MAIMUUTTUNUT US009809653B2 HIU LI HUWULUHAN (12 ) United States Patent ( 10 ) Patent No. : US 9 ,809 ,653 B2 Baudat et al. (45 ) Date of Patent: Nov . 7 , 2017 (54 ) ANTI- LAMP1 ANTIBODIES AND ANTIBODY ( 58) Field of Classification Search DRUG CONJUGATES , AND USES THEREOF None See application file for complete search history . ( 71 ) Applicant: SANOFI, Paris (FR ) (72 ) Inventors: Yves Baudat, Paris (FR ); Francis ( 56 ) References Cited Blanche, Paris ( FR ); Béatrice Cameron , Paris (FR ) ; Tarik Dabdoubi, U . S . PATENT DOCUMENTS Le Coudray Montceaux (FR ) ; 4 , 179 , 337 A 12 / 1979 Davis et al. Anne -Marie LeFebvre , Le Plessis Pâté 4 ,256 ,746 A 3 / 1981 Miyashita et al . ( FR ) ; Magali Mathieu , Choisy le Roi 4 , 294 , 757 A 10 / 1981 Asai 4 , 301 , 144 A 11/ 1981 Iwashita et al. (FR ) ; Ana Merino - Trigo , Paris (FR ) ; 4 , 307 ,016 A 12 / 1981 Asai et al. Manoel Nunes, Paris (FR ) 4 ,313 ,946 A 2 / 1982 Powell et al . 4 , 315 , 929 A 2 / 1982 Freedman et al. 4 ,322 , 348 A 3 / 1982 Asai et al. (73 ) Assignee : SANOFI, Paris (FR ) 4 ,331 , 598 A 5 / 1982 Hasegawa et al. 4 , 361, 650 A 11/ 1982 Asai et al . ( * ) Notice : Subject to any disclaimer, the term of this 4 , 362 ,663 A 12 / 1982 Kida et al. patent is extended or adjusted under 35 4 , 364 , 866 A 12 / 1982 Asai et al. U .S .C . 154 (b ) by 0 days . 4 ,371 , 533 A 2 / 1983 Akimoto et al . 4 ,424 ,219 A 1 / 1984 Hashimoto et al. 4 ,450 , 254 A 5 / 1984 Isley et al. (21 ) Appl. No. : 14/ 751 ,598 4 ,496 ,689 A 1 / 1985 Mitra 4 ,640 ,835 A 2 / 1987 Shimizu et al. ( 22 ) Filed : Jun . 26 , 2015 (Continued ) (65 ) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2016 /0280793 A1 Sep . 29, 2016 EP 0125023 Al 11/ 1984 EP 0173494 A2 3 /1986 Related U .S . Application Data ( Continued ) (63 ) Continuation of application No . PCT/ EP2013 / 078017 , filed on Dec. 26 , 2013 . OTHER PUBLICATIONS George et al. (Circulation . 1998 ; 97: 900 -906 ). * ( 30 ) Foreign Application Priority Data (Continued ) Dec . 27 , 2012 ( EP ) . . 12306691 Dec. 27 , 2012 (EP ) .. .. 12306694 Primary Examiner — Sheela J Huff (51 ) Int . Ci. ( 74 ) Attorney, Agent, or Firm — Sandra A . Brockman -Lee A61K 39 /395 (2006 .01 ) CO7K 16 / 28 ( 2006 . 01 ) A61K 47/ 48 ( 2006 .01 ) (57 ) ABSTRACT GOIN 33 /574 ( 2006 .01 ) Antibodies are provided which specifically bind human and COOK 16 / 30 ( 2006 .01 ) C120 1 /68 ( 2006 . 01 ) Macaca fascicularis lysosomal- associated membrane pro A61K 39/ 00 ( 2006 .01 ) tein 1 (LAMP1 ) and immunoconjugates comprising (52 ) U .S . CI. said antibodies conjugated or linked to a growth inhibitory CPC . .. CO7K 16 /2896 ( 2013 .01 ) ; A61K 47/ 48384 agent . Pharmaceutical compositions comprising antibodies (2013 .01 ) ; A61K 47 /48561 ( 2013 . 01 ) ; A61K or immunoconjugates of the invention and use of the anti 47 /48569 (2013 .01 ); A61K 47 / 48584 bodies or immunoconjugates for the treatment of are ( 2013 .01 ) ; A61K 47/ 48592 (2013 .01 ) ; A6IK also provided , as well as LAMP1 antibodies , isolated 47/ 48615 ( 2013 .01 ) ; CO7K 16 /30 (2013 .01 ) ; nucleic acids, vectors and host cells comprising a sequence CO7K 16 / 3023 ( 2013 .01 ) ; C07K 16 / 3046 encoding said antibodies and the use of said antibody as a ( 2013 . 01 ) ; CO7K 16 /3069 (2013 .01 ) ; C120 diagnostic tool. The application further provides for the 1 /6886 ( 2013 .01 ) ; GOIN 33 /574 ( 2013 .01 ) ; detection of LAMP1 amplification or gain in cancer A61K 2039 / 505 ( 2013 .01 ) ; COZK 2317 / 24 cells leading to the determination if patients with cancer are ( 2013 . 01 ) ; CO7K 2317 / 33 ( 2013 .01 ) ; COOK likely to respond to anti -LAMP1 therapy. Anti- LAMP1 2317 /34 ( 2013 . 01 ) ; COZK 2317 /41 ( 2013 .01 ) ; therapeutic agent for use for treating cancer in a patient CO7K 2317/ 565 ( 2013 .01 ) ; CO7K 2317 / 73 harboring LAMP1 gene copy number gain in cancer cells is (2013 .01 ) ; CO7K 2317 / 732 ( 2013 .01 ) ; COOK further provided . 2317 / 77 ( 2013 .01 ) ; CO7K 2317 / 92 ( 2013 .01 ) ; C12Q 2600 /106 (2013 . 01 ) ; C12Q 2600/ 156 (2013 .01 ) 17 Claims, 47 Drawing Sheets US 9, 809 , 653 B2 Page 2

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5 VDK o PSVDKY : 00 ! SVNTEKVWVQ360SLRALQATVGNSYKCNAEEHVRVIKAFLOGIQLNTILPDARDPAFKAANG301 Transmembranedomain 1MAAPGSARRPLLLLLLLLLLGLMHCASAAMEMVKNGNGTACHMANFSAAFSVNYDTKSGP60 SVNYDTKSGP58THCASAAMFIVKNGNGTACIMANFSAAFSLLLLLLLLGL9MAAPGSARR:- 61KNMTFDLPSDATVVLNRSSCGKENISDPSLVTAFGRGHTLILNETRNATRYSVOLMSFVY120 VTLHDATIQAYLS180TRADIDKKYRCVSGTQVHMNNVT:SDTHLEPNASSKEIKTVESITD121NL 119NLSDTHLÉPNASSKEIKIVESITDTRADIDKKYRCVSGTQVHMNNVTVTLHDATIQAYLS179 241NLTYERKDNITVIRLLNINPNKTSASOSCOAHLVTLELHSEQTTVLLFQFOMNASSSRFF300 239NLTYERKONTIVIRLLNINPNKTLASGSCGAHLVTLELHSEGSTVLLFOFGMNASSSRFF298 299?QGIQLNTTLPDARDPAFKAANSSERALQATVGNSYKCNAEEHVRVTKAFSVNIFKVWVQ358 FIG.1 .59KNMTEDLPSDAKVVLNSSSCGKENTSDPSLVIAFGRGQTLTLNFTRNATRYSVOLMSEVY118 RCEQDRPSPTTAPPAPPSPSPSPVPK 361AFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIVLTAYLVGRKRSHAGYOT 369ÁFKVEGGQFGSVEECLLDENNALIPLAVGGALAOLVLIVLTAYLVGRKRSHAGYOTT Signalpeptide GET NSSESREETRCEQDRPSPTTAPPAPPS ?

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FIG . 2 U . S . Patent Nov. 7, 2017 Sheet 3 of 47 US 9 , 809 ,653 B2

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FIG . 23 U . S . Patent Nov . 7 , 2017 Sheet 28 of 47 US 9 ,809 ,653 B2

www Control + DM4- SPDB -HUMAb1 _ 1 - 5 mg/kg DM4 - SPDB -HUMAb1 _ 1 - 2 ,5 mg/ kg may im DM4 - SPDB -HUMAb1 _ 1 - 1 , 25 mg/ kg mm3) DM4- SPDB -HUMAb1 _ 1 - 0, 62 mg/ kg Mediantumorvolume(

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FIG . 24 U . S . Patent Nov . 7 ,2017 Sheet 29 of 47 US 9 ,809 , 653 B2

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FIG . 25A U . S . Patent Nov . 7 , 2017 Sheet 30 of 47 US 9 ,809 ,653 B2

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FIG . 25B U . S . Patent Nov . 7 , 2017 Sheet 31 of 47 US 9, 809 ,653 B2

mm3sizetumor šejese move samacowe wewe have theme . 20 25 30 35 40 days post implantation contols DM4- SPDB - CHMAD2 10 mg /kg DM4- SPDB - CHMAD2 5 mg /kg DM4 - SPDB - CHMAD2 2 . 5 mg /kg

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FIG . 27 U . S . Patent Nov . 7 , 2017 Sheet 33 of 47 US 9 ,809 ,653 B2

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FIG . 28 U . S . Patent Nov . 7, 2017 Sheet 34 of 47 US 9 ,809 ,653 B2

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FIG . 33 U . S . Patent Nov. 7 , 2017 Sheet 40 of 47 US 9 ,809 ,653 B2

Target cells : Pengacyized phagocytice nimiento 28 .87 % 3 B3 manteelwwwwwwwwwwwwwwww PKH67 KA PKH67 varmendet ili ** & .. . . : : . . . . : 58 .58 % triV! macrocages $ 4 .68 % donkerbintenimento die tandenstimmen perkawinannammide berikan interdunninummi di tutundinamentoin withich entry pomarang yangterpanjang dan penggu na ts 35 9 04 * *CD14 - - 07 C014 -207

FIG . 34 U . S . Patent Nov . 7 , 2017 Sheet 41 of 47 US 9 ,809 ,653 B2

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FIG . 35 U . S . Patent Nov. 7 , 2017 Sheet 42 of 47 US 9 ,809 ,653 B2

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FIG . 36 U . S . Patent Nov. 7 , 2017 Sheet 43 of 47 US 9 ,809 ,653 B2

153786153471 Ab:antiLAMP1SAR427972m=147376 WhyttalerendWohlmanatau SNPP+TomayA=1110 D5 Intact DAR=2 151600152479 D4 151438 |150580 D3 VWhimbaton 147000147500148000148500149000149500150000150500151000151500152000152500153000153500154000 FIG.37 150417 ????????TITTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTmass 149562 149739 W D2 149404 148550 148592 www D1 148390 wwwwww 147530 whowiwname Abnu 147376

