked or otherwise associated with transcriptional and translation control sequences , and the selected cytotoxic agents , polymers , biological response 30 like . Antagonists may also include small molecule inhibi modifiers (BRMs ) or the like to provide directed immuno - tors , fusion proteins, receptor molecules and derivatives therapies with various ( and optionally multiple ) mechanisms which bind specifically to the protein thereby sequestering of action . As alluded to above such antibodies may be its binding to its substrate target , antagonist variants of the pan - SEZ6 antibodies and associate with two or more SEZ6 protein , antisense molecules directed to the protein , RNA family members ( e . g . , SEZ6 and SEZ6L as shown in FIG . 35 aptamers , and ribozymes against the protein . 11A ) or immunospecific antibodies that selectively react As used herein and applied to two or more molecules or with one or both isoforms of SEZ6 . In yet other embodi- compounds , the terms " recognizes” or “ associates” shall be ments the modulators may operate on the genetic level and held to mean the reaction , binding , specific binding , com may comprise compounds as antisense constructs , siRNA , bination , interaction , connection , linkage , uniting, coales miRNA and the like that interact or associate with the 40 cence , merger or joining , covalently or non - covalently , of genotypic component of a SEZ6 determinant . the molecules whereby one molecule exerts an effect on the It will further be appreciated that the disclosed SEZ6 other moleculebule . modulators may deplete , silence , neutralize , eliminate or Moreover , as demonstrated in the examples herein ( e . g . , inhibit growth , propagation or survival of tumor cells , see FIG . 11 ) , some modulators of human SEZ6 may , in including TPC , and / or associated neoplasia through a vari - 45 certain cases , cross - react with SEZ6 from a species other ety of mechanisms, including agonizing or antagonizing than human ( e . g ., rat or cynomolgus monkey ) . In other cases selected pathways or eliminating specific cells depending, exemplary modulators may be specific for one or more for example , on the form of SEZ6 modulator, any associated isoforms of human SEZ6 and will not exhibit cross -reactiv payload or dosing and method of delivery . Thus , while ity with SEZ6 orthologs from other species . Of course , in preferred embodiments disclosed herein are directed to the 50 conjunction with the teachings herein such embodiments depletion , inhibition or silencing of specific tumor cell may comprise pan -SEZ6 antibodies that associate with two subpopulations such as tumor perpetuating cells or to modu - or more SEZ6 family members from a single species or lators that interact with a specific epitope or domain , it must antibodies that exclusively associate with SEZ6 . be emphasized that such embodiments are merely illustra - In any event, and as will be discussed in more detail tive and not limiting in any sense . Rather, as set forth in the 55 below , those skilled in the art will appreciate that the appended claims, the present invention is broadly directed to disclosed modulators may be used in a conjugated or uncon SEZ6 modulators and their use in the treatment, manage jugated form . That is , the modulator may be associated with ment or prophylaxis of various SEZ6 associated disorders or conjugated to ( e . g . covalently or non - covalently ) phar irrespective of any particular mechanism , binding region or maceutically active compounds, biological response modi target tumor cell population . 60 fiers , anticancer agents , cytotoxic or cytostatic agents , diag Regardless of the form of the modulator selected it will be nostic moieties or biocompatible modifiers . In this respect it appreciated that the chosen compound may be antagonistic will be understood that such conjugates may comprise in nature . As used herein an “ antagonist ” refers to a mol peptides , polypeptides, proteins , fusion proteins , nucleic ecule capable of neutralizing , blocking , inhibiting, abrogat- acid molecules , small molecules, mimetic agents , synthetic ing , reducing or interfering with the activities of a particular 65 drugs , inorganic molecules, organic molecules and radioiso or specified target ( e .g . , SEZ6 ) , including the binding of topes . Moreover, as indicated herein the selected conjugate receptors to ligands or the interactions of enzymes with may be covalently or non - covalently linked to the SEZO US 9 ,993 , 566 B2 29 30 modulator in variousmolar ratios depending, at least in part, aqueous environment. Thus, each naturally occurring IgG on the method used to effect the conjugation . antibody comprises two identical binding sites proximal to the amino - terminus of each arm of the Y . V. Modulator Fabrication and Supply It will be appreciated that the position of CDRs can be 5 readily identified by one of ordinary skill in the art using A . Antibody Modulators standard techniques . Also familiar to those in the art is the 1. Overview numbering system described in Kabat et al. ( 1991, NIH As previously alluded to particularly preferred embodi Publication 91 -3242 , National Technical Information Ser ments of the instant invention comprise SEZ6 modulators in vice, Springfield , Va . ) . In this regard Kabat et al. defined a the form of antibodies that preferentially associate with one 10 numbering system for variable domain sequences that is or more isoforms of SEZ6 (and , optionally , may cross -react applicable to any antibody . One of ordinary skill in the art with other SEZ6 family members ) . Those of ordinary skill in can unambiguously assign this system of “ Kabat number the art will appreciate the well developed knowledge base on ing ” to any variable domain sequence , without reliance on antibodies such as set forth , for example , in Abbas et al. , any experimental data beyond the sequence itself. Unless Cellular and Molecular Immunology , 6th ed ., W . B . Saunders 15 otherwise specified , references to the numbering of specific Company ( 2010 ) or Murphey et al. , Janeway ' s Immunobi amino acid residue positions in an antibody are according to ology , gth ed . , Garland Science ( 2011 ) , each of which is the Kabat numbering system . incorporated herein by reference in its entirety . Thus, according to Kabat, in the VH , residues 31 - 35 The term “ antibody ” comprises polyclonal antibodies , comprise CDR1, residues 50 -65 make up CDR2, and 95 - 102 multiclonal antibodies , monoclonal antibodies , chimeric 20 comprise CDR3 , while in the V?, residues 24 - 34 are CDR1, antibodies , humanized and primatized antibodies , human 50 -56 comprise CDR2, and 89 - 97 make up CDR3. For antibodies, recombinantly produced antibodies, intrabodies , context, in a VH , FR1 corresponds to the domain of the multispecific antibodies , bispecific antibodies, monovalent variable region encompassing amino acids 1 - 30 ; FR2 cor antibodies , multivalent antibodies , anti -idiotypic antibodies , responds to the domain of the variable region encompassing synthetic antibodies, including muteins and variants thereof; 25 amino acids 36 - 49 ; FR3 corresponds to the domain of the antibody fragments such as Fab fragments , F ( ab ') fragments , variable region encompassing amino acids 66 - 94 , and FR4 single -chain FvFcs , single - chain Fvs ; and derivatives corresponds to the domain of the variable region from amino thereof including Fc fusions and other modifictaions , and acids 103 to the end of the variable region . The FRs for the any other immunologically active molecule so long as they light chain are similarly separated by each of the light chain exhibit the desired biological activity ( i. e ., antigen associa - 30 variable region CDRs. tion or binding ). Moreover , the term further comprises all Note that CDRs vary considerably from antibody to classes of antibodies (i . e . IgA , IgD , IgE , IgG , and IgM ) and antibody ( and by definition will not exhibit homology with all isotypes ( i. e . , IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , the Kabat consensus sequences ) . In addition , the identity of as well as variations thereof unless otherwise dictated by certain individual residues at any given Kabat site number context. Heavy - chain constant domains that correspond to 35 may vary from antibody chain to antibody chain due to the different classes of antibodies are denoted by the corre interspecies or allelic divergence . Alternative numbering is sponding lower case Greek letter a , d , e , Y , and u , respec set forth in Chothia et al ., J . Mol . Biol. 196 : 901- 917 ( 1987 ) tively . Light chains of the antibodies from any vertebrate and MacCallum et al. , J . Mol. Biol. 262 :732 - 745 ( 1996 ) , species can be assigned to one of two clearly distinct types although as in Kabat, the FR boundaries are separated by the called kappa ( K ) and lambda ( a ) , based on the amino acid 40 respective CDR termini as described above . See also sequences of their constant domains . Chothia et al ., Nature 342 , pp . 877 -883 ( 1989 ) and S . Dubel , While all such antibodies are within the scope of the ed ., Handbook of Therapeutic Antibodies, 3a ed ., WILEY present invention , preferred embodiments comprising the VCH Verlag GmbH and Co . (2007 ) , where the definitions IgG class of immunoglobulin will be discussed in some include overlapping or subsets of amino acid residues when detail herein solely for the purposes of illustration . It will be 45 compared against each other. Each of the aforementioned understood that such disclosure is , however, merely demon references is incorporated herein by reference in its entirety strative of exemplary compositions and methods of practic and the amino acid residues which comprise binding regions ing the present invention and not in any way limiting of the scope of the invention or the claims appended hereto . or CDRs as defined by each of the above cited references As is well known , the variable domains of both the light 50 and are set forth for comparison in Table 1 below . (VL ) and heavy (VH ) chain portions determine antigen recognition and specificity and the constant domains of the TABLE 1 light chain (C ) and the heavy chain (CH1 , CH2 or C 3 ) CDR DEFINITIONS confer and regulate important biological properties such as secretion , transplacental mobility , circulation half- life , 55 Kabat Chothia MacCallum3 complement binding , and the like . VH CDR1 31 - 35 26 - 32 30 - 35 The “ variable” region includes hypervariable sites that VH CDR2 50 - 65 50 - 58 47 - 58 VH CDR3 95 - 102 95 - 102 93 - 101 manifest themselves in three segments commonly termed V CDR1 24 - 34 23 - 34 30 - 36 complementarity determining regions (CDRs ) , in both the V , CDR2 50 -56 50 - 56 46 - 55 light - chain and the heavy - chain variable domains . The more 60 89 - 97 89 - 96 highly conserved portions of variable domains flanking the V? CDR3 89 - 97 Residue numbering follows the nomenclature of Kabat et al. , supra CDRs are termed framework regions (FRs ) . For example , in Residue numbering follows the nomenclature of Chothia et al ., supra naturally occurring monomeric immunoglobulin G (IgG ) PResidue numbering follows the nomenclature ofMacCallum et al. , supra antibodies , the six CDRs present on each arm of the “ Y ” are short, non - contiguous sequences of amino acids that are 65 More practically variable regions and CDRs in an anti specifically positioned to form the antigen binding site as the body sequence can be identified ( i) according to general antibody assumes its three dimensional configuration in an rules that have been developed in the art such as those US 9 ,993 ,566 B2 31 32 discussed above or ( ii ) by aligning the sequences against a In FIGS. 10A and 10B the annotated CDRs are defined database of known variable regions. Methods for identifying using Kabat numbering . However, as discussed herein and these regions are described in Kontermann and Dubel, eds. , demonstrated in Example 8 below , one skilled in the art Antibody Engineering, Springer , New York , N . Y ., 2001 , and could readily define, identify , derive and /or enumerate the Dinarello et al. , Current Protocols in Immunology , John 5 CDRs as defined by Chothia et al ., MacCallum et al. or one Wiley and Sons Inc . , Hoboken , N . J ., 2000 . Exemplary of the website databases such as Abysis or VBase2 for each databases of antibody sequences are described in , and can be respective heavy and light chain sequence set forth in FIG . accessed through , the “ Abysis ” website at www .bioin 10A or FIG . 