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( 12 ) United States Patent ( 10) Patent No .: US 10,851,160 B2 Baumert Et Al

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( 12 ) United States Patent ( 10) Patent No .: US 10,851,160 B2 Baumert Et Al US010851160B2 ( 12 ) United States Patent ( 10 ) Patent No .: US 10,851,160 B2 Baumert et al . ( 45 ) Date of Patent : Dec. 1 , 2020 ( 54 ) HUMANIZED ANTI - CLAUDIN - 1 ( 56 ) References Cited ANTIBODIES AND USES THEREOF FOREIGN PATENT DOCUMENTS ( 71 ) Applicants : INSERM ( Institut National de la Santé et de la Recherche Médicale ) , WO 2010/034812 A1 4/2010 Paris ( FR ) ; Université de Strasbourg , WO 2010/039801 A2 4/2010 Strasbourg ( FR ) ; Chu Strasbourg , Les Hôpitaux Universitaires de OTHER PUBLICATIONS Strasbourg , Strasbourg ( FR ) Owen et al . ( Journal of Immunological Methods, 1994 , p . 149 165 ) . * ( 72 ) Inventors : Thomas Baumert , Strasbourg ( FR ) ; Mailly et al . , “ Clearance of persistent hepatitis C virus infection in Rajeevkumar Tawar, Oxfordshire humanized mice using a claudin - 1 - targeting monclonal antibody , " (GB ) Nature Biotechnology, 33 : 549-554 ( 2015 ) . Yamashita et al . , “ Discovery of Anti - Claudin - 1 Antibodies as Can ( 73 ) Assignees : Institut National de la Sante et de la didate Therapeutics against Hepatitis C Virus , ” Journal of Pharma Recherche Medicale , Paris ( FR ) ; cology and Experimental Therapeutics, 353 : 112-118 ( 2015 ) . Universite de Strasbourg , Strasbourg Paciello et al . , “ Novel human anti - cluadin 1 mAbs inhibit hepatitis ( FR ) ; Chu Strasbourg , les Hopitaux C virus infection and may synergize with anti - SRB1 mAb , ” Journal Universitaires de Strasbourg , of General Virology, 97 : 82-94 ( 2016 ) . Strasbourg ( FR ) Safdari et al . , “ Antibody humanization methods — a review and update , ” Biotechnology and Genetic Engineering Reviews , 29 : ( * ) Notice: Subject to any disclaimer , the term of this 175-186 ( 2013 ) . International Search Report issued in corresponding International patent is extended or adjusted under 35 Patent Application No. PCT / EP2017 / 056703 dated Jun . 8 , 2017 . U.S.C. 154 ( b ) by 0 days . Written Opinion issued in corresponding International Patent Appli ( 21 ) Appl . No .: 16 / 086,934 cation No. PCT / EP2017 / 056703 dated Jun . 8 , 2017 . ( 22 ) PCT Filed : Mar. 21 , 2017 * cited by examiner Primary Examiner — Agnieszka Boesen ( 86 ) PCT No .: PCT /EP2017 / 056703 ( 74 ) Attorney, Agent, or Firm - Morgan , Lewis & $ 371 ( c ) ( 1 ) , Bockius LLP ( 2 ) Date : Sep. 20 , 2018 ( 57 ) ABSTRACT ( 87 ) PCT Pub . No .: WO2017 / 162678 The present invention relates to humanized anti - claudin - 1 PCT Pub . Date : Sep. 28 , 2017 antibodies and uses thereof. Hepatitis C virus infection is a leading cause of chronic liver disease and a major indication ( 65 ) Prior Publication Data for liver transplantation . The tight junction protein claudin - 1 ( CLDN1 ) is an essential entry factor for HCV and a prom US 2019/0100586 A1 Apr. 4 , 2019 ising target for therapy. For clinical development, the inven Foreign Application Priority Data tors have humanized a rat anti -CLDN1 antibody produced ( 30 ) by genetic immunization that prevent HCV infection and 16305317 also cure chronically infected human liver chimeric mice . Mar. 22 , 2016 ( EP ) The lead humanized anti - CLDN1 antibody ( H3L3 ) pan ( 51 ) Int . Cl . genotypically inhibited HCV pseudoparticle infection of CO7K 16/28 ( 2006.01 ) primary human hepatocytes ( PHH ) without detectable A61P 31/14 ( 2006.01 ) escape . H3L3 efficiently inhibited infection by diverse HCV A61P 1/16 ( 2006.01 ) genotype 3 strains and exhibited marked synergy with C12N 15/62 ( 2006.01 ) direct- acting antivirals ( DAAs ) . The inventors also demon A61K 39/00 ( 2006.01 ) strate that anti -CLDN1 H3L3 cures persistent HCV infec ( 52 ) U.S. CI . tion in human - liver chimeric uPA - SCID mice in mono CPC C07K 16/28 ( 2013.01 ) ; A61P 1/16 therapy. Thus, the present invention relates to humanized ( 2018.01 ) ; A61P 31/14 ( 2018.01 ) ; C12N 15/62 anti -claudin - 1 antibodies and uses thereof, in particular for ( 2013.01 ) ; A6IK 2039/505 ( 2013.01 ) ; A61K the prevention and treatment of hepatitis C virus infection, 2039/545 ( 2013.01 ) ; CO7K 2317/24 ( 2013.01 ) ; virus - induced liver diseases , hepatocellular carcinoma CO7K 2317/55 ( 2013.01 ) ; CO7K 2317/565 ( HCC ) , nonalcoholic fatty liver disease (NAFLD ) and non ( 2013.01 ) ; CO7K 2317/567 ( 2013.01 ) alcoholic steatohepatitis (NASH ) . ( 58 ) Field of Classification Search None 12 Claims , 6 Drawing Sheets See application file for complete search history . Specification includes a Sequence Listing . U.S. Patent Dec. 1 , 2020 Sheet 1 of 6 US 10,851,160 B2 ( A ) FR1 CDR1 FR2 CDR2 OM - 7D3 - B3 VH EVOLVESGGGLVOPGRSLKLSCLGSGFSFSSYGMNWIRQAPGKGLESVASISPSGSYFYY IGHV5-34 * 01 EVOLVESGGGLVOPGRSLKLSCVASGFTFSDYRMNWIRQAPGKGLEWVASISSSSSYIYY FR3 CDR3 J - region ADSVKGRFTISRENAKNTLYLOMTSLRAEDTALYYCARLPGFNPPFDHWGQGVVVTYSS ADTVKG - FTISRDNAKDTLYLQMTSLRSEDTALYYCAR ----- DYFDYWGQGVMVTVSS FR1 CDRI FR2 CDR2 OM - 7D3 - B3 VL NTVMTQSPTSMFMSVGDRVTMNCKASONVGGNVDWYQWKPGOSPKLLMYGASNRYTGVPD IGKV6S11 * 01 NTVMTQSPTSMFISVGDRVTMNCKASQNVGTNVDWYQOKTGQSPKLLIYGASNRYTGVPD RFRGSGSGTDFTLTISNMQTEDLAVYYCLQYKNNPWTFGGGTKVELK RFTGSGSGTDE TLTISNMQAEDLAVYYCLQYNYNPWTFGGGTKLELK Figure 1A ( B ) FR1 CDR1 FR2 CDR2 OM - 7D3 - B3 VH EVQLVESGGGLVQPGRSLKLSCLGSGESESSYGMNWIRQAPGKGLEWVASTSPSGSYTYY IGHV3-21 * 01 EVOLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYY Humanized H1 EVOLVESGGGLVKPGGSLRLSCAASGFSFSSYGMNWVRQAPGKGLEWVSSISPSGSYFYY Humanized H2 QVQLVESGGGWOPGRSLRISCAASGFSFSSYGMNWVRQAPGKGLEWVTSISPSGSYTYY Humanized H3 QVQLVESGGGVVQPGRSLRLSCLGSGESESSYGMNWVRQAPGKGLEWVASISPSGSYFYY FR3 CDR3 J - region ADSVKGRFTISRENAKNTLYLQMTSLRAEDTALYYCARLPGFNPPFDHWGQGVVVTVSS ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR ------ DAFDVWGQGTMVTVSS ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLPGFNPPFDHWGQGTLVTVSS ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARLPGFNPPFDHWGQGTLVTVSS ADSVKGRFTISRDNSKNTLYLGMTSLRAEDTAIYYCARLPGFNPPEDHWGQGTLVIVSS FR1 CDR1 FR2 CDR2 OM - 7D3 - B3 VL NTVMTQSPTSMFMSVGDRVTMNCKASONVGGNVDWYQWKPGQSPKLLMYGASNRYTGVPD IGKV3-15 * 01 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA Humanized L1 DIQMTOSPATLSVSPGERATLSCKASONVGGNVDWYQWKPGQAPRLLIYGASNRYTGIPA Humanized L2 DIQMTQSPSSLSASVGDRVTITCKASONVGGNVDWYQWKPGKAPKLLIYGASNRYTGVPS Humanized L3 DIOMTQSPSSLSASVGDRVTITCKASQNVGGNVDWYQWKPGKAPKLLIYGASNRYTGVPD FR3 CDR3 J - region RFRGSGSGTDFTLTISNMQTEDLAVYYCLQYKNNPWTFGGGTKVELK RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK RFRGSGSGTEFTLTISSLOSEDFAVYYCLQYKNNPWTFGQGTKVEIK RFRGSGSGTDFTLTISSLQPEDVATYYCLQYKNNPWTFGQGTKVEIK RFRGSGSGTDFTLTISSLOPEDVATYYCLQYKNNPWTFGGGTKVEIK Figure 1B U.S. Patent Dec. 1 , 2020 Sheet 2 of 6 US 10,851,160 B2 ( A ) 3000 ... 