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wwwwwwwwwwwwwwwwwwwwwwwww * * 300 TEW SOUS u put FIG . 41 US 9 ,809 , 653 B2 ANTI -LAMP1 ANTIBODIES AND ANTIBODY Immunol Immunopathol. 67 ( 1 ) : 31 - 39 ) and (Sil DRUG CONJUGATES , AND USES THEREOF verstein , R . L . and Febbraio , M ., 1992 , Blood 80 : 1470 1475 ) . BACKGROUND Increased cell surface expression of LAMP1 and LAMP2 5 has been observed in tumor cell lines , for example in highly Antibodies are provided which specifically bind human metastatic colonic carcinoma L4 cells (Saitoh , O . et al. , and Macaca fascicularis lysosomal- associated membrane 1992 , J Biol Chem 267 : 5700 - 5711 ) , on human metastasiz protein 1 (LAMP1 ) proteins and immunoconjugates com ing A2058 , HT1080 ( human ) , prising said antibodies conjugated or linked to a growth CaCo - 2 (human colon -adenocarcinoma ) cells and in col inhibitory agent. Pharmaceutical compositions comprising " orectal neoplasms (Furuta , K . et al . , 2001 , J Pathol 159 ( 2 ) : antibodies or immunoconjugates of the invention and use of 449 - 455 ) . the antibodies or immunoconjugates for the treatment of The chromosomal region 13734 in which LAMP1 is cancer are also provided , as well as LAMP1 antibodies , located has recently been linked to amplification events isolated nucleic acids , vectors and host cells comprising a 16 including a larger amplicon that involves CUL4A , LAMP1 , sequence encoding said antibodies and the use of said TFDP1, and GAS6 in human breast cancer ( Abba, Martin C . antibody as a diagnostic tool. The application further pro et al. ; Cancer Res 2007 ; 4104 ). TFDP1 and perhaps CUL4A vides for the detection of LAMP1 gene amplification or gain were identified in the above mentioned publication as the in cancer cells leading to the determination if patients with leading genes driving the amplification phenomenon . In cancer are likely to respond to anti- LAMP1 therapy . There - 20 particular, analysis of publicly available breast cancer gene fore , it is proposed an in vitro method of selecting patients expression (microarrays ) data sets indicated that TFDP1 with cancer which comprises determining , in a biological overexpression is associated with estrogen receptor (ER ) sample of a patient with cancer which includes cancer cells , negative and high - grade breast carcinomas, as well as if said patient harbors a LAMP1 gene copy number gain ; and shorter overall survival, relapse - free survival, and metasta selecting the patient based on the presence of LAMP1 gene 25 sis - free interval. Conversely, LAMP1 expression did not copy number gain . Anti -LAMP1 therapeutic agent for use significantly correlate with tumor grade. In the end , Abba et for treating cancer in a patient harboring LAMP1 gene copy al. did not report that LAMP1 amplification translated into LAMP1al. overexpression in human breast cancer cells . number gain in cancer cells is further provided . The 11 amino -acid cytoplasmic tail of LAMP1 contains a Lysosome- associated membrane protein 1 (LAMP1 ) , also 30 7 amino -acid linker sequence and a 4 amino acid long known as CD107 antigen - like family member A ( CD107a ) , tyrosine motif (YQTI ) . It was shown that small changes in is a single -pass type I membrane protein , which belongs to the spacing of this motif relative to the membrane dramati the LAMP family . LAMP2 is the closest member of the cally impair sorting in the early / sorting endosomes . Muta family and both proteins are the most abundant glycopro tions within said tyrosine motif were shown to have an teins within the lysosomal membrane (Sawada , R .. et al . , , 3525 impact on the binding of LAMP1 to adaptor proteins leading 1993 , J Biol Chem 268 : 12675 - 12681) . as well to impaired sorting (Obermuller , S . et al . , 2002 , Although encoded by separate genes , with LAMP1 Journal of Cell Science 115: 185 - 194 ; Rohrer , J . et al. , 1996 , located on chromosome 13q34 and LAMP2 on Xq24 - 25 , the Journal of Cell Biology 132 ( 4 ) : 565 - 576 ) . Therefore , the proteins are similar in their primary structure, with ~ 36 % abnormal cell surface expression of LAMP1 in different (Mattei , M . G . et al. , 1990 , J Biol Chem 40 cancer cell lines might be related to mutations in the 265 : 7548 - 7551 ) . LAMP1 and LAMP2 consist of a polypep - cytoplasmic tail even though the mechanism is still unclear. tide core of approximately 40 kDa; they are both anchored Furthermore , it has been shown that certain point mutations via their C - terminus to the lysosomalmembrane and expose in the cytoplasmic tail lead to plasma membrane accumu the largest part, extensively glycosylated , to the lumenal side lation (Gough , N . R . et al. , 1999 , Journal of Cell Science 112 of lysosomes . Both proteins are among the most heavily 45 ( 23 ) : 4257 - 4269 ) . glycosylated of cellular proteins with ~ 50 % of their mass as Due to the fact that LAMP1 is a marker for endosomes and these seem to be the key and lysosomes, numerous commercially available anti for maintaining lysosome acidity and protecting the lyso - LAMP1 antibodies were developed for research purposes . somal membranes from autodigestion . However, the full These antibodies are either polyclonal or monoclonal and biological function of these two highly glycosylated proteins 50 are restricted to some biochemical application such as in particular LAMP1 still needs to be elucidated ( Fukuda , immunohistochemistry (IHC ) , Western blots (WB ) , Fluores M ., 1991 , J Biol Chem , 266 :21327 - 21330 ; Winchester, B . , cence activated cell sorter (FACS ) analysis, Immunoprecipi 2001, European Journal of Paediatry Neurology , 5 : 11 - 19 ; tation (IP ) and Enzyme - linked immunosorbent assay Gasnier, B . , 2009 Biochimica et Biophysica Acta 1793 :636 - (ELISA ) . 649 ). 55 LAMP1 protein also has been detected at the cell mem LAMP1 is highly expressed in late endosomes and lyso - brane of tumor cells . somes designating LAMP1 as marker for these two organ - E . Venetsanakos (WO 2005 /012912 ) suggested that elles (Cook , N . R . et al. , 2004 , Traffic , 5 (9 ): 685 -699 ). Thus, LAMP1 is expressed on the surface of colon cancer cells but most of the literature on LAMP1 relates to endocytosis , not on the surface of normal colon cells and proposed that pinoscyosis , or phagocytosis (Cook , N . R . et al. , 2004 , 60 tumor growth might be reduced by targeting a cytotoxic Traffic , 5 ( 9 ) : 685 -699 ) . agent to LAMP1 via an anti- LAMP1 antibody. Venetsana Although the majority of LAMP1 and LAMP2 reside in kos did not describe , however, preparation of anti -LAMP1 the lysosome, some LAMP1 and LAMP2 immunoreactivity antibodies or conjugates thereof with cytotoxic or cytostatic is also observed at low levels at the plasma membrane . The agent or any data supporting his hypothesis . Indeed , though LAMP1 found in the plasma membrane represents only 65 a decade has passed since Venetsanakos ' initial filing and no 1 - 2 % of total LAMP1. This is for example true for periph - anti -LAMP1 antibodies or their use as immunoconjugates in eral blood (Holcombe , R . F . et al. 1993 , Clin an anti -LAMP1 therapy has entered clinical development, so US 9 ,809 , 653 B2 far. Accordingly , a great need exists for anti- LAMP1 anti the mutation N55R in its heavy chain sequence of SEQ ID bodies or immunoconjugates for the treatment of cancer. NO : 53 and the interaction with LAMP1. FIG . 18 : HRMS data of DM4- SPDB - huMAb1 _ 3 conju BRIEF DESCRIPTION OF THE DRAWINGS gate . 5 FIG . 19 : HRMS data of DM4- SPDB -huMAb1 _ 1 conju FIG . 1 : sequence alignment of human (SEQ ID NO : 24 ) gate . and Macaca fascicularis LAMP1 (SEQ ID NO : 39 ) full FIG . 20 : HRMS data of DM4 -SPDB -huMAb1 _ 2 conju length proteins . gate . FIG . 2 : Expression Profile of LAMP1 derived from FACS FIG . 21 : HRMS data of DM4- SPDB -chMAb2 conjugate . analysis with the monoclonal mouse antibodies Mab1 and 10 FIG . 22 : HRMS data of DM4 - SPDB - chMAb3 VLR24 MAD2 . R93 conjugate . FIG . 3 : Reactivity of MAb1 with human LAMP1 and FIG . 23 : Evaluation of the anti -tumor activity of DM4 cynomolgus monkey LAMP1. SPDB -huMAb1 _ 1 against primary human colon adenocar FIG . 4 : Evaluation of the competition ofMAD2 (murine ) 15 cinoma CR -LRB -010P in SCID female mice. with MAb1 ( chimeric ) for binding to LAMP1. With 2nd anti FIG . 24 : Evaluation of the anti - tumor activity of DM4 hFc being a secondary antibody anti- human Fc and 2nd anti SPDB -huMAb1 _ 1 against human primary invasive ductal mFc being secondary antibody anti -mouse Fc. carcinoma BRE - IGR -0159 in SCID female mice . FIG . 5 : Evaluation of the anti -tumor activity of DM4- FIG . 25A and FIG . 25B : Evaluation of the anti- tumor SPDB - chMAb1 conjugate against primary human colon 20 activity of DM4- SPDB - huMAb1 _ 1 against primary primary adenocarcinoma CR -LRB -010P in SCID female mice . human lung tumor LUN -NIC -0070 in SCID female mice . FIG . 6 : Evaluation of the anti -tumor activity of DM4- FIG . 26 : Evaluation of the anti - tumor activity of DM4 SPDB -chMAb1 conjugate against primary lung tumor SPDB - chMAb2 against primary human colon adenocarci LUN - NIC - 0014 in SCID female mice . noma CR - LRB -010P in SCID female mice . FIG . 7 : HRMS data of DM4- SPDB - chMAb1 conjugate . 25 FIG . 27 : Evaluation of the anti - tumor activity of DM4 FIG . 8A : Box Plot of RNA Intensity expression of SPDB -chMAb2 against human primary invasive ductal car LAMP1 by Copy Number Changer category . cinoma BRE - IGR - 0159 in SCID female mice . FIG . 8B : Plot of LAMP1 Copy Number according to FIG . 28 : Evaluation of the anti - tumor activity of DM4 LAMP1 mRNA expression on colon tumors. Points repre SPDB -chMAb3 against human primary invasive ductal car sent individualmRNA expression , bars corresponds to mean 30 cinoma BRE - IGR - 0159 in SCID female mice . values. FIG . 29 : Graphical representation of the in vitro ADCC FIG . 9 : A Sperman Correlation analysis of LAMP1 mediated by chMAbi , chMAL2 and chMAb3 . mRNA and Copy Number Change data . FIG . 30 : Graphical representation of the in vitro ADCC FIG . 10A : Box Plot of RNA Intensity expression and dependency on LAMP1 antigen density with a ) HCT hu LAMP1 by Copy Number Change in Soft Tissue Sarcoma . 35 LAMP1 clone 8 LAMP1 antigen density : 160000 b ) HCT hu FIG . 10B : Box Plot of RNA Intensity expression and LAMP1 clone 4 LAMP1 antigen density : 2000 and c ) HCT LAMP1 by Copy Number Change in Corpus Endometrioid hu LAMP1 clone 12 LAMP1 antigen density : 5000 . Carcinoma. FIG . 31A : Comparison of in vitro ADCC of chMAb1 and FIG . 10C : Box Plot of RNA Intensity expression and DM4- SPDB - chMAb1 . LAMP1 by Copy Number Change in Breast Invasive Car- 40 FIG . 31B : Comparison of in vitro ADCC of chMAb2 and cinoma. DM4- SPDB -chMA ) 2 . FIG . 11A : Histogram of LAMP1 Copy number by FIG . 32 : In vitro ADCC mediated by humAb1 _ 1 . LAMP1 membrane expression ( IHC category scoring ) for FIG . 33 : In vitro ADCC mediated by DM4- SPDB colon tumor PDX . huMAb1 _ 1. FIG . 11B : Histogram of LAMP1 Copy number by 45 FIG . 34 : Flow cytometry analysis of ADCP with a ) LAMP1 membrane expression ( IHC category scoring ) for Macrophages and target cells without Mab1 _ 1 and b ) Mac lung and stomach tumor PDXs. rophages and target cells and Mab1 _ 1 . FIG . 12 : Expression profile oMAb1, 2 and 3 onto three FIG . 35 : In vitro ADCP of huMAb1_ negB . PDXs (CR - IGR - 034P , LUN -N1c - 014 and BRE - IGR -0159 ) . FIG . 36 : Loss of in vitro ADCP for huMAB1_ negA . FIG . 13 : Graphical representation showing the residues of 50 FIG . 37 : HRMS data of humAb1_ 1 conjugate modified Fabl that are part of the paratope ( ie residues with atoms with SNPP with ( 2E , 2 ' E , 11aS , 11a' S ) - 8 , 8 - ( ( ( 4 - ( 2 -( 2 - ( 2 - ( ( 2 within 4A of the antigen atoms) . mercapto - 2 -methylpropyl ) (methyl ) amino ) ethoxy ) ethoxy ) FIG . 14 :Graphical representation showing the residues of ethoxy )pyridine - 2 ,6 - diyl) bis (methylene )) bis (oxy ) ) bis ( 2 HLAMP1 forming the epitope for Fabl ( ie residues with ethylidene - 7 -methoxy - 2, 3 -dihydro - 1Hbenzo [ e] pyrrolo [ 1, 2 atoms within 4A of the antigen atoms ). 55 a ] [ 1, 4 ] diazepin -5 ( 11aH )- one ) DM4 -SPDB - chMA62 FIG . 15 : Graphical representation showing the overlay of conjugate . the residues of hLAMP1 and a model of G187E correspond - FIG . 38 : Immunohistochemistry staining ( IHC ) on FFPE ing to cLAMP1 . Differences in orientation of Lys151 of sample of colon adenocarcinoma patient derived xenograft hLAMP1 and of Tyr32 of Fab1 - LC are indicated , necessary CR -LRB -010P and human breast carcinoma with the poly to accommodate the mutation from Glycine to Glutamine at 60 clonal rabbit rAb4 Antibody. The Negative controls were position 187 . performed by omission of the primary antibody. Further FIG . 16 : Graphical representation showing an overlay of more , other irrelevant antibodies were negative or displayed the heavy chain residues of Fab1 and a model of Fabl with intracellular immunostaining the mutation 128Q in its heavy chain sequence of SEQ ID FIG . 39 : Immunocytochemistry ( ICC ) in FFPE format NO : 53 and the interaction with LAMP1. 65 with the polyclonal rabbit rAb4 Antibody at 1 ug/ mL . FIG . 17 : Graphical representation showing an overlay of FIG . 40 : Immunohistochemistry staining ( IHC ) on FFPE the heavy chain residues of Fab1 and a model of Fabl with sample of adenocarcinoma patient derived xenograft US 9 ,809 ,653 B2 CR -LRB -010P with MAb4 obtained from hybridoma domains separated by a hinge - like structure enriched with 88LAMP1- 2 . The negative control was performed by omis - prolines and serines many being linked to 0 - . sion of the primary antibody. LAMP1 has one transmembrane domain consisting of 24 FIG . 41 : Binding affinity by ELISA of MAb4 towards hydrophobic amino acids near the COOH terminus, and LAMP1 ( black ) or LAMP2 (grey ) . 5 contains a short cytoplasmic segment composed of 11 amino acid residues at the COOH - terminal end . DETAILED DESCRIPTION The nomenclature of the two domains of LAMP1, “ the first lumenal domain ” and the “ second lumenal domain " are Definitions based on the orientation of LAMP1 within its original 10 As used herein “ LAMP1” designates the “ Lysosomal localization , the lysosome. Nevertheless , when LAMP1 is associated membrane protein 1” , a member of a family of expressed at the cell surface , the two lumenal domains glycoproteins that is also known as LAMPA , CD107a or become extracellular domains, and therefore exposed at the LGP120 . LAMP1 is , according to protein expression data cell surface . Therefore , in one embodiment “ extracellular” for human tumoral samples in comparison to non tumoral is15 min context of the invention refers to LAMP1 protein con samples presented in the following Example 5 , expressed at structs comprising the first and / or second luminal domain ( s ) the cell surface of colon adenocarcinomas, gastrointestinal of LAMP1 as defined below and / or variants thereof. The tumors (small intestine, rectum , parotid gland ), vital organs domain organisation of human LAMP1 according to tumors (lung , liver, stomach , pancreas and kidney ) , repro NP _ 005552 . 3 ( SEQ ID NO : 24 ) has been mapped in ductive organ tumors ( breast , ovary and prostate ) as well as 20 example 6 . 1 and will be used in this document as follows: skin , larynx and soft tissue tumors . The human gene LAMP1 is found on chromosome 13734 TABLE 1 (113 , 951, 469 - 113 ,977 ,441 ) and has a total length of 26 ,273 kb . Description of human LAMP1 domains A reference sequence of the cDNA coding for full- length 25 Positions in NP _ 005552 human LAMP1, including the sequence encoding the signal LAMP1 Domains ( SEQ ID NO : 24 ) peptide , is available from the GenBank database under Peptide signal Met1 - Ala28 accession number NM _ 005561. 3 (SEQ ID NO : 23 ) and the First lumenal domain Ala29 - Arg195 representative protein sequence , including the signal peptide Loop 1 , Ala29 - Leu100 ( positions 1 - 28 ) is available under NP _ 005552 . 3 ( SEQ ID 30 Loop 2 , L2 Thr101- Arg195 Hinge Pro196 - Thr 227 NO : 24 ). One potential isoform of LAMP1 has been Second lumenal domain Asn228 -Met382 reported which would miss the amino acids at positions Loop 3 , L3 Asn228 - Ile309 136 - 188 of SEQ ID NO : 24 , corresponding to exon 4 of the Loop 4 , L4 Leu310 -Met382 gene coding for human LAMP1. No synonymous SNPs Transmembrane domain Leu383 -Gly406 have been identified in Caucasian population of at least 60 35 Lysosome targeting motif Arg407- Ile417 individuals . Concerning its orthologs , human LAMP1 shares 66 % Accordingly , the domain consisting of the first to third sequence identity with respectively mouse LAMP1 loops of human LAMP1 consists of amino acids at positions (NP _ 034814 , SEQ ID NO : 25 ) and rat LAMP1 29 - 309 of SEO ID NO : 24 . (NP 036989 , SEQ ID NO : 26 ), and human and Macaca 40 Domain organisation of Macaca fascicularis LAMPI mulatta LAMP1 (XP _ 001087801, SEQ ID NO : 27 ) share 96 % sequence identity . according to the predicted sequence ( SEQ ID NO : 39 ) is as The sequence of LAMP1 from Macaca mulatta (SEQ ID follows: NO : 27 ) and the predicted sequence of Macaca fascicularis (SEQ ID NO : 39) are identical to 99 % , said sequences 45 TABLE 2 differing by one additional leucine at position 11 of Macaca Description of Macaca fascicularis LAMP1 domains mulatta LAMP1 ( SEQ ID NO : 27 ), i .e . in the signal peptide . Accordingly the sequences of mature LAMP1 from Macaca Positions in mulatta and Macaca fascicularis are identical . LAMP1 Domains SEQ ID NO : 39 The closest member of the LAMP family is LAMP 2 50 Peptide signal Metl - Ala26 First lumenal domain Ala27 - Arg193 ( P13473 , human LAMP2 , soluble LAMP2 protein SEQ ID Loop 1 , L1 Ala27 - L98 NO : 40 ) . Human LAMP1 and LAMP2 proteins share ~ 36 % Loop 2 , L2 Thr99 - Arg193 sequence identity , and comprise some conserved glycosy Hinge Pro194 - Thr 225 lation sites. Second lumenal domain Asn226 -Met380 A " domain ” may be any region of a protein , generally 55 Loop 3 , L3 Asn226 - Thr307 Loop 4 , L4 Leu308 -Met380 defined on the basis of sequence homologies and often Transmembrane domain Leu381 -Gly404 related to a specific structural or functional entity . The Lysosome targeting motif Arg405 - Ile415 domain organization of LAMP1 has not been entirely pub - lished so far. Human LAMP1 consists of 417 amino acid residues and 60. Accordingly, the domain consisting of first to third loops 28 amino -terminal residues corresponding to a cleavable of Macaca fascicularis LAMP1 consists of amino acids at signal peptide. The major portion ofLAMP1 resides on the positions 27 -307 of SEQ ID NO : 39 . lumenal side of the lysosome and is heavily glycosylated by A sequence alignment of human and Macaca fascicularis N - glycans . LAMP1 contains 18 potential N - glycosylation LAMP1 full - length proteins is shown on FIG . 1 . sites of which 5 are occupied with poly - N -acetyllactosamine 65 The loop region 4 of human and Macaca fascicularis glycans (Carlsson , S . R . and Fukuda , M . , 1990 , J . Biol. LAMP1 do not contain any glycosylation site , which dis Chem . 265 ( 33 ): 20488 - 20495 ) . They are clustered into two tinguishes Loop 4 from Loops 1- 3 of LAMP1. US 9 ,809 , 653 B2 Loops 1 - 4 have been defined from the primary amino acid is = 2 . In particular, the genome displays two alleles for each sequence , and has been mapped in example 6 . 1 , but not from gene , one on each chromosome of a pair of homologous the 3D structure of LAMP1 since the structure was not ( except for the genes localized on sexual solved prior to this work . chromosomes ) . A " coding sequence ” or a sequence " encoding ” an expres - 5 Herein the word “ gene number ” and “ gene copy number " sion product, such as a RNA , polypeptide, protein , or can be used interchangeably . enzyme, is a nucleotide sequence that, when expressed . In the context of the invention , a " copy " of a sequence results in the production of that RNA , polypeptide, protein , encompasses a sequence identical to said sequence but also or enzyme, i . e . , the nucleotide sequence encodes an amino allelic variations of said sequence . acid sequence for that polypeptide , protein or enzyme. A 10 One example to measure DNA copy number and therefore coding sequence for a protein may include a start codon DNA copy number change is array - based CGH which is a ( usually ATG ) and a stop codon . A region encoding an high -throughput technique to measure DNA copy number expression product present in the DNA is called " coding change across the genome. The DNA fragments or “ clones " DNA sequence ” or “ CDS ” . of test and reference samples are hybridized to mapped array As used herein , references to specific proteins (e . g. , 15 fragments . Log 2 intensity ratios of test to reference provide antibodies ) can include a polypeptide having a native amino useful information about genome -wide profiles in copy acid sequence , as well as variants and modified forms number. regardless of their origin or mode of preparation . A protein The “ Log 2 ” or “ Log 2 ratio " value is used to describe the which has a native amino acid sequence is a protein having copy number of a gene or a DNA fragment in a cell genome. the same amino acid sequence as obtained from nature. Such 20 In an ideal situation , the log 2 ratio of normal (copy native sequence proteins can be isolated from nature or can variation neutral) clones is log 2 ( 2 / 2 ) = 0 , single copy losses be prepared using standard recombinant and/ or synthetic is log 2 ( 1 / 2 ) = - 1 , and single copy gains is log 2 ( 3 / 2 ) = 0 .58 . methods. Native sequence proteins specifically encompass Multiple copy gains or amplifications would have values of naturally occurring truncated or soluble forms, naturally log 2 ( 4 / 2 ) , log 2 ( 5 / 2 ), . . . . occurring variant forms ( e . g ., alternatively spliced forms) , 25 As used herein , the term " gain ” of a sequence refers in naturally occurring allelic variants and forms including general to the presence of a copy number 22 . 5 (alternatively post- translational modifications . A native sequence protein a Log 2 ratio 0 . 32 ) of said sequence in the diploid genome includes proteins following post- translational modifications of a subject. These > 2 . 5 copies may be adjacent or not on the such as glycosylation , or phosphorylation , or other modifi - genome; in particular they may be present in different cations of some amino acid residues . 30 regions of a pair of chromosomes or on chromosomes As used herein , the term “ marker ” refers to any biologi- belonging to distinct pairs of chromosomes of the genome. cal, chemical or physical mean allowing identifying the Accordingly , the term " gene copy number gain ” refers to presence , and possibly quantifying the expression of a target the presence of copy numbers (alternatively a Log 2 gene and / or protein in a biological sample . Such markers are ratio 0 .32 ) of a specific gene in the diploid genome of a well known from one skilled in the art. Advantageously , the 35 subject. When the copy number = 2 . 5 , 50 % of the cells used markers according to the invention are genetic markers for defining the copy number contain the usual 2 copies of and / or protein markers . the gene in a diploid organism and 50 % of the cells used for The term " gene ” means a DNA sequence that codes for, defining the copy number contain the usual 2 copies and 1 or corresponds to , a particular sequence of amino acids additional copy more of said gene ( in total 3 copies of said which comprises all or part of one or more proteins or 40 gene ) . enzymes , and may or may not include regulatory DNA The term “ low gain " of a sequence refers in general to the sequences , such as promoter sequences , which determine for presence of a copy number 22 . 5 but < 5 ( alternatively example the conditions under which the gene is expressed . 0 .32slog 2 ratio < 1 . 32 ) of said sequence in the diploid Some genes, which are not structural genes, may be tran genome of a subject. The terms " amplification ” , “ Amp ” , or scribed from DNA to RNA , but are not translated into an 45 “ high gain ” refer herein to the presence of a copy amino acid sequence . Other genes may function as regula - number 25 , or alternatively a Log 221. 32 , of a specific tors of structural genes or as regulators of DNA transcrip - sequence in the diploid genome of a subject. Accordingly , tion . In particular , the term gene may be intended for the the term " gene number amplification ” refers to the presence genomic sequence encoding a protein , i . e . a sequence com of 25 copy numbers of a specific gene in the diploid genome prising regulator, promoter, intron and exon sequences. 50 of a subject As used herein , the terms “ copy number variation ” , “ copy As used herein , a “ fragment of a sequence ” corresponds number variant" and " CNV ” are used indifferently and refer to a portion of said sequence , for instance of a nucleotide to a DNA segment of 1 kb or larger and present at variable sequence . Said fragment is preferably at least 10 bp long . copy number in comparison with a reference genome. The More preferably said fragment is at least 15 bp long , in terms “ structural variant" , " duplicon ” , “ indel ” , “ intermedi- 55 particular at least 20 bp long. Most preferably, said fragment ate - sized structural variant ( ISV ) ” , “ low copy repeat is at least 25 bp long , at least 30 bp long, in particular at least ( LCR ) ” , “ multisite variant (MSV ) ” , “ paralogous sequence 33 bp long . A fragment of the above sequence may be in variant (PSV ) ” , “ segmental duplication ” , “ interchromo10 particular a primer or probe . somal duplication ” , and “ intrachromosomal duplication ” . In the context of the invention , a “ mutated sequence ” of found in the literature , are included herein in the term 60 a reference sequence refers to a sequence including “ CNV " . insertion (s ), deletion ( s ) or substitution ( s ) of one or more Furthermore, copy number variation can refer to a single nucleotide( s ), wherein said mutated sequence is at least 75 % gene , or include a contiguous set of genes. identical to the reference sequence . The percentage of As used herein " gene number ” describes the numbers of sequence identity is calculated by comparing the mutated genes present in the cell . In diploid organisms, in a normal 65 sequence optimally aligned with the reference sequence , state , two copies of each nucleic sequence are naturally determining the number of positions at which the identical present in the genome , therefore , the copy number (CN ) nucleic acid base ( e . g ., A , T , C , G , U , or I ) occurs in both US 9 ,809 , 653 B2 10 sequences to yield the number of matched positions, divid CH ) . The variable regions of both (VL ) and heavy ing the number of matched positions by the total number of ( VH ) chains determine binding recognition and specificity to positions of the reference sequence , and multiplying the the antigen . The constant region domains of the light (CL ) result by 100 to yield the percentage of sequence identity . and heavy (CH ) chains confer important biological proper Preferably , the mutated sequence is at least 80 % , 85 % , 90 % , 5 ties such as antibody chain association , secretion , trans 95 % identical to the reference sequence . placental mobility , complement binding , and binding to Fc Preferably said mutated sequence of a reference sequence receptors (FcR ). The Fv fragment is the N -terminal part of is an allelic variant of said reference sequence . As used the Fab fragment of an immunoglobulin and consists of the herein , an “ allelic variant" denotes any of two or more variable portions of one light chain and one heavy chain . The alternative forms of a gene occupying the same chromosome 10 specificity of the antibody resides in the structural comple . mentarity between the antibody combining site and the A sequence " at least 85 % identical to a reference antigenic determinant. Antibody combining sites are made sequence ” is a sequence having, on its entire length , 85 % , or up of residues that are primarily from the hypervariable or more , in particular 90 % , 91 % 92 % 93 % 94 % , 95 % , 96 % , complementarity determining regions (CDRs ) . Occasion 97 % , 98 % or 99 % sequence identity with the entire length 15 ally , residues from nonhypervariable or framework regions of the reference sequence . (FR ) influence the overall domain structure and hence the A percentage of “ sequence identity ” may be determined combining site . Complementarity Determining Regions or by comparing the two sequences , optimally aligned over a CDRs refer to amino acid sequences which together define comparison window , wherein the portion of the polynucle - the binding affinity and specificity of the natural Fv region otide or polypeptide sequence in the comparison window 20 of a native immunoglobulin binding site . The light and may comprise additions or deletions ( i. e . gaps) as compared heavy chains of an immunoglobulin each have three CDRs, to the reference sequence (which does not comprise addi designated CDR1- L , CDR2 - L , CDR3 - L and CDR1 - H , tions or deletions ) for optimal alignment of the two CDR2- H , CDR3- H , respectively . A conventional antibody sequences . The percentage is calculated by determining the antigen -binding site , therefore , includes six CDRs, compris number of positions at which the identical nucleic acid base 25 ing the CDR set from each of a heavy and a light chain V or amino acid residue occurs in both sequences to yield the region . number of matched positions, dividing the number of “ Framework Regions ” (FRs ) refer to amino acid matched positions by the total number of positions in the sequences interposed between CDRs, i. e . to those portions window of comparison and multiplying the result by 100 to of immunoglobulin light and heavy chain variable regions yield the percentage of sequence identity . Optimal alignment 30 that are relatively conserved among different immunoglobu of sequences for comparison is conducted by global pairwise lins in a single species . The light and heavy chains of an alignment, e .g . using the algorithm of Needleman and Wun - immunoglobulin each have four FRs , designated FR1- L , sch J. Mol. Biol. 48 : 443 ( 1970 ) . The percentage of sequence FR2 -L , FR3 -L , FR4 - L , and FR1- H , FR2 -H , FR3 - H , FR4 - H , identity can be readily determined for instance using the respectively . program Needle , with the BLOSUM62 matrix , and the 35 As used herein , a “ human framework region ” is a frame following parameters gap -open = 10 , gap - extend = 0 .5 . work region that is substantially identical ( about 85 % , or A " conservative amino acid substitution ” is one in which more , in particular 90 % , 95 % , 97 % , 99 % or 100 % ) to the an amino acid residue is substituted by another amino acid framework region of a naturally occurring human antibody. residue having a side chain R group with similar chemical In the context of the invention , CDR /FR definition in an properties ( e . g ., charge or hydrophobicity ) . In general, a 40 immunoglobulin light or heavy chain is to be determined conservative amino acid substitution will not substantially based on IMGT definition ( Lefranc , M . P . et al ., 2003 , Dev change the functional properties of a protein . Examples of Comp Immunol. 27 ( 1 ) : 55 - 77 ; on the world wide web at groups of amino acids that have side chains with similar imgt. org ) . chemical properties include 1 ) aliphatic side chains: glycine , As used herein , the term “ antibody ” denotes conventional alanine , valine , leucine , and isoleucine ; 2 ) aliphatic -hy - 45 antibodies and fragments thereof , as well as single domain droxyl side chains: serine and threonine ; 3 ) amide -contain - antibodies and fragments thereof, in particular variable ing side chains : asparagine and glutamine ; 4 ) aromatic side heavy chain of single domain antibodies , and chimeric , chains : phenylalanine , tyrosine , and tryptophan ; 5 ) basic humanised , bispecific or multispecific antibodies . side chains : lysine , arginine , and histidine; 6 ) acidic side As used herein , antibody or immunoglobulin also chains: aspartic acid and glutamic acid ; and 7 ) sulfur- 50 includes " single domain antibodies ” which have been more containing side chains : cysteine and methionine. Conserva - recently described and which are antibodies whose comple tive amino acids substitution groups are: valine - leucine mentary determining regions are part of a single domain isoleucine , phenylalanine - tyrosine -tryptophane , lysine - polypeptide . Examples of single domain antibodies include arginine, alanine -valine , glutamate -aspartate , and heavy chain antibodies, antibodies naturally devoid of light asparagine - glutamine . 55 chains, single domain antibodies derived from conventional An “ antibody” may be a natural or conventional antibody four -chain antibodies, engineered single domain antibodies . in which two heavy chains are linked to each other by Single domain antibodies may be derived from any species bonds and each heavy chain is linked to a light including , but not limited to mouse , human , camel, llama, chain by a disulfide bond. There are two types of light chain , goat, rabbit and bovine . Single domain antibodies may be lambda ( 1 ) and kappa ( k ) . There are five main heavy chain 60 naturally occurring single domain antibodies known as classes (or isotypes ) which determine the functional activity heavy chain antibody devoid of light chains . In particular, of an antibody molecule: IgM , IgD , IgG , IgA and IgE . Each Camelidae species , for example camel, dromedary , llama , chain contains distinct sequence domains . The light chain alpaca and guanaco , produce heavy chain antibodies natu includes two domains or regions, a variable domain (VL ) rally devoid of light chain . Camelid heavy chain antibodies and a constant domain (CL ) . The heavy chain includes four 65 also lack the CH1 domain . domains , a variable domain (VH ) and three constant The variable heavy chain of these single domain antibod domains (CH1 , CH2 and CH3, collectively referred to as ies devoid of light chains are known in the art as “ VHH ” or US 9 ,809 ,653 B2 11 12 " nanobody ” . Similar to conventional VH domains , VHHs scFvs, or can be generated by coupling monovalentscFvs by contain four FRs and three CDRs . Nanobodies have advan - a peptide linker, such as divalent sc (Fv ) . “ dsFv ” is a tages over conventional antibodies : they are about ten times VH : :VL heterodimer stabilised by a disulphide bond . smaller than IgG molecules , and as a consequence properly “ ( dsFv ) 2 ” denotes two dsFv coupled by a peptide linker. folded functional nanobodies can be produced by in vitro 5 The term “ bispecific antibody ” or “ BsAb ” denotes an expression while achieving high yield . Furthermore , nano - antibody which combines the antigen - binding sites of two bodies are very stable , and resistant to the action of pro - antibodies within a single molecule . Thus , BsAbs are able to teases. The properties and production of nanobodies have bind two different antigens simultaneously. Genetic engi been reviewed by Harmsen and De Haard H J (Appl . neering has been used with increasing frequency to design , Microbiol. Biotechnol. 2007 November ; 77 ( 1 ) : 13 - 22 ). 10 modify , and produce antibodies or antibody derivatives with The term “ monoclonal antibody ” or “ mAb ” as used herein a desired set of binding properties and effector functions as refers to an antibody molecule of a single amino acid described for instance in EP 2 050 764 A1. composition that is directed against a specific antigen , and is The term “ multispecific antibody ” denotes an antibody not to be construed as requiring production of the antibody which combines the antigen -binding sites of two or more by any particular method . A monoclonal antibody may be 15 antibodies within a single molecule . produced by a single clone of B cells or hybridoma, butmay The term “ diabodies ” refers to small antibody fragments also be recombinant, i . e . produced by protein engineering with two antigen -binding sites, which fragments comprise a The term " chimeric antibody ” refers to an engineered heavy - chain variable domain (VH ) connected to a light antibody which in its broadest sense contains one or more chain variable domain ( VL ) in the same polypeptide chain regions from one antibody and one or more regions from on 20 (VH - VL ). By using a linker that is too short to allow pairing or more other antibody ( ies ) . In particular a chimeric anti - between the two domains on the same chain , the domains are body comprises a VH domain and a VL domain of an forced to pair with the complementary domains of another antibody derived from a non - human animal, in association chain and create two antigen - binding sites . with a CH domain and a CL domain of another antibody , in The term " hybridoma" denotes a cell , which is obtained particular a human antibody. As the non -human animal, any 25 by subjecting a B cell prepared by immunizing a non -human animal such as mouse , rat, hamster , rabbit or the like can be mammal with an antigen to cell fusion with a myeloma cell used . A chimeric antibody may also denote a multispecific derived from a mouse or the like which produces a desired antibody having specificity for at least two different anti - monoclonal antibody having an antigen specificity . gens. In an embodiment, a chimeric antibody has variable By “ purified ” and “ isolated ” it is meant, when referring to domains of mouse origin and constant domains of human 30 a polypeptide ( i . e . the antibody of the invention ) or a origin nucleotide sequence , that the indicated molecule is present The term "humanised antibody ” refers to an antibody in the substantial absence of other biological macromol which is initially wholly or partially of non -human origin ecules of the same type. The term " purified ” as used herein and which has been modified to replace certain amino acids, in particular means at least 75 % , 85 % , 95 % , or 98 % by in particular in the framework regions of the heavy and light 35 weight, of biological macromolecules of the same type are chains , in order to avoid or minimize an immune response present . An “ isolated ” nucleic acid molecule which encodes in humans . The constant domains of a humanized antibody a particular polypeptide refers to a nucleic acid molecule are most of the time human CH and CL domains . In an which is substantially free of other nucleic acid molecules embodiment, a humanized antibody has constant domains of that do not encode the subject polypeptide ; however, the human origin . 40 molecule may include some additional bases or moieties " Fragments” of (conventional ) antibodies comprise a por - which do not deleteriously affect the basic characteristics of tion of an intact antibody, in particular the antigen binding the composition . region or variable region of the intact antibody . Examples of As used herein , the term “ subject” denotes a mammal , antibody fragments include Fv , Fab , F (ab ' ) 2 , Fab ', dsFv, such as a rodent, a feline , a canine , and a primate . In ( dsFv ) 2 , scFv , sc (Fv ) 2 , diabodies , bispecific and multispe - 45 particular a subject according to the invention is a human . cific antibodies formed from antibody fragments . A frag - Throughout the instant application , the term “ comprising " ment of a conventional antibody may also be a single is to be interpreted as encompassing all specifically men domain antibody, such as a heavy chain antibody or VHH . tioned features as well optional, additional, unspecified The term “ Fab ” denotes an antibody fragment having a ones . As used herein , the use of the term " comprising ” also molecular weight of about 50 ,000 and antigen binding 50 discloses the embodiment wherein no features other than the activity , in which about a half of the N -terminal side of H specifically mentioned features are present ( i. e . “ consisting chain and the entire L chain , among fragments obtained by of” ) . treating IgG with a , papaine , are bound together Throughout the instant application , the term “ and / or ” is a through a disulfide bond . grammatical conjunction that is to be interpreted as encom The term “ F ( ab ) 2 ” refers to an antibody fragment having 55 passing that one or more of the cases it connects may occur. a molecular weight of about 100 ,000 and antigen binding For example , the sentence “ quantifying the expression of a activity , which is slightly larger than the Fab bound via a target gene and / or protein in a biological sample ” indicates disulfide bond of the hinge region , among fragments the expression of a target gene may be quantified (mRNA ) , obtained by treating IgG with a protease , pepsin . or the expression of a protein or the expression of a target A single chain Fv (“ scFv ” ) polypeptide is a covalently 60 gene (mRNA ) and the protein together may be quantified . linked VH :: VL heterodimer which is usually expressed from Accordingly , the wording " a variable domain of heavy a gene fusion including VH and VL encoding genes linked chain of sequence SEQ ID NO : 1 or a sequence at least 85 % by a peptide - encoding linker . The human scFv fragment of identical thereto and/ or a variable domain of light chain of the invention includes CDRs that are held in appropriate sequence of sequence SEQ ID NO : 5 , or a sequence at least conformation , in particular by using gene recombination 65 85 % identical thereto ” is to b interpreted as “ a variable techniques . Divalent and multivalent antibody fragments domain of heavy chain of sequence SEQ ID NO : 1 or a can form either spontaneously by association of monovalent sequence at least 85 % identical thereto ” or “ a variable US 9 ,809 ,653 B2 13 14 domain of light chain of sequence of sequence SEQ ID NO : vivo in mice bearing the primary human colon adenocarci 5 , or a sequence at least 85 % identical thereto ” or “ a variable noma xenograft derived from patient CR -LRB -010P , when domain of heavy chain of sequence SEQ ID NO : 1 or a used at a dose of 10 mg/ kg , 5 mg/kg and 2 . 5 mg/ kg , with a sequence at least 85 % identical thereto and a variable single injection , as described in example 10 . 1 . 1 . domain of light chain of sequence of sequence SEQ ID NO : 5 Furthermore, the inventors showed that this immunocon 5 , or a sequence at least 85 % identical thereto ” . jugate induces a marked anti - tumor activity in vivo in mice The term “ cancer ” , “ ” , “ tumor ” , and “ carci bearing the primary human lung tumor xenograft derived noma” are used interchangeably herein to refer to cells that from patient LUN -NIC -0014 , when used at a dose of 10 exhibit relatively autonomous growth , so that they can mg/ kg , 5 mg/kg and 2 . 5 mg/kg , with a single injection , as exhibit an aberrant growth characterized by sig - 10 described in example 10 . 1 . 2 . nificant loss of control of cell proliferation . In general, cells They have also shown that the immunoconjugates DM4 of interest for detection or treatment in the present applica SPDB -huMAb1 _ 1 , DM4 -SPDB -chMAD2 , and DM4 tion include precancerous ( e . g . benign ) , malignant, meta SPDB - chMAb3 induce a marked anti - tumor activity in vivo static , and non -metastatic cells . 15 in different patient- derived xenograft as shown in example Immunoconjugates 10 . 2 - 10 . 4 . For example , it was shown the immunoconjugate DM4 For therapeutic purposes , it is advantageous to create an SPDB - huMAb1 _ 1 induces a marked anti - tumor activity in antibody with optimal characteristics for use as an antibody vivo in a primary human invasive ductal carcinoma xeno drug conjugate , i . e . an antibody which specifically recog - 20 graft and primary human lung tumor xenograft derived from nizes a target present on the surface of cancer cells and patient , when used at a dose of 10 mg/ kg , 5 mg/kg , 2 . 5 which is capable of efficiently triggering internalization once mg/kg , or 1 .25 mg/ kg with a single injection , as described in bound to said target. example 10 . 2 . 2 and 10 . 2 . 3 . The inventors raised antibodies against colon tumor cells Also the immunoconjugates DM4- SPDB -chMAb2 and or lung tumor cells and screened resulting clones for the 25 DM4- SPDB -chMAb3 induced a marked anti -tumor activity differential binding to tumor cells and non - tumor tissue . in vivo in a murine model of primary human invasive ductal The inventors identified in this way antibodies distin carcinoma xenograft derived from patient, when used at a guishing tumoral from non - tumoral tissues . Three of those dose of 10 mg/kg , 5 mg/ kg and 2 .5 mg/ kg or 5 mg/ kg , 2 .5 antibodies were selected ( the so -called antibodies “MAb1 ” , “ MAb2” and “ MAb3” ), fulfilling the expected features 30 mg/ kg and 1. 25 mg/ kg , respectively , with a single injection , necessary for therapeutical application , in particular in the 30 as described in example 10 . 3 . 2 and 10 .4 . form of ADC . Those three antibodies showed high binding Altogether , for the first time, these results validly identify affinity (within the nanomolar range ) to cell surface LAMP1 as a therapeutic target for the treatment of cancer . expressed LAMP1 in cancer cells . Furthermore, those three Accordingly, the invention relates to an immunoconjugate anti -LAMP1 antibodies showed high capacity to trigger 35 comprising an antibody which : internalization of the LAMP1 /anti - LAMP1 antibody com a ) binds to human and Macaca fascicularis LAMP1 plex , as shown in example 4 . 4 and 4 . 3 . proteins ; and The inventors demonstrated that the chimeric antibodies b ) is linked or conjugated to at least one growth inhibitory derived from MAD1, MAD2 , MAD3 ( chMAbi , chMAV2 . agent. chMAb3 ), combined with a cytotoxic maytansinoid (DM4 ) 40 Any antibody which binds to human and Macaca fas showed as well a high but slightly different binding affinity cicularis LAMP1 proteins , as described throughout the to human LAMP1 or cynomologus monkey LAMP1 then instant application ( e . g . M?b4 , fragments thereof, or chi the naked antibody as shown in example 8 . 1 . 7 and 8 . 1 . 8 . meric or humanised version thereof ) , can be incorporated in Accordingly in one embodiment the immunoconjugate in the immunoconjugate according to the invention . context of the invention has an affinity (EC50 ) for full length 45 As used herein , " conjugate ” , “ immunoconjugate ” , “ anti human LAMPI and cynomologues monkey LAMP1 body -drug conjugate” or “ ADC ” have the samemeaning and expressed at the cell surface of a recombinant cell line , are interchangeable . wherein the cell line may be HCT116 and the apparent A " growth inhibitory agent” , or “ anti -proliferative agent” , affinity measured via Flow Cytometry is $ 30 nM , for which can be used indifferently , refers to a compound or example s20 nM or s15 nM . 50 composition which inhibits growth of a cell , especially TheMethods to measure the affinity (EC5 . ) for full length tumour cell, either in vitro or in vivo . A growth inhibitory human LAMP1 and cynomologues monkey LAMP1 are agent denotes in particular a cytotoxic agent or a radioactive further explained in the chapter “ antibodies ” . isotope . The inventors additionally demonstrated that a chimeric The term “ radioactive isotope " is intended to include antibody derived from MAbi (chMAbl ) , combined with a 55 radioactive isotopes suitable for treating cancer , such as cytotoxic maytansinoid (DM4 ) , induces cytotoxic activity in At211 , Ac225, Bi212 , Bi213, Pb212 , Er169, 1131 , 1124 , 1125 , Y90 , vitro on human HCT116 tumor cells containing a stable In ! 11, P32 , Re186 ,Re188 , Sm153 , Sr89 , Zr89 , Tc99m ,Gas , Cu64 integration of the LAMP1 coding DNA sequence in the and radioactive isotopes of Lu such as Lul77 . Such radio genomic DNA and expressing LAMP1 on their surface . isotopes generally emit mainly beta -radiation . In an embodi Furthermore , the inventors demonstrated that humanized 60 ment the radioactive isotope is alpha - emitter isotope , more antibodies derived from MAbl (huMAb1 _ 1 , huMAb1_ 2 , precisely Thorium 227 ( Th227) which emits alpha - radiation . huMAb1 _ 3 ) , combined with a cytotoxic maytansinoid The immunoconjugates according to the present invention ( DM4 ) induce cytotoxic activity in vitro on human HCT116 can be prepared as described in the application WO2004 / tumor cells containing a stable integration of the LAMPI 091668 . coding DNA sequence in the genomic DNA . 65 In one embodiment, a radioactive isotope is selected from They have also shown that the immunoconjugate DM4- the group consisting of At211, Ac225 , Bi213, Pb212, Erl69 , SPDB -chMAb1 induces a marked anti- tumor activity in 1124 , 1125 , In !! !, P32 , Re186, Sm153 , Sr89 , Zr89 , Tc99m , Ga68 , US 9 ,809 , 653 B2 15 16 Cu64 and radioactive isotopes of Lu, for instance from At211 , LAMP1 protein consists of amino acids Ala29 to lle309 of Er169 , 1125, Inl11, p32 , Re186 , Sm153 , Sr89 , radioactive iso SEQ ID NO : 24 and the domain consisting of the first to topes of Lu , and Th227. third loops of Macaca fascicularis LAMP1 protein consists The term " cytotoxic agent” as used herein refers to a of amino acids Ala27 to Thr307 of SEQ ID NO : 39 substance that inhibits or prevents the function of cells 5 and / or causes destruction of cells . The term " cytotoxic According to a second embodiment, the invention relates agent” is intended to include chemotherapeutic agents , to an immunoconjugate wherein the antibody binds to a enzymes , antibiotics , and toxins such as small molecule domain consisting of the first to third loops of human and toxins or enzymatically active toxins of bacterial, fungal, Macaca fascicularis LAMP1 proteins ; wherein the domain plant or animal origin , including fragments and / or variants 10 consisting of the first to third loops of human LAMP1 thereof, and the various antitumor or anticancer agents protein consists of amino acids Ala29 to Ile309 of SEQ ID disclosed below . In some embodiments , the cytotoxic agent NO : 24 and the domain consisting of the first to third loops is a drug or a pro - drug of a compound consisting in an of Macaca fascicularis LAMP1 protein consists of amino anti - tubulin agent such as taxoids or taxanes , a vinca - acids Ala27 to Thr307 of SEQ ID NO : 39 . alkaloid , a maytansinoid or maytansinoid analog such as 15 Therefore , according to this second embodiment, the DM1 or DM4, a cryptophycin derivative , an auristatin or immunoconiugate comórises an antibody which dolastatin analog ; a DNA alkylating agent, such as a tomay immunoconjugate comprises an antibody which mycin or pyrrolobenzodiazepine derivative , a CC - 1065 or a ) binds to a domain consisting of the first to third loops of CC - 1065 analog ; a leptomycin derivative ; a topoisomerase human and Macaca fascicularis LAMP1 proteins ; II inhibitor, an RNA polymerase II inhibitor such as alpha - 20 wherein the domain consisting of the first to third loops of amanitin . human LAMP1 protein consists of amino acids Ala29 to According to a first embodiment, said at least one growth Ile309 of SEQ ID NO : 24 and the domain consisting of inhibitory agent is neither an undefined radioactive isotope , the first to third loops of Macaca fascicularis LAMP1 a chemotherapeutic drug , a protein or lectin , nor pokeweed protein consists of amino acids Ala27 to Thr307 of SEQ antiviral protein , abrin , ricin and each of their A chains, 25 ID NO : 39 ; and doxorubicin , cisplastin , Iodine - 131, Yttrium - 90 , Rheniumhenium - b ) is linked or conjugated to said at least one growth 188, Bismuth - 212 , Taxol, 5 - Fluorouracil , VP - 16 ( etopo inhibitory agent. side) , bleomycin , methotrexate , vindesine , adriamycin , vin cristine , vinblastine , bis - chloroethylnitrosourea (BCNU ) , Although not compulsory, in said second embodiment , the mitomycin , cyclophosphamide and a cytokine such as TNF 30 at least one growth inhibitory agent may be different from an and TNF - B . undefined radioactive isotope , a chemotherapeutic drug , a According to this first embodiment , the invention relates protein or lectin , in particular from pokeweed antiviral in particular to an immunoconjugate comprising an antibody protein , abrin , ricin and each of their A chains , doxorubicin , which : cisplastin , Iodine - 131 , Yttrium - 90 , Rhenium - 188 , Bismuth a ) binds to human and Macaca fascicularis LAMP1 pro - 35 212 , Taxol, 5 - Fluorouracil , VP - 16 ( etoiposide) , bleomycin , teins; and methotrexate , vindesine , adriamycin , vincristine , vinblas b ) is linked or conjugated to at least one growth inhibitory tine, BCNU ,mitomycin , cyclophosphamide and a cytokine agents such as TNF and TNF - B . (i ) a cytotoxic agent selected from the group consisting of Accordingly , said at least one growth inhibitory agent enzymes other than from pokeweed antiviral protein ; 40 may be a radioactive isotopes selected from the group antibiotics other than from bleomycin and mitomycin ; consisting of At211 , Ac225 , Bi213 , Pb212 , Er169 , 1124 , 1125 , toxins of bacterial, fungal, or animal origin or of plant In111 , p32 , Re186 , Sm153, Sr89 , Zr89 , Tc99m , Ga 8 , Cu64 and origin other than from abrin and ricin , including frag - radioactive isotopes of Lu such as Lul77 , and Th227, for ments and /or variants thereof; a drug or a pro -drug of instance At211 , Er169 , 1125 , In111 , 232, Rel86, Sm153, Sr89, a compound consisting in an anti -tubulin agent such as 45 radioactive isotopes of Lu such as Lu177, and Th227, or a a maytansinoid ormaytansinoid analog such as DM1 or cytotoxic agent as defined in said first embodiment. DM4 , a taxoid or taxane other than from paclitaxel In said first and second embodiments , said at least one ( Taxol) , a vinca - alkaloid other than from vindesine , growth inhibitory agent may be in particular drug or a vincristine and vinblastine , a cryptophycin derivative , an auristatin or dolastatin analog ; a DNA alkylating 50 pro - drug of a compound consisting in a maytansinoid or agent other than from BCNU and cyclophosphamide, maytansinoid analog such as DM1 or DM4, a tomaymycin such as a tomaymycin or pyrrolobenzodiazepine or pyrrolobenzodiazepine derivative , a cryptophycin deriva derivative , a CC - 1065 or CC - 1065 analog ; a leptomy tive , a leptomycin derivative, an auristatin or dolastatin cin derivative; a topoisomerase II inhibitors other than analog , or a CC - 1065 or CC - 1065 analog , a RNA poly doxorubicin (adriamycin ) and etoposide, a RNA poly - 55 merase II inhibitor such as alpha -amanitin . merase II inhibitor such as alpha -amanitin , or In one embodiment , a suitable tomamycin is a tomamy Güla radioactive isotope selected from the group consist - cine dimer. Said tomamycin dimer is for instance ( 2E , 2 'E , ing of At211, Ac225 , Bi213 , Pb212, Er169, 1124 , 1125 , In ! ll . 11aS , 11a' S ) - 8 , 8 ' - ( ( ( 4 - ( 2 - ( 2 - ( 2 - ( ( 2 -mercapto - 2 -methylpro p3z , Re186, Sm153, Sp89, Zr89 , Tc99m , Ga68, Cu64 and pyl ) (methyl ) amino ) ethoxy )ethoxy )ethoxy )pyridine - 2 ,6 radioactive isotopes of Lu such as Lu177 , and Th227. 60 diyl) bis (methylene ) )bis (oxy ) ) bis ( 2 - ethylidene - 7 -methoxy In one embodiment a radioactive isotope is selected from 2 , 3 -dihydro -1Hbenzo [ e ]pyrrolo [ 1 , 2 - a ] [ 1 , 4 ] diazepin - 5 the group consisting of At211 , Er169 , 1125 , In111, P32 , Re186 . (llaH ) -one ). Sm153 , Sr89, radioactive isotopes of Lu , and Th227. The structural formula of (2E ,2 ' E , 11aS , 11a' S ) -8 , 8' - ( (( 4 In said first embodiment, the antibody may bind in ( 2 - ( 2 - ( 2 - ( ( 2 -mercapto - 2 -methylpropyl ) (methyl ) amino ) particular to a domain consisting of the first to third loops of 65 ethoxy ) ethoxy ) ethoxy )pyridine - 2 ,6 - diyl) bis (methylene )) bis human and Macaca fascicularis LAMP1 proteins ; wherein (oxy )) bis ( 2 - ethylidene - 7 -methoxy - 2 , 3 - dihydro - 1Hbenzo [ e ] the domain consisting of the first to third loops of human pyrrolo [ 1 , 2 - a ][ 1, 4 ] diazepin -5 ( 11aH ) -one ) is US 9, 809, 653 B2 17 US9 , 809 , 63 B2