10B . Accordingly , each of the subject CDRs f .org .uk / abs (maintained by A . C . Martin in the Department and antibodies comprising CDRs defined by all such nomen of Biochemistry & Molecular Biology University College 10 clature are expressly included within the scope of the instant London , London , England ) and VBASE2 website at invention . More broadly , the terms “ variable region CDR www . vbase2 .org , as described in Retter et al . , Nucl. Acids amino acid residue” or more simply “ CDR ” includes amino Res ., 33 (Database issue ): D671- D674 ( 2005 ). In this regard acids in a CDR as identified using any sequence or structure the Abysis database website will automatically designate based method as set forth above . and annotate CDRs and framework regions ( as per any of the 15 For any heavy chain constant region amino acid positions commonly used numbering systems) upon entry of the discussed in the present application , numbering is according subject heavy or light chain variable region nucleic acid or to the Eu index first described in Edelman et al. , 1969 , Proc , amino acid sequence . Moreover, the Abysis database web Natl. Acad . Sci. USA 63 ( 1) : 78 - 85 describing the amino acid site also includes general rules that have been developed for sequence of myeloma protein Eu, which reportedly was the readily identifying CDRs which can be used in accordance 20 first human IgG1 sequenced . The Eu index of Edelman is with the teachings herein . In the context of the instant also set forth in Kabat et al . , 1991 ( supra . ) . Thus , the terms invention it will be appreciated that any of the disclosed light “ EU index as set forth in Kabat” or “ EU index of Kabat” in and heavy chain CDRs derived from the murine variable the context of the heavy chain refers to the residue num region amino acid sequences set forth in FIG . 10A or FIG . bering system based on the human IgG1 Eu antibody of 10B may be combined or rearranged to provide optimized 25 Edelman et al. as set forth in Kabat et al. , 1991 ( supra . ) . The anti - SEZ6 ( e . g . humanized , CDR grafted or chimeric anti - numbering system used for the light chain constant region hSEZ6 ) antibodies in accordance with the instant teachings. amino acid sequence is similarly set forth in Kabat 1991 . That is , one or more of the CDRs derived from the light Exemplary kappa C , and IgG1 heavy chain constant region chain variable region amino acid sequences set forth in FIG . amino acid sequences compatible with the instant invention 10A (SEQ ID NOS : 20 - 168 , even numbers ) or the heavy 30 are set forth as SEQ ID NOS : 403 and 404 in the appended chain variable region amino acid sequences set forth in FIG . sequence listing . Those of skill in the art will appreciate that 10B (SEO ID NOS: 21 - 169 , odd numbers ) may be incor the disclosed constant region sequences may be joined with porated in a SEZ6 modulator and , in particularly preferred the disclosed heavy and light chain variable regions using embodiments , in a CDR grafted or humanized antibody that standard molecular biology techniques to provide full- length immunospecifically associates with one or more SEZ6 iso - 35 antibodies that may be incorporated in the SEZ6 antibodies forms. Examples of light ( SEQ ID NOS : 170 - 192 , even and ADCs of the instant invention . numbers ) and heavy (SEQ ID NOS: 171 - 193, odd numbers As set forth below in the Examples , selected embodi and 194 - 199 ) chain variable region amino acid sequences of ments of the invention comprise murine antibodies that such humanized modulators are also set forth in FIGS. 10A immunospecifically bind to SEZ6 , which can be considered and 10B . 40 “ source” antibodies or “ reference ” antibodies . In other Note that hSC17 . 200vL1 (SEQ ID NO : 192 ) is a variant embodiments , antibodies contemplated by the invention of the humanized light chain construct hSC17 .200 (SEQ ID may be derived from such “ source” or “ reference” antibod NO : 190 ), hSC17 . 155vH1- vH6 (SEQ ID NOS : 193 - 198 ) are ies through optional modification of the constant region or variants of the heavy chain construct hSC . 155 (SEQ ID NO : the epitope -binding amino acid sequences of the source 184 ) which is derived from SC17 . 90 ( SEQ ID NO : 127) and 45 antibody . In one embodiment an antibody is " derived ” from that hSC161VH1 (SEQ ID NO : 199 ) is a variant of the heavy a source antibody if selected amino acids in the source chain construct hSC17 . 161 (SEQ ID NO : 189 ) . As will be antibody are altered through deletion , mutation , substitution , discussed in more detail below these variants were con - integration or combination . In another embodiment , a structed and tested to optimize one or more biochemical " derived ” antibody is one in which fragments of the source properties of the parent antibody . The full length amino acid 50 antibody ( e . g . , one or more CDRs or the entire variable sequences of exemplary humanized antibodies, hSC17 .200 region ) are combined with or incorporated into an acceptor and hSC17 . 200vL1 are set out in FIG . 10C as SEQ ID NOs: antibody sequence to provide the derivative antibody ( e . g . 400 - 402 . The humanized antibody variant hSC17 . 200vL1 is chimeric , CDR grafted or humanized antibodies ) . These derived from humanized antibody hSC17 .200 and shares a " derived ” ( e . g . humanized or CDR - grafted ) antibodies can common HC with the hSC17 . 200 antibody. Thus the full 55 be generated using standard molecular biology techniques length LC and HC of hSC17 . 200 correspond to SEQ ID for various reasons such as, for example , to improve affinity NOs: 400 and 401 , respectively ; and the full length LC and for the determinant; to improve production and yield in cell HC of hSC17 .200vL1 correspond to SEQ ID NOs: 403 and culture ; to reduce immunogenicity in vivo ; to reduce toxic 401, respectively . ity ; to facilitate conjugation of an active moiety ; or to create Taken together these novel amino acid sequences depict 60 a multispecific antibody. Such antibodies may also be seventy - five murine and eleven humanized exemplary derived from source antibodies through modification of the modulators ( along with reported variants ) in accordance mature molecule ( e . g . , glycosylation patterns or pegylation ) with the instant invention . Moreover, corresponding nucleic by chemical means or post- translational modification . acid sequences of each of the seventy - five exemplary murine Examples of source ” murine antibodies of the invention are modulators and eleven humanized modulators and variants 65 SC17 . 155 , SC17 . 161 and SC17 .200 and examples of anti set forth in FIGS. 10A and 10B are included in the sequence bodies that are derived from such source antibodies are listing of the instant application (SEQ ID NOS : 220 - 399 ) . hSC17 . 155 , hSC17 . 155vH1- vH5 ( derived from SC17. 155 ) ; US 9 , 993 ,566 B2 33 34 hSC17 . 161vL1 ( derived from SC17 . 161 ) andand b . Monoclonal Antibodies hSC17 . 200vL1 ( derived from hSC17 .200 ) . In addition , the invention contemplates use ofmonoclonal 2 . Antibody Modulator Generation antibodies . As known in the art , the term “monoclonal a . Polyclonal Antibodies antibody ” ( or mAb ) refers to an antibody obtained from a The production of polyclonal antibodies in various host 5 population of substantially homogeneous antibodies, i . e ., the animals , including rabbits , mice , rats , etc . is well known in individual antibodies comprising the population are identi the art . In some embodiments , polyclonal anti -SEZ6 anti cal except for possible mutations ( e . g . , naturally occurring body - containing serum is obtained by bleeding or sacrificing m utations ) , thatmay be present in minor amounts . In certain the animal. The serum may be used for research purposes in embodiments , such a monoclonal antibody includes an the form obtained from the animal or, in the alternative , the 10 antibody comprising a polypeptide sequence that binds or anti - SEZ6 antibodies may be partially or fully purified to associates with an antigen wherein the antigen -binding provide immunoglobulin fractions or homogeneous anti - polypeptide sequence was obtained by a process that body preparations. includes the selection of a single target binding polypeptide Briefly the selected animal is immunized with a SEZ6 sequence from a plurality of polypeptide sequences . immunogen ( e . g . , soluble SEZ6 or SEZO ) which may , for 15 More generally , and as exemplified in Example 6 herein , example , comprise selected isoforms, domains and / or pep - monoclonal antibodies can be prepared using a wide variety tides , or live cells or cell preparations expressing SEZ6 or of techniques known in the art including hybridoma, recom immunoreactive fragments thereof . Art known adjuvants binant techniques , phage display technologies , transgenic that may be used to increase the immunological response animals ( e . g ., a XenoMouse® ) or some combination thereof. depending on the inoculated species include , but are not 20 For example , monoclonal antibodies can be produced using limited to , Freund ' s (complete and incomplete ), mineral gels hybridoma and art- recognized biochemical and genetic such as aluminum hydroxide , surface active substances such engineering techniques such as described in more detail in as lysolecithin , pluronic polyols , polyanions, peptides, oil An , Zhigiang ( ed . ) Therapeutic Monoclonal Antibodies : emulsions , keyhole limpet hemocyanins, dinitrophenol, and From Bench to Clinic , John Wiley and Sons, 1st ed . 