( 12 ) United States Patent
`Beaulieu et al .
`
`( 10 ) Patent No . :
`( 45 ) Date of Patent :
`
`US 9 , 891 , 223 B2
`* Feb . 13 , 2018
`
`US009891223B2
`
`( 54 ) METHOD OF ASSAYING LEUKOCYTE
`ELASTASE INHIBITOR FOR THE IN VITRO
`DIAGNOSIS OF COLORECTAL CANCER
`( 75 ) Inventors : Corinne Beaulieu , Rillieux la Pape
`( FR ) ; Jean - Philippe Charrier , Tassin
`la Demi - Lune ( FR ) ; Genevieve
`Choquet - Kastylevsky , Francheville
`( FR ) ; Odile Mejan - Letourneur , Lyons
`( FR ) ; Dominique Rolland , Saint Genis
`les Ollieres ( FR )
`( 73 ) Assignee : BIOMERIEUX , Marcy l ' Etoile ( FR )
`Subject to any disclaimer , the term of this
`( * ) Notice :
`patent is extended or adjusted under 35
`U . S . C . 154 ( b ) by 0 days .
`This patent is subject to a terminal dis
`claimer .
`( 21 ) Appl . No . :
`12 / 452 , 035
`( 22 ) PCT Filed :
`Jul . 10 , 2008
`PCT / FR2008 / 051291
`( 86 ) PCT No . :
`$ 371 ( c ) ( 1 ) ,
`( 2 ) , ( 4 ) Date : Dec . 14 , 2009
`( 87 ) PCT Pub . No . : W02009 / 024691
`PCT Pub . Date : Feb . 26 , 2009
`Prior Publication Data
`US 2010 / 0129828 A1 May 27 , 2010
`Foreign Application Priority Data
`( 30 )
`Jul . 19 , 2007 ( FR ) .
`. . . . . . . . . 07 05202
`( 51 )
`Int . Ci .
`GOIN 33 / 53
`( 2006 . 01 )
`GOIN 30 / 72
`( 2006 . 01 )
`( 2006 . 01 )
`GOIN 33 / 574
`U . S . CI .
`CPC . GOIN 33 / 57419 ( 2013 . 01 ) ; GOIN 2333 / 811
`( 2013 . 01 )
`( 58 ) Field of Classification Search
`None
`See application file for complete search history .
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`
`( Continued )
`Primary Examiner – Hong Sang
`( 74 ) Attorney , Agent , or Firm — Oliff PLC
`( 57 )
`ABSTRACT
`The present invention relates to a method for the in vitro
`diagnosis of colorectal cancer by determining the presence
`of the Leukocyte Elastase Inhibitor tumor marker in a
`biological sample taken from a patient suspected of having
`colorectal cancer . Said method can be used for early diag
`nosis , screening , therapeutic follow - up and prognosis , and
`also for relapse diagnosis in relation to colorectal cancer .
`15 Claims , 12 Drawing Sheets
`
`*
`
`( 65 )
`
`( 52 )
`
`( 56 )
`
`Geneoscopy Exhibit 1075, Page 1
`
`