SH

A “ maytansinoid ” as used herein denotes maytansinoids and maytansinoid analogs . Maytansinoids are drugs that inhibit microtubule formation and that are highly toxic to mammalian cells . Examples of suitable maytansinoids include maytansinol 20 and maytansinol analogs . SH . Examples of suitable maytansinol analogues include cil those having a modified aromatic ring and those having modifications at other positions . Such suitable maytansi Me noids are disclosed in U . S . Pat. Nos. 4 ,424 ,219 ; 4 ,256 , 746 ; 4 ,294 ,757 ; 4 , 307 ,016 ; 4 , 313 , 946 ; 4 ,315 ,929 ; 4 ,331 ,598 ; 4 , 361, 650 ; 4 , 362 ,663 ; 4 ,364 , 866 ; 4 , 450 , 254 ; 4 , 322 , 348 ; 4 , 371, 533 ; 6 , 333, 410 ; 5 ,475 , 092 ; 5 , 585 , 499 ; and 5 , 846 ,545 . Specific examples of suitable analogues of maytansinol 30 INH?? TO having a modified aromatic ring include : OH ( 1) C -19 -dechloro ( U . S . Pat . No . 4, 256 ,746 ) (prepared by Meo LAH reduction of ansamytocin P2 ) ; In another embodiment, the cytotoxic conjugates of the ( 2 ) C - 20 - hydroxy ( or C - 20 - demethyl ) + / - C - 19 - dechloro present! invention utilize the thiol- containing maytansinoid ( U . S . Pat. Nos. 4 ,361 , 650 and 4 ,307 ,016 ) (prepared by 35 DM4, formally termed N ' -deacetyl - N2 - (4 -methyl - 4 -mer demethylation using Streptomyces or Actinomyces or capto - 1- oxopentyl) - maytansine , as the cytotoxic agent. dechlorination using LAH ) ; and DM4 is represented by the following structural formula (II ) : ( 3 ) C -20 -demethoxy , C - 20 - acyloxy GOCOR ), ( II) +/ - dechloro ( U . S . Pat . No . 4 , 294 ,757 ) (prepared by a acylation using acyl chlorides ) . Specific examples of suitable analogues of maytansinol SH having modifications of other positions include : ( 1) C - 9 -SH ( U . S . Pat . No. 4 ,424 , 219 ) (prepared by the etsi reaction ofmaytansinol with H2S or P2S3) ; 45 MeoMeO ., ( 2 ) C - 14 - alkoxymethyl (demethoxy / CH _OR ) (U .S . Pat. No . 4 , 331, 598 ); ( 3 ) C - 14 -hydroxymethyl or acyloxymethyl (CH OH or CH OAC ) (U .S . Pat . No . 4 ,450 ,254 ) (prepared from 50 Nocardia ); N ( 4 ) C - 15 -hydroxy /acyloxy (U .S . Pat. No . 4 ,364 , 866 ) (pre OH H pared by the conversion of maytansinol by Streptomy Meo ces ); In further embodiments of the invention , other may ( 5 ) C - 15 -methoxy ( U . S . Pat. Nos. 4 , 313 , 946 and 4 , 315 , 55 tansines , including thiol and disulfide - containing maytansi noids bearing a mono or di -alkyl substitution on the carbon 929 ) ( isolated from Trewia nudiflora ) ; atom bearing the sulfur atom , may be used . These include a (6 ) C - 18 - N -demethyl (U .S . Pat . Nos . 4 ,362 ,663 and maytansinoid having , at C - 3 , C - 14 hydroxymethyl, C - 15 4 ,322 , 348 ) (prepared by the demethylation of may hydroxy , or C - 20 desmethyl, an acylated amino acid side tansinol by Streptomyces ) ; and chain with an acyl group bearing a hindered sulfhydryl group , wherein the carbon atom of the acyl group bearing ( 7 ) 4 ,5 - deoxy ( U . S . Pat. No . 4 , 371, 533 ) (prepared by the the thiol functionality has one or two substituents, said titanium trichloride /LAH reduction of maytansinol) . substituents being CH3, C2H5, linear or branched alkyl or In a specific embodiment, the cytotoxic conjugates of the alkenyl having from 1 to 10 reagents and any aggregate present invention utilize the thiol- containing maytansinoid which may be present in the solution . (DM1 ) , formally termed Na' - deacetyl -N2 ' - ( 3 -mercapto - 1 - 65 Examples of these cytotoxic agents and of methods of oxopropyl) -maytansine , as the cytotoxic agent. DM1 is conjugation are further given in the application WO2008 / represented by the following structural formula (I ) : 010101 which is incorporated by reference. US 9 ,809 , 653 B2 20 In some embodiments of the present invention , the anti synthesis . The length of DNA may comprise respective body is covalently attached , directly or via a cleavable or regions encoding the two portions of the conjugate either non - cleavable linker , to the at least one growth inhibitory adjacent one another or separated by a region encoding a agent. linker peptide which does not destroy the desired properties “ Linker ” , as used herein , means a chemical moiety com - 5 of the conjugate . prising a covalent bond or a chain of atoms that covalently The antibodies of the present invention may also be used attaches a polypeptide to a drug moiety . in Dependent Enzyme Mediated Prodrug Therapy by con The conjugates may be prepared by in vitro methods. In jugating the polypeptide to a prodrug -activating enzyme order to link a drug or prodrug to the antibody, a linking which converts a prodrug ( e . g . a peptidyl chemotherapeutic group is used . Suitable linking groups are well known in the 10 agent, see WO81701145 ) to an active anti -cancer drug ( See , art and include disulfide groups , thioether groups, acid labile for example , WO 88 /07378 and U . S . Pat . No . 4 ,975 ,278 ). groups, photolabile groups, peptidase labile groups and The enzyme component of the immunoconjugate useful for esterase labile groups. Conjugation of an antibody of the ADEPT includes any enzyme capable of acting on a prodrug invention with cytotoxic agents or growth inhibitory agents in such a way so as to convert it into its more active , may be made using a variety of bifunctional protein cou - 15 cytotoxiccyto form . Enzymes that are useful in the method of pling agents including but not limited to N - succinimidyl - this invention include , but are not limited to , alkaline pyridyldithiobutyrate ( SPDB ) , butanoic acid 4 - [ ( 5 -nitro - 2 phosphatase useful for converting phosphate -containing pyridinyl) dithio ] - 2 , 5 -dioxo - 1 -pyrrolidinyl ester (nitro prodrugs into free drugs ; arylsulfatase useful for converting SPDB ) , 4 - ( Pyridin - 2 - yldisulfanyl) - 2 -sulfo -butyric acid sulfate - containing prodrugs into free drugs ; cytosine deami (sulfo - SPDB ), N -succinimidyl (2 -pyridyldithio ) propionate nase useful for converting non - toxic fluorocytosine into the (SPDP ) , SNPP ( N -succinimidyl 4 - ( 5 -nitro - 2 -pyridyldithio ) 20 anticancer drug , 5 - fluorouracil; , such as serratia pentanoate ) , succinimidyl ( N -maleimidomethyl ) cyclo protease , thermolysin , subtilisin , carboxypeptidases and hexane - 1 -carboxylate (SMCC ), iminothiolane ( IT ), bifunc - cathepsins ( such as cathepsins B and L ), that are useful for tional derivatives of imidoesters ( such as dimethyl converting peptide - containing prodrugs into free drugs ; adipimidate HCL ), active esters ( such as disuccinimidyl D - alanylcarboxypeptidases , useful for converting prodrugs suberate ), aldehydes (such as glutaraldehyde ) , bis -azido 25 that contain D - amino acid substituents ; - cleav compounds (such as bis ( p -azidobenzoyl ) -hexanediamine ) ing enzymes such as 0 -galactosidase and neuraminidase bis - diazonium derivatives (such as bis - ( p - diazoniumben useful for converting glycosylated prodrugs into free drugs; zoyl) - ethylenediamine ), diisocyanates (such as toluene 2 ,6 P - lactamase useful for converting drugs derivatized with diisocyanate ) , and bis - active fluorine compounds ( such as P - lactams into free drugs; and penicillin amidases , such as 1 , 5 - difluoro - 2 , 4 - dinitrobenzene ) . For example , a ricin zo penicillin V amidase or penicillin G amidase , useful for immunotoxin can be prepared as described in Vitetta et al converting drugs derivatized at their amine nitrogens with (1987 ) . Carbon labeled 1- isothiocyanatobenzyl methyldieth phenoxyacetyl or phenylacetyl groups, respectively , into ylene triaminepentaacetic acid (MX -DTPA ) is an exemplary free drugs. The enzymes can be covalently bound to the chelating agent for conjugation of radionucleotide to the polypeptides of the invention by techniques well known in antibody (WO 94 / 11026 ) . the art such as the use of the heterobifunctional crosslinking The linker may be a “ cleavable linker” facilitating release 35 reagents discussed above . of the cytotoxic agent or growth inhibitory agent in the cell . According to said first and second embodiments , in the For example , an acid -labile linker, a peptidase -sensitive conjugate of the invention , the growth inhibitory agent may linker . anesterase labile linker, a photolabile linker or a be a maytansinoid , in particular DM1 or DM4 . disulfide - containing linker ( See e. g . U . S . Pat. No . 5 ,208 , In such a conjugate , the antibody is conjugated to said at 020 ) may be used . The linker may be also a “ non -cleavable 40 least one growth inhibitory agent by a linking group . In linker ” (for example SMCC linker) that might lead to better particular said linking group is a non -cleavable linker, such tolerance in some cases . as SPDB , sulfo - SPDB , or SMCC . Alternatively , a fusion protein comprising the antibody of In particular , the conjugate may be selected from the the invention and a cytotoxic or growth inhibitory polypep group consisting of: tide may be made, by recombinant techniques or peptide i ) an antibody - SPDB - DM4 conjugate of formula (III )

( III)

Lys

Antibody

tul **111011 011

- IITTIIN NH ÕH Ab - SPDB - DM4 US 9 ,809 , 653 B2 21 iiii) ) an antibodyantibodu. - sulfo .. - SPDB -DM4 conjugate of formula 22 ( IV )

OH (IV ) OSSS= 0 ZI Lys

tyyAntibody

II N **# 111 > +1011111

11lll NH OH Ab - SulfoSPDB - DM4

iii ) an antibody - SMCC -DM1 conjugate of formula (V )

( V )