2009 ; potentially useful human adjuvants such as BCG ( bacille 25 Shire et . al. ( eds. ) Current Trends in Monoclonal Antibody Calmette -Guerin ) and corynebacterium parvum . Such adju - Development and Manufacturing , Springer Science + Busi vants may protect the antigen from rapid dispersal by n ess Media LLC , 1st ed . 2010 ; Harlow et al ., Antibodies : A sequestering it in a local deposit , or they may contain Laboratory Manual, Cold Spring Harbor Laboratory Press, substances that stimulate the host to secrete factors that are 2nd ed . 1988 ; Hammerling, et al ., in :Monoclonal Antibodies chemotactic for macrophages and other components of the 30 and T - Cell Hybridomas 563 -681 (Elsevier , N . Y . , 1981) each immune system . Preferably the immunization schedule will of which is incorporated herein in its entirety by reference . involve two or more administrations of the selected immu - It should be understood that a selected binding sequence can nogen spread out over a predetermined period of time. be further altered , for example , to improve affinity for the The amino acid sequence of a SEZ6 protein as shown in target, to humanize the target binding sequence , to improve FIG . 1C or 1D can be analyzed to select specific regions of 35 its production in cell culture , to reduce its immunogenicity the SEZ6 protein for generating antibodies. For example , in vivo , to create a multispecific antibody, etc. , and that an hydrophobicity and hydrophilicity analyses of a SEZ6 antibody comprising the altered target binding sequence is amino acid sequence are used to identify hydrophilic regions also an antibody of this invention . in the SEZ6 structure . Regions of a SEZ6 protein that show c . Chimeric Antibodies immunogenic structure, as well as other regions and 40 In another embodiment, the antibody of the invention may domains , can readily be identified using various other meth - comprise chimeric antibodies derived from covalently ods known in the art, such as Chou - Fasman , Garnier - joined protein segments from at least two different species or Robson , Kyte -Doolittle , Eisenberg , Karplus- Schultz or types of antibodies . As known in the art , the term “ chimeric ” Jameson -Wolf analysis . Average Flexibility profiles can be antibodies is directed to constructs in which a portion of the generated using the method of Bhaskaran R . , Ponnuswamy 45 heavy and / or light chain is identical with or homologous to P . K ., 1988 , Int. J . Pept . Protein Res. 32 : 242 - 255. Beta - turn corresponding sequences in antibodies derived from a par profiles can be generated using the method of Deleage, G ., ticular species or belonging to a particular antibody class or Roux B ., 1987 , Protein Engineering 1 :289 - 294 . Thus , each subclass, while the remainder of the chain ( s ) is identical SEZ6 region , domain or motif identified by any of these with or homologous to corresponding sequences in antibod programs or methods is within the scope of the present 50 ies derived from another species or belonging to another invention and may be isolated or engineered to provide antibody class or subclass , as well as fragments of such immunogens giving rise to modulators comprising desired antibodies, so long as they exhibit the desired biological properties . Preferred methods for the generation of SEZ6 activity ( U . S . Pat. No . 4 ,816 , 567 ; Morrison et al ., Proc . Natl . antibodies are further illustrated by way of the Examples Acad . Sci . USA , 81: 6851 -6855 ( 1984 )) . provided herein . Methods for preparing a protein or poly - 55 In one embodiment, a chimeric antibody in accordance peptide for use as an immunogen are well known in the art. with the teachings herein may comprise murine Vy and V , Also well known in the art are methods for preparing amino acid sequences and constant regions derived from immunogenic conjugates of a protein with a carrier , such as human sources. In other compatible embodiments a chime BSA , KLH or other carrier protein . In some circumstances , ric antibody of the present invention may comprise a human direct conjugation using , for example , carbodiimide 60 ized antibody as described below . In another embodiment, reagents are used ; in other instances linking reagents are the so - called “ CDR - grafted ” antibody , the antibody com effective . Administration of a SEZ6 immunogen is often prises one or more CDRs from a particular species or conducted by injection over a suitable time period and with belonging to a particular antibody class or subclass , while use of a suitable adjuvant, as is understood in the art . During the remainder of the antibody chain ( s ) is /are identical with the immunization schedule , titers of antibodies can be taken 65 or homologous to a corresponding sequence in antibodies as described in the Examples below to determine adequacy derived from another species or belonging to another anti of antibody formation . body class or subclass . For use in humans , selected rodent US 9 ,993 ,566 B2 35 36 CDRs may be grafted into a human antibody, replacing one 95 % of the humanized antibody variable region residues will or more of the naturally occurring variable regions or CDRs correspond to those of the recipient FR and CDR sequences . of the human antibody . These constructs generally have the e . Human Antibodies advantages of providing full strength modulator functions In another embodiment, the antibodies may comprise ( e. g ., CDC (complement dependent cytotoxicity ), ADCC 5 fully human antibodies . The term “ human antibody” refers (antibody -dependent cell -mediated cytotoxicity ), etc . ) while to an antibody which possesses an amino acid sequence that reducing unwanted immune responses to the antibody by the corresponds to that of an antibody produced by a human subject. and /or has been made using any of the techniques for making human antibodies . d . Humanized Antibodies J » 10 Human antibodies can be produced using various tech Similar to the CDR - grafted antibody is a “ humanized ” niques known in the art. One technique is phage display in antibody . As used herein , " humanized ” forms of non - human which a library of (preferably human ) antibodies is synthe ( e . g . ,murine ) antibodies are chimeric antibodies that contain sized on phages, the library is screened with the antigen of a minimal sequence derived from one or more non - human interest or an antibody -binding portion thereof, and the immunoglobulins. In one embodiment, a humanized antianti 15 phage that binds the antigen is isolated , from which one may body is a human immunoglobulin (recipient or acceptor obtain the immunoreactive fragments . Methods for prepar antibody ) in which residues from a CDR of the recipient are ing and screening such libraries are well known in the art replaced by residues from a CDR of a non -human species and kits for generating phage display libraries are commer (donor antibody ) such as mouse , rat, rabbit , or nonhuman cially available ( e . g ., the Pharmacia Recombinant Phage primate having the desired specificity , affinity , and / or capac - 20 Antibody System , catalog no . 27 - 9400 -01 ; and the Strata ity . In certain preferred embodiments , residues in one or gene SurfZAPTM phage display kit, catalog no . 240612 ) . more FRs in the variable domain of the human immuno There also are other methods and reagents that can be used globulin are replaced by corresponding non -human residues in generating and screening antibody display libraries ( see , from the donor antibody to help maintain the appropriate e. g. , U . S . Pat. No . 5 ,223 ,409 ; PCT Publication Nos. WO three -dimensional configuration of the grafted CDR (s ) and 25 92 / 18619, WO 91 /17271 , WO 92 /20791 , WO 92/ 15679 , thereby improve affinity . Furthermore , humanized antibod WO 93 /01288 , WO 92 /01047 , WO 92 /09690 ; and Barbas et ies may comprise residues that are not found in the recipient al. , Proc. Natl. Acad . Sci. USA 88 :7978 -7982 (1991 )) . antibody or in the donor antibody to , for example, further In one embodiment, recombinant human antibodies may refine antibody performance . be isolated by screening a recombinant combinatorial anti CDR grafting and humanized antibodies are described , 30 body library prepared as above . In one embodiment, the for example , in U . S . Pat. Nos . 6 , 180 ,370 and 5, 693 , 762 . The library is a scFv phage display library, generated using humanized antibody optionally may also comprise at least a human V , and Vh cDNAs prepared from mRNA isolated portion of an immunoglobulin Fc , typically that of a human from B - cells . immunoglobulin . For further details , see , e . g . , Jones et al . , The antibodies produced by naive libraries ( either natural Nature 321 : 522 - 525 ( 1986 ) ; and U . S . Pat . Nos. 6 , 982, 321 35 or synthetic ) can be of moderate affinity ( K , of about 10 % to and 7 , 087 ,409 . Still another method is termed “ humaneer - 10 M - ) , but affinity maturation can also be mimicked in ing” which is described , for example , in U . S . P . N . 2005 / vitro by constructing and reselecting from secondary librar 0008625 . Additionally, a non -human antibody may also be ies as described in the art . For example , mutation can be modified by specific deletion of human T - cell epitopes or introduced at random in vitro by using error -prone poly “ deimmunization ” by the methods disclosed in WO 40 merase reported in Leung et al ., Technique , 1 : 11 - 15 98 /52976 and WO 00 /34317 . Each of the aforementioned ( 1989 ) ) . Additionally , affinity maturation can be performed references are incorporated herein in their entirety . by randomly mutating one or more CDRs, e. g . using PCR Humanized antibodies may also be bioengineered using with primers carrying random sequence spanning the CDR common molecular biology techniques, such as isolating , of interest , in selected individual Fv clones and screening for manipulating, and expressing nucleic acid sequences that 45 higher -affinity clones . WO 9607754 described a method for encode all or part of immunoglobulin variable regions from inducing mutagenesis in a CDR of an immunoglobulin light at least one of a heavy or light chain . In addition to the chain to create a library of light chain genes. Another sources of such nucleic acid noted above, human germline effective approach is to recombine the Vy or V , domains sequences are available as disclosed , for example , in Tom - selected by phage display with repertoires of naturally linson , I . A . et al . ( 1992 ) J. Mol. Biol. 227 :776 - 798 ; Cook , 50 occurring V domain variants obtained from unimmunized G . P . et al. ( 1995 ) Immunol. Today 16 : 237 - 242 ; Chothia , D . donors and to screen for higher affinity in several rounds of et al . ( 1992 ) J . Mol. Biol. 227 :799 -817 ; and Tomlinson et al. chain reshuffling as described in Marks et al. , Biotechnol. , ( 1995 ) EMBO J 14: 4628 - 4638 . The V - BASE directory 10 : 779 -783 (1992 ). This technique allows the production of ( VBASE2 - Retter et al . , Nucleic Acid Res. 33 ; 671 -674 , antibodies and antibody fragments with a dissociation con 2005 ) provides a comprehensive directory of human immu - 55 stant K , (kokon ) of about 10 - 9 M or less . noglobulin variable region sequences ( compiled by Tomlin - In other embodiments , similar procedures may be son , I. A . et al . MRC Centre for Protein Engineering , employed using libraries comprising eukaryotic cells ( e . g ., Cambridge , UK ) . Consensus human FRs can also be used , yeast) that express binding pairs on their surface . See , for e .g ., as described in U . S . Pat. No. 6 , 300 ,064 . example , U . S . Pat . No . 