2937 HepG2 Huh7.5.1 2000 MFA CD81 Rats H1L1 H1L2 H1L3 H2L1 H2L2 H2L3 H3L1 H3L2 H3L34 Controls Humanized a - CLDN1 variants ( B ) 293T ( C ) 1500 293T - CLDN1 107 106 . 1000 HCVccInfection(Log10RLU) 105. AMFI 104 500, ZOL Rat. DOU CD81 Rat H3L31 Mock H1L1 H1L2-1 H1L3- H2L11 H2L2 H2L3 H3L1 H3L2 H3L34 ( D ) 125 HCVpp 1a 100 10O HCVpp 1b infection,%HCVpp 75 50 Mock Rat H1L1 H1L2 H1L3 H2L1 4212H2L2 H2L3 H3L1 H3L2 313H3L3 Figure 2 U.S. Patent Dec. 1 , 2020 Sheet 3 of 6 US 10,851,160 B2 (A) H3L3 OM -7D3 - B3 125 125 hlgG4 control rlgG2 control 1004 HCVccinfection,% 75m Jc1 HCVccinfection,% 75 . Jc1 O Jc1-4156S 50 Choo JC1 - A156S JC1 -R155K Jct - R155K 25 25 -4 -3 -2 -1 0 1 2 3 4 -3 -2 -1 0 1 2 3 Log ( H3L3 , ug /mL ) Log ( OM - 7D3 - B3 , ug /mL ) ( B ) Control H3L3 163 34 % 3 % PE-A le FITC ( C ) 40 TT Infectedtargetcells,% 30 20 10 ControlOM- 7D3 -B3 H3L3 Figure 3A - C U.S. Patent Dec. 1 , 2020 Sheet 4 of 6 US 10,851,160 B2 Log H3L3(,ug/mL) 70072 -1 -2 -4-3 DCV,uM)Log( -5 -6 60 40 20 0 -20 Andron . 01 -1 Log(SOF,uM) -2 -3 -4 09 40 20 0 -20 expected below (D) or above inhibition % Figure 3D U.S. Patent Dec. 1 ,? 2020 Sheet 5 of 6 US 10,851,160 B2 ( A ) ( B ) 60 N7-3-3 CLON - 1 H33 CLON - 6 400 ? 40 CLDN - 9 T 200 ?? 20 Huh7.5.1 PHH Huh7.5.1 218 235 283 S2310 * PHH ( C ) ?? PDF 283 PHH S1443 ??? PHH 223 PR 216 PHP 233 ?? PH S2310 O PHH S1437 ? PHH228 PH 235 PHF 288 350 GT 1a ( H77 ) 150 GT 1b ( HCV - J ) GT 2b ( 18 ) 175 12 ??? 12 ?? 10 10 75 ??? 75 5 5 50 25 25 2 ? ' ' ' ' ' ' ' ' ' ' ? ? ? 7 2 4 -3 -2 2 3 GT 3a (UKN3A1.28 ) GT 3a ( S52 ) GT 3a ( gt3SXB1) 450 150 150 12 ? 125 12 ? HCVppentry,% 10 10 75 7 5 50 ?? 5 ???? 25 ? .4 3 2 3 GT 4 (UKMN4.21.16 ) GT 5 ( UKN5.14.4 ) ??? GT 6 UKN6.5.340 ) 125 12 ? 125 100 10 ? 100 7 50 ?? 25 ? 25 - ? 3 2- . 3 .4 .3 .2 . Log ( antibody , pg /mL ) Figure 4 U.S. Patent Dec. 1 , 2020 Sheet 6 of 6 US 10,851,160 B2 (A) DO CTRL H3L3 ++++ HCVviralload copies/mL)(Logio LOQ 1/100 Gooo LOQ 1/20 -3 0 3 6 9 12 Treatment time (weeks ) ( B ) CTRL H3L3 mL) Humanalbumin (Log10ug/ 3 6 9 12 Treatment time (weeks ) ( C ) 1200 * CTRL H3L3 1000 HumanIgG4(ug/mL) 800 600 400 200 3 6 9 12 TreatmentA time (weeks ) Figure 5 US 10,851,160 B2 1 2 HUMANIZED ANTI - CLAUDIN - 1 CLDN1 co - receptor complex . Furthermore, they recently ANTIBODIES AND USES THEREOF reported that the lead rat anti - CLDN1 mAb ( OM - 7D3 - B3 ) prevents de novo HCV infection and clears chronic HCV SEQUENCE LISTING SUBMISSION VIA infection without inducing any toxicity in human - liver chi EFS - WEB 5 meric uPA SCID mice ( 21 ) . Given these most promising findings, humanization of this antibody represents the next A computer readable text file , entitled “ SequenceLis step in its clinical development. ting.txt " created on or about Sep. 20 , 2018 with a file size of about 13 kb contains the sequence listing for this application SUMMARY OF THE INVENTION and is hereby incorporated by reference in its entirety. 10 The present invention relates to humanized anti - claudin - 1 FIELD OF THE INVENTION antibodies
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