N On all ** *011 Antibody

| NH TO ÕH

Ab - SPDB - DM1

In a further embodiment , in the conjugate of the inven In general, the conjugate can be obtained by a process tion , the growth inhibitory agent may be tomamycin , for comprising the steps of: instance à tomamycin dimer, for example (2E , Ž 'E , 11aS , 55 ( i) bringing into contact an optionally -buffered aqueous 11a' S ) - 8 , 8 ' - ( ( ( 4 - ( 2 - ( 2 - ( 2 - ( ( 2 -mercapto - 2 -methylpropyl ) solution of a cell - binding agent ( e . g . an antibody (methyl ) amino )ethoxy ) ethoxy ) ethoxy )pyridine - 2 ,6 - diyl) bis according to the invention ) with solutions of a linker (methylene )) bis (oxy ) ) bis ( 2 - ethylidene - 7 -methoxy - 2 , 3 and a cytotoxic compound ; dihydro - 1Hbenzo [ e ]pyrrolo [1 , 2 -a ][ 1 ,4 ] diazepin -5 ( 11aH )- ( ii ) then optionally separating the conjugate which was one ) . formed in (i ) from the unreacted cell -binding agent. In such a conjugate , the antibody is conjugated to said at The aqueous solution of cell- binding agent can be buff least one growth inhibitory agent by a linking group , for e red with buffers such as, e . g . potassium phosphate , acetate , instance by SNPP. citrate or N -2 -Hydroxyethylpiperazine -N - 2 -ethanesulfonic Accordingly, in one embodiment the conjugate may an acid (Hepes buffer ) . The buffer depends upon the nature of antibody -SNPP - ( 2 ,2 ' E , 11aS , 11a' S ) -8 , 8' - ( (( 4 - (2 -( 2 -( 2 - ( (2 - 65 the cell- binding agent. The cytotoxic compound is in solu mercapto - 2 -methylpropyl ) (methyl ) amino )ethoxy )ethoxy ) tion in an organic polar solvent, e . g . dimethyl sulfoxide ethoxy )pyridine -2 ,6 - diyl) . (DMSO ) or dimethylacetamide (DMA ). US 9 ,809 , 653 B2 23 24 between molecules . A method of DAR calculation may be derived 20° C . and 40° C . The reaction time can vary from 1 to 24 from Antony S . Dimitrov (ed ) , LLC , 2009, Therapeutic hours . The reaction between the cell- binding agent and the Antibodies and Protocols , vol 525 , 445 , Springer Science : cytotoxic agent can be monitored by size exclusion chro The absorbances for the conjugate at îd ( and ) and at 280 matography (SEC ) with a refractometric and / or UV detector. 5 nm (ango ) are measured using a classic spectrophotometer If the conjugate yield is too low , the reaction time can be apparatus (allowing to calculate the “ DAR parameter ” ) . The extended . absorbances can be expressed as follows: A number of different chromatography methods can be used by the person skilled in the art in order to perform the And =( CDXEDAD ) +( C4x6AD ) separation of step ( ii ) : the conjugate can be purified e . g . by 10 SEC , adsorption chromatography ( such as ion exchange A280 =( CDX€D280 )+ (CAX€ 4280 ) chromatography, IEC ) , hydrophobic interaction chromatog wherein : raphy (HIC ) , affinity chromatography , mixed -support chro cp and ca are respectively the concentrations in the solu matography such as hydroxyapatite chromatography, or high tion of the drug and of the antibody performance liquid chromatography (HPLC ) . Purification 15 Edad and ED280 are respectively the molar extinction by dialysis or diafiltration can also be used . coefficients of the drug at hp and 280 nm As used herein , the term “ aggregates” means the associa eard and € 4280 are respectively the molar extinction coef tions which can be formed between two or more cell -binding ficients of the antibody at A ) and 280 nm . agents , said agents being modified or not by conjugation . Resolution of these two equations with two unknowns The aggregates can be formed under the influence of a great 20 leads to the following equations: number of parameters , such as a high concentration of CD = [ (€1280XAND ) - (EANDXA280 ) ]/ [( EDXDX€ 4280) – cell -binding agent in the solution , the pH of the solution , (EADXED280 ) ] high shearing forces, the number of bonded dimers and their hydrophobic character , the temperature (see Wang , L . and CA= [ 4280 - ( CoXED280) ]/ € A280 Gosh , R ., 2008 , J . Membr Sci. 318 : 311 - 316 , and references 25 The average DAR is then calculated from the ratio of the cited therein ) ; note that the relative influence of some of drug concentration to that of the antibody : DAR = JCA these parameters is not clearly established . In the case of In the immunoconjugate according to the invention, the proteins and antibodies , the person skilled in the art will antibody is in particular specific for human and Macaca refer to Cromwell , M . E . et al. ( 2006 , AAPS Journal 8 ( 3 ) : fascicularis LAMP1 proteins . E572 -E579 ). The content in aggregates can be determined 30 The antibody is in particular a chimeric or humanised with techniques well known to the skilled person , such as antibody . The antibody may also be an antibody fragment, or SEC (see Walter et al. , 1993 , Anal. Biochem ., 212 ( 2 ) : a bispecific or multispecific antibody. 469 -480 ). Antibodies binding specifically to human and Macaca After step ( i ) or ( ii ) , the conjugate - containing solution can fascicularis LAMP1 proteins which are particularly contem be submitted to an additional step ( iii ) of chromatography, 35 plated to be included in the immunoconjugates of the ultrafiltration and /or diafiltration . invention are described in further details in the following The conjugate is recovered at the end of these steps in an “ Antibodies ” section . aqueous solution . According to an embodiment , the conjugate according to Antibodies the invention is characterised by a " drug - to -antibody ratio " 40 (or “ DAR ” ) as measured by DAR UV ranging from 1 to 10 , The inventors identified four antibodies ( the so -called for instance from 2 to 5 , in particular from 3 to 4 . This is antibodies “ MAbl ” , “ MAB2 ” , “ MAb3 ” and “ MA64 ” ) that generally the case of conjugates including maytansinoid bind specifically to human LAMP1 and distinguish tumoral molecules . from non - tumoral tissues . The antibodies MAb1, MAD2 , This DAR number can vary with the nature of the 45 MAb3 allowed for the first time to detect extracellularly antibody and of the drug ( i . e . the growth - inhibitory agent) expressed LAMP1 and thus to perform IHC analysis on used along with the experimental conditions used for the Frozen -OCT ( from Optimal Cutting Temperature ) speci conjugation ( like the ratio growth - inhibitory agent/ antibody, mens and AFA ( Alcohol Formalin Acetic acid Fixative ) to the reaction time, the nature of the solvent and of the distinguish cancerous from non - cancerous tissue . cosolvent if any ) . Thus the contact between the antibody and 50 However, IHC analysis of tumor tissues from biobanks or the growth - inhibitory agent leads to a mixture comprising from hospitals before or during patient treatment is routinely several conjugates differing from one another by different done with formalin - fixed paraffin -embedded (FFPE ) drug - to -antibody ratios ; optionally the naked antibody ; samples . Although MAB1, MAb2 and MAb3 allow LAMP1 optionally aggregates . The DAR that is determined is thus a membrane reinforcement in frozen -OCT and AFA ( Alcohol mean value . 55 Formalin Acetic acid Fixative ) sample format, they can not A method which can be used to determine the DAR , lead to the detection of LAMP1 reinforcement in FFPE herein called DAR UV, consists in measuring spectropho - format. One of the reasons is probably the effect of the tometrically the ratio of the absorbance at of a solution of formalin fixative combined to the complexity of the protein . substantially purified conjugate at ?y, and 280 nm . 280 nm The inventors discovered that allowed the produc is a wavelength generally used for measuring protein con - 60 tion of a monoclonal antibody M?b4 that can be further centration , such as antibody concentration . The wavelength more used for IHC experiments on the FFPE the format and ay is selected so as to allow discriminating the drug from the thus allows the application of the herein presented methods antibody, i. e . as readily known to the skilled person , ay is a on FFPE tumor biobanks and FFPE hospital samples . wavelength at which the drug has a high absorbance and ap Those four antibodies showed a high binding affinity is sufficiently remote from 280 nm to avoid substantial 65 (within the nanomolar range ) to cell surface expressed overlap in the absorbance peaks of the drug and antibody . an LAMP1 in cancer cells . Furthermore , at least the anti may be selected as being 252 nm in the case of maytansinoid LAMP1 MADI , MAb2 and MAb3 antibodies showed a high US 9 ,809 , 653 B2 25 26 capacity to trigger internalization of the LAMP1/ anti positions 97 to 111 (SEQ ID NO : 11 ), and FR4 - H spans LAMP1 antibody complex , as shown in example 4 . 4 . amino acid positions 112 to 122 , and a variable domain of light chain consisting of sequence Additionally , the four antibodies are cross - reactive with NIVLTQSPVSLAVSLGQRATISCRASESVDINGNTFM Macaca fascicularis LAMP1 but do not display any cross 5 HWYQQKPGQSPKLVIYAASNIESGV PARF reactivity with human LAMP2 protein . SGSGSSTDFTÈTIDPVEADDVATYYCQQFNIEDPWTF The binding sites of antibodies MAb1, MAb2 and MAb3 GGGTKVEIK (SEQ ID NO : 12 , with CDRs shown in bold characters ) have been mapped to a domain consisting of the first to third in which FR1 - L spans amino acid positions 1 to 26 , CDR1 -L loops of human and Macaca fascicularis LAMP1 proteins, spans amino acid positions 27 to 36 (SEQ ID NO : 13 ) , in particular to the first lumenal domain of human LAMP1. 10 FR2- L spans amino acid positions 37 to 53 , CDR2 - L spans More specifically the binding site of MAbl was mapped in amino acid positions 54 to 56 , FR3 - L spans amino acid loops 1 - 2 and the binding site of MAb2 and MAb3 was positions 57 to 92 , CDR3- L spans amino acid positions 93 mapped in loop1. The antibodies MAb1 and MAD2 do not to 101 (SEQ ID NO : 14 ), and FR4 - H spans amino acid compete with each other for binding to human LAMP1 . positions 102 to 111. Therefore at least two epitopes on LAMP1 have been found 15 A variant of antibody MAB2 , called herein “MAB2 " to interact with the antibodies of the invention . was also generated by introducing canonical residues by substitution of A116T in the variable domain of the heavy The binding site of Antibody M?b4 has been mapped to chain and by substitution of V9A , V51L , 158L , S72G and a domain consisting of the third to fourth loop of human and A108T in the variable domain of the light chain . Macaca fascicularis LAMP1 proteins , in particular to the 20 The so - called " antibody MAD2 " comprises : fourth loop of human LAMP1. More specifically the anti - a variable domain of heavy chain consisting of sequence body M?b4 binds to a region of Loop 4 comprising the OVOLOOSAAELARPGASVKMSCKASGYTFTSYT amino acids 360 to 375 of human LAMP1 that consists of MHWVKORPGOGLEWIGYFNPS SGYPEYNOKFKDK sequences SEQ ID NO : 82 . TTLTADKSSNTAFIQLNSLTSEDSAVYYCSRGYYYG The inventors have determined the sequence of variable 25 SRGYALDFWGQ GTSVTVSS (SEQ ID NO : 15 ). a variable domain of light chain consisting of sequence heavy and light chains of these monoclonal antibodies which NIVLTOSPASLAVSLGQRATISCRASESVDINGNTF are directed against the human and Macaca fascicularis MHWYQQKPGQSPKLLIYAASN LESGVPARFSGSGS LAMP1 proteins . GTDFTFTIDPVEADDVATYYCOONIEDPWTFGGGT The so - called antibody “MAbl ” comprises : KLEIK (SEQ ID NO : 16 ) . a variable domain of heavy chain consisting of sequence Both “MAb2 . can " and “MAb2 ” , under chimeric form , have the same affinity for human LAMP1 ( see Table 13 ). QVQLQQSGAELVKPGASVKMSCKASGYIFTNYNIH The so - called antibody “MAb3 ” comprises : WVKKSPGOGLEWIGAIYPGNGDAPY SOKFKDKATL a variable domain of heavy chain consisting of sequence TADKSSSTTYMOLSRLTSEDSAVYYCVRANWDVA QIQLVOSGPELKKPGETVKISCKASGYIFTNYGMN FAYWGOGTLVSVSA (SEQ ID NO : 1 , with CDRs shown 35 WVKOAPGKGLKWMGWINTYTGESRY ADDEKGR in bold characters ) in which FR1 - H spans amino acid FALSLETSASTAYLOINNLENEDMATYFCAREDYYG positions 1 to , 25 , CDR1- H spans amino acid positions 26 to NSPWFFDVWGAGTTVTV SS 33 (SEQ ID NO : 2 ) , FR2 - H spans amino acid positions 34 ( SEQ ID NO : 42 , with CDRs shown in bold characters ) to 50 , CDR2 - H spans amino acid positions 51 to 58 ( SEQ ID in which FR1- H spans amino acid positions 1 to 25 , NO : 3 ) , FR3- H spans amino acid positions 59 to 96 , 40 CDR1- H spans amino acid positions 26 to 33 (SEQ ID NO : CDR3- H spans amino acid positions 97 to 107 ( SEQ ID NO : 43 ) , FR2 - H spans amino acid positions 34 to 50 , CDR2 - H 4 ) , and FR4 - H spans amino acid positions 108 to 118 , and spans amino acid positions 51 to 58 (SEQ ID NO : 44 ) , a variable domain of light chain consisting of sequence FR3- H spans amino acid positions 59 to 96 , CDR3 - H spans DIQMTOSPPSLSASLGGKVTITCKASODIDRYMAWY amino acid positions 97 to 111 ( SEQ ID NO : 45 ) , and FR4- H ODKPGKGPRLLIHDTSTLQPGIPSRF SGSGSGRDYSF - 4543 spans amino acid positions 112 to 122, and SISNLEPEDIATYYCLQYDNLWTFGGGTKLEIK a variable domain of light chain consisting of sequence DIQDIQMTQTTSSLSASLGDRVTISCNASQGINKYLNWY (SEQ ID NO : 5 , with CDRs shown in bold characters ) in OOKPDGTVKLLIYYTSTLHSGVPSRF SGSGSGTDYS which FR1 - L spans amino acid positions 1 to 26 , CDR1 -L LTINNLEPEDIATYYCOOYTKLPFTFGSGTKLEIK spans amino acid positions 27 to 32 ( SEQ ID NO : 6 ) , FR2 - L 50 (SEO ID NO : 46 , with CDRs shown in bold characters ) spans amino acid positions 33 to 49 , CDR2- L spans amino in which FR1- L spans amino acid positions 1 to 26 , CDR1- L acid positions 50 to 52, FR3 - L spans amino acid positions 53 spans amino acid positions 27 to 32 (SEQ ID NO : 47) , to 88 , CDR3- L spans amino acid positions 89 to 96 (SEQ ID FR2- L spans amino acid positions 33 to 49 , CDR2- L spans NO : 7 ) , and FR4 - H spans amino acid positions 97 to 106 . amino acid positions 50 to 52 , FR3 - L spans amino acid The so -called antibody “MAB2 ” comprises: 55 positions 53 to 88 , CDR3- L spans amino acid positions 89 to 97 ( SEQ ID NO : 48 ), and FR4- H spans amino acid a variable domain of heavy chain consisting of sequence positions 98 to 107 . QVQLQQSAAELARPGASVKMSCKASGYTFTSYTMH A variant of MAb3 (“ MAB3 VL _ R24 _ R93 " ) was gener WVKORPGQGLEWIGYFNPSSGYPE YNQKFKDKTTL ated by introducing into VL sequence of MAb3 the follow TADKSSNTAFIQLNSLTSEDSAVYYCSRGYYYGSRG - 60 ing amino acid substitutions: N24R and K93R . Accordingly . YALDFWGQGASVT VSS the variable domain of light chain of MAb3 VL _ R24 _ R93 (SEQ ID NO : 8 , with CDRs shown in bold characters ) in consist of which FR1 -H spans amino acid positions 1 to 25 , CDR1- H DIQMTQTTSSLSASLGDRVTISCRASQGINKYLN spans amino acid positions 26 to 33 (SEQ ID NO : 9 ) , FR2 - H WYQQKPDGTVKLLIYYTSTLHSG VPSRFSGSGSGT spans amino acid positions 34 to 50 , CDR2 - H spans amino 65 DYSLTINNLEPEDIATYYCQQYTRLPFTFGSGTKLEIK acid positions 51 to 58 (SEQ ID NO : 10 ), FR3- H spans (SEQ ID NO : 51 ) ( the mutated residues as compared with amino acid positions 59 to 96 , CDR3- H spans amino acid VL of MAb3 being shown in enlarged characters ). US 9 ,809 , 653 B2 27 28 CDR3- L of MAb3 VL _ R24 _R93 thus consists of lysine residue in the case of ADC . N -linked glycosylation QQYTRLPFT (SEQ ID NO : 52 ) . occurs by attachment of an oligosaccharide chain to an The so called “ MAB4 ” comprises : asparagine residue in the tripeptide sequence Asn - X -Ser or a variable domain of heavy chain consisting of sequence Asn - X - Thr, where X may be any amino acid except Pro . QVQLQQSGAELVRPGTSVKVSCKASGYAFT 5 Removal of an N - glycosylation site may be achieved by NYLIEVWVKQRPGQGLEWIGVINPGS GGTNYNEK - mutating either the Asn or the Ser / Thr residue to a different FKGKATLTADKSSSTAYMOLSSLTSDDSAVYF residue , in particular by way of conservative substitution . CARYRSYDWYFDVWGAGTT VTVSS (SEQ ID NO : 88 , Deamidation of asparagine and glutamine residues can occur with CDRs shown in bold characters ) in which FR1- H spans depending on factors such as pH and surface exposure . amino acid positions 1 to 25 , CDR1- H spans amino acid 10 Asparagine residues are particularly susceptible to deami positions 26 to 33 (SEQ ID NO : 83 ) , FR2 - H spans amino d ation , primarily when present in the sequence Asn - Gly , and acid positions 34 to 50 , CDR2- H spans amino acid positions to a lesser extent in other dipeptide sequences such as 51 to 58 (SEQ ID NO : 84 ) , FR3 - H spans amino acid Asn - Ala . When such a deamidation site , in particular Asn positions 59 to 96 , CDR3 - H spans amino acid positions 97 Gly, is present in a CDR sequence , it may therefore be to 108 ( SEQ ID NO : 85 ) , and FR4 - H spans amino acid 15 desirable to remove the site , typically by conservative sub positions 109 to 119 , and stitution to remove one of the implicated residues . In the a variable domain of light chain consisting of sequence case of ADC , attachment of a cytotoxic to mAb could be DIQMTQSPASLSASVGETVTITCRVSGNIH prepared via covalent linkage to lysine side chain residue . NYLAWYQQKQGKSPQLLVYNAKTLAD GVPSRF This steric hindrance may interfere with mAb binding to SGSGSGTOYSLKINSLOPEDFGSYYCQHFWSNPYTF - 20 antigen . It may therefore be desirable to remove the lysine GGGTKLEIK (SEQ ID NO : 89 , with CDRs shown in bold residue , typically by an arginine conservative substitution . characters ) in which FR1 - L spans amino acid positions 1 to Substitution in a CDR sequence to remove one of the 26 , CDR1- L spans amino acid positions 27 to 32 (SEQ ID implicated residues is also intended to be encompassed by NO : 86 ) , FR2 - L spans amino acid positions 33 to 49 , the present invention . The inventors further generated CDR2 - L spans amino acid positions 50 to 52 , FR3 - L spans 25 humanized antibodies “ huMAb1 _ 1 ” , “ huMAb1_ 2 " , amino acid positions 53 to 88 , CDR3- L spans amino acid " huMAb1_ 3 ” based on CDR grafting and / or on Molecular positions 89 to 97 (SEQ ID NO : 87 ), and FR4- H spans Dynamic Trajectories (4D humanization protocol) as amino acid positions 98 to 107 . described in example 7 . 2 . 1 and herein below . The antibody may also be a humanised antibody or a Accordingly , in one embodiment, the anti- LAMP1 anti fragment of a humanised antibody . For example , the anti - 30 bodies in context of the invention are humanized anti body of the invention may result from humanisation of any LAMP1 antibodies obtained through CDR grafting and /or of the antibodies defined above. based on Molecular Dynamic Trajectories (4D humanization Numerous methods for humanisation of an antibody protocol) . sequence are known in the art ; see e . g . the review by Accordingly, in an embodiment, the humanized anti Almagro & Fransson ( 2008 ) Front Biosci . 13 : 1619 - 1633 . 35 LAMP1 antibody “ humAb1 _ 1 " comprises : One commonly used method is CDR grafting , or antibody the variable domain (VHL ) of heavy chain chain consist reshaping , which involves grafting of the CDR sequences of ing of sequence a donor antibody , generally a mouse antibody , into the QVOLVOSGAEVKKPGSSVKVSCKASGYIFTNYNI framework scaffold of a human antibody of different speci- HWVKKSPGQGLEWIGAIYPGNG DAPYSQKFQG ficity . Since CDR grafting may reduce the binding specific - 40 KATLTADTSTSTTYMELSSLRSEDTAVYYCVRANWD ity and affinity , and thus the biological activity of the parent VAFAYWGQGTLVTV SS (SEQ ID NO : 53 ) and antibody , back mutations may be introduced at selected the variable domain (VL1 ) of light chain of huMAb1 _ 1 positions of the CDR grafted antibody in order to retain the consisting of sequence binding specificity and affinity of the parent antibody . Iden - DIQMTQSPSSLSASVGDRVTITCKASQDIDRY tification of positions for possible back mutations can be 45 MAWYODKPGKAPRLLIHDTSTLOS GVPSRFSGSGS performed using information available in the literature and GRDYTLTISNLEPEDFATYYCLQYDNLWTFGGGTK in antibody databases . An alternative humanization tech - VEIK (SEQ ID NO : 56 ) . nique to CDR grafting and back mutation is resurfacing , in The humanized antibody “ huMAb1 _ 2 " comprises: which non - surface exposed residues of non - human origin a variable domain (VH2 ) of heavy chain consisting of are retained , while surface residues are altered to human 50 sequence residues . Another alternative technique is known as “ guided QVQLVOSGAELVKPGASVKMSCKASGYIFTNYNI selection ” (Jespers , L . S . et al ., 1994 , Biotechnology 12 ( 9 ) : HWVKKSPGQGLEWIGAIYPGNG DAPYSQK 899 - 993 ) and can be used to derive from a murine antibody FODRATLTADTSSSTTYMELSSLTSEDSAVYYCVRAN a fully human antibody conserving the epitope and binding WDVAFAYWGQGTLVS VSS ( SEQ ID NO : 54 ) , and characteristics of the parental antibody . The technique of 55 a variable domain of light chain (VL2 ) consisting of humanization based on molecular dynamic calculations as sequence disclosed in the application WO2009 /032661 may be used . DIQMTQSPPSLSASVGGKVTITCKASODIDRY Thus in one embodiment humanized antibodies may also be MAWYQDKPGKGPKLLIHDTSTLQP GIPSRFSGSGS called “ resurfaced ” antibodies . GRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGT For chimeric antibodies , humanisation typically involves 60 KLEIK (SEQ ID NO : 57 ) modification of the framework regions of the variable region The humanized antibody “ huMAb1 _ 3 ” comprises : sequences . a variable domain (VH3 ) of heavy chain consisting of Amino acid residues that are part of a CDR will typically sequence not be altered in connection with humanisation , although in OVOLVOSGAELVKPGASVKMSCKASGYIFTNYNI certain cases it may be desirable to alter individual CDR 65 HWVRQAPGQGLEWIGAIYPGN GDAPYAQK amino acid residues , for example to remove a glycosylation FQGRATLTADTSSSTTYMELSSLTSEDTAVYYCVRAN site , a deamidation site , an undesired cysteine residue , a WDVAFAYWGQGTLV TVSS (SEQ ID NO : 55 ) , and US 9 ,809 , 653 B2 29 30 a variable domain of light chain (VL3 ) consisting of In one embodiment, the antibody according to the inven sequence tion has an affinity ( EC ) for full length human and cyno DIQMTOSPSSLSASVGGKVTITCKASODIDRY molgus monkey LAMP1 expressed at the cell surface of a MAWYQQKPGKGPKLLIHDTSTLQP GVPSRFSGSGS recombinant cell line , wherein the cell line may be HCT116 GRDYSLTISSLEPEDIATYYCLQYDNLWTFGGGT- 5 and the apparent affinity measured via Flow Cytometry is KLEIK (SEQ ID NO : 58 ) 520 nM , in particular s10 nM , s8 nM or s7 nM . The invention relates to an antibody which binds specifi In one example , the antibody according to the invention cally to human and Macaca fascicularis LAMP1 proteins. has an affinity (EC50 ) for cynomolgus monkey LAMP1 “ Affinity ” is defined , in theory , by the equilibrium asso expressed at the cell surface of a recombinant cell line , ciation between the whole antibody and the antigen . It can 10 wherein the cell line may be HEK293 and the apparent be experimentally assessed by a variety of known methods, affinity measured via Flow Cytometry is 550 nM , for such as measuring association and dissociation rates with example s40 nM or s35 nM . surface plasmon resonance or measuring the EC50 in an In another example , the antibody according to the inven immunochemical assay (ELISA , FACS ) . Enzyme- linked tion has a KD for full purified human LAMP1 ( SEQ ID NO immunosorbent assay ( ELISA ) is a biochemistry assay that 15 28 ) expressed in HEK293 cells measured via surface plas uses a solid - phase enzyme immunoassay to detect the pres - mon resonance ( SPR ) is < 70 nM , for example s60 nM , 550 ence of a substance , usually an antigen , in a liquid sample or nM , < 40 nM , s30 nM , s20 nM or s10 nM . wet sample . Antigens from the sample are attached to a The use of surface plasmon resonance to determine is surface . Then , a further specific antibody is applied over the known to the skilled in the art . In one example the binding surface so it can bind to the antigen . This antibody is linked 20 kinetics of for example the murine , chimer or humanized to an enzyme, and , in the final step , a substance containing anti -LAMP1 mAbs were determined by surface plasmon the enzyme' s substrate is added . The subsequent reaction resonance assay using typically a BIAcore 2000 (BIAcore produces a detectable signal, most commonly a color change Inc . , Uppsala , N . J . ) . Therefore , for example a CM5 BIAcore in the substrate . Fluorescence -activated cell sorting (FACS ) biosensor chip was docked into the instrument and activated provides a method for sorting a heterogeneous mixture of 25 with for example 70 uL of 1 : 1 NHS / EDC at room tempera biological cells into two or more containers , one cell at a ture . Typically , a mouse anti - ahuman Fc IgG1 (BIAcore time, based upon the specific light scattering and fluorescent # BR - 1008 - 39 ) and rabbit anti- amurine Fc IgG1 (BIAcore characteristics of each cell. In these assays , the EC50 is the # BR - 1008 - 38 ) ( 50 ug/ mL in 1 M acetate buffer , pH5 ) were concentration of the antibody which induces a response immobilized on the activated chips in all flow cells . The halfway between the baseline and maximum after some 30 immobilization was carried out at a flow rate of for example specified exposure time on a defined concentration of anti - 10 uL /min up to saturation . The chip was then blocked by for gen by ELISA ( enzyme- linked immuno - sorbent assay ) or example injection of 70 uL of ethanolamine -HCl , pH 8 . 5 , cell expressing the antigen by FACS ( Fluorescence Acti - followed by one wash with 3 M MgCl, for anti - ahuman Fc vated Cell Sorting ) . IgG1 and one wash with 10 mM Glycine -HC1 pH 1 . 7 for A monoclonal antibody binding to antigen 1 (Agl ) is 35 anti -amurine Fc IgGl. To measure the binding of for " cross- reactive” to antigen 2 (Ag2 ) when the ECsos are in a example anti- LAMP1 mAbs to LAMP1 , antibodies were similar range for both antigens. In the present application , a used at 1 - 5 ug/ mL in BIAcore running buffer (HBS -EP ) . The monoclonal antibody binding to Agl is cross - reactive to Ag2 antigen for example (Sequence ID No 28 protein produced when the ratio of affinity of Ag2 to affinity of Agl is equal as described in example 6 . 2 ) was injected from for example or less than 10 ( in particular 5 , 2 , 1 or 0 . 5 ) , affinities being 40 1 to 256 nM . Following completion of the injection phase , measured with the same method for both antigens . dissociation was monitored in a BIAcore running buffer at A monoclonal antibody binding to Agl is “ not signifi- the same flow rate for typically 600 sec . The surface was cantly cross - reactive ” to Ag2 when the affinities are very typically regenerated between injections using for example different for the two antigens. Affinity for Ag2 may not be 2x5 uL 3 M MgCl2 (2x30 s ) or anti- ahuman Fc IgG1 and measurable if the binding response is too low . In the present 45 1x30 UL 10 mM Glycine -HC1 pH 1. 7 for anti -amurine Fc application , a monoclonal antibody binding to Agl is not IgG1 ( 180 s ) . Individual sensorgrams were typically ana significantly cross - reactive to Ag2 , when the binding lyzed using BIAevaluation software . response of the monoclonal antibody to Ag2 is less than 5 % Thus, the polypeptide according to the invention may be of the binding response of the same monoclonal antibody to used in toxicological studies performed in monkeys , Agl in the same experimental setting and at the same 50 wherein the toxicity profile obtained from those studies is antibody concentration . In practice , the antibody concentra - relevant to anticipate potential adverse effects in humans . tion used can be the EC o or the concentration required to Alternatively , or furthermore , the antibody according to reach the saturation plateau obtained with Agl. the invention has an affinity ( EC50 ) for LAMP1 expressed on A monoclonal antibody “ binds specifically ” to Agl when the surface of advanced human primary colon tumor it is not significantly cross - reactive to Ag2 . 55 CR - IGR - 034P and measured via Flow Cytometry is s50 The antibody according to the invention binds specifically nM , s40 nM , in particular s30 nM , 520 nM or s5 nM . to human and Macaca fascicularis LAMP1 proteins . It does Antibody binding capacity or ABC is the quantification of not significantly cross - react with human LAMP2 (SEQ ID cell surface antigen . ABC can be measured using QIFIKIT® NO : 40 ) . (Registered trademark of BIOCYTEX ) . The antibodies In one embodiment, the antibody according to the inven - 60 according to the invention have a high ABC on many Patient tion has an affinity ( EC ) for human and / or cynomolgus derived Xenografts of different origin ( 220 . 000 , in particular monkey LAMP1 expressed at the cell surface of a recom - 250 .000 , 2100 .000 , 2150 .000 ABC ) and tumor cell lines , in binant cell line , wherein the cell line may be HEK293 and /or particular colon tumor cells such as Colo205, SW480 or HCT116 and the apparent affinity measured via Flow LS174T ( 21 . 