7 ,700 ,302 and U .S . Ser . No . 12 /404 , In selected embodiments , and as detailed in Example 8 60 059 . In one embodiment, the human antibody is selected below , at least 60 % , 65 % , 70 % , 75 % , or 80 % of the from a phage library, where that phage library expresses humanized antibody heavy or light chain variable region human antibodies (Vaughan et al . Nature Biotechnology amino acid residues will correspond to those of the recipient 14 : 309- 314 ( 1996 ) : Sheets et al . Proc . Natl. Acad . Sci. USA FR and CDR sequences. In other embodiments at least 85 % 95 :6157 -6162 ( 1998 ) . In other embodiments , human bind or 90 % of the humanized antibody variable region residues 65 ing pairs may be isolated from combinatorial antibody will correspond to those of the recipient FR and CDR libraries generated in eukaryotic cells such as yeast . See e . g . , sequences. In a further preferred embodiment, greater than U .S . Pat. No. 7, 700 ,302 . Such techniques advantageously US 9 ,993 , 566 B2 37 38 allow for the screening of large numbers of candidate acids may encode one or both chains of an antibody of the modulators and provide for relatively easy manipulation of invention , or a fragment or derivative thereof. The nucleic candidate sequences ( e . g ., by affinity maturation or recom acid molecules of the invention also include polynucleotides binant shuffling ) . sufficient for use as hybridization probes , PCR primers or Human antibodies can also be made by introducing 5 sequencing primers for identifying , analyzing , mutating or human immunoglobulin loci into transgenic animals , e . g ., amplifying a polynucleotide encoding a polypeptide ; anti mice in which the endogenous immunoglobulin genes have sense nucleic acids for inhibiting expression of a polynucle been partially or completely inactivated and human immu otide , and as well as complementary sequences . The nucleic noglobulin genes have been introduced . Upon challenge , acids can be any length . They can be , for example , 5 , 10 , 15 , human antibody production is observed , which closely 10 20 , 25 , 30 , 35 , 40 , 45 , 50 , 75 , 100 , 125, 150 , 175, 200 , 250 , resembles that seen in humans in all respects , including gene 300 , 350 , 400 , 450 , 500 , 750 , 1 ,000 , 1 ,500 , 3 ,000 , 5 , 000 or rearrangement, assembly , and antibody repertoire . This more nucleotides in length , and /or can comprise one or more approach is described , for example , in U . S . Pat. Nos . 5 ,545 , additional sequences , for example , regulatory sequences , 807 ; 5 ,545 , 806 ; 5 , 569 ,825 ; 5 ,625 , 126 ; 5 ,633 ,425 ; 5 ,661 , and /or be part of a larger nucleic acid , for example , a vector. 016 , and U .S . Pat . Nos . 6 ,075 , 181 and 6 , 150 ,584 regarding 15 It will be appreciated that such nucleic acid sequences can XenoMouse® technology ; and Lonberg and Huszar, Intern . further be manipulated to create modulators including chi Rev. Immunol. 13 :65 - 93 ( 1995 ) . Alternatively, the human meric , humanized or fully human antibodies . More particu antibody may be prepared via immortalization of human B larly , isolated nucleic acid molecules (which may be modi lymphocytes producing an antibody directed against a target fied ) can be used to clone constant and variable region antigen ( such B lymphocytes may be recovered from an 20 sequences for the manufacture antibodies as described in individual suffering from a neoplastic disorder or may have U . S . Pat. No . 7 ,709 , 611 . been immunized in vitro ) . See, e . g . , Cole et al ., Monoclonal The term “ isolated nucleic acid ” means a that the nucleic Antibodies and Cancer Therapy, Alan R . Liss , p . 77 ( 1985 ) ; acid was ( i ) amplified in vitro , for example by polymerase Boerner et al. , J . Immunol, 147 ( 1 ) : 86 -95 ( 1991 ) ; and U . S . chain reaction (PCR ), ( ii ) recombinantly produced by clon Pat . No. 5 , 750 ,373 . 25 ing , ( iii ) purified , for example by cleavage and gel - electro 3 . Further Processing phoretic fractionation , or ( iv ) synthesized , for example by No matter how obtained ,modulator -producing cells ( e . g . , chemical synthesis . An isolated nucleic acid is a nucleic acid hybridomas , yeast colonies, etc .) may be selected , cloned that is available for manipulation by recombinant DNA and further screened for desirable characteristics including , techniques . for example , robust growth , high antibody production and , 30 Whether the source of the nucleic acid encoding the as discussed in more detail below , desirable antibody char desired immunoreactive portion of the antibody is obtained acteristics. Hybridomas can be expanded in vivo in synge - or derived from phage display technology , yeast libraries, neic animals , in animals that lack an immune system , e . g . , hybridoma -based technology or synthetically , it is to be nude mice , or in cell culture in vitro . Methods of selecting , understood that the present invention encompasses the cloning and expanding hybridomas and / or colonies , each of 35 nucleic acid molecules and sequences encoding the antibod which produces a discrete antibody species , are well known ies or antigen - binding fragments or derivatives thereof . to those of ordinary skill in the art . Further , the instant invention is directed to vectors and host B . Recombinant Modulator Production cells comprising such nucleic acid molecules . 1 . Overview Once DNA fragments encoding Vh and V , segments are Once the source is perfected DNA encoding the desired 40 obtained , these DNA fragments can be further manipulated SEZ6 modulators may be readily isolated and sequenced by standard recombinant DNA techniques , for example to using conventional procedures ( e . g . , by using oligonucle convert the variable region genes to full -length antibody otide probes that are capable of binding specifically to genes chain genes , to Fab fragment genes or to a scFv gene . In encoding antibody heavy and light chains ) . Isolated and these manipulations , a Ve- or Vy - encoding DNA fragment subcloned hybridoma cells ( or phage or yeast derived colo - 45 is operatively linked to another DNA fragment encoding nies ) may serve as a preferred source of such DNA if the another protein , such as an antibody constant region or a modulator is an antibody. If desired , the nucleic acid can flexible linker. The term “ operatively linked ” , as used in this further be manipulated as described herein to create agents context, means that the two DNA fragments are joined such including fusion proteins , or chimeric , humanized or fully that the amino acid sequences encoded by the two DNA human antibodies . More particularly , isolated DNA (which 50 fragments remain in - frame. may be modified ) can be used to clone constant and variable The isolated DNA encoding the Vy region can be con region sequences for the manufacture antibodies . verted to a full - length heavy chain gene by operatively Accordingly , in exemplary embodiments antibodies may linking the Vy - encoding DNA to another DNA molecule be produced recombinantly , using conventional procedures encoding heavy chain constant regions (CH1 , CH2 and CH3) . ( such as those set forth in Al- Rubeai; An , and Shire et . al. all 55 The sequences of human heavy chain constant region genes supra , and Sambrook J. & Russell D .Molecular Cloning : A are known in the art (see e .g . , Kabat , E . A ., et al. (1991 ) Laboratory Manual, 3rd ed ., Cold Spring Harbor Laboratory Sequences of Proteins of Immunological Interest, Fifth Press, Cold Spring Harbor, N . Y . (2000 ) ; Ausubel et al. , Short Edition , U . S . Department of Health and Human Services, Protocols in Molecular Biology : A Compendium ofMethods NIH Publication No . 91 - 3242 ) and DNA fragments encom from Current Protocols in Molecular Biology , Wiley, John & 60 passing these regions can be obtained by standard PCR Sons , Inc . ( 2002 ) ) in which the isolated and subcloned amplification . The heavy chain constant region can be an hybridoma cells ( or phage or yeast derived colonies ) serve IgG1, IgG2, IgG3, IgG4, IgA , IgE , IgM or IgD constant as a preferred source of nucleic acid molecules. region , but most preferably is an IgGl or IgG4 constant The term “ nucleic acid molecule ” , as used herein , is region . As discussed in more detail below an exemplary intended to include DNA molecules and RNA molecules and 65 IgG1 constant region that is compatible with the teachings artificial variants thereof ( e . g . , peptide nucleic acids ), herein is set forth as SEQ ID NO : 403 in the appended whether single - stranded or double -stranded . The nucleic sequence listing . For a Fab fragment heavy chain gene, the US 9 ,993 ,566 B2 39 40 VH - encoding DNA can be operatively linked to another assigned to various substitutions, deletions and other modi DNA molecule encoding only the heavy chain CH1 constant fications, including conservative amino acid substitutions . region . For instance , the sequence analysis tool GCG (Accelrys The isolated DNA encoding the V? region can be con Software Inc . ) contains programs such as “GAP ” and verted to a full - length light chain gene ( as well as a Fab light 5 “ BEST- FIT ” which can be used with default parameters to chain gene ) by operatively linking the V , - encoding DNA to determine sequence homology or sequence identity between another DNA molecule encoding the light chain constant closely related polypeptides , such as homologous polypep region , C , . The sequences of human light chain constant tides from different species of organisms or between a wild region genes are known in the art ( see e . g ., Kabat, E . A . , et type protein and a mutein thereof. (See , e . g . , GCG Version al. ( 1991 ) Sequences of Proteins of Immunological Interest, 10 6 . 1 or Durbin et. Al. , Biological Sequence Analysis : Proba Fifth Edition , U . S . Department of Health and Human Ser bilistic models of proteins and nucleic acids ., Cambridge vices , NIH Publication No . 