`

`US 9 , 891 , 223 B2
`Page 2
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`WO
`WO
`WO
`WO
`WO
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`WO
`WO
`W0
`WO
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`WO
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`( abstract only ) .
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`of Early Stage Ovarian Cancer ” , Proteomics , 2005 , vol . 5 , pp .
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`20023818 , accessed Nov . 9 , 2015 .
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`Antigenic Site on Protein Binding to Monoclonal Antibodies of
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`* cited by examiner
`
`Geneoscopy Exhibit 1075, Page 4
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 1 of 12
`
`US 9 , 891 , 223 B2
`
`Serum LEI assay
`
`w
`
`CRC +
`
`s
`
`tor
`CRC
`Figure 1
`
`Serum ezrin assay
`
`45007
`4000
`3500
`3000
`52500
`LEI ( ng / ml ) . 20004
`1500
`1000
`500
`
`Ezrin ( ng / ml )
`
`CRC +
`
`CRC
`Figure 2
`
`Geneoscopy Exhibit 1075, Page 5
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 2 of 12
`
`US 9 , 891 , 223 B2
`
`Serum aminoacylase assay
`.
`
`Aminoacylase ( ng / ml )
`
`.
`
`CRC +
`
`CRC
`Figure 3
`
`Serum L - FABP assay
`:
`
`L - FABP ( ng / mi )
`f * * * * *
`
`që
`
`XLV
`
`CRC +
`
`Figure 4
`
`CRC
`
`Geneoscopy Exhibit 1075, Page 6
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 3 of 12
`
`US 9 , 891 , 223 B2
`
`Serum I - FABP assay
`
`1 - FABP ( pg / ml )
`
`CRC +
`
`Figure 5
`
`CRC
`
`Geneoscopy Exhibit 1075, Page 7
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 4 of 12
`
`US 9 , 891 , 223 B2
`
`Serum apoprotein Al assay
`( microplate ELISA )
`
`Apo A1 ( ug / ml )
`
`CRC +
`
`CRC
`
`Serum apoprotein Al assay
`( Lincoplex kit )
`15004
`
`še
`Apo A1 ( ng / ml )
`
`še
`
`e
`
`CRC +
`
`CRC
`
`Figure 6
`
`Geneoscopy Exhibit 1075, Page 8
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 5 of 12
`
`US 9 , 891 , 223 B2
`
`Serum apolipoprotein A2 assay
`
`Apo A2 ( wg / ml )
`
`* : * * * *
`
`CRC +
`Figure 7
`
`CRC
`
`Serum I - plastin assay
`
`1 - Plastin ( RFV )
`
`CRC
`
`CRC +
`
`CRC +
`Figure 8
`
`Geneoscopy Exhibit 1075, Page 9
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 6 of 12
`
`US 9 , 891 , 223 B2
`
`Serum B2 - microglobulin assay
`
`B2 - Microglobulin ( ng / ml )
`
`CRC +
`Figure 9
`
`CRC
`
`1000
`
`Serum CEA assay
`
`CEA ( ng / ml ) P? CRC +
`
`Figure 10
`
`CRC
`
`Geneoscopy Exhibit 1075, Page 10
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 7 of 12
`
`US 9 , 891 , 223 B2
`
`1000 ,
`
`CA 19 - 9 ( U / ml )
`
`PS
`
`Testosterone ( ng / ml )
`
`2 1 P
`
`Serum CA 19 - 9 assay
`
`CRC +
`Figure 11
`
`CRC
`
`Serum testosterone assay
`
`???
`CRC +
`Figure 12
`
`CRC
`
`Geneoscopy Exhibit 1075, Page 11
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 8 of 12
`
`US 9 , 891 , 223 B2
`
`Serum E - cadherin assay
`90009 . 8000
`7000 E - Cadherin ( ng / ml )
`60001
`50004
`40004
`3000
`2000
`1000
`
`CRC +
`Figure 13
`
`CRC
`
`Serum PAPI assay
`. .
`
`Ï
`
`PAP1 ( ng / ml )
`
`Ï
`
`. . .
`
`CRC +
`
`CRC
`
`Figure 14
`
`Geneoscopy Exhibit 1075, Page 12
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 9 of 12
`
`US 9 , 891 , 223 B2
`
`Serum galectin - 3 assay
`
`140007
`Galectin - 3 ( RFV )
`12000
`8000 Y . . . .
`10000
`
`CRC
`
`CRC +
`Figure 15
`
`CRC CRC
`
`Serum LDH assay
`
`2250
`2000
`1750
`1500
`LDH ( ng / ml )
`
`750
`500
`250
`
`:
`
`promet
`
`i potencia
`
`CRC +
`Figure 16
`
`CRC
`
`Geneoscopy Exhibit 1075, Page 13
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 10 of 12
`
`US 9 , 891 , 223 B2
`
`Serum Proteasome 205 assay
`100009
`
`Proteasome 205 ( ng / ml ) AO 1000
`
`CRC +
`Figure 17
`
`CRC
`
`Fecal aminoacylase assay
`
`Aminoacylase ( ng / ml )
`
`0 . 125
`
`0 . 0625
`
`Ley
`
`CRC +
`Figure 18
`
`CRC
`
`Geneoscopy Exhibit 1075, Page 14
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 11 of 12
`
`US 9 , 891 , 223 B2
`
`Fecal galectin - 3 assay
`
`=
`CRC
`CRC +
`Figure 19
`
`Fecal proteasome 205 assay
`
`Galectin - 3 ( RFV )
`
`
`
`Proteasome 205 ( RFV )
`
`CRC +
`Figure 20
`
`CRC
`
`Geneoscopy Exhibit 1075, Page 15
`
`

`

`U . S . Patent
`
`Feb . 13 , 2018
`
`Sheet 12 of 12
`
`US 9 , 891 , 223 B2
`
`12 000
`? ?
`10 000
`
`
`
`?i
`8 000
`
`6 000
`
`
`
`Tumor marker secretion by cancer cells
`60 000
`
`Caco - 2
`HT - 29
`HT29 - 16
`
`50 000
`
`40 000
`
`30 000
`
`20 000
`
`10 000
`
`-
`
`- -
`
`. .
`
`.
`
`-
`
`-
`
`. .
`
`: : . . .
`
`.
`
`! ! .
`
`.
`
`' .
`
`. . .
`
`. .
`
`.
`
`.
`
`.
`
`Gal - 3
`
`
`
`Spots per 109 cells ?i
`
`?i
`4 000
`
`j
`2 000
`
`.
`h
`
`Ezrin
`
`Figure 21
`
`Geneoscopy Exhibit 1075, Page 16
`
`

`

`US 9 , 891 , 223 B2
`
`nificant risk ( 0 . 1 % ) of perforation and bleeding of the colon
`METHOD OF ASSAYING LEUKOCYTE
`and of death ( 1 / 10000 ) , and it is also very expensive for
`ELASTASE INHIBITOR FOR THE IN VITRO
`public health . Furthermore , colonoscopy requires a very
`DIAGNOSIS OF COLORECTAL CANCER
`restrictive prior colonic preparation , which in large part
`The present invention relates to the cancerology field . 5 explains the poor compliance .
`Tumor markers that can be assayed by immunoassays
`More particularly , the subject of the present invention is a
`method for the in vitro diagnosis of colorectal cancer in a
`have for a long time been described in the context of
`human patient , by determining the presence of the Leuko
`colorectal cancer . They are in particular the carcinoembry
`cyte Elastase Inhibitor ( LEI ) in a biological sample taken
`onic antigen ( CEA ) and CA19 - 9 .
`from this patient , it being possi