500 , 22 . 500 , 24 . 000 ABC ) . Cytometry is s70 nM , for example s60 nM , 550 nM , s45 65 Alternatively , or furthermore , the antibody according to nM , s40 nM , 535 nM , 530 nM , s25 nM , 520 nM , s15 nM the invention has the ability to internalize and recycle or s10 nM . LAMP1 to the cell membrane . In particular, when bound by US 9 ,809 , 653 B2 32 an antibody according to the invention , a molecule of Quantification of internalization can also be performed by LAMP1 at the membrane of cancer cell has the capacity fluorescence -based quenching methods. In particular, a fluo undergo at least 1 , 4 , 7 or 9 recyclings at the cell membrane. rescence -based Alexa488 -quenching method has been In other words, one molecule of LAMP1 expressed at the described to analyze internalisation of targeting agents surface of a cancer cell can be bound by , and therefore 5 (Frejd et al. 2010 , International Journal of Oncology , 36 : internalize , at least 2 , 5 , 8 or 10 molecules of antibody 757 - 763 ). According to said description , internalization is according to the invention . Still in other words , according to calculated as the Mean fluorescence intensity (MFI ) value of an embodiment, at least 2 , 5 , 8 or 10 molecules of antibody quenched cells ( intracellular compartments only ) divided by according to the invention are internalized by one molecule the MFI value of unquenched cells (both cell surface and of LAMP1 expressed at the surface of a cancer cell . 10 intracellular compartments ) at 37° C . , according to the Internalization may be assayed for instance by determin - following formula : ing an internalization score or by a fluorescence -based quenching method . FL of quenched cells at 37° C . The internalization score ( IS ) is defined as a ratio of the 15 Percentage of iternalized fraction - X100 fluorescence intensity inside the cell to the intensity of the 15 - FL of unquenched cells at 37° C . * entire cell . It may be measured as described by using the imaging flow cytemeter ImageStream * ( from the supplier Cells incubated with Alexa488 - labelled compounds at 4° Amnis® Corporation , 2505 Third Avenue , Suite 210 , C . are used as a control since internalization of antibodies Seattle , Wash . 98121 - 1480 , on the world wide web at 20 does not take place at 4° C . amnis . com ) . The higher the score , the greater the fluores - The inventors showed that after quenching , the total cence intensity is inside the cell . As described by Amnis (see fluorescence of Alexa488 -MAb1 measured from cells the world wide web at amnis . com ), the inside of the cell is labelled at 37° C . (both cell surface and intracellular com defined by an erosion of a mask that fits the membrane of the partments ) was 10 - fold higher than the fluorescence of cells cell . The score is invariant to cell size and can accommodate 35 labelled at 40 C ( cell surface ) after 4 h ( example 4 . 4 ) . concentrated bright regions and small dim spots . The ratio is Accordingly , these results also indicate that the LAMP1 mapped to a logarithmic scale to increase the dynamicnamic rangerange protein is quickly and repeatedly recycling at cell mem to values between { - inf, inf } . The thickness of the mem brane . brane (in pixels ) determines which pixels are used to define Thus, the inventors showed for the first time that LAMP1 the boundary and the membrane portions of the cell . The 30 can function as a receptor mediating the internalization of user supplies an ‘ internal mask based on the brightfield antibodies and suggest that availability of specific internal image that covers the inside of the cell, the thickness of the izing antibodies should aid in developing novel therapeutic membrane in pixels and the fluorescent channel of interest . methods to target toxins, drugs or short- range isotopes to be The cell is divided into 2 regions: External ( B ) and internal delivered specifically to the interior of the cancer cells . ( 1 ) . The user supplies the internal region as the mask . The 35 Furthermore the inventors could show that the results external region is determined by : 1 . Dilating the internal from example 4 . 4 taken together indicate that each LAMP1 mask by the membrane thickness . 2 . Combining 1 with the molecule is involved in several ( at least up to 10 on average ) object mask of the channel of interest. 3 . External region internalization cycles via recycling at cell membrane during equals mask 2 and not the internal mask . Next, the mean the course of the experiment. intensity of the upper quartile of the pixels in each region is 40 The antibody of the invention binds specifically to a determined . The Internalization Score ( IS ) is then computed domain consisting in particular of the first to third loops of as follows: human and Macaca fascicularis LAMP1 proteins. The domain consisting of the first to third loops of human LAMP1 protein is defined by the amino acids Ala29 to IS = log a ) ,where a = - mi Pi 45 Ile309 of SEQ ID NO : 24 , and the domain consisting of the 1 - 0 ) m ; + mB PB first to third loops ofMacaca fascicularis LAMP1 protein is defined by the amino acids Ala27 to Thr307 of SEQ ID NO : ml =Mean intensity of upper quartile pixels in I, 39. According to an embodiment , the antibody of the inven mB = Mean intensity of upper quartile pixels in B , tion binds specifically to the first lumenal domain of human PI = Peak intensity of upper quartile pixels in I , pB = Peak 50 and Macaca fascicularis LAMP1 proteins. intensity of upper quartile pixels in B . The first lumenal domain of human LAMP1 is defined by In the case of transferrin , (Williams A . et al. , 1996 , the amino acids at positions Ala29 to Arg195 of SEO ID NO : Biomembranes, 4 : 255 - 287) the authors have obtained an IS 24, and the first lumenal domain of Macaca fascicularis of 0 when the cells were left on ice and an IS of 0 . 9 when LAMP1 protein is defined by the amino acids at positions the cells were incubated at 37° C . for one hour. 55 Ala27 to Arg193 of SEQ ID NO : 39. More specifically , the For the antibodies of the invention , the inventors have antibody can bind to the human and Macaca fascicularis shown that the internalization scores (IS ) at 37° C . were first lumenal domain indifferently whether expressed as a 10 - fold higher than at 4° C . Since internalization of anti - soluble extracellular domain ( e . g . amino acids Ala29 bodies does not take place at 4° C . , the internalization scores Met382 for human LAMP1 ( SEQ ID NO : 24 ) or Ala27 obtained at 4° C . reflect the density of LAMP1 molecules at 60 Met380 for Macaca fascicularis LAMP1 (SEQ ID NO : 39 ) ) , the cell surface . A 10 - fold higher value of the IS parameter or as a membrane - anchored full- length LAMP1 protein at 37° C . than at 4° C . therefore means that the LAMP1 recombinantly expressed at the surface of a cell line , for protein is quickly and repeatedly recycling at the cell instance HT29, Colo205 and HCT116 , HEK293 cell line . membrane . In other words the antibodies according to the The inventors demonstrated that MAb1 binds to the amino invention have a very high internalization capacity , much 65 acids 101 to 195 of SEQ ID NO : 24 corresponding to Loop higher than the capacity calculated from the density of the 2 of human LAMP1 , for example to the amino acids 101 to LAMP1 protein at the cell surface . 110 (SEQ ID NO : 72 ) , 144 to 157 (SEQ ID NO :73 ) and 174 US 9 ,809 , 653 B2 33 34 to 188 (SEQ ID NO : 74 ) of SEQ ID NO : 24 as herein the amino acids 1149, D150 and R186 of SEQ ID NO : 24 , described in example 4 .8 . It has been further identified by or crystallography, that the binding site of MAbl further the amino acids G38 and D70 of SEQ ID NO : 24 , or encompasses the amino acids Asn35 , Cys80 , Gly 81, Glu83 , The invention also provides for an antibody which com Asn84 located in loop 1 of SEQ ID NO : 24 . 5 petes for binding to a domain consisting of the first to third Accordingly , MAbl also binds to the amino acids at loops of human and Macaca fascicularis LAMP1 proteins positions 29 to 100 of SEQ ID NO : 24 corresponding to with an antibody selected from the group consisting of the so -called antibodies Mab1, Mab2 , MAb2can ,MAD3 ,MAb3 Loop 1 of human LAMP1 , for example to a region that VL _ R24 _ R93 , huMAb1 _ 1 and huMAb1_ 2 , huMAb1 _ 3 i . e . : consists of the amino acids at positions 35 to 84 of SEQ ID f 10 ( i) an antibody comprising a variable domain of heavy NO : 24 ( SEQ ID NO : 97 ) , or to two regions that consists of chain of sequence SEQ ID NO : 1 and /or a variable Asn35 of SEQ ID NO : 24 and amino acids at positions 80 - 84 domain of light chain of sequence of sequence SEQ ID of SEQ ID NO : 24 . NO : 5 ; or Furthermore both MAB2 and MAb3 bind to the amino ( ii ) an antibody comprising a variable domain of heavy acids 29 to 100 (SEQ ID NO : 77 ) of SEQ ID NO : 24 15 chain of sequence SEQ ID NO : 8 and / or a variable corresponding to Loop 1 of human LAMP1 , for instance domain of light chain of sequence of sequence SEQ ID MAb2 and MAb3 both bind to the the amino acids 29 to 41 NO : 12 ; or ( SEQ ID NO : 75 ) and 68 to 80 (SEQ ID NO : 76 ) of SEQ ID ( iii ) an antibody comprising a variable domain of heavy NO : 24 . chain of sequence SEQ ID NO : 15 and/ or a variable In a further antibody, the antibody of the invention binds 20 domain of light chain of sequence of sequence SEQ ID specifically to the second luminal domain of human and NO : 16 ; or Macaca fascicularis LAMP1 proteins, for instance to the ( iv ) an antibody comprising a variable domain of heavy fourth loop . chain of sequence SEQ ID NO : 42 and / or a variable The fourth loop of human LAMP1 protein consists of domain of light chain of sequence of sequence SEQ ID amino acids at positions Leu310 to Met382 of SEQ ID NO : 25 NO : 46 ; or 24 and the fourth loop of Macaca fascicularis LAMP1 ( v ) an antibody comprising a variable domain of heavy protein consists of amino acids at positions Leu 308 to chain of sequence SEQ ID NO : 42 and /or a variable Met380 of SEQ ID NO : 39 . domain of light chain of sequence of sequence SEQ ID More specifically , the antibody binds to a region of Loop NO : 51 ; or 4 comprising the amino acids 360 to 375 of human LAMP1 30 ( vi) an antibody comprising a variable domain of heavy that consists of sequences SEQ ID NO : 82. chain of sequence SEQ ID NO : 53 and /or a variable In another embodiment, the antibody of the invention domain of light chain of sequence of sequence SEQ ID binds specifically to human and Macaca fascicularis NO : 56 ; or LAMP1 proteins indifferently whether in non - glycosylated ( vii) an antibody comprising a variable domain of heavy or glycosylated form . chain of sequence SEQ ID NO : 54 and /or a variable Accordingly , in an embodiment, the invention relates to domain of light chain of sequence of sequence SEQ ID an antibody which binds to : NO : 57 ; or three regions of Loop 2 of human LAMP1 that consist of ( viii ) an antibody comprising a variable domain of heavy sequences SEQ ID NO : 72 , SEQ ID NO : 73 and SEQ ID chain of sequence SEQ ID NO : 55 and /or a variable NO : 74 , respectively , and optionally further to a region of 40 domain of light chain of sequence of sequence SEQ ID Loopl of human LAMP1 that consists of sequence (SEQ ID NO : 58 . NO : 97 ) ; or In an embodiment, said antibody competes for binding to two regions of Loop 1 of human LAMP1 that consist of the first lumenal domain of human and Macaca fascicularis sequences SEQ ID NO : 75 and SEQ ID NO : 76 , respec - LAMP1 proteins. For instance the invention provides for an tively ; or 45 antibody which competes for binding to : a region of Loop4 of human LAMP1 that consist of three regions of Loop 2 of human LAMP1 that consist of sequence SEQ ID NO : 82 . sequences SEQ ID NO : 72 , SEQ ID NO : 73 and SEO ID Furthermore , the inventors identified the residues R146 , NO : 74 , respectively ; or D150 , K152 , R106 , A108 , N181 , S182 , S183 , R186 and two regions of Loop 1 of human LAMP1 that consist of G187 of SEQ ID NO : 24 as likely to interact with MAb1 as 50 sequences SEQ ID NO :75 and SEQ ID NO : 76 , respectively , described in example 6 . 5 . They further identified the resi- with an antibody comprising a variable domain of heavy dues A29 , M30 , M32 , G36 , A40 , S69, 170 , 172 , 174 , 175 , chain and a variable domain of light chain as defined and R77 of SEQ ID NO : 24 as likely to interact with MAb2 according to i -viii ) above, as appropriate (i .e . with said three and / or MAb3. Those residues have been individually regions of Loop 2 for an antibody as defined according to i replaced by an alanine residue in the LAMP1 sequence 55 and vi- viii ) above , or with said two regions of Loop 1 for an derived from hLAMP1_ AGYQTI and encoded in plasmid antibody as defined according to ii - v ) ) . pXL5626 as described in example 6 . 6 . The inventors In one embodiment the competition is determined by use observed loss of binding to MAbl for alanine mutations at of an ELISA as described in Example 4 .8 of the specifica positions 1149 , D150 and R186 of SEQ ID NO : 24 in the tion , wherein competition is defined by a signal of less than LAMP1 protein , indicating that these positions are impor - 60 80 % of signal compared to mAb control alone as assessed by tant for MAb1 binding to LAMP1. Furthermore , loss of absorption , when the two competing antibodies are in solu binding was demonstrated for LAMP1 to MAb3 for alanine tion at similar molarity , and wherein competition is defined mutations at positions G38 and D70 of SEQ ID NO : 24 due by a signal of less than 80 % , for instance less than 70 % , to Ala substitution in LAMP1 protein indicating that these 60 % , 50 % , 40 % , 30 % , 20 % , 10 % . positions are important for MAb3 binding to LAMP1. 65 The ability of an antibody to compete for binding to a Accordingly , in an embodiment, the invention relates to domain consisting of the first to third loops , in particular to an antibody which binds to : the first lumenal domain , of human and Macaca fascicularis US 9 ,809 , 653 B2 35 36 LAMP1 proteins with an antibody comprising the variable ( ii ) a CDR1- H of sequence SEQ ID NO : 9 or a sequence heavy and light chains of an antibody selected from the differing from SEQ ID NO : 9 by one amino acid substitu group consisting of the so - called antibodies MAb1, MAD2, tion , a CDR2- H of sequence SEQ ID NO : 10 or a sequence MAD2 MAD3 , MAb3 _ VLR24 - R93 , huMAb1 _ 1 , differing from SEQ ID NO : 10 by one amino acid substi huMAb1 _ 2 and huMAb1_ 3 (hereafter a " reference ” anti - 5 tution , a CDR3- H of sequence SEQ ID NO : 11 or a sequence body ) may be readily assayed , for instance, by competitive differing from SEQ ID NO : 11 by one amino acid substi ELISA wherein the antigen ( i. e . a polypeptide comprising or tution ; consisting of a fragment of human or Macaca fascicularis and/ or LAMP1 including the first to third loops of LAMP1, or the a CDR1- L of sequence SEQ ID NO : 13 or a sequence first lumenal domain , in particular a protein containing the 10 differing from SEQ ID NO : 13 by one amino acid substi first lumenal domain of LAMP1 from human and cynomol- tution , a CDR2- L of sequence AAS or a sequence differing gus origin such as presented in example 6 .3 ) is bound to a from AAS by one amino acid substitution , and a CDR3 -L of solid support and two solutions containing the candidate sequence SEQ ID NO : 14 or a sequence differing from SEQ antibody and the reference antibody, respectively, are added ID NO : 14 by one amino acid substitution ; or and the antibodies are allowed to compete for binding to the 15 ( iii) a CDR1- H of sequence SEQ ID NO : 43 or a sequence antigen . The amount of reference antibody bound to the differing from SEQ ID NO : 43 by one amino acid substi antigen may then be measured , and compared to the amount tution , a CDR2- H of sequence SEQ ID NO : 44 or a sequence of reference antibody bound to the antigen when measured differing from SEQ ID NO : 44 by one amino acid substi against a negative control. An amount of bound reference tution , and a CDR3 - H of sequence SEQ ID NO : 45 or a antibody in presence of the candidate antibody decreased as 20 sequence differing from SEQ ID NO : 45 by one amino acid compared to the amount of bound reference antibody in substitution ; and /or presence of the negative control indicates that the candidate a CDR1 - L of sequence SEQ ID NO : 49 or a sequence antibody has competed with the reference antibody. Conve differing from SEQ ID NO : 47 by one amino acid substi niently , the reference antibody may be labeled ( e . g . fluores - tution , a CDR2 - L of sequence YTS or a sequence differing cently ) to facilitate detection of bound reference antibody . 25 from YTS by one amino acid substitution , and a CDR3 - L of Repeated measurements may be performed with serial dilu - sequence SEQ ID NO : 48 or SEQ ID NO : 52 or a sequence tions of the candidate and /or reference antibody . differing from SEQ ID NO : 48 or SEQ ID NO : 52 by one In another example binding competition between MAb1 amino acid substitution . and MAD2 or MAb3 can be typically measured between two In a further embodiment, the antibody according to the anti - LAMP1 mAbs by ELISA with recombinant human 30 invention comprises the CDR sequences of the heavy and /or LAMP1 coated on plate (as described in example 6 . 2 ) . light chains of so - called anti- LAMP1 antibody M?b4 . More Briefly , typically two mAbs were added simultaneously at specifically , the antibody can comprise the CDR sequences concentrations of for example 0 . 06 and 15 mg/ L , the con - of the heavy light chain , or the the CDR sequences of the centration of typically 0 . 06 mg/ L being close to the EC50 heavy and light chains, of the so - called anti - LAMP1 anti MAb format was chosen so that the two mAbs had different 35 body M?b4 . Fc domains ( either human or murine ). Individual measure - Accordingly , the antibody of the invention may comprise ments ofmAb binding could be performed typically by their a CDR1- H of sequence SEQ ID NO : 83 , a CDR2- H of unique specific binding to Fc ( for example with Peroxidase - sequence SEQ ID NO : 84 , a CDR3 - H of sequence SEQ ID AffiniPure Goat Anti -Human IgG Ab , Fcy Fragment Specific NO : 85 , a CDR1- L of sequence SEQ ID NO : 86 , a CDR2 - L ( Jackson 109 - 035 - 098 ) or with Peroxidase - AffiniPure Goat 40 of sequence NAK , and a CDR3 - L of sequence SEQ ID NO : Anti -Mouse IgG Ab , Fcy Fragment Specific ( Jackson 115 - 87. 035 - 164 )) . The results were reported as a percentage of the Furthermore , the antibody of the invention may comprise , value obtained from the mAb alone at the same concentra - or consist of, a heavy chain of sequence SEQ ID NO : 98 tion . and /or a light chain of sequence of sequence SEQ ID NO : 99 In particular , the antibody according to the invention 45 ( i . e . heavy and / or light chain of MAb4 as described in comprises the CDR sequences of the heavy and / or light example 17 . 2 . 3 ) . chains of one of so -called anti -LAMP1 antibodies MAbi , In one embodiment this antibody may be chimeric , MAD2 and MAb3 . More specifically , the antibody can humanized , or an antibody fragment. comprise the CDR sequences of the heavy light chain , or the In the antibody of the invention , one individual amino the CDR sequences of the heavy and light chains , of one of 50 acid may be altered by substitution , in particular by conser so - called anti -LAMP1 antibodies MAb1 , MAD2 , MAb3 and vative substitution , in one or more ( in particular in only one ) MAD3 VL _ R24 _ R93 . of the above CDR sequences. Such an alteration may be Accordingly, the antibody of the invention may comprise : intended for example to remove a glycosylation site or a a CDR1 - H of sequence SEQ ID NO : 2 or a sequence deamidation site , in connection with humanisation of the differing from SEQ ID NO : 2 by one amino acid substitu - 55 antibody. Another alteration could also be intended to tion , a CDR2- H of sequence SEQ ID NO : 3 or a sequence remove a lysine in a CDR , since covalent attachment to differing from SEQ ID NO : 3 by one amino acid substitu - cytotoxic via lysine side chain residue may interfere with tion , and a CDR3 - H of sequence SEQ ID NO : 4 or a binding to antigen in the case of ADC . For instance, SEQ ID sequence differing from SEQ ID NO : 4 by one amino acid NO : 48 and SEQ ID NO : 52 are CDR3- L sequences that substitution ; 60 differ by one amino acid substitution at their position 5 . and /or According to an embodiment, the antibody comprises a CDR1 - L of sequence SEQ ID NO : 6 or a sequence (i ) a CDR1- H of sequence SEQ ID NO : 2 , a CDR2- H of differing from SEQ ID NO : 6 by one amino acid substitu - sequence SEQ ID NO : 3 , and a CDR3 - H of sequence SEQ tion , a CDR2 - L of sequence DTS or a sequence differing ID NO : 4 ; and / or from DTS by one amino acid substitution and a CDR3- L of 65 a CDR1- L of sequence SEQ ID NO : 6 , a CDR2- L of sequence SEQ ID NO : 7 or a sequence differing from SEQ sequence DTS , and a CDR3 - L of sequence SEQ ID ID NO : 7 by one amino acid substitution ; or NO : 7 ; or US 9 ,809 ,653 B2 37 38 ( ii ) a CDR1- H of sequence SEQ ID NO : 9 , a CDR2 - H of that consist of sequences SEQ ID NO : 72, SEQ ID NO : 73 sequence SEQ ID NO : 10 , a CDR3- H of sequence SEQ ID and SEQ ID NO : 74 , respectively ; and comprises NO : 11 ; and / or a ) a CDR1- L consisting of sequence X X X DRY (SEQ a CDR1- L of sequence SEO ID NO : 13 . a CDR2 - L of ID NO : 93 ) wherein each of X , and X , is any amino acid and sequence AAS , and a CDR3 - L of sequence SEQ ID NO : 14 ; 5 X3 is selected from Ile , Leu and Val; and or a CDR2- L consisting of sequence DX X , wherein X , a is ( iii ) a CDR1 - H of sequence SEO ID NO : 43 , a CDR2 - H selected from T or S and X2 is any amino acid ; and a CDR3 - L consisting of the sequence LQYX X X3WT, in of sequence SEQ ID NO : 44 , and a CDR3 -H of sequence which X , X , and X2 is any amino acid ; and / or SEQ ID NO : 45 , and /or of 10 b ) a CDR1- H consisting of sequence X X2IFX3NYN a CDR1 - L of sequence SEQ ID NO : 47, a CDR2 - L of ( SEQ ID NO : 82 ) wherein each of X and X2 are any amino sequence YTS , and a CDR3 - L of sequence SEQ ID NO : 48 acid and X , is selected from Tyr or Phe ; and a CDR2 - H or SEQ ID NO : 52 . consisting of SEQ ID NO : 3 ; and In particular , the antibody can comprise: CDR3 - H consisting of sequence VRANWX , X , X FAY (i ) a CDR1- H of sequence SEQ ID NO : 2 , a CDR2 - H of 15 ( s sequence SEQ ID NO : 3 , a CDR3 - H of sequence KZSEQ -HOT ID 15 (acidSEQ . ID NO : 84 ) wherein each of X1, X2, X3, is any amino NO : 4 , and /or In one embodiment said antibody retains the ability to a CDR1- L of sequence SEQ ID NO : 6 , a CDR2 - L of bind to loop 2 . sequence DTS , and a CDR3 - L of sequence SEQ ID NO : 7 ; The skilled in the art knows methods to verify if the or 20 antibody according to the definition retains its ability to bind ( ii ) a CDR1- H of sequence SEQ ID NO : 9 , a CDR2 - H of to three regions of loop 2 of human LAMP1 that consist of sequence SEQ ID NO : 10 , a CDR3- H of sequence SEQ ID sequences SEQ ID NO : 72 , SEQ ID NO : 73 and SEO ID NO : 11 , and / or NO : 74 , respectively , and thus does not suffer from a a CDR1- L of sequence SEQ ID NO : 13 , a CDR2 - L of disturbed canonical structure . sequence AAS , and a CDR3 - L of sequence SEQ ID NO : 14 ; 25 The invention also provides antibodies as defined above or further comprising at least the variable domain of heavy ( iii) a CDR1 - H of sequence SEO ID NO : 43 , a CDR2 - H chain and / or the variable domain of light chain of one of the of sequence SEQ ID NO : 44 , a CDR3- H of sequence SEQ so -called anti- LAMP1 antibodies MAb1, MAB2 , MAb2. com , ID NO : 45 , and /or MAD3 , MAb3 VL _ R24 _ R93 , huMAb1 _ 1 and huMAb1 _ 2 , CDR1 - L of sequence SEQ ID NO : 47 , a CDR2 - L of 30 huMAb1 _ 3 , for instance MAbi, MAB2 , MAb2. com , MAD3 , sequence YTS , and a CDR3 - L of sequence SEQ ID NO : 48 MAb3 VL _ R24 _ R93 . or SEQ ID NO : 52 , or Thus the invention relates in particular to an antibody ( iv ) a fragment of an antibody as defined in (i ), ( ii ), or ( iii ). which comprises : The inventors crystallized recombinant Fab from ( i ) a variable domain of heavy chain of sequence SEQ ID huMAb1 _ 1 that was identified to bind to loop 1 and loop 2 35 NO : 1 or a sequence at least 85 % identical thereto and / or a in a complex with non - glycosylated LAMP1 protein accord - variable domain of light chain of sequence of sequence SEQ ing to the protocol described in example 7 . 3 . 1 . Based on the ID NO : 5 , or a sequence at least 85 % identical thereto ; or determination of the tridimensional structure of huMab1_ 1 ( ii ) a variable domain of heavy chain of sequence SEQ ID in complex with LAMP1 , most of its CDRs can be associ- NO : 8 , or a sequence at least 85 % identical thereto , and /or ated to specific canonical structure as referenced in Al- 40 a variable domain of light chain of sequence of sequence Lazikini, Lesk and Chothia (1997 ) J . Mol. Biol. 273 : 927 SEQ ID NO : 12 , or a sequence at least 85 % identical thereto ; 948 mentioned above. The crystal structure allowed or determining mutations that can be introduced into the CDRS ( iii ) a variable domain of heavy chain of sequence SEQ ID without disturbing said canonical structure . It is known to NO : 15 , or a sequence at least 85 % identical thereto , and /or the skilled in the art that disturbation of said canonical 45 a variable domain of light chain of sequence of sequence structure would result in a modified binding behavior . They SEQ ID NO : 16 , or a sequence at least 85 % identical thereto ; thus identified by analyzing the crystallographic structure, or that Q27 and D28 of SEQ ID NO : 68 located in CDR1- L can ( iv ) a variable domain of heavy chain of sequence SEQ ID be replaced by any amino acid as long as the loop retains the NO : 42 , or a sequence at least 85 % identical thereto , and /or canonical structure K2B and 129 of SEQ ID NO : 68 can be 50 a variable domain of light chain of sequence of sequence replaced by an equivalent hydrophobic residues , for instance SEQ ID NO : 46 or SEQ ID NO : 51, or a sequence at least Leu or Val. T51 of SEQ ID NO : 68 and S52 of SEQ ID NO : 85 % identical thereto ; or 68 , both located in CDR2- L can be replaced by a Ser, in case (v ) a variable domain of heavy chain of sequence SEQ ID of T51 and by any amino acid , in the case of S52 , as long NO : 53 or a sequence at least 85 % identical thereto and /or as this loop retains the classic y - turn conformation . Residues 55 a variable domain of light chain of sequence of sequence D92 , N93 , L94 of SEQ ID NO : 68 , located in CDR3- L can SEQ ID NO : 56 , or a sequence at least 85 % identical thereto ; be replaced by any amino acids as long as the loop retains or canonical structure à 1B . Furthermore , G26 of SEQ ID NO : (vi ) a variable domain of heavy chain of sequence SEQ ID 69, located in CDR - 1H can be replaced by any amino acid , NO : 54 or a sequence at least 85 % identical thereto and /or Y27 of SEQ ID NO : 69 , located in CDR - 1H by a phenyl - 60 a variable domain of light chain of sequence of sequence alanine , T30 of SEQ ID NO : 69 , located in CDR - 1H by any SEQ ID NO : 57 , or a sequence at least 85 % identical thereto ; amino acid , as long as the loop retains the canonical struc or ture 1 . Residues D102 , V103 and A104 of SEQ ID NO : 69, ( vii) a variable domain of heavy chain of sequence SEQ located in CDR - 3H can be replaced by any amino acid of ID NO : 55 or a sequence at least 85 % identical thereto similar sizes and properties . 