91 - 3242) and DNA fragments Press ( 1998 ) ) . encompassing these regions can be obtained by standard Polypeptide sequences can also be compared using PCR amplification . The light chain constant region can be a FASTA using default or recommended parameters, a pro kappa or lambda constant region , but most preferably is a 15 gram in GCG Version 6 .1 . FASTA (e . g ., FASTA2 and kappa constant region . In this respect an exemplary com - FASTA3 ) provides alignments and percent sequence identity patible kappa light chain constant region is set forth as SEQ of the regions of the best overlap between the query and ID NO : 404 in the appended sequence listing . search sequences ( Pearson (2000 ) supra ) . Another preferred 2 . Hybridization and Sequence Identity algorithm when comparing a sequence of the invention to a As indicated , the invention further provides nucleic acids 20 database containing a large number of sequences from that hybridize to other nucleic acids under particular hybrid different organisms is the computer program BLAST, espe ization conditions. More specifically the invention encom - cially blastp or tblastn , using default parameters . See , e . g ., passes nucleic acidsmolecules that hybridize under moder Altschul et al. ( 1990 ) J. Mol. Biol . 215 : 403 410 and ate or high stringency hybridization conditions ( e . g ., as Altschul et al . ( 1997 ) Nucleic Acids Res . 25 : 3389 402 , each defined below ) , to the nucleic acid molecules of the inven - 25 of which is herein incorporated by reference. tion . Methods for hybridizing nucleic acids are well -known In this regard the invention also includes nucleic acid in the art . As is well known , a moderately stringent hybrid molecules that encode polypeptides that are “ substantially ization conditions comprise a prewashing solution contain identical" with respect to an antibody variable region poly ing 5x sodium chloride / sodium citrate (SSC ) , 0 . 5 % SDS , 1 . 0 peptide sequence ( e . g ., either the donor light or heavy chain mM EDTA (pH 8 . 0 ), hybridization buffer of about 50 % 30 variable region , acceptor light or heavy chain variable region formamide , 6xSSC , and a hybridization temperature of 55° or resulting humanized construct ) . As applied to such poly C . (or other similar hybridization solutions , such as one peptides , the term " substantial identity " or " substantially containing about 50 % formamide , with a hybridization identical” means that two peptide sequences , when opti temperature of 42° C .) , and washing conditions of 60° C ., in mally aligned , such as by the programsGAP or BEST- FIT 0 .5xSSC , 0 . 1 % SDS . By way of comparison hybridization 35 using default gap weights , share at least 60 % or 65 % under highly stringent hybridization conditions comprise sequence identity , preferably at least 70 % , 75 % , 80 % , 85 % , washing with 6xSSC at 45° C ., followed by one or more or 90 % sequence identity , even more preferably at least washes in 0 . 1xSSC , 0 . 2 % SDS at 68° C . Furthermore , one 93 % , 95 % , 98 % or 99 % sequence identity. Preferably , of skill in the art can manipulate the hybridization and /or residue positions which are not identical differ by conser washing conditions to increase or decrease the stringency of 40 vative amino acid substitutions . A " conservative amino acid hybridization such that nucleic acids comprising nucleotide substitution ” is one in which an amino acid residue is sequences that are at least 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , substituted by another amino acid residue having a side 95 % , 98 % or 99 % identical to each other typically remain chain ( R group ) with similar chemical properties ( e . g . , hybridized to each other. charge or hydrophobicity ) . In general , a conservative amino The invention also includes nucleic acid molecules that 45 acid substitution will not substantially change the functional are “ substantially identical ” to the described nucleic acid properties of a protein . In cases where two or more amino molecules . In one embodiment, the term substantially iden - acid sequences differ from each other by conservative sub tical with regard to a nucleic acid sequence means may be stitutions , the percent sequence identity or degree of simi construed as a sequence of nucleic acid molecules exhibiting larity may be adjusted upwards to correct for the conserva at least about 65 % , 70 % , 75 % , 80 % , 85 % , or 90 % sequence 50 tive nature of the substitution . identity. In other embodiments , the nucleic acid molecules 3 . Expression exhibit 95 % or 98 % sequence identity to the reference The varied processes of recombinant expression , i. e . , the nucleic acid sequence . production of RNA or of RNA and protein /peptide , are well The basic parameters affecting the choice of hybridization known as set forth , for example , in Berger and Kimmel , conditions and guidance for devising suitable conditions are 55 Guide to Molecular Cloning Techniques , Methods in Enzy set forth by, for example , Sambrook , Fritsch , and Maniatis mology volume 152 Academic Press , Inc ., San Diego , Calif. ; ( 1989 , Molecular Cloning : A Laboratory Manual, Cold Sambrook et al. , Molecular Cloning - A Laboratory Manual Spring Harbor Laboratory Press , Cold Spring Harbor, N . Y . , (3rd Ed . ) , Vol . 1 - 3 , Cold Spring Harbor Laboratory , Cold chapters 9 and 11 ; and Current Protocols in Molecular Spring Harbor, N . Y ., (2000 ) ; and Current Protocols in Biology , 1995 , Ausubel et al. , eds. , John Wiley & Sons , Inc ., 60 Molecular Biology , F . M . Ausubel et al. , eds ., Current sections 2 . 10 and 6 . 3 - 6 . 4 ) , and can be readily determined by Protocols , a joint venture between Greene Publishing Asso those having ordinary skill in the art based on , for example , ciates , Inc . and John Wiley & Sons , Inc . , (supplemented the length and /or base composition of the nucleic acid . through 2006 ). Sequence similarity for polypeptides , which is also Certain terms of interest include “ expression control referred to as sequence identity , is typically measured using 65 sequence ” which comprises promoters, ribosome binding sequence analysis software . Protein analysis software sites, enhancers and other control elements which regulate matches similar sequences using measures of similarity transcription of a gene or translation of mRNA . As is well US 9 , 993 ,566 B2 41 42 known, a “ promoter ” or “ promoter region ” relates to a P3X63 mouse myeloma cells , PER cells , PER .C6 cells or nucleic acid sequence which generally is located upstream hybridoma cells , yeast cells , insect cells , and plant cells , to ( 5 ') to the nucleic acid sequence being expressed and con - name only a few , but also cells comprised within a trans trols expression of the sequence by providing a recognition genic animal, transgenic plant or cultured plant or animal and binding site for RNA - polymerase . 5 tissue . Exemplary promoters which are compatible according to For long -term , high - yield production of recombinant pro the invention include promoters for SP6 , 13 and 17 poly - teins stable expression is preferred . Accordingly , cell lines merase , human U6 RNA promoter, CMV promoter , and artificial hybrid promoters thereof ( e . g . CMV) where a part that stably express the selected modulator may be engi or parts are fused to a part or parts of promoters of genes of 10 neered using standard art- recognized techniques . Rather other cellular proteins such as e . g . human GAPDH ( glycer than using expression vectors that contain viral origins of aldehyde - 3 - phosphate dehydrogenase ), and including or not replication , host cells can be transformed with DNA con including (an ) additional intron ( s ) . trolled by appropriate expression control elements (e . g. , In certain embodiments , the nucleic acid molecule may be promoter , enhancer , sequences, transcription terminators , present in a vector, where appropriate with a promoter. 15 polyadenylation sites, etc . ) , and a selectable marker. Any of which controls expression of the nucleic acid . The well the selection systems well known in the art may be used , known term “ vector ” comprises any intermediary vehiclevehicle for including the glutamine synthetase gene expression system a nucleic acid which enables said nucleic acid , for example , ( the GS system ) which provides an efficient approach for to be introduced into prokaryotic and /or eukaryotic cells enhancing expression under certain conditions. The GS and , where appropriate , to be integrated into a genome. 20 system is discussed in whole or part in connection with EP Methods of transforming mammalian cells are well known patents 0 216 846 , 0 256 055 , 0 323 997 and 0 338 841 and in the art . See, for example , U . S . Pat . Nos . 4 , 399 , 216 , U . S . Pat. Nos. 5 ,591 , 639 and 5 , 879 , 936 each of which is 4 ,912 ,040 , 4 ,740 ,461 , and 4 , 959 , 455 . The vectors may incorporated herein by reference . Another preferred expres include a nucleotide sequence encoding an antibody of the sion system , the FreedomTM CHO - S Kit is commercially invention ( e. g ., a whole antibody, a heavy or light chain of 25 provided by Life Technologies (Catalog Number A13696 an antibody , a Vy or V? of an antibody, or a portion thereof, 01) also allows for the development of stable cell lines that or a heavy - or light - chain CDR , a single chain Fv , or may be used for modulator production . fragments or variants thereof) , operably linked to a promoter Such host -expression systems represent vehicles by ( see , e . g . , PCT Publication WO 86 /05807 ; PCT Publication which the coding sequences of interest may be produced and WO 89 /01036 ; and U . S . Pat . No . 5 , 122 ,464 ). 30 subsequently purified , but also represent cells which may, A variety of host -expression vector systems are commer - when transformed or transfected with the appropriate cially available , and many are compatible with the teachings nucleotide coding sequences , express a molecule of the herein and may be used to express the modulators of the invention in situ . The host cell may be co - transfected with invention . Such systems include , but are not limited to , two expression vectors of the invention , for example , the microorganisms such as bacteria ( e . g ., E . coli , B . subtilis , 35 first vector encoding a heavy chain derived polypeptide and streptomyces ) transformed with recombinant bacteriophage the second vector encoding a light chain derived polypep DNA , plasmid DNA or cosmid DNA expression vectors tide . containing modulator coding sequences ; yeast ( e . g ., Saccha - Thus , in certain embodiments , the present invention pro romyces, Pichia ) transfected with recombinant yeast expres vides recombinant host cells allowing for the expression of sion vectors containing modulator coding sequences ; insect 40 antibodies or portions thereof . Antibodies produced by cell systems infected with recombinant virus expression expression in such recombinant host cells are referred to vectors ( e . g ., baculovirus ) containing modulator coding herein as recombinant antibodies . The present invention also sequences ; plant cell systems ( e . g . , Nicotiana , Arabidopsis, provides progeny cells of such host cells , and antibodies duckweed , corn , wheat, potato , etc . ) infected with recom - produced by the same. binant viral expression vectors ( e . g . , cauliflower mosaic 45 C . Chemical Synthesis virus ; tobacco mosaic virus ) or transfected with recombinant In addition , the modulators may be chemically synthe plasmid expression vectors ( e . g ., Ti plasmid ) containing sized using techniques known in the art ( e . g ., see Creighton , modulator coding sequences ; or mammalian cell systems 1983 , Proteins : Structures and Molecular Principles , W . H . ( e. g ., COS, CHO , BHK , 293 , 3T3 cells , etc .) harboring Freeman & Co ., N . Y ., and Hunkapiller, M ., et al ., 1984 , recombinant expression constructs containing promoters 50 Nature 310 : 105 - 111 ) . Furthermore , if desired , nonclassical derived from the genome of mammalian cells ( e . g ., metal- amino acids or chemical amino acid analogs ( such as lothionein promoter ) or from mammalian viruses ( e . g . , the D - isomers of the common amino acids , 2 ,4 - diaminobutyric adenovirus late promoter ; the vaccinia virus 7 .5K promoter ) . acid , a -amino isobutyric acid , 4 -aminobutyric acid , and the As used herein , the term “ host cell ” covers any kind of like ) can be introduced as a substitution or addition into a cellular system which can be engineered to generate the 55 polypeptide sequence . polypeptides and antigen - binding molecules of the present D . Transgenic Systems invention . In one embodiment, the host cell is engineered to In other embodiments modulators may be produced trans allow the production of an antigen binding molecule with genically through the generation of a mammal or plant that modified glycoforms. In a preferred embodiment, the anti - is transgenic for recombinant molecules such as the immu gen binding molecule , or variant antigen binding molecule , 60 noglobulin heavy and light chain sequences and that pro is an antibody, antibody fragment, or fusion protein . In duces the desired compounds in a recoverable form . This certain embodiments, the host cells have been further includes , for example , the production of protein modulators manipulated to express increased levels of one or more ( e . g . , antibodies ) in , and recovery from , the milk of goats , polypeptides having N -acetylglucosaminyltransferase III cows, or other mammals . See, e . g . , U . S . Pat. Nos. 5 , 827 ,690 , (GnTI11 ) activity . Compatible host cells include cultured 65 5 , 756 ,687 , 5 ,750 , 172, and 5 , 741, 957 . In some embodiments , cells , e . g ., mammalian cultured cells , such as CHO cells , non -human transgenic animals that comprise human immu BHK cells , NSO cells , SP2 / 0 cells , YO myeloma cells , noglobulin loci are immunized to produce antibodies. US 9 ,993 ,566 B2 43 44 Other transgenic techniques are set forth in Hogan et al. , comprises an antibody or fragment, construct or derivative Manipulating the Mouse Embryo : A Laboratory Manual 2nd thereof such associations may be through one or more ed ., Cold Spring Harbor Press ( 1999 ) ; Jackson et al. , Mouse “ binding sites ” or “ binding components ” expressed on the Genetics and Transgenics : A Practical Approach , Oxford antibody, where a binding site comprises a region of a University Press ( 2000 ) ; and Pinkert , Transgenic Animal 5 polypeptide that is responsible for selectively binding to a Technology : A Laboratory Handbook , Academic Press target molecule or antigen of interest . Binding domains ( 1999 ) and U . S . Pat. No. 6 ,417 ,429 . In some embodiments , comprise at least one binding site (e .g . , an intact IgG the non -human animals are mice , rats , sheep , pigs , goats , antibody will have two binding domains and two binding cattle or horses, and the desired product is produced in sites) . Exemplary binding domains include an antibody blood , milk , urine, saliva , tears , mucus and other bodily 110 varvariable domain , a receptor - binding domain of a ligand, a fluids from which it is readily obtainable using art -recog - ligand -binding domain of a receptor or an enzymatic nized purification techniques. Other compatible production systems include methods for domain . making antibodies in plants such as described , for example, A . Antibodies in U . S . Pat. Nos . 6 , 046 ,037 and 5 , 959, 177 which are incor - 15 As noted above, the term “ antibody ” is intended to cover, porated herein with respect to such techniques . at least , polyclonal antibodies , multiclonal antibodies , chi E . Isolation / Purification meric antibodies , CDR grafted antibodies , humanized and Once a modulator of the invention has been produced by primatized antibodies, human antibodies , recombinantly recombinant expression or any other of the disclosed tech produced antibodies, intrabodies , multispecific antibodies , niques, it may be purified by any method known in the art 20 bispecific antibodies , monovalent antibodies , multivalent for purification of immunoglobulins or proteins. In this antibodies , anti - idiotypic antibodies, as well as synthetic respect the modulator may be “ isolated ” which means that it antibodies . has been identified and separated and /or recovered from a B . Fragments component of its natural environment. Contaminant com Regardless of which form of themodulator ( e . g . chimeric , ponents of its natural environment are materials that would 25 humanized , etc .) is selected to practice the invention it will interfere with diagnostic or therapeutic uses for the poly - be appreciated that immunoreactive fragments of the same peptide and may include enzymes , hormones , and other may be used in accordance with the teachings herein . An proteinaceous or nonproteinaceous solutes. Isolated modu - “ antibody fragment” comprises at least a portion of an intact lators include a modulator in situ within recombinant cells antibody . As used herein , the term “ fragment of an antibody because at least one component of the polypeptide' s natural 30 molecule includes antigen - binding fragments of antibodies, environment will not be present. and the term “ antigen - binding fragment” refers to a poly If the desired molecule is produced intracellularly, as a peptide fragment of an immunoglobulin or antibody that first step , the particulate debris, either host cells or lysed immunospecifically binds or reacts with a selected antigen fragments , may be removed , for example , by centrifugation or immunogenic determinant thereof or competes with the or ultrafiltration . Where the modulator is secreted into the 35 intact antibody from which the fragments were derived for medium , supernatants from such expression systems are specific antigen binding . generally first concentrated using a commercially available Exemplary fragments include : V , Vy , scFv, F ( ab ') 2 protein concentration filter, for example , an Amicon or fragment, Fab fragment, Fd fragment, Fv fragment, single Pellicon ultrafiltration unit (Millipore Corp . ) . Once the domain antibody fragments , diabodies, linear antibodies , insoluble contaminants are removed the modulator prepa - 40 single - chain antibody molecules and multispecific antibod ration may be further purified using standard techniques ies formed from antibody fragments. In addition , an active such as, for example , hydroxylapatite chromatography, gel fragment comprises a portion of the antibody that retains its electrophoresis , dialysis , and affinity chromatography, with ability to interact with the antigen / substrates or receptors affinity chromatography of particular interest . In this regard and modify them in a manner similar to that of an intact protein A can be used to purify antibodies that are based on 45 antibody ( though maybe with somewhat less efficiency ). human IgG1, IgG2 or IgG4 heavy chains (Lindmark , et al. , In other embodiments , an antibody fragment is one that J Immunol Meth 62 : 1 ( 1983 ) ) while protein G is recom comprises the Fc region and that retains at least one of the mended for all mouse isotypes and for human IgG3 (Guss , biological functions normally associated with the Fc region et al ., EMBO J 5 : 1567 ( 1986 ) ) . Other techniques for protein when present in an intact antibody, such as FcRn binding , purification such as fractionation on an ion - exchange col- 50 antibody half - life modulation , ADCC function and comple umn , ethanol precipitation , reverse phase HPLC , chroma - ment binding . In one embodiment, an antibody fragment is tography on silica , chromatography on heparin , sepharose a monovalent antibody that has an in vivo half - life substan chromatography on an anion or cation exchange resin (such tially similar to an intact antibody . For example , such an as a polyaspartic acid column ), chromatofocusing, SDS antibody fragment may comprise an antigen binding arm PAGE and ammonium sulfate precipitation are also avail - 55 linked to an Fc sequence capable of conferring in vivo able depending on the antibody to be recovered . In particu stability to the fragment. larly preferred embodiments the modulators of the instant As would be well recognized by those skilled in the art , invention will be purified , at least in part, using Protein A or fragments can be obtained via chemical or enzymatic treat Protein G affinity chromatography . ment ( such as papain or pepsin ) of an intact or complete 60 antibody or antibody chain or by recombinant means . See , VI. SEZ6 Modulator Fragments and Derivatives e . g . , Fundamental Immunology, W . E . Paul, ed . , Raven Press , N . Y . (1999 ) , for a more detailed description of anti Whatever generation and production methodology is body fragments . selected , modulators of the instant invention will react, bind , C . Derivatives combine, complex , connect , attach , join , interact or other - 65 The invention further includes immunoreactivemodulator wise associate with a target determinant ( e . g . , antigen ) and derivatives and antigen binding molecules comprising one thereby provide the desired results . Where the modulator or more modifications . US 9 , 993 ,566 B2 45 46 1 . Multivalent Antibodies increased production , altered Fc ligand binding to an Fc In one embodiment, the modulators of the invention may receptor (FcR ) , enhanced or reduced “ ADCC ” ( antibody be monovalent or multivalent ( e . g ., bivalent, trivalent, etc. ) . dependent cell mediated cytotoxicity ) or " CDC ” ( comple As used herein , the term “ valency ” refers to the number of ment -dependent cytotoxicity ) activity , altered glycosylation potential target binding sites associated with an antibody . 