65 and / or a variable domain of light chain of sequence of Accordingly , the invention provides for an antibody sequence SEQ ID NO : 58 , or a sequence at least 85 % which binds to three regions of Loop 2 of human LAMP1 identical thereto . US 9 ,809 , 653 B2 39 40 In one embodiment , the invention relates to an isolated According to an embodiment, the antibody according to anti -LAMP - 1 antibody which comprises : the invention comprises or consists of an IgG , or a fragment ( i) a CDR1- H of sequence SEQ ID NO : 2 , a CDR2- H of thereof. sequence SEQ ID NO : 3 , and a CDR3- H of sequence SEQ The antibody of the invention and a fragment thereofmay ID NO : 4 ; and / or 5 be, respectively , a murine antibody and a fragment of a a CDR1- L of sequence SEQ ID NO : 6 , a CDR2- L of murine antibody . sequence DTS , and a CDR3 - L of sequence SEQ ID NO : 7 ; The antibody may also be a chimeric antibody, and in or particular a murine/ human antibody, e . g . an antibody com (ii ) a CDR1 - H of sequence SEQ ID NO : 9 , a CDR2 - H of prising murine variable domains of heavy and light chains sequence SEQ ID NO : 10 , a CDR3 - H of sequence SEQ ID " and a CH domain and a CL domain from a human antibody . NO : 11 ; and / or The antibody may be a fragment of such an antibody . a CDR1 - L of sequence SEQ ID NO : 13 , a CDR2- L of According to an embodiment , the antibody of the inven sequence AAS , and a CDR3 - L of sequence SEQ ID NO : 14 ; tion is : or a ) a chimeric antibody comprising , or consisting of, a (iii ) a CDR1 - H of sequence SEQ ID NO : 43 , a CDR2H - h eavy chain of sequence SEQ ID NO : 17 and / or a light chain of sequence SEQ ID NO : 44 , and a CDR3- H of sequence of sequence of sequence SEQ ID NO : 18 (i . e . heavy and /or SEQ ID NO : 45 , and / or light chain of chMAbl as described in example 7 ) ; or a CDR1 - L of sequence SEQ ID NO : 47 , a CDR2 - L of b ) a chimeric antibody comprising , or consisting of, a sequence YTS , and a CDR3 -L of sequence SEQ ID NO : 48 20 heavy chain of sequence SEQ ID NO : 19 and /or a light chain or SEQ ID NO : 52 ; or of sequence of sequence SEQ ID NO : 20 ; ( i. e . heavy and /or ( iv ) CDR1- H of sequence SEQ ID NO : 83 , a CDR2 - H of light chain of chMAb2 as described in example 7 ); or sequence SEQ ID NO : 84 , a CDR3- H of sequence SEQ ID c ) a chimeric antibody comprising , or consisting of, a NO : 85 , and /or heavy chain of sequence SEQ ID NO : 21 and / or a light chain a CDR1 -L of sequence SEQ ID NO : 86 , a CDR2 - L of 25 of sequence of sequence SEQ ID NO : 22 ; ( i . e . heavy and /or sequence NAK , and a CDR3 -L of sequence SEQ ID NO : 87 ; light chain of chMAb2can as described in example 7 ); or or a heavy chain of sequence SEQ ID NO : 60 or a light chain d ) a chimeric antibody comprising , or consisting of, a of sequence SEQ ID NO : 59 ; or heavy chain of sequence SEQ ID NO : 49 and / or a light chain ( v ) a heavy chain of sequence SEQ ID NO : 62 or a light of sequence of sequence SEQ ID NO : 50 ; ( i. e . heavy and / or chain of sequence SEQ ID NO : 61 ; or 30 light chain of chMAb3 ) ; or (vi ) a heavy chain of sequence SEQ ID NO : 64 or a light e ) a chimeric antibody comprising , or consisting of, a chain of sequence SEQ ID NO : 63 . heavy chain of sequence SEO ID NO : 49 and /or a light chain For instance , the sequence of the variable domain of of sequence of sequence SEQ ID NO :81 ; ( i. e . heavy and / or heavy or light chain may differ from the reference sequence light chain of chMAb3 _ VLR24 - R93 ; or SEQ ID NO : 1 , 5 , 8 , 12, 15 , 16 , 42 , 46 or 51, 53 , 56 , 54 , 57, 35 f ) a fragment of the chimeric antibody defined in a ) , b ) , c ) , 55 , 58 , for instance from SEQ ID NO : 1 , 5 , 8 , 12 , 15 , 16 , 42 , d ) and e ). 46 or 51 as appropriate , by one or more amino acid substi - The antibody of the invention may also be a humanized tution ( s ) , in particular by one or more conservative amino a ntibody . Thus , according to an embodiment, the antibody of acid substitution ( s ) and / or substitution ( s ) with canonical the invention comprises, or consists of: residues . 40 i ) a heavy chain of sequence SEQ ID NO : 60 or a In particular, the sequence of the variable domain of sequence at least 85 % identical thereto and/ or a light chain heavy or light chain may differ from the reference sequence of sequence of sequence SEQ ID NO : 59 or a sequence at SEQ ID NO : 1 , 5 , 8, 12 , 15 , 16 , 42 , 46 or 51, 53 , 56 , 54 , 57 , least 85 % identical thereto ( i. e . heavy and /or light chain of 55 , 58 , for example from SEQ ID NO : 1 , 5 , 8 , 12 , 15 , 16 , 42 , huMAb1 _ 1 as described in example 7 . 2 ) ; or 46 or 51 by conservative amino acid substitution ( s ), only . 45 ii ) a heavy chain of sequence SEQ ID NO : 62 or a The sequence alterations as compared with sequence SEQ sequence at least 85 % identical thereto and / or a light chain ID NO : 1, 5 , 8 , 12 , 15 , 16 , 42 , 46 or 51 , 53 , 56 , 54 , 57 , 55 , of sequence of sequence SEQ ID NO : 61 or a sequence at 58 , for example from SEQ ID NO : 1 , 5 , 8, 12 , 15 , 16 , 42 , 46 least 85 % identical thereto ( i. e . heavy and /or light chain of or 51 will in particular be present essentially in one or more humAb1 _ 2 as described in example 7 . 2 ) ; or of the framework regions, FR1 - L , FR2 - L , FR3 - L , FR4 - L 50 iii ) a heavy chain of sequence SEQ ID NO : 64 a or a and /or FR1- H , FR2 - H , FR3 -H , FR4 - H . sequence at least 85 % identical thereto nd /or a light chain of However , amino acid substitutions in one or more CDRs sequence of sequence SEQ ID NO : 63 or a sequence at least are also possible . 85 % identical thereto (i . e . heavy and /or light chain of The invention also provides antibodies as defined above huMAb1 _ 3 as described in example 7 . 2 ) . further comprising at least the variable domain of heavy 55 In one embodiment the antibody of the invention is a chain and / or the variable domain of light chain of one of the humanized antibody . In a further embodiment said human so - called anti -LAMP1 antibodies MAb4. ized antibody is obtained through the resurfacing technol Thus the invention relates in particular to an antibody ogy . Such antibodies may also be called “ resurfaced ” anti which comprises a variable domain of heavy chain of bodies . sequence SEQ ID NO : 88 or a sequence at least 85 % 60 The antibody according to the invention may also be a identical thereto and /or a variable domain of light chain of single domain antibody or a fragment thereof. In particular, sequence of sequence SEQ ID NO : 89, or a sequence at least a single domain antibody fragment may consist of a variable 85 % identical thereto . heavy chain (VHH ) which comprises the CDR1- H , CDR2- H The antibody according to the invention is in particular a and CDR3 - H of the antibodies as described above . The conventional antibody, in particular a conventional mono - 65 antibody may also be a heavy chain antibody, i. e . an clonal antibody, or an antibody fragment, a bispecific or antibody devoid of light chain , which may or may not multispecific antibody . contain a CH1 domain . US 9 ,809 , 653 B2 The single domain antibody or a fragment thereof may Any expression vector for animal cell can be used, so long also comprise the framework regions of a camelid single as a gene encoding the human antibody C region can be domain antibody , and optionally the constant domain of a inserted and expressed . Examples of suitable vectors include camelid single domain antibody. PAGE107 (Miyaji , H . et al. , 1990 , Cytotechnology 3 ( 2 ) : The antibody according to the invention may also be an 5 133 - 140 ) , PAGE103 (Mizukami , T . and Itoh , S . et al ., 1987 , antibody fragment, in particular a humanised antibody frag - J Biochem . 101 ( 5 ) : 1307 - 1310 ) , pHSG274 ( Brady , G . et al. , ment, selected from the group consisting of Fv , Fab , F ( ab ') 2 , 1984 , Gene 27 (2 ) : 223 - 232 ), PKCR (O 'Hare , K . et al ., 1981 , Fab' , dsFv, (dsFv )2 , scFv , sc (Fv ) 2 , and diabodies . Proc Natl Acad Sci USA . 78 ( 3 ) : 1527 - 1531) , PSG1 beta The antibody may also be a bispecific or multispecific d2 - 4 - (Miyaji , H . et al. , 1990 , Cytotechnology 4 : 173 - 180 ) one 10 and the like. antibody formed from antibody fragments , at least one Other examples of plasmids include replicating plasmids antibody fragment being an antibody fragment according to comprising an origin of replication pCEP5, or integrative the invention . Multispecific antibodies are polyvalent pro plasmids, such as for instance PUC , pcDNA , PBR , and the tein complexes as described for instance in EP 2 050 764 A1 like . or US 2005 /0003403 A1 . Other examples of viral vector include adenoviral , retro The bispecific or multispecific antibodies according to the viral. herpes virus and AAV vectors . Such recombinant invention can have specificity for ( a ) the first to third loops , viruses may be produced by techniques known in the art, in particular to the first lumenal domain on human /Macaca such as by transfecting packaging cells or by transient fascicularis LAMP1 targeted by one of the so - called MAb1 , transfection with helper plasmids or viruses. Typical MAD2 , MAD2Can, MAb3 , MAb3 _ VLR24 -R93 antibodies 20 examples of virus packaging cells include PA317 cells , and ( b ) at least one other antigen . According to an embodi PsiCRIP cells , GPenv + cells , 293 cells , etc . Detailed proto ment the at least one other antigen is not a human or Macaca cols for producing such replication - defective recombinant fascicularis LAMP1 family member, and in particular not at viruses may be found for instance in WO 95 / 14785 , WO least one or all of human and Macaca fascicularis LAMP2 . 96 / 22378 , U . S . Pat. No . 5 ,882 , 877 , U . S . Pat. No. 6 ,013 ,516 , According to another embodiment, the at least one other 25 U . S . Pat. No . 4 , 861 ,719 , U . S . Pat . No. 5 ,278 , 056 and WO antigen may be an epitope on human Macaca fascicularis 94 / 19478 . LAMP1 other than said first to third loops, in particular first further object of the present invention relates to a cell lumenal domain targeted by one of the so -called MAb1 , which has been transfected , infected or transformed by a MAB2, MAD2 Con and MAb3 antibodies . nucleic acid and / or a vector according to the invention . Said antibodies can be produced by any technique well 30 The term “ transformation ” means the introduction of a known in the art. In particular said antibodies are produced “ foreign ” ( i . e . extrinsic ) gene , DNA or RNA sequence to a by techniques as hereinafter described . host cell , so that the host cell will express the introduced Antibodies and fragments thereof according to the inven - gene or sequence to produce a desired substance, typically tion can be used in an isolated ( e . g ., purified ) from or a protein or enzyme coded by the introduced gene or contained in a vector , such as a membrane or lipid vesicle 35 sequence . A host cell that receives and expresses introduced ( e . g . a liposome) . DNA or RNA bas been “ transformed ” . The antibodies of the invention may represent any com The nucleic acids of the invention may be used to produce bination of the above mentioned features . a recombinant antibody of the invention in a suitable expres Nucleic Acids , Vectors and Recombinant Host Cells sion system . The term " expression system ” means a host cell A further object of the invention relates to a nucleic acid 40 and compatible vector under suitable conditions , e .g . for the sequence comprising or consisting of a sequence encoding expression of a protein coded for by foreign DNA carried by an antibody of the invention as defined above . the vector and introduced to the host cell . Typically , said nucleic acid is a DNA or RNA molecule , Common expression systems include E . coli host cells which may be included in any suitable vector , such as a and plasmid vectors , insect host cells and Baculovirus plasmid , cosmid , episome, artificial chromosome , phage or 45 vectors , and mammalian host cells and vectors . Other a viral vector. examples of host cells include , without limitation , prokary The terms “ vector " , " cloning vector " and " expression otic cells ( such as bacteria ) and eukaryotic cells ( such as vector” mean the vehicle by which a DNA or RNA sequence yeast cells, mammalian cells , insect cells , plant cells , etc . ) . ( e . g . a foreign gene ) can be introduced into a host cell , so as Specific examples include E . coli, Kluyveromyces or Sac to transform the host and promote expression ( e . g . transcrip - 50 charomyces yeasts , mammalian cell lines ( e . g . , Vero cells , tion and translation ) of the introduced sequence . CHO cells , 3T3 cells , COS cells , HEK293 cells etc . ) as well So , a further object of the invention relates to a vector as primary or established mammalian cell cultures ( e . g ., comprising a nucleic acid of the invention . produced from lymphoblasts, fibroblasts , embryonic cells , Such vectors may comprise regulatory elements , such as epithelial cells , nervous cells , adipocytes, etc . ). Examples a promoter, enhancer, terminator and the like , to cause or 55 also include mouse SP2 / 0 -Ag14 cell (ATCC CRL1581 ) , direct expression of said polypeptide upon administration to mouse P3X63 - Ag8 .653 cell (ATCC CRL1580 ) , CHO cell in a subject . Examples of promoters and enhancers used in the which a dihydrofolate reductase gene (hereinafter referred to expression vector for animal cell include enhancer and as “ DHFR gene” ) is defective (Urlaub , G . et al . ; 1980 , Proc promoter of human cytomegalovirus (Nelson , J. , 1996 J . Natl Acad Sci USA . 77 ( 7 ) : 4216 -4220 ), rat YB2 / Virology 70 : 3207 - 3986 ), early promoter and enhancer of 60 3HL .P2 .G11 . 16Ag. 20 cell (ATCC CRL1662 , hereinafter SV40 (Mizukami , T. and Itoh , S . et al ., 1987 , J Biochem . referred to as “ YB2/ 0 cell” ), and the like . The YB2/ 0 cell is 101( 5 ) : 1307 - 1310 ) , LTR promoter and enhancer of Molo - of interest , since ADCC activity of chimeric or humanised ney mouse leukemia virus (Kuwana Y . et al. , 1987, Biochem antibodies is enhanced when expressed in this cell. Biophys Res Commun . 149 : 960 - 968 ) , promoter (Mason , J . In particular , for expression of humanised antibody, the O . et al ., 1985 , Cell 41: 479 - 487 ) and enhancer (Gillies , S . 65 expression vector may be either of a type in which a gene D . et al ., 1983 , Cell 33 : 717 -728 ) of immunoglobulin H encoding an antibody heavy chain and a gene encoding an chain and the like . antibody light chain exists on separate vectors or of a type US 9 ,809 , 653 B2 43 44 in which both genes exist on the same vector ( tandem type ) . nucleic sequences encoding humanised VL and VH domains In respect of easiness of construction of a humanised anti as previously described , constructing a humanised antibody body expression vector, easiness of introduction into animal expression vector by inserting them into an expression cells , and balance between the expression levels of antibody vector for animal cell having genes encoding human anti H and L chains in animal cells , humanised antibody expres- 5 body CH and human antibody CL , and expressing the sion vector of the tandem type is preferred (Shitara , K . et al. , coding sequence by introducing the expression vector into 1994 , J Immunol Methods. January 3 : 167( 1 - 2 ) : 271 - 8 ) . an animal cell . Examples of tandem type humanised antibody expression As the CH domain of a humanized or chimeric antibody, vector include pKANTEX93 (WO 97 / 10354 ) , pEE18 and it may be any region which belongs to human immuno the like . 10 globulin heavy chains , but those of IgG class are suitable The present invention also relates to a method of produc - and any one of subclasses belonging to IgG class , such as ing a recombinant host cell expressing an antibody accord - IgG1, IgG2, IgG3 and IgG4, can also be used . Also , as the ing to the invention , said method comprising the steps CL of a human chimeric antibody , it may be any region consisting of: ( i ) introducing in vitro or ex vivo a recombi - which belongs to human immunoglobulin light chains, and nant nucleic acid or a vector as described above into a 15 those of kappa class or lambda class can be used . competent host cell , ( ii ) culturing in vitro or ex vivo the Antibodies can be humanised using a variety of tech recombinant host cell obtained and (iii ) , optionally , selecting niques known in the art including , for example , the tech the cells which express and /or secrete said antibody . nique disclosed in the application WO2009 /032661 , CDR Such recombinant host cells can be used for the produc - grafting ( EP 239, 400 ; PCT publication W091 /09967 ; U . S . tion of antibodies of the invention . 20 Pat . Nos . 5 , 225 ,539 ; 5 ,530 , 101; and 5 ,585 ,089 ) , veneering or resurfacing (EP 592 , 106 ; EP 519 ,596 ; Padlan E A ( 1991 ) ; Methods of Producing Antibodies of the Invention Studnicka , G . M . et al. , 1994 , Protein Eng . 7 ( 6 ) : 805 - 814 ; Roguska , M . A . et al . , 1994 , Proc Natl Acad Sci USA 91 ( 3 ) : Antibodies of the invention may be produced by any 969 - 973 ) , and chain shuffling (U . S . Pat. No. 5 ,565 , 332 ) The technique known in the art, such as , without limitation , any 25 general recombinant DNA technology for preparation of chemical, biological, genetic or enzymatic technique, either such antibodies is also known (see European Patent Appli alone or in combination . cation EP 125023 and International Patent Application WO Knowing the amino acid sequence of the desired 96 / 02576 ) . sequence , one skilled in the art can readily produce said Antibodies of the invention are suitably separated from antibodies or immunoglobulin chains, by standard tech - 30 the culture medium by conventional immunoglobulin puri niques for production of polypeptides . For instance , they can fication procedures such as , for example protein A affinity be synthesized using well -known solid phase method , in chromatography , ceramic hydroxyapatite chromatography , particular using a commercially available peptide synthesis mixed -mode chromatography, size -exclusion chromatogra apparatus ( such as that made by Applied Biosystems, Foster phy etc . City, Calif. ) and following the manufacturer ' s instructions. 35 The Fab of the present invention can be obtained by Alternatively , antibodies and immunoglobulin chains of the treating an antibody which specifically reacts with LAMP1 invention can be synthesized by recombinant DNA tech - with a protease , such as papaine . Also , the Fab can be niques as is well -known in the art. For example , these produced by inserting DNA sequences encoding both chains fragments can be obtained as DNA expression products after of the Fab of the antibody into a vector for prokaryotic incorporation of DNA sequences encoding the desired (poly ) 40 expression , or for eukaryotic expression , and introducing the peptide into expression vectors and introduction of such vector into procaryotic or eukaryotic cells ( as appropriate ) to vectors into suitable eukaryotic or prokaryotic hosts that will express the Fab . express the desired polypeptide , from which they can be The F ( ab ' ) 2 of the present invention can be obtained later isolated using well -known techniques. treating an antibody which specifically reacts with LAMP1 In particular , the invention further relates to a method of 45 with a protease , pepsin . Also , the F (ab ') 2 can be produced by producing an antibody of the invention , which method binding Fab ' described below via a thioether bond or a comprises the steps consisting of: (i ) culturing a transformed disulfide bond . host cell according to the invention ; ( ii ) expressing said The Fab ' of the present invention can be obtained treating antibody or polypeptide ; and (iii ) recovering the expressed F ( ab ') 2 which specifically reacts with LAMP1 with a reduc antibody or polypeptide . 50 ing agent, such as dithiothreitol. Also , the Fab ' can be Methods for producing humanised or chimeric antibodies produced by inserting DNA sequences encoding Fab ' chains involve conventional recombinant DNA and gene transfec - of the antibody into a vector for prokaryotic expression , or tion techniques are well known in the art ( See Morrison , S . a vector for eukaryotic expression , and introducing the L . and Oi, V . T ., 1984 , Annu Rev Immunol 2 : 239 - 256 and vector into prokaryotic or eukaryotic cells ( as appropriate ) to patent documents U . S . Pat. No . 5 , 202 , 238 ; and U . S . Pat. No. 55 perform its expression . 5 , 204 , 244 ) . The scFv of the present invention can be produced by In a particular embodiment, a chimeric antibody of the taking sequences of the CDRs or VH and VL domains as present invention can be produced by obtaining nucleic previously described , constructing a DNA encoding an scFv sequences encoding the murine VL and VH domains as fragment, inserting the DNA into a prokaryotic or eukaryotic previously described , constructing a chimeric antibody 60 expression vector , and then introducing the expression vec expression vector by inserting them into an expression tor into prokaryotic or eukaryotic cells (as appropriate ) to vector for animal cell having genes encoding human anti - express the scFv . To generate a humanised scFv fragment, a body CH and human antibody CL , and expressing the well known technology called CDR grafting may be used , coding sequence by introducing the expression vector into which involves selecting the complementary determining an animal cell. 65 regions ( CDRs ) according to the invention , and grafting In another particular embodiment, a humanised antibody them onto a human scFv fragment framework of known of the present invention can be produced by obtaining three dimensional structure ( see , e. g ., WO98 /45322 ; WO US 9 ,809 , 653 B2 45 46 87 / 02671 ; U . S . Pat . No . 5 , 859, 205 ; U . S . Pat . No . 5 , 585 ,089 ; It is known in the art that certain amino acids may be U . S . Pat. No. 4 ,816 , 567; EP0173494 ) . substituted by other amino acids having a similar hydro The single chain antibody or VHH directed against pathic index or score and still result in a protein with similar LAMP1 may be obtained for instance by a method com - biological activity, i. e . still obtain a biological functionally prising the steps of ( a ) immunizing a mammal belonging to 5 equivalent protein . It is also possible to use well - established the Camelidae with LAMP1 or a fragment thereof, so as to technologies , such as alanine -scanning approaches, to iden elicit antibodies ( and in particular heavy chain antibodies ) tify , in an antibody or polypeptide of the invention , all the against LAMP1 ; ( b ) obtaining a biological sample from the amino acids that can be substituted without significant loss Camelidae thus immunized , said sample comprising heavy of binding to the antigen . Such residues can be qualified as chain antibody sequences and / or Vyy sequences that are 10 neutral , since they are not involved in antigen binding or in directed against LAMP1 ; and (c ) recovering ( e . g . isolating ) maintaining the structure of the antibody. One or more of heavy chain antibody sequences and / or VH , sequences that these neutral positions can be substituted by alanine or by are directed against LAMP1 from said biological sample . another amino acid can without changing the main charac Suitable single chain antibody or VHH may also be obtained teristics of the antibody or polypeptide of the invention . by screening a library comprising heavy chain antibody 15 As outlined above , amino acid substitutions are generally sequences and / or VHH sequences for heavy chain antibody therefore based on the relative similarity of the amino acid sequences and /or VHH sequences that compete for binding side - chain substituents , for example , their hydrophobicity , to the first to third loops, in particular to the first lumenal hydrophilicity , charge , size , and the like . Exemplary substi domain of human and Macaca fascicularis LAMP1 proteins tutions which take several of the foregoing characteristics with an antibody comprising the variable heavy and light 20 into consideration are well known to those of skill in the art chains of an antibody selected from the group consisting of and include : arginine and lysine ; glutamate and aspartate ; the so - called antibodies MAb1, MAD2, MAD2C , MAB3 , serine and threonine ; glutamine and asparagine ; and valine , huMAb1_ 1 and humAb1 _ 2 , huMAb1 _ 3 , for instance leucine and isoleucine . MAb1 , MAD2 , MAD2 Can , MAb3 . It may be also desirable to modify the antibody of the 25 invention with respect to effector function , e . g . so as to Modification of the Antibodies of the Invention enhance antigen -dependent cell- mediated cytotoxicity (ADCC ) and /or complement dependent cytotoxicity (CDC ) Amino acid sequence modification (s ) of the antibodies of the antibody. This may be achieved by introducing one or described herein are contemplated . For example , it may be more amino acid substitutions in an Fc region of the anti desirable to improve the binding affinity and / or other bio - 30 body . Alternatively or additionally , cysteine residue ( s ) may logical properties of the antibody. be introduced in the Fc region , thereby allowing inter - chain Modifications and changes may be made in the structure disulfide bond formation in this region . The homodimeric of the antibodies of the present invention , and in the DNA antibody thus generated may have improved internalization sequences encoding them , and still result in a functional capability and /or increased complement- mediated cell kill antibody or polypeptide with desirable characteristics. 35 ing and / or antibody - dependent cellular cytotoxicity ( ADCC ) In making the changes in the amino sequences of poly - (Caron , P . C . et al. , 1992, J Exp Med. 176 ( 4 ) : 1191- 1195 and peptide , the hydropathic index of amino acids may be Shopes B ., 1992 , J Immunol. 148 ( 9 ) : 2918 - 2922 ) considered . The importance of the hydropathic amino acid Another type of amino acid modification of the antibody index in conferring interactive biologic function on a protein of the invention may be useful for altering the original is generally understood in the art . It is accepted that the 40 glycosylation pattern of the antibody , i. e . by deleting one or relative hydropathic character of the amino acid contributes more carbohydrate moieties found in the antibody , and/ or to the secondary structure of the resultant protein , which in adding one or more glycosylation sites that are not present turn defines the interaction of the protein with other mol - in the antibody. The presence of either of the tripeptide ecules, for example , enzymes, substrates, receptors, DNA , sequences asparagine - X - serine , and asparagine - X - threo antibodies , antigens , and the like . Each amino acid has been 45 nine , where X is any amino acid except proline , creates a assigned a hydropathic index on the basis of their hydro - potential glycosylation site . Addition or deletion of glyco phobicity and charge characteristics these are : isoleucine sylation sites to the antibody is conveniently accomplished ( + 4 . 5 ) ; valine ( + 4 . 2 ) ; leucine ( + 3 . 8 ) ; phenylalanine ( + 2 . 8 ) ; by altering the amino acid sequence such that it contains one cysteine / cystine ( + 2 . 5 ) ; methionine ( + 1 . 9 ) ; alanine ( + 1 . 8 ) ; or more of the above -described tripeptide sequences ( for glycine ( - 0 .4 ) ; threonine (- 0 . 7 ) ; serine ( - 0 . 8 ); tryptophane 50 N - linked glycosylation sites ). ( - 0 . 9 ) ; tyrosine ( - 1 . 3 ) ; proline ( - 1 . 6 ) ; histidine ( - 3 . 2 ) ; Another type of modification of the antibody of the glutamate ( - 3 . 5 ) ; glutamine ( - 3 . 5 ) ; aspartate - 3 . 5 ) ; aspara - invention may be to remove a lysine in a CDR or spacially gine ( - 3 . 5 ) ; lysine ( - 3 . 9 ) ; and arginine ( - 4 . 5 ) . close to a CDR since covalent attachment to cytotoxic via A further object of the present invention also encompasses lysine side chain residue may interfere with binding to function -conservative variants of the polypeptides of the 55 antigen in the case of ADC . present invention . Another type of modification involves the removal of For example , certain amino acids may be substituted by sequences identified , either in silico or experimentally, as other amino acids in a protein structure without appreciable potentially resulting in degradation products or heterogene loss of activity . Since the interactive capacity and nature of ity of antibody preparations. As examples , deamidation of a protein define its biological functional activity , certain 60 asparagine and glutamine residues can occur depending on amino acid substitutions can be made in a protein sequence , factors such as pH and surface exposure . Asparagine resi and of course in its DNA encoding sequence , while never - dues are particularly susceptible to deamidation , primarily theless obtaining a protein with like properties . It is thus when present in the sequence Asn -Gly , and to a lesser extent contemplated that various changes may be made in the in other dipeptide sequences such as Asn -Ala . When such a antibodies sequences of the invention , or corresponding 65 deamidation site , in particular Asn -Gly , is present in an DNA sequences which encode said polypeptides, without antibody or polypeptide of the invention , it may therefore be appreciable loss of their biological activity . desirable to remove the site , typically by conservative sub US 9 ,809 , 653 B2 48 stitution to remove one of the implicated residues . Such In particular , the pharmaceutical compositions contain substitutions in a sequence to remove one or more of the vehicles which are pharmaceutically acceptable for a for implicated residues are also intended to be encompassed by mulation capable of being injected . These may be in par the present invention . ticular isotonic , sterile , saline solutions (monosodium or Another type of covalent modification involves chemi- 5 disodium phosphate , sodium , potassium , calcium or mag cally or enzymatically coupling glycosides to the antibody. nesium chloride and the like or mixtures of such salts ), or These procedures are advantageous in that they do not dry , especially freeze - dried compositions which upon addi require production of the antibody in a host cell that has tion , depending on the case , of sterilized water or physi glycosylation capabilities for N - or O -linked glycosylation . ological saline , permit the constitution of injectable solu Depending on the coupling mode used , the sugar ( s ) may be The doses used for the administration can be adapted as attached to ( a ) arginine and histidine , ( b ) free carboxyl a function of various parameters , and in particular as a groups , ( c ) free sulfhydryl groups such as those of cysteine , function of the mode of administration used , of the relevant ( d ) free hydroxyl groups such as those of serine , threonine , pathology , or alternatively of the desired duration of treat orhydroxyproline , ( e ) aromatic residues such as those of 1815 ment. phenylalanine , or tyrosine , ( f) the amide group of glutamine . To prepare pharmaceutical compositions , an effective For example , such methods are described in WO87 /05330 . amount of the antibody or immunoconjugate of the inven Removal of any carbohydrate moieties present on the tion may be dissolved or dispersed in a pharmaceutically antibody may be accomplished chemically or enzymatically acceptable carrier or aqueous medium . Chemical deglycosylation requires exposure of the antibody 20 The pharmaceutical forms suitable for injectable use to the compound trifluoromethanesulfonic acid , or an include sterile aqueous solutions or dispersions; and sterile equivalent compound . This treatment results in the cleavage powders for the extemporaneous preparation of sterile of most or all sugars except the linking sugar ( N - acetylglu - injectable solutions or dispersions. In all cases, the form cosamine or N - acetylgalactosamine ) , while leaving the anti - must be sterile and must be fluid to the extent that easy body intact . Chemical deglycosylation is described by 25 syringability exists . It must be stable under the conditions of Sojahr, H . et al. ( 1987 , Arch Biochem Biophys. 259 ( 1 ) : manufacture and storage and must be preserved against the 52 -57 ) and by Edge , A . S . et al. ( 1981 , Anal Biochem . contaminating action of microorganisms, such as bacteria 118 ( 1 ) : 131 - 137 ) . Enzymatic cleavage of carbohydrate moi - and fungi. eties on antibodies can be achieved by the use of a variety The carrier can be a solvent or dispersion medium con of endo - and exo - glycosidases as described by Thotakura , N 30 taining, for example , water, ethanol, polyol ( for example , R . et al. ( 1987 , Methods Enzymol 138 : 350 -359 ) . glycerol, propylene glycol, and liquid polyethylene glycol, Another type of covalent modification of the antibody and the like ) , suitable mixtures thereof. The proper fluidity comprises linking the antibody to one of a variety of non can be maintained , for example , by the use of a coating , such proteinaceous polymers , e . g ., polyethylene glycol, polypro as lecithin , by the maintenance of the required particle size pylene glycol, or polyoxyalkylenes, in the manner set forth 35 in the case of dispersion and by the use of surfactants , in U . S . Pat. Nos. 4 ,640 , 835 ; 4 ,496 , 689 ; 4 , 301, 144 ; 4 ,670 , stabilizing agents , cryoprotectants or antioxidants . The pre 417 ; 4 ,791 , 192 or 4 ,179 , 337 . vention of the action of microorganisms can be brought about by antibacterial and antifungal agents . In many cases , Pharmaceutical Compositions it will be preferable to include isotonic agents , for example , 40 sugars or sodium chloride . The antibodies or immunoconjugates of the invention Sterile injectable solutions are prepared by incorporating may be combined with pharmaceutically acceptable excipi- the active compounds in the required amount in the appro ents , and optionally sustained - release matrices , such as priate solvent with several of the other ingredients enumer biodegradable polymers, to form therapeutic compositions . ated above, as required , followed by filtered sterilization . Thus, another object of the invention relates to a phar - 45 Generally , dispersions are prepared by incorporating the maceutical composition comprising an antibody or an various sterilized active ingredients into a sterile vehicle immunoconjugate of the invention and a pharmaceutically which contains the basic dispersion medium and the acceptable?? carrier. required other ingredients from those enumerated above . In The invention also relates to a polypeptide or an immu - the case of sterile powders for the preparation of sterile noconjugate according to the invention , for use as a medi- 50 injectable solutions , the preferred methods of preparation cament. are vacuum - drying and freeze -drying techniques which “ Pharmaceutically ” or “ pharmaceutically acceptable ” yield a powder of the active ingredient plus any additional refers to molecular entities and compositions that do not desired ingredient from a previously sterile - filtered solution produce an adverse , allergic or other untoward reaction thereof. when administered to a mammal, especially a human , as 55 Upon formulation , solutions will be administered in a appropriate . A pharmaceutically acceptable carrier or excipi- manner compatible with the dosage formulation and in such ent refers to a non -toxic solid , semi- solid or liquid filler, amount as is therapeutically effective . The formulations are diluent, encapsulating material or formulation auxiliary of easily administered in a variety of dosage forms, such as the any type . type of injectable solutions described above , but drug The form of the pharmaceutical compositions, the route of 60 release capsules and the like can also be employed . administration , the dosage and the regimen naturally depend For parenteral administration in an aqueous solution , for upon the condition to be treated , the severity of the illness , example , the solution should be suitably buffered if neces the age , weight, and gender of the patientotient, eteetc . sary and the liquid diluent first rendered isotonic with The pharmaceutical compositions of the invention can be sufficient saline or glucose . These particular aqueous solu formulated for a topical, oral, parenteral, intranasal, intra - 65 tions are especially suitable for intravenous, intramuscular, venous , intramuscular , subcutaneous or intraocular admin - subcutaneous and intraperitoneal administration . In this con istration and the like . nection , sterile aqueous media which can be employed will US 9 ,809 ,653 B2 49 50 be known to those of skill in the art in light of the present The inventors have also shown that the immunoconju disclosure . For example , one dosage could be dissolved in 1 gates of DM4- SPDB -huMAb1 _ 1 , DM4- SPDB - chMA ) 2 , mL of isotonic NaCl solution and either added to 1000 mL DM4- SPDB - chMAb3 induce a marked anti- tumor activity of hypodermoclysis fluid or injected at the proposed site of in vivo in different murine model of different cancer xeno infusion , ( see for example , “ Remington 's Pharmaceutical 5 graft models as shown in example 10 . 2 - 10 .4 . Sciences ” 15th Edition , pages 1035 - 1038 and 1570 - 1580 ) . For example , it was shown the immunoconjugate DM4 Some variation in dosage will necessarily occur depending SPDB - huMAb1 _ 1 induces a marked anti -tumor activity in vivo primary human invasive ductal carcinoma xenograft on the condition of the subject being treated . The person and primary human lung tumor xenograft derived from responsible for administration will , in any event, determine 10 patient, when used at a dose of 10 mg/ kg , 5 mg/ kg and 2 . 5 the appropriate dose for the individual subject. mg/ kg , with a single injection , as described in example The antibody or immunoconjugate of the invention of the 10 . 2 . 2 and 10 . 2 . 3 . invention may be formulated within a therapeutic mixture to Also the immunoconjugates DM4- SPDB -chMAb2 and comprise about 0 .01 to 100 milligrams, per dose or so . DM4- SPDB -chMAb3 induce a marked anti - tumor activity 15 in primary human invasive ductal carcinoma xenograft Therapeutic Methods and Uses derived from patient, when used at a dose of 10 mg/ kg , 5 mg/ kg and 2 . 5 mg/ kg or 5 mg/ kg , 2 . 5 mg/kg and 1 . 25 mg/ kg , The inventors have shown that an antibody directed respectively , with a single injection , as described in example against the first to third loops of LAMP1, in particular 1032 and 10 . 4 against the first lumenal domain of LAMP1, in particular 20 Thus , polypeptides , antibodies , immunoconjugates , or MAb1 and MAb2 and Mab3 , is able to actively internalize pharmaceutical compositions of the invention may be useful the LAMP1 receptor -antibody complex after binding and for treating cancer. accumulate probably via coated pits . Internalized antibodies The invention further relates to an anti- LAMP1 therapeu MAb1, MAb2 and Mab3 localized to early endosomes and tic agent for use for treating cancer in a patient harboring subsequently trafficked to and accumulation in lysosomal 25 LAMP1 gene copy number gain in cancer cells . compartments . In an embodiment, said patient harboring LAMP1 gene ImageStream multispectral imaging flow cytometer (Am - copy number gain in cancer cells has been selected by the in nis corp .) reveals that the internalized antibodies accumulate vitro method of selecting patients with cancer according to in lysosomal compartments . Immunofluorescence analysis the invention . In particular , the use comprises selecting said of viable Colo205 cells incubated with MAb1, MAD2 and 30 patient harboring LAMP1 gene copy number gain in cancer MAb3 at 4° C . showed distinct plasma membrane staining cells by a method of selecting patients with cancer according Incubation of cells at 37° C . with MAB1, MAD2 and MAb3 to the invention . revealed labeling of both plasmamembrane and intracellular The invention also relates to a method of treating a patient vesicles after 4 hours incubation . Since the internalization with cancer which comprises score ( IS ) revealing the fluorescence inside cells (as mea - 35 a ) selecting a patient with cancer who is likely to respond sured at 37° C . ) is 10 - fold higher than the fluorescence at the to anti -LAMP1 therapy by a in vitro method of selecting cell surface ( as measured at 4° C . ), this means that the patients with cancer according to the invention ; and LAMP1 protein is quickly recycling at cell membrane . All b ) administering anti - LAMP1 therapy to said selected together , our results show for the first time that LAMP1 patient. might function as a receptormediating the internalization of 40 The invention further relates to a method of selecting a antibodies and suggest that availability of specific internal patient with cancer for anti -LAMP1 therapy , comprising : izing antibodies should aid in developing novel therapeutic ( a ) determining , in a biological sample of a patient with methods to target toxins, drugs or short- range isotopes to be cancer which includes cancer cells, if said patient harbors a delivered specifically to the interior of the cancer cells . LAMP1 gene copy number gain ; and Furthermore , they have shown that an antibody according 45 (b ) administering to said patient anti -LAMP1 therapy , if to the invention , combined with a cytotoxic maytansinoid said patient harbors a LAMP1 gene copy number gain . ( DM4) , induces cytotoxic activity in vitro on human The invention also relates to a method of treating cancer HCT116 tumor or HEK293 cells containing a stable inte in a patient, comprising : gration of the LAMP1 coding DNA sequence in the genomic ( a ) determining , in a biological sample of a patient with DNA wherein individual clones present different intensities 50 cancer which includes cancer cells, if said patient harbors a of LAMP1 on the cell surface . LAMP1 gene copy number gain ; and In another example 9 . 4 , the inventors showed that an ( b ) administering to said patient anti -LAMP1 therapy, if antibody according to the invention , combined with a cyto - said patient harbors a LAMP1 gene copy number gain . toxic tomamycin dimer , induces cytotoxic activity in vitro . The cancer to be treated with antibodies , immunoconju They have also shown that an antibody combined with a 55 gates , or pharmaceutical compositions of the invention is a cytotoxic maytansinoid (DM4 ) induces a marked anti- tumor cancer expressing LAMP1 on the cell surface , in particular activity in vivo in a murine model of primary human colon overexpressing LAMP1 on the cell surface as compared to adenocarcinoma xenografts derived from patient, when used normal ( i . e . non tumoral) cells of the same tissular origin . at a dose of 10 mg/ kg , 5 mg/ kg and 2 . 5 mg/ kg , with a single Expression of LAMP1 by cancer cells may be readily injection at day 17 post tumor implantation as described in 60 assayed for instance by using an antibody according to the example 10 . 1 . 1 . invention , as described in the following section “ Diagnostic Furthermore , they have also shown that this immunocon - uses” , and in particular by an immunohistochemical method jugate induces a marked anti - tumor activity in vivo in a fo instance as described in Example 5 . murine model of primary human lung tumor xenografts In particular the cancer may be colon adenocarcinomas derived from patient, when used at a dose of 10 mg/ kg , 5 65 but also gastrointestinal tumors ( small intestine , rectum , mg/ kg and 2 . 5 mg/ kg , with a single injection at day 26 post parotid gland ), vital organs tumors ( lung , liver, pancreas , tumor implantation as described in example 10 . 1 . 2 . stomach and kidney ) , reproductive organ tumors (breast , US 9 ,809 , 653 B2 51 ovary and prostate ) as well as skin , larynx and soft tissue Immunol. , 157: 4963- 4969 , 1996 ; Shields et al. , J . Biol . tumors , for instance the cancer is selected from the group Chem . , 276 ( 1 ) : 6591 -6604 , 2001) . consisting of colon adenocarcinoma, gastrointestinal tumors In one example the mutation of 266A of for example SEQ (small intestine, rectum , parotid gland ) , vital organs tumors ID NO : 56 in the hulgG1 corresponds to the D265A muta ( lung , liver , pancreas and kidney ) , reproductive organ 5 tion mentioned above and thus significantly decrease bind tumors (breast , ovary and prostate ) as well as skin , larynx or ing to Fc Rs and ADCC . soft tissue tumors . Thus, an object of the invention relates to a method for In one embodiment gastrointestinal tumors are small treating a cancer comprising administering a subject in need intestine tumor, rectum tumor and / or parotid gland tumor. thereof with a therapeutically effective amount of a poly In one embodiment reproductive organ tumors gastroin - peptide , an antibody, an immunoconjugate or a pharmaceu testinal tumors are lung tumor, liver tumor, pancreas tumor, tical composition of the invention . stomach tumor and kidney tumor. “ Antibody - dependent cellular phagocytosis ” or “ ADCP ” In one embodiment reproductive organ tumors are breast refers to a form of cytotoxicity in which antibodies bound tumor, ovary tumor or prostate tumor. 15 onto Fc receptors ( FcRs) present on certain cytotoxic cells Screening of a panel of human tumors by immunohisto ( e . g . macrophages ) enable these cytotoxic effector cells to chemistry using a mouse anti -human LAMP1 antibody bind specifically to an antigen -bearing target cell and sub according to the invention indeed showed antibody staining sequently kill the target cell by phagocytosis . To assess in these types of cancers , as described in further details in ADCP activity of a molecule of interest , an in vitro ADCP Example 5 . 20 assay , such as that described in McEarchem et al. , 2007 , In particular, LAMP1 expressing human tumoral cells Blood 109: 1185 . may be selected from the group consisting of colon , stom In the context of the invention , the term “ treating” or ach , rectum , lung squamous cell carcinoma, breast invasive " treatment” , as used herein , means reversing, alleviating , ductal and lobular carcinoma and prostate adenocarcinoma inhibiting the progress of, or preventing the disorder or cells . These tumors were indeed found to display more than 25 condition to which such term applies, or one or more 50 % of cells positive for LAMP1 expression at the cell symptoms of such disorder or condition . By the term “ treat membrane ( see example 5 ) . ing cancer” as used herein is meant the inhibition of the The antibodies or immunoconjugates of the invention growth of malignant cells of a tumour and / or the progression may be used alone or in combination with any suitable of metastases from said tumor. Such treatment can also lead growth - inhibitory agent. 30 to the regression of tumor growth , i . e . , the decrease in size The antibodies of the invention may be conjugated or of a measurable tumor . In particular , such treatment leads to linked to a growth inhibitory agent, cytotoxic agent, or a the complete regression of the tumor or metastase . prodrug -activating enzyme as previously described . Anti - According to the invention , the term “ patient " or " patient bodies of the invention may be indeed useful for targeting in need thereof” is intended for a human or non -human said growth inhibitory agent , cytotoxic agent, or a prodrug 35 mammal affected or likely to be affected with a malignant to the cancerous cells expressing or over - expressing LAMP1 tumor. In particular, said patient may be a patient who has on their surface . been determined to be susceptible to a therapeutic agent It is also well known that therapeutic monoclonal anti - targeting LAMP1 , in particular to an antibody or immuno bodies can lead to the depletion of cells bearing the antigen conjugate according to the invention , for instance according specifically recognized by the antibody . This depletion can 40 to a method as described herebelow . be mediated through at least three mechanisms: antibody As disclosed above, “ anti -LAMP1 therapy ” is a therapy mediated cellular cytotoxicity , complement dependent lysis , which involves a therapeutic agent targeting LAMP1. and direct anti - tumour inhibition of tumour growth through According to the invention , the term “ therapeutic agent signals given via the antigen targeted by the antibody. targeting LAMP1” or “ anti -LAMP1 therapeutic agent” " Complement dependent cytotoxicity ” or “ CDC ” refers to 45 describe an agent binding to LAMP1 and having cytotoxic the lysis of a target cell in the presence of complement and / or cytostatic activity . Activation of the classical complement pathway is initiated As used herein , the term “ binding agent” refers to a by the binding of the first component of the complement molecule that exhibits specific binding activity towards system to antibodies which are bound to their cognate LAMP1. Such a binding molecule can include a variety of antigen . To assess complement activation , a CDC assay, e . g . 50 different types ofmolecules including , for example, macro as described in Gazzano - Santoro et al. (1997 ) may be molecules and small organic molecules . Small molecule performed . binding agents can include , for example , receptor ligands, “ Antibody - dependent cell -mediated cytotoxicity ” or antagonists and agonists . Macromolecules can include , for “ ADCC ” refers to a form of cytotoxicity in which secreted example , peptide , polypeptide and protein , nucleic acids antibodies bound onto Fc receptors ( FcRs) present on certain 55 encoding polypeptide binding agents , lectins , carbohydrate cytotoxic cells ( e . g . Natural Killer (NK ) cells , neutrophils , and lipids. It is understood that the term includes fragments and macrophages ) enable these cytotoxic effector cells to and domains of the agent so long as binding function is bind specifically to an antigen -bearing target cell and sub - retained . Similarly , the boundaries of the domains are not sequently kill the target cell . To assess ADCC activity of a critical so long as binding activity is maintained . In the molecule of interest , an in vitro ADCC assay, such as that 60 specific example where the binding agent is a peptide, described in U . S . Pat . No. 5 ,500 , 362 or 5 ,821 ,337 was also polypeptide or protein , such binding proteins can include contemplated . It is known to the skilled in the art that monomeric or multimeric species . Heteromeric binding pro specific mutations such as the D265A mutation according to teins are a specific example of multimeric binding proteins . the nomenclature described by Kabat et al. (Sequences of It is understood that when referring to multimeric binding Proteins of Immunological Interest, 5th edition , National 65 proteins that the term includes fragments of the subunits so Institute of Health , Bethesda , Md ., 1991) significantly long as assembly of the polypeptides and binding function of decrease binding to Fc Rs and ADCC ( Lund et al. , J. the assembled complex is retained . Heteromeric binding US 9 ,809 , 653 B2 53 54 proteins include , for example, antibodies and fragments tumors , for example gastrointestinal tumors ( small intestine , thereof such as Fab and F (ab ') 2 portions. rectum , parotid gland ), vital organs tumors ( lung, liver , According to an embodiment, the anti -LAMP1 therapeu pancreas and kidney ) , reproductive organ tumors (breast , tic agent is an anti -LAMP1 antibody or an immunoconjugate ovary and prostate ) as well as skin , larynx and soft tissue comprising an anti -LAMP1 antibody and at least one growth 5 tumors . Therefore, LAMP1 constitutes a marker of certain inhibitory agent. cancers and , therefore , has the potential to be used to By a “ therapeutically effective amount of the polypep - indicate the effectiveness of an anti -cancer therapy or detect tide of the invention is meant a sufficient amount of the ing recurrence of the disease . polypeptide to treat said cancer disease , at a reasonable In particular, LAMP1 is highly expressed at the surface of benefit /risk ratio applicable to any medical treatment . It will 10 carcinomas selected from the group consisting of colon , be understood , however, that the total daily usage of the rectum , lung squamous cell carcinoma, stomach , breast polypeptides and compositions of the present invention will invasive ductal and lobular carcinoma and prostate adeno be decided by the attending physician within the scope of carcinoma cells, more particularly colon , rectum , lung sound medical judgment. The specific therapeutically effec - squamous cell carcinoma, breast invasive ductal and lobular tive dose level for any particular patient will depend upon a 15 carcinoma and prostate adenocarcinoma cells. variety of factors including the disorder being treated and As described above in the chapter ‘ antibodies ' , the inven the severity of the disorder ; activity of the specific polypep - tors developped antibodies MAb1, MAD2 , MAb3 allowing tide employed ; the specific composition employed , the age , for the first time to detect extracellularly expressed LAMP1 body weight, general health , sex and diet of the patient; the and thus to perform IHC analysis on Frozen -OCT ( from time of administration , route of administration , and rate of 20 Optimal Cutting Temperature ) specimens and AFA ( Alcohol excretion of the specific polypeptide employed ; the duration Formalin Acetic acid Fixative ) and MAb4 allowing LAMP1 of the treatment; drugs used in combination or coincidental reinforcement in FFPE format and thus allows to distinguish with the specific polypeptide employed ; and like factors well cancerous from non -cancerous tissue . known in the medical arts. For example , it is well known In a preferred embodiment, the antibody of the invention within the skill of the art to start doses of the compound at 25 is used as component of an assay in the context of a therapy levels lower than those required to achieve the desired targeting LAMP1 expressing tumours , in order to determine therapeutic effect and to gradually increase the dosage until susceptibility of the patient to the therapeutic agent, monitor the desired effect is achieved . Another object of the inven the effectiveness of the anti- cancer therapy or detect recur tion relates to a polypeptide , an antibody , an immunocon - rence of the disease after treatment. In particular, the same jugate or a pharmaceutical composition of the invention for 30 antibody of the invention is used both as component of the use in the treatment of a malignant tumour. therapeutic agent and as component of the diagnostic assay . In particular, the polypeptide , antibody, immunoconjugate Thus, a further object of the invention relates to an or pharmaceutical composition may be used for inhibiting antibody according to the invention for use for in vivo the progression of metastases of a malignant tumour. detecting LAMP1 expression in a subject, or for use for ex Polypeptides of the invention may be used in combination 35 vivo detecting LAMP1 expression in biological sample of a with any other therapeutical strategy for treating malignant subject. Said detection may be intended in particular for tumour ( e . g . adjuvant therapy ) , and / or for reducing the a ) diagnosing the presence of a cancer in a subject , or growth of the metastatic tumour. b ) determining susceptibility of a patient having cancer to Efficacy of the treatment with an antibody or immuno - a therapeutic agent targeting LAMP1, in particular an immu conjugate according to the invention may be readily assayed 40 noconjugate according to the invention , or in vivo , for instance on a mouse model of cancer and by c ) monitoring effectiveness of anti - LAMP1 cancer measuring e . g . changes in tumor volume between treated therapy or detecting cancer relapse after anti -LAMP1 cancer and control groups , % tumor regression , partial regression therapy , in particular for therapy with an immunoconjugate and / or complete regression as defined in Example 10 . according to the invention by detecting expression of the In one embodiment, the antibody is one of the anti - 45 surface protein LAMP1 on tumor cells . LAMP1 antibodies developed by the applicant ( the so -called In an embodiment, the antibody is intended for an in vitro antibodies “ MAb1” , “MAb2 " , " MAb3 ” , huMAb1 1 and or ex vivo use . For example , LAMP1 may be detected in huMAb1 _ 2 , huMAb1 _ 3 ) that bind specifically to human vitro or ex vivo in a biological sample obtained from a LAMP1 and distinguish tumoral from non -tumoral tissues subject, using an antibody of the invention . The use accord as further described in the section “ Antibodies " above . 