5 and / or disulfide bonds and modified binding specificity . In Each target binding site specifically binds one target mol- this regard it will be appreciated that these Fc variants may ecule or specific position or locus on a target molecule . advantageously be used to enhance the effective anti -neo When an antibody is monovalent , each binding site of the plastic properties of the disclosed modulators . molecule will specifically bind to a single antigen position or To this end certain embodiments of the invention may epitope . When an antibody comprises more than one target 10 comprise substitutions or modifications of the Fc region , for binding site (multivalent ) , each target binding site may example the addition of one or more amino acid residue , specifically bind the same or different molecules ( e . g ., may substitutions, mutations and / or modifications to produce a bind to different ligands or different antigens , or different compound with enhanced or preferred Fc effector functions. epitopes or positions on the same antigen ) . See , for example , For example, changes in amino acid residues involved in the U . S . P . N . 2009 /0130105 . In each case at least one of the 15 interaction between the Fc domain and an Fc receptor ( e . g . , binding sites will comprise an epitope , motif or domain FcyRI, FcyRIIA and B , FcyRIII and FcRn ) may lead to associated with a SEZ6 isoform . increased cytotoxicity and / or altered pharmacokinetics, such In one embodiment, the modulators are bispecific anti as increased serum half - life ( see , for example , Ravetch and bodies in which the two chains have different specificities , Kinet , Annu Rev. Immunol 9 : 457 - 92 ( 1991 ) ; Capel et al. , as described in Millstein et al. , 1983 , Nature , 305 :537 -539 . 20 Immunomethods 4 : 25 - 34 ( 1994 ) ; and de Haas et al. , J . Lab . Other embodiments include antibodies with additional Clin .Med . 126 :330 -41 (1995 ) each of which is incorporated specificities such as trispecific antibodies. Other more herein by reference ). sophisticated compatible multispecific constructs and meth - In selected embodiments , antibodies with increased in ods of their fabrication are set forth in U . S . P . N . 2009 / vivo half - lives can be generated by modifying ( e .g ., substi 0155255 , as well as WO 94 /04690 ; Suresh et al. , 1986 , 25 tuting , deleting or adding ) amino acid residues identified as Methods in Enzymology, 121 :210 ; and W096 / 27011. involved in the interaction between the Fc domain and the As alluded to above, multivalent antibodies may immu - FcRn receptor ( see , e . g . , International Publication Nos . WO nospecifically bind to different epitopes of the desired target 97/ 34631 ; WO 04 /029207 ; U .S . Pat. No . 6 , 737 ,056 and molecule or may immunospecifically bind to both the target U . S . P . N . 2003 /0190311 . With regard to such embodiments , molecule as well as a heterologous epitope, such as a 30 Fc variants may provide half- lives in a mammal, preferably heterologous polypeptide or solid support material. While a human , of greater than 5 days , greater than 10 days , greater preferred embodiments of the anti -SEZ6 antibodies only than 15 days, preferably greater than 20 days, greater than 25 bind two antigens ( i .e . bispecific antibodies ) , antibodies with days , greater than 30 days, greater than 35 days, greater than additional specificities such as trispecific antibodies are also 40 days, greater than 45 days, greater than 2 months, greater encompassed by the instant invention . Bispecific antibodies 35 than 3 months , greater than 4 months , or greater than 5 also include cross -linked or " heteroconjugate " antibodies . months . The increased half - life results in a higher serum titer For example , one of the antibodies in the heteroconjugate which thus reduces the frequency of the administration of can be coupled to avidin , the other to biotin . Such antibodies the antibodies and / or reduces the concentration of the anti have , for example , been proposed to target immune system bodies to be administered . Binding to human FcRn in vivo cells to unwanted cells ( U . S . Pat . No . 4 ,676 , 980 ) , and for 40 and serum half life of human FcRn high affinity binding treatment of HIV infection (WO 91/ 00360 , WO 92 /200373 , polypeptides can be assayed , e. g ., in transgenic mice or and EP 03089 ) . Heteroconjugate antibodies may be made transfected human cell lines expressing human FcRn , or in using any convenient cross - linking methods . Suitable cross - primates to which the polypeptides with a variant Fc region linking agents are well known in the art , and are disclosed are administered . WO 2000 /42072 describes antibody vari in U . S . Pat. No. 4 ,676 , 980 , along with a number of cross - 45 ants with improved or diminished binding to FcRns. See linking techniques. also , e . g . , Shields et al. J. Biol. Chem . 9 ( 2 ) : 6591- 6604 In yet other embodiments , antibody variable domains ( 2001 ) . with the desired binding specificities ( antibody - antigen In other embodiments , Fc alterations may lead to combining sites ) are fused to immunoglobulin constant enhanced or reduced ADCC or CDC activity. As in known domain sequences , such as an immunoglobulin heavy chain 50 in the art , CDC refers to the lysing of a target cell in the constant domain comprising at least part of the hinge , C2, presence of complement, and ADCC refers to a form of and / or Cy3 regions , using methods well known to those of cytotoxicity in which secreted Ig bound onto FcRs present ordinary skill in the art . on certain cytotoxic cells ( e . g ., Natural Killer cells , neutro 2 . Fc Region Modifications phils , and macrophages ) enables these cytotoxic effector In addition to the various modifications, substitutions , 55 cells to bind specifically to an antigen -bearing target cell and additions or deletions to the variable or binding region of the subsequently kill the target cell with cytotoxins . In the disclosed modulators ( e . g ., Fc -SEZ6 or anti - SEZ6 antibod context of the instant invention antibody variants are pro ies ) set forth above , those skilled in the art will appreciate vided with “ altered ” FcR binding affinity , which is either that selected embodiments of the present invention may also enhanced or diminished binding as compared to a parent or comprise substitutions or modifications of the constant 60 unmodified antibody or to an antibody comprising a native region ( i . e . the Fc region ) . More particularly , it is contem sequence FcR . Such variants which display decreased bind plated that the SEZ6 modulators of the invention may i ng may possess little or no appreciable binding, e . g ., 0 - 20 % contain inter alia one or more additional amino acid residue binding to the FcR compared to a native sequence, e . g . as substitutions , mutations and / or modifications which result in determined by techniques well known in the art . In other a compound with preferred characteristics including, but not 65 embodiments the variant will exhibit enhanced binding as limited to : altered pharmacokinetics , increased serum half compared to the native immunoglobulin Fc domain . It will life , increase binding affinity , reduced immunogenicity , be appreciated that these types of Fc variants may advan US 9 ,993 ,566 B2 48 tageously be used to enhance the effective anti- neoplastic fluorescent, radioisotopic or affinity label to allow for detec properties of the disclosed antibodies. In yet other embodi- tion and isolation of the modulator. ments , such alterations lead to increased binding affinity , reduced immunogenicity, increased production , altered gly VII . Modulator Characteristics cosylation and / or disulfide bonds ( e . g ., for conjugation 5 sites ), modified binding specificity , increased phagocytosis ; No matter how obtained or which of the aforementioned and/ or down regulation of cell surface receptors ( e . g . B cell forms the modulator takes, various embodiments of the disclosed modulators may exhibit certain characteristics . In receptor ; BCR ) , etc. selected embodiments , antibody - producing cells ( e . g . , 3 . Altered Glycosylation ed 10 hybridomas or yeast colonies ) may be selected , cloned and Still other embodiments comprise one or more engineered further screened for favorable properties including , for glycoforms, i. e. , a SEZ6 modulator comprising an altered example , robust growth , high modulator production and , as glycosylation pattern or altered carbohydrate composition discussed in more detail below , desirable modulator char that is covalently attached to the protein ( e . g . , in the Fc acteristics. In other cases characteristics of the modulator domain ). See , for example , Shields, R . L . et al . ( 2002 ) J. 15 may be imparted or influenced by selecting a particular Biol. Chem . 277 : 26733 - 26740 . Engineered glycoforms may antigen ( e . g ., a specific SEZ6 isoform or fragment thereof) be useful for a variety of purposes, including but not limited or immunoreactive fragment of the target antigen for inocu to enhancing or reducing effector function , increasing the lation of the animal . In still other embodiments the selected affinity of the modulator for a target or facilitating produc modulators may be engineered as described above to tion of the modulator. In certain embodiments where 20 enhance or refine immunochemical characteristics such as reduced effector function is desired , the molecule may be affinity or pharmacokinetics . engineered to express an aglycosylated form . Substitutions A . Neutralizing Modulators that may result in elimination of one or more variable region In certain embodiments , the modulators will comprise framework glycosylation sites to thereby eliminate glyco - " neutralizing ” antibodies or derivatives or fragments sylation at that site are well known ( see e . g . U . S . Pat. Nos . 25 thereof . That is , the present invention may comprise anti 5 ,714 , 350 and 6 ,350 ,861 ) . Conversely , enhanced effector body molecules that bind specific domains, motifs or functions or improved binding may be imparted to the Fc epitopes and are capable of blocking , reducing or inhibiting containing molecule by engineering in one or more addi the biological activity of SEZ6 . More generally the term tional glycosylation sites . " neutralizing antibody ” refers to an antibody that binds to or can 30 interacts with a target molecule or ligand and prevents Other embodiments include an Fc variant that has an 30 binding or association of the target molecule to a binding altered glycosylation composition , such as a hypofucosy partner such as a receptor or substrate , thereby interrupting lated antibody having reduced amounts of fucosyl residues a biological response that otherwise would result from the or an antibody having increased bisecting GlcNAc struc interaction of the molecules. tures . Such altered glycosylation patterns have been dem - ,35 It will be appreciated that competitive binding assays onstrated to increase the ADCC ability of antibodies . Engi known in the art may be used to assess the binding and neered glycoformsmay be generated by any method known specificity of an antibody or immunologically functional to one skilled in the art, for example by using engineered or fragment or derivative thereof. With regard to the instant variant expression strains , by co -expression with one or invention an antibody or fragment will be held to inhibit or more enzymes ( for example N -acetylglucosaminyltrans - 40 reduce binding of SEZ6 to a binding partner or substrate ferase III (GnT111 ) ) , by expressing a molecule comprising (e . g ., a neurotrophic ligand ) when an excess of antibody an Fc region in various organisms or cell lines from various reduces the quantity of binding partner bound to SEZ6 by at organisms or by modifying carbohydrate (s ) after the mol- least about 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 85 % , ecule comprising Fc region has been expressed ( see , for 90 % , 95 % , 97 % , 99 % or more as measured , for example , by example , WO 2012 / 117002 ). 45 impaired neurotrophic ligand activity or in an in vitro 4 . Additional Processing competitive binding assay . In the case of antibodies to SEZO The modulators may be differentially modified during or for example , a neutralizing antibody or antagonist will after production , e . g . , by glycosylation , acetylation , phos preferably alter ligand activity by at least about 20 % , 30 % , phorylation , amidation , derivatization by known protecting 40 % , 50 % , 60 % , 70 % , 80 % , 85 % , 90 % , 95 % , 97 % , 99 % or anti - 50 more . It will be appreciated that this modified activity may blocking groups, proteolytic cleavage , linkage to an anti be measured directly using art- recognized techniques ormay body molecule or other cellular ligand , etc . Any of numerous be measured by the impact the altered activity has down chemical modifications may be carried out by known tech stream ( e . g . , oncogenesis , cell survival or pathway activa niques , including but not limited , to specific chemical cleav tion ) . age by cyanogen bromide, trypsin , chymotrypsin , papainull , 55 B . Internalizing Modulators V8 protease , NaBH4, acetylation , formylation , oxidation , While evidence indicates that SEZ6 or selected isoforms reduction , metabolic synthesis in the presence of tunicamy thereof may be present in a soluble form , at least some SEZO cin , etc . likely remains associated with the cell surface thereby Various post - translational modifications also encom - allowing for internalization of the disclosed modulators . passed by the invention include , for example , N - linked or 60 Accordingly , the anti- SEZ6 antibodies of the instant inven O - linked carbohydrate chains, processing of N - terminal or tion may be internalized , at least to some extent, by cells that C -terminal ends, attachment of chemical moieties to the express SEZ6 . For example , an anti- SEZ6 antibody that amino acid backbone, chemical modifications of N - linked or binds to SEZ6 on the surface of a tumor- initiating cell may O - linked carbohydrate chains, and addition or deletion of an be internalized by the tumor- initiating cell . In particularly N - terminalmethionine residue as a result of prokaryotic host 65 preferred embodiments such anti - SEZ6 antibodies may be cell expression . Moreover, the modulators may also be associated with or conjugated to anticancer agents such as modified with a detectable label, such as an enzymatic , cytotoxic moieties that kill the cell upon internalization . In US 9 , 993 ,566 B2 49 50 particularly preferred embodiments themodulator will com antigen in a complex mixture of proteins and /or macromol prise an internalizing antibody drug conjugate . ecules . In preferred embodiments , an antibody is said to As used herein , a modulator that “ internalizes” is one that specifically bind an antigen when the equilibrium dissocia is taken up ( along with any payload ) by the cell upon tion constant (KD ) is less than or equal to 10 - * M or less than binding to an associated antigen or receptor. As will be 5 or equal to 10 - ' M , more preferably when the equilibrium appreciated , the internalizing modulator may , in preferred dissociation constant is less than or equal to 10 - 8M , and embodiments , comprise an antibody including antibody even more preferably when the dissociation constant is less fragments and derivatives thereof, as well as antibody con - than or equal to 10 - ' M jugates . Internalization may occur in vitro or in vivo . For More directly the term “ epitope ” is used in its common therapeutic applications , internalization will preferably 10 biochemical sense and refers to that portion of the target occur in vivo in a subject in need thereof. The number of antigen capable of being recognized and specifically bound antibody molecules internalized may be sufficient or by a particular antibody modulator. When the antigen is a adequate to kill an antigen - expressing cell , especially an polypeptide such as SEZ6 , epitopes may generally be antigen - expressing cancer stem cell. Depending on the formed from both contiguous amino acids and noncontigu potency of the antibody or antibody conjugate , in some 15 ous amino acids juxtaposed by tertiary folding of a protein instances, the uptake of a single antibody molecule into the " conformational epitopes ” ) . In such conformational cell is sufficient to kill the target cell to which the antibody epitopes the points of interaction occur across amino acid binds. For example , certain toxins are so highly potent that residues on the protein that are linearly separated from one the internalization of a few molecules of the toxin conju - another. Epitopes formed from contiguous amino acids gated to the antibody is sufficient to kill the tumor cell. 20 ( sometimes referred to as “ linear " or " continuous ” epitopes ) Whether an antibody internalizes upon binding to a mam - are typically retained upon protein denaturing , whereas malian cell can be determined by various assays including epitopes formed by tertiary folding are typically lost upon those described in the Examples below (e . g ., Example 15 , 17 protein denaturing . In any event an antibody epitope typi and 18 ) . Methods of detecting whether an antibody inter - cally includes at least 3 , and more usually , at least 5 or 8 - 10 nalizes into a cell are also described in U . S . Pat. No . 25 amino acids in a unique spatial conformation . 7 ,619 , 068 which is incorporated herein by reference in its In this respect it will be appreciated that, in certain entirety . embodiments , an epitope may be associated with , or reside C . Depleting Modulators in , one or more regions, domains or motifs of the SEZO In other embodiments the antibodies will comprise deplet - protein (e . g ., amino acids 1 - 906 of mature isoform 1 ) . As ing antibodies or derivatives or fragments thereof. The term 30 discussed in more detail herein the extracellular region of " depleting ” antibody refers to an antibody that preferably the SEZ6 protein comprises a series of generally recognized binds to or associates with an antigen on or near the cell domains including five Sushi domains and two CUB surface and induces , promotes or causes the death or elimi- domains along with an N - terminal domain . For the purposes nation of the cell ( e . g ., by CDC , ADCC or introduction of a of the instant disclosure the term " domain ” will be used in cytotoxic agent ) . In some embodiments , the selected deplet - 35 accordance with its generally accepted meaning and will be ing antibodies will be associated or conjugated to a cytotoxic held to refer to an identifiable or definable conserved struc agent. tural entity within a protein that exhibits a distinctive Preferably a depleting antibody will be able to remove, secondary structure content. In many cases , homologous incapacitate , eliminate or kill at least 20 % , 30 % , 40 % , 50 % , domains with common functions will usually show 60 % , 70 % , 80 % , 85 % , 90 % , 95 % , 97 % , or 99 % of SEZ6 40 sequence similarities and be found in a number of disparate tumorigenic cells in a defined cell population . In some proteins ( e . g . , Sushi domains are reportedly found in a large embodiments the cell population may comprise enriched , number of different proteins ) . Similarly , the art -recognized sectioned , purified or isolated tumor perpetuating cells . In term “ motif " will be used in accordance with its common other embodiments the cell population may comprise whole meaning and shall generally refer to a short , conserved tumor samples or heterogeneous tumor extracts that com - 45 region of a protein that is typically ten to twenty contiguous prise tumor perpetuating cells . Those skilled in the art will amino acid residues . As discussed throughout, selected appreciate that standard biochemical techniques as embodiments comprise modulators that associate with or described in the Examples below ( e . g . , Examples 14 and 15 ) bind to an epitope within specific regions, domains ormotifs may be used to monitor and quantify the depletion of of SEZ6 . tumorigenic cells or tumor perpetuating cells in accordance 50 In any event once a desired epitope on an antigen is with the teachings herein . determined , it is possible to generate antibodies to that D . Binning and Epitope Binding epitope, e .g ., by immunizing with a peptide comprising the It will further be appreciated the disclosed anti -SEZ6 epitope using techniques described in the present invention . antibody modulators will associate with , or bind to , discrete Alternatively, during the discovery process , the generation epitopes or immunogenic determinants presented by the 55 and characterization of antibodies may elucidate informa selected target or fragment thereof. In certain embodiments , tion about desirable epitopes located in specific domains or epitope or immunogenic determinants include chemically motifs . From this information , it is then possible to com active surface groupings of molecules such as amino acids, petitively screen antibodies for binding to the same epitope . sugar side chains , phosphoryl groups, or sulfonyl groups, An approach to achieve this is to conduct competition and , in certain embodiments , may have specific three - 60 studies to find antibodies that competitively bind with one dimensional structural characteristics, and /or specific charge another, i . e . the antibodies compete for binding to the characteristics . Thus, as used herein the term " epitope” antigen . A high throughput process for binning antibodies includes any protein determinant capable of specific binding based upon their cross -competition is described in WO to an immunoglobulin or T - cell receptor or otherwise inter - 03 / 48731 . Other methods of binning or domain level or acting with a molecule . In certain embodiments , an antibody 65 epitope mapping comprising modulator competition or anti is said to specifically bind ( or immunospecifically bind or g en fragment expression on yeast is set forth in Examples 9 react) an antigen when it preferentially recognizes its target and 10 below .