50 ing to the invention may also be an in vivo use . For example , an antibody according to the invention is administered to the Diagnostic Uses subject and antibody - cell complexes are detected and / or quantified , whereby the detection of said complexes is The antibody according to the invention revealed that indicative of a cancer. some LAMP1 expression occurred at the membrane of 55 The invention further relates to an in vitro or ex vivo non - tumoral cells but was restricted to stomach epithelial method of detecting the presence of a cancer in a subject, cells , oesophageal epithelial cells , breast epithelial cells , comprising the steps consisting of: prostate epithelial cells , testicular epithelial cells and limited a ) contacting a biological sample of a subject with an to a few cells . Nevertheless , prevalence and mean intensities antibody according to the invention , in particular in condi for LAMP1 expression at the membrane of non - tumoral 60 tions sufficient for the antibody to form complexes with said samples were lower than those found in tumours . biological sample , Instead , LAMP1 is highly expressed at the surface of a b ) measuring the level of antibody bound to said biologi variety other carcinomas than colon adenocarcinomas , cal sample , including gastrointestinal tumors ( small intestine , rectum , c ) detecting the presence of a cancer by comparing the parotid gland ) , vital organs tumors ( lung , liver, stomach , 65 measured level of bound antibody with a control, an pancreas and kidney ) , reproductive organ tumors (breast , increased level of bound antibody compared to control being ovary and prostate ) as well as skin , larynx and soft tissue indicative of a cancer . US 9 ,809 , 653 B2 55 56 The invention also relates to an in vitro or ex vivo method which was obtained from the subject previously in time, of determining susceptibility of a patient having cancer to a upon or after completion of the anti -LAMP1 cancer therapy . therapeutic agent targeting LAMP1, in particular to an Said anti- LAMP1 cancer therapy is in particular a therapy immunoconjugate according to the invention , which method using an antibody or immunoconjugate according to the comprises the steps consisting of: 5 invention . Said anti -LAMP1 cancer therapy targets a a ) contacting a biological sample sample of a patient LAMP1 expressing cancer, in particular a colon adenocar having cancer with an antibody according to the invention , in particular in conditions sufficient for the antibody to form cinoma, gastrointestinal tumors (small intestine , rectum , complexes with said biological sample, parotid gland ) , vital organs tumors ( lung , liver , pancreas, b ) measuring the level of antibody bound to said biologi- 10 stomach and kidney ), reproductive organ tumors ( breast , cal sample sample , ovary and prostate ) as well as skin , larynx and soft tissue c ) comparing the measured level of bound antibody to tumors . said biological sample sample with the level of antibody In an embodiment, antibodies of the invention may be bound to a control, labelled with a detectable molecule or substance , such as a wherein an increased level of bound antibody to said 1515 nuofluorescent molecule , a radioactive molecule or any other biological sample sample compared to control is indicative labels known in the art that provide ( either directly or of a patient susceptible to a therapeutic agent targeting indirectly ) a signal. LAMP1 . As used herein , the term " labeled ” , with regard to the In the above methods, said control can be a normal, non antibody according to the invention , is intended to encom cancerous , biological sample of the same type , or a reference 20 pass direct labeling of the antibody by coupling ( i. e ., physi value determined a representative of the antibody binding cally linking ) a detectable substance, such as a radioactive level in normal biological sample of the same type . In an agent or a fluorophore ( e .g . fluorescein isothiocyanate embodiment, the antibodies of the invention are useful for (FITC ) or phycoerythrin (PE ) or Indocyanine (Cy5 ) ) to the diagnosing a LAMP1 expressing cancer, such as a colon polypeptide , as well as indirect labeling of the polypeptide adenocarcinoma, gastrointestinal tumors ( small intestine , 25 by reactivity with a detectable substance . rectum , parotid gland ) , vital organs tumors ( lung , liver, An antibody of the invention may be labelled with a pancreas and kidney ) , reproductive organ tumors ( breast, radioactive molecule by any method known to the art . For ovary and prostate ) as well as skin , larynx and soft tissue example radioactive molecules include but are not limited tumors . radioactive atom for scintigraphic studies such as 1123 , 1124 , The invention further relates to an in vitro or ex vivo 30 1125, In111 , Rel86 , Re188 , Tc99, and isotopes for Positron method ofmonitoring effectiveness of anti -LAMP1 cancer Emission Tomography such as Zr89 , 1124 , Gas or Cu04 . therapy, comprising the steps consisting of : A " biological sample " encompasses a variety of sample a ) contacting a biological sample of a subject undergoing types obtained from a subject and can be used in a diagnostic anti - LAMP1 cancer therapy, with an antibody according to or monitoring assay. Biological samples include but are not the invention , in particular in conditions sufficient for the 35 limited to blood and other liquid samples of biological antibody to form complexes with said biological sample , origin , solid tissue samples such as a biopsy specimen or b ) measuring the level of antibody bound to said biologi - tissue cultures or cells derived therefrom , and the progeny cal sample , thereof. Therefore , biological samples encompass clinical c ) comparing the measured level of bound antibody with samples, cells in culture , cell supernatants, cell lysates , the level of antibody bound to a control; 40 serum , plasma, biological fluid , and tissue samples, in wherein a decreased level of bound antibody to said particular tumor sample . biological sample compared to control is indicative of In particular, the biological tissues may be prepared as effectiveness of said anti -LAMP1 cancer therapy . frozen -OCT (Optimal Cutting Temperature ) or AFA ( acetic In said method , an increased level of bound antibody to formalin alcohol) samples . Indeed , antibodies according to said biological sample compared to control is indicative of 45 the invention can advantageously be used on AFA sample ineffectiveness of said anti -LAMP1 cancer therapy. which is a format used by hospitals to collect and archive Said control is in particular a biological sample of the tissue samples . same type as the biological sample submitted to analysis, but Measuring or determining the level of antibody bound the which was obtained from the subject previously in time, said biological sample may be performed by any suitable during the course of the anti -LAMP1 cancer therapy . 50 method known in the art such as FACS or IHC , for instance . The invention further relates to an in vitro or ex vivo The invention also relates to an in vivo method of method ofdetecting cancer relapse after anti -LAMP1 cancer detecting the presence of a cancer in a subject , comprising therapy, comprising the steps consisting of: the steps consisting of: (a ) contacting a biological sample of a subject having a ) administering an antibody according to the invention completed anti -LAMP1 cancer therapy , with an antibody 55 detectably labelled to a patient, according to the invention , in particular in conditions suf b ) detecting localisation of said detectably labelled anti ficient for the antibody to form complexes with said bio - body in the patient by imaging . logical sample , Antibodies of the invention may be useful for staging of ( b ) measuring the level of antibody bound to said bio - cancer ( e . g . , in radioimaging ) . They may be used alone or in logical sample , 60 combination with other cancer markers . ( c ) comparing the measured level of bound antibody with The terms “ detection ” or “ detected ” as used herein the level of antibody bound to a control, includes qualitative and / or quantitative detection (measuring wherein a increased level of bound antibody to said levels ) with or without reference to a control . biological sample compared to control is indicative of In the content of the invention , the term “ diagnosing ” , as cancer relapse after anti -LAMP1 cancer therapy. 65 used herein , means the determination of the nature of a Said control is in particular a biological sample of the medical condition intended to identify a pathology which same type as the biological sample submitted to analysis , but affects the subject from a number of collected data . US 9 ,809 , 653 B2 57 58 In said method , the cancer is a LAMP1 expressing cancer , methods to compare genomic sequences and nucleotide in particular a colon adenocarcinoma, gastrointestinal positions are well known to the one skilled in the art . tumors (small intestine , rectum , parotid gland ) , vital organs There are numerous methods allowing determining the tumors ( lung , liver , pancreas and kidney ), reproductive presence of a LAMP1 gene copy number change in biologi organ tumors (breast , ovary and prostate ) as well as skin , 5 cal samples which are well known from the one skilled in the larynx and soft tissue tumors . art. These methods include , without being limited , hybrid ization methods with DNA probes specific of marker Method of Selecting Patients with Cancer sequences , such as comparative genomic hybridization The invention relates to an in vitro method of selecting 10 ( CGH ) , matrix - CGH , array - CGH , oligonucleotide arrays , patients with cancer which comprises : representational oligonucleotide microarray (ROMA ) , high a ) determining , in a biological sample of a patient with throughput technologies for SNP genotyping , for example cancer which includes cancer cells , if said patient harbors a Affymetrix SNP chips, and amplification methods such as LAMP1 gene copy number gain , and quantitative PCR . b ) selecting the patient based on the presence of LAMP1 15 in particular, the presence of said marker LAMPI gene gene copy number gain . copy number change is determined by amplification , or by In an embodiment, said method is for selecting a patient hybridization with DNA probes specific for LAMP1 gene or with cancer who is likely to respond to anti- LAMP1 therapy , genes included in the LAMP1 amplicon . In an embodiment, and said patient is selected as likely to respond to anti the method of the invention is implemented by Fluorescence LAMP1 therapy if said patient harbors a LAMP1 gene copy 20 In Situ Hybridization (FISH ), Comparative Genomic number gain . If said patient does not harbor a LAMP1 gene Hybridization (CGH ), New Generation Sequencing (NGS ) copy number gain , the patients may nevertheless be selected and /or Polymerase Chain Reaction (PCR ) . as likely to respond to anti - LAMP1 therapy based , for Accordingly , the invention relates to a method , wherein instance , on the detection of cell surface expression of LAMP1 gene copy number gain is determined with a LAMP1 as expression or overexpression of LAMP1 at the 25 method selected from the group consisting of FISH , CGH , surface of tumor cells may have other causes than LAMPI N GS and / or PCR . gene copy number gain . Methods of quantitative PCR are well - known in the art The LAMP1 gene gain can be related to a focal somatic and include real- time PCR , competitive PCR and radioac gain or amplification , a somatic large region gain or ampli tive PCR . For instance , a quantitative PCT method to fication on 139, a somatic chromosome duplication , a 30 somatic chromosome triplication or polyploidy. LAMP1 enumerate DNA copy number has been described in the U . S . gene copy number gain or amplification is included in a Pat . No . 6 , 180 , 349 . larger amplicon . The term “ amplicon ” as used herein refers As used herein , the term “ primer ” refers to an oligonucle to a segment of the genome that forms multiple linear otide which is capable of annealing to a target sequence and copies . According to the invention , the amplicon which 35 serving as a point of initiation of DNA synthesis under might undergo copy number variation leading to a LAMP1 conditions suitable for amplification of the primer extension gene copy number gain will be called herein LAMP1 product which is complementary to said target sequence . amplicon . The primer is typically single stranded for maximum effi Said “ LAMP1 amplification ” comprises a DNA region ciency in amplification . In particular, the primer is an which can measure between 378 kb and 34 . 2 MB . Said 40 oligodeoxyribonucleotide . The length of the primer depends “ LAMP1 gain " comprises a DNA region wich can mesaure on several factors, including temperature and sequence of between 523 kb and 95 . 8 MB the primer, but must be long enough to initiate the synthesis In one embodiment, the LAMP1 gene copy number gain of amplification products . In an embodiment the primer is can be signified by the CNV of a LAMP1 amplicon in colon from 15 to 35 nucleotides in length . A primer can further PDX which comprises at least 454 kb from base 113785387 45 contain additional features which allow for detection , immo to base 114240314 on human bilization , or manipulation of the amplified product. The (NC _ 000013 ) . Said minimal LAMP1 amplicon contains primer may furthermore comprise covalently -bound fluo others genes than LAMP1, for example the genes rescent dyes, which confer specific fluorescence properties ADPRHL1, CUL4A , DCUNID2, GRTP1 , LAMP1 , to the hybrid consisting of the primer and the target LOC100130463 , PCID2, PRO7, TFDP1, TMCO3 and F10 . 50 sequence or non covalently -bound fluorescent dyes which In another embodiment said LAMP1 amplicon comprises can interact with the double - stranded DNA/ RNA to change at least the genes ADPRHLI, ATP11A , ATP4B , CUL4A , the fluorescence properties . Fluorescent dyes which can be DCUNID2 , F10 , F7, FAM70B , FLJ41484 , FLJ44054 , used are for example SYBR - green or ethidium bromide . GAS6 , GRK1, GRTP1, LAMP1, LINC00552 , A “ pair of primers ” or “ primer pair ” as used herein refers LOC100128430 , LOC100130463 , LOC100506063, 55 to one forward and one reverse primer as commonly used in LOC100506394 , MCF2L , MCF2L - AS1, PCID2, PROZ , the art of DNA amplification such as in PCR amplification . RASA3, TFDP1 and TMCO3C13orf35 . As used herein , a “ probe ” refers to an oligonucleotide In a further embodiment the LAMP1 amplicon comprises capable of binding in a base - specific manner to a comple 95 . 8 Mb from base 19, 296 ,544 to base 115 , 107 ,245 on mentary strand of nucleic acid . A probe may be labeled with human chromosome 13 (NC _ 000013 ) . 60 a detectable moiety . Various labeling moieties are known in In the context of the present invention , the positions of the the art. Said moiety may, for example , either be a radioactive nucleotides are indicated accordingly to the NCBI human compound , a detectable enzyme (e . g ., horse radish peroxi genome sequence (Build 37 , February 2009 ). It is known to d ase (HRP ) ) or any other moiety capable of generating a the one skilled in the art, that a genome sequence is variable detectable signal such as calorimetric , fluorescent, chemilu from an individual to another. Therefore, the positions 65 minescent or electrochemiluminescent signal. The detect defined herein for the LAMP1 amplicon may slightly change able moiety may be detected using known methods . A probe according to the sequence used . However, may vary in length from about 5 to 100 nucleotides , for US 9 ,809 , 653 B2 59 instance from about 10 to 50 nucleotides, or from about 20 The inventors showed that, LAMP1 gain is detected in 28 to 40 nucleotides . In an embodiment, a probe comprises 33 tumor types , including Colorectal adenocarcionoma, Stom nucleotides . ach , Liver, Lung ( Adenocarcinoma and Squamous ) , Breast The terms “ hybridize” or “ hybridization , ” as is known to (Basal , BRCA , LUMA , LUMB and HER2) , Ovary , Head & those skilled in the art , refer to the binding of a nucleic acid 5 neck , Kidney (Kidney Chromophobe , Kidney Renal Clear molecule to a particular nucleotide sequence under suitable Cell Carcinoma, Kidney Renal Papilliary ) , Cell Carcinoma, conditions , namely under stringent conditions . Cervical squamous Cell , Pancreatic , Prostate , Bladder The term “ stringent condition ” or “ high stringency con urothelial, Glioma (Low grade glyoma and Glioblastoma dition ” as used herein corresponds to conditions that are multiform ) , Uterus, Thyroid , Leukemia , Lymphoma, suitable to produce binding pairs between nucleic acids 10 Esophageal, Melanoma and Soft tissue sarcoma. High gain or amplification is detected in , breast , cervical , colorectal, having a determined level of complementarity , while being glioblastoma, head and neck , liver, lung , glioma, ovarian , unsuitable to the formation of binding pairs between nucleic stomach and uterine cancer . acids displaying a complementarity inferior to said deter Accordingly , in an embodiment of the method of the mined level. Stringent conditions are the combination of 1015 invention , the patient is having a cancer selected from the both hybridization and wash conditions and are sequence group consisting of colorectal , stomach , liver , lung , breast , dependent . These conditionsmay be modified according to ovarian , head and neck , kidney , pancreatic , prostate, uterine , methods known from those skilled in the art ( Tijssen , 1993 , glioma, bladder , thyroid cancer and leukemia , lymphoma, Laboratory Techniques in Biochemistry and Molecular Biol esophageal, melanoma and soft tissue sarcoma, for instance ogy - Hybridization with Nucleic Acid Probes, Part 1, Chap - 20 colorectal, stomach , liver, lung , ovarian , head and neck , ter 2 " Overview of principles of hybridization and the kidney, pancreatic , prostate , uterine , glioma, bladder , thy strategy of nucleic acid probe assays ” , Elsevier , New York ). roid cancer and leukemia , lymphoma, esophageal , mela Generally , high stringency conditions are selected to be noma and soft tissue sarcoma . about 5° C . lower than the thermal melting point ( Tm ) , for In a further embodiment the patient is having a cancer instance at a temperature close to the Tm of perfectly 25 selected from the group consisting of, breast, cervical, base -paired duplexes (Andersen , Nucleic acid Hybridiza - colorectal, glioblastoma, head and neck , liver, lung, glioma, tion , Springer , 1999 , p . 54 ) . Hybridization procedures are ovarian , stomach , and uterine cancer ; or in particular from well known in the art and are described for example in the group consisting of cervical , colorectal , glioblastoma, Ausubel, F. M . , Brent, R . , Kingston , R . E . , Moore , D . D ., head and neck , liver , lung , glioma, ovarian , stomach , thy Seidman , J. G ., Smith , J. A . , Struhl, K . eds. (1998 ) Current 30 roid , and uterine cancer; or still more particularly from the protocols in molecular biology. V . B . Chanda, series ed . New group consisting of colon and lung cancer . York : John Wiley & Sons . LAMP1 gene copy number gain and high expression of High stringency conditions typically involve hybridizing LAMP1 could be detected at the surface of cancers selected at about 50° C . to about 68° C . in 5xSSC /5xDenhardt ' s from the group consisting of colon , lung , liver, pancreatic , solution / 1 . 0 % SDS , and washing in 0 .2xSSC / 0 . 1 % SDS at 35 kidney breast, ovarian , prostate, stomach cancer. about 60° C . to about 68° C . Thus in one embodiment the cancer may be selected from In one embodiment, the invention relates to a method colon , lung, liver, pancreatic , kidney , ovarian , prostate , wherein the mean LAMP1 gene copy number in cancer cells stomach cancer , for example from colon , lung, liver, pan is 22 . 5 . In particular the mean LAMP1 gene copy number in creatic , kidney , ovarian , prostate , stomach cancer . cancer cells may be 22 .5 and < 5 . 40 Furthermore , LAMP1 gene copy gain is correlated with In one embodiment, the invention relates to a method the LAMP1 mRNA expression in bladder, breast , colon , wherein the mean LAMP1 gene copy number in cancer cells lung , stomach and ovarian cancer . A significant association is > 5 . is shown between LAMP1 gene copy number gain / ampli The method of the invention can further comprise deter - fication and the expression of LAMP1 at the plasma mem mining if LAMP1 is expressed at the surface of cancer cells 45 brane of tumor cells for colon , stomach and lung tumor of the patient, and i ) said patient is selected as likely to PDX . respond to anti- LAMP1 therapy if said patient harbors a Accordingly , in a further embodiment of the method of LAMP1 gene copy number gain and if said cancer cells of the invention , the patient is having a cancer selected from the patient express LAMP1 at their surface or ii ) said patient the group consisting of breast , colon , lung , stomach , and is selected as unlikely to respond to anti -LAMP1 therapy if 50 ovarian . In another embodiment, the patient is having a said cancer cells of the patient do not express LAMP1 at cancer selected from the group consisting of colon , lung, their surface . stomach and ovarian . There are numerous methods allowing determining if An “ anti -LAMP1 therapy ” is a therapy which involves a LAMP1 is expressed at the surface of cancer cells , or therapeutic agent targeting LAMP1 . In one embodiment, overexpressed as compared with normal cells ( i . e . non 55 such an anti -LAMP1 therapy is an anti -LAMP1 antibody or tumoral ) of the same tissular origin , as which are well immunoconjugate . Anti -LAMP1 therapy is described in known from the one skilled in the art . These methods include further details hereafter. for example , without being limited , IHC , Western Blot The cancer may be in particular bladder , cervical , col (WB ) , Fluorescence activated cell sorting (FACS ) analysis , orectal, glioblastoma, head and neck , kidney , liver , lung , immunofluorescence ( IF ) , immunoprecipitation ( IP ) and 60 glioma, ovarian , pancreatic , prostate , stomach , thyroid , and Enzyme - linked immunosorbent assay (ELISA ). uterine cancer. In another example the cancer is particularly In an embodiment, immunohistochemistry ( IHC ) is used colorectal or lung cancer . for determining if LAMP1 is expressed or over- expressed at the surface of cancer cells . Kits Expression of LAMP1 by cancer cells may be readily 65 assayed for instance by using an anti -LAMP1 antibody as Finally , the invention also provides kits comprising at described in example 3 . least one antibody or immunoconjugate of the invention . US 9 ,809 ,653 B2 Kits containing antibodies of the invention find use in SEQ ID NO : 29 shows the sequence of cynomologous detecting the surface protein LAMP1, or in therapeutic or monkey LAMP1 extracellular domain without Peptide Sig diagnostic assays. Kits of the invention can contain a poly - nal , followed by C - terminal tag including 6 amino acid peptide or antibody coupled to a solid support, e . g . a tissue His - sequence . culture plate or beads ( e . g . sepharose beads ). Kits can be 5 SEQ ID NO : 30 shows the sequence of a human and provided which contain antibodies for detection and quan mouse LAMP1 chimer containing mouse Loop1 region of tification of the surface protein LAMP1 in vitro , e . g . in an LAMP1 and human Loop2 - 4 of LAMP1 without Peptide ELISA or a Western blot. Such an antibody useful for Signal , followed by C -terminal 6 amino acid His - Tag . detection may be provided with a label such as a fluorescent SEQ ID NO : 31 shows the sequence of a human and or radiolabel. 10 mouse LAMP1 chimer containing mouse Loop1 -2 region of The invention will be further illustrated in light of the LAMP1 and human Loop3 - 4 of LAMP1 without Peptide following Figures and Examples . Signal , followed by C - terminal 6 amino acid His - Tag . SEQ ID NO : 32 shows the sequence of a human and BRIEF DESCRIPTION OF THE SEQUENCES 15 mouse LAMP1 chimer containing human Loop1 - 2 region of LAMP1 and mouse Loop3 - 4 of LAMP1 without Peptide SEQ ID NO : 1 shows the VH sequence of the “MAb1 ” Signal, followed by C - terminal tag including 6 amino acid antibody . His sequence SEQ ID NO : 2 - 4 show the sequences of the CDR1- H , SEQ ID NO : 33 shows the sequence of a human and CDR2 -H , CDR3- H of the “MAbl ” antibody . 20 mouse LAMP1 chimer containing human Loop1- 3 region of SEQ ID NO : 5 shows the VL sequence of the “MAb1 ” LAMP1 and mouse Loopt of LAMP1 without Peptide antibody . Signal , followed by C - terminal tag including 6 amino acid SEO ID NO : 6 - 7 show the sequences of the CDR1- L , His sequence . CDR3 - L of the “ MAbl” antibody . SEQ ID NO : 34 shows the sequence of mouse LAMP1 SEQ ID NO : 8 shows the VH sequence of the “ MAb2 ” 25 extracellular domain without Peptide Signal, followed by antibody . C - terminal tag including 6 amino acid His sequence . SEQ ID NO : 9 - 11 show the sequences of the CDR1- H , SEQ ID NO : 35 shows the light chain sequence of the CDR2- H , CDR3- H of the “ MAb2 ” antibody . " MAb1” antibody . SEO ID NO : 12 shows the VL sequence of the “MAb2 ” SEQ ID NO : 36 shows the heavy chain sequence of the antibody . 30 “ MAb1” antibody . SEQ ID NO : 13 - 14 show the sequences of the CDR1- L SEQ ID NO : 37 shows the light chain sequence of the “MAB2 " antibody. CDR3 - L of the “MAb2 ” antibody . SEQ ID NO : 38 shows the heavy chain sequence of the SEQ ID NO : 15 shows the VH sequence of the so - called “ MAB2” antibody. “ Mab2can ” antibody. 35 SEQ ID NO : 39 shows the predicted full -length LAMP1 SEQ ID NO : 16 shows the VL sequence of the so -called protein sequence of Macaca fascicularis . “ Mab2can " antibody . SEQ ID NO : 40 shows the sequence of human LAMP2 SEQ ID NO : 17 shows the sequence of the heavy chain of extracellular domain without Peptide Signal, followed by the chimeric antibody " chMAbl” antibody. C - terminal 10 amino acid His - Tag . SEO ID NO : 18 shows the sequence of the light chain of 40 SEO ID NO : 41 shows the full - length protein sequence of the chimeric antibody “ chMAbl ” antibody. human LAMP2 . SEQ ID NO : 19 shows the sequence of the heavy chain of SEQ ID NO : 42 shows the VH sequence of the “ MAb3" the chimeric antibody " chMAb2 ” antibody . antibody. SEQ ID NO : 20 shows the sequence of the light chain of SEQ ID NO : 43 -45 show the sequences of the CDR1 - H , the chimeric antibody " chMAb2 ” antibody . 45 CDR2 - H , CDR3- H of the “MAb3 ” antibody . SEQ ID NO : 21 shows the sequence of the heavy chain of SEQ ID NO : 46 shows the VL sequence of the “MAb3 ” the chimeric antibody “ chMab2can ” antibody. antibody . SEQ ID NO : 22 shows the sequence of the light chain of SEQ ID NO : 47 and 48 show the sequences of the the chimeric antibody " chMab2can ” antibody . CDR1 - L and CDR3- L of the “MAb3 ” antibody . SEQ ID NO : 23 shows the DNA sequence of full - length 50 SEQ ID NO : 49 shows the sequence of the heavy chain of human LAMP1 as available from GenBank database under the chimeric antibody “ chMAb3 ” antibody . accession number NM _ 005561. 3 . SEQ ID NO : 50 shows the sequence of the light chain of SEQ ID NO : 24 shows the Protein sequence of full- length the chimeric antibody “ chMAb3 ” antibody . human LAMP1 as available from GenBank database under SEQ ID NO : 51 shows the sequence of the variable NP _ 005552 . 3 . 55 domain of light chain of antibody “MAb3 VL R24 R93 ” . SEQ ID NO : 25 shows the Protein sequence of full - length SEQ ID NO : 52 shows the sequence of CDR3 - L of mouse LAMP1 as available from GenBank database under antibody “MAD3 VL R24 R93 ” . NP _ 034814 SEQ ID NO : 53 shows the VH1 sequence of the human SEQ ID NO : 26 shows the Protein sequence of full - length ized antibody " huMAb1_ 1 ” antibody. rat LAMP1 as available from GenBank database under 60 SEQ ID NO : 54 shows the VH2 sequence of the human NP _ 036989 . ized antibody “ huMAb1 _ 2 " antibody . SEQ ID NO : 27 shows the Protein sequence of full - length SEQ ID NO : 55 shows the VH3 sequence of the human Macaca mulatta LAMP1 as available from GenBank data ized antibody “ huMAb1 _ 3 ” antibody . base under XP _ 002723509 . SEQ ID NO : 56 shows the VL1 sequence of the human SEQ ID NO : 28 shows the sequence of human LAMP1 65 ized antibody " huMAb1_ 1 ” antibody . extracellular domain without Peptide Signal , followed by SEQ ID NO : 57 shows the VL2 sequence of the human C - terminal 6 amino acid His - Tag . ized antibody " huMAb1 _ 2" antibody . US 9 , 809, 653 B2 63 64 SEQ ID NO : 58 shows the VL3 sequence of the human SEQ ID NO : 92 shows the amino acid sequence corre ized antibody " huMAbi 3 ” antibody. sponding to the amino acids 307 to 321 of SEQ ID NO : 24 . SEQ ID NO : 59 shows the light chain variant 1 sequence SEQ ID NO : 93 shows a consensus sequence for CDR1- L of the “ huMAb1 _ 1 ” antibody . of MAbi/ huMAb1 _ 1/ huMAb1 _ 2/ huMAb1 _ 3 antibody SEQ ID NO : 60 shows the heavy chain variant 1 sequence 5 family based on residues identified as important for the of the “ huMAb1 _ 1 ” antibody . canonical structure and thus the binding of human LAMP1 SEQ ID NO : 61 shows the light chain variant 2 sequence using crystallography . of the “ humAb1 _ 2 ” antibody. SEQ ID NO : 94 shows a consensus sequence for CDR3 - L SEQ ID NO : 62 shows the heavy chain variant 2 sequence of MAb1 /huMAbi 1 /huMAbi 2 /huMAb1 3 antibody of the “ huMAb1 _ 2 " antibody . 10 family based on residues identified as important for the SEQ ID NO : 63 shows the light chain variant 3 sequence canonical structure and thus the binding of human LAMP1 of the " huMAb1 _ 3 ” antibody . using crystallography . SEQ ID NO : 64 shows the heavy chain variant 3 sequence SEQ ID NO : 95 shows a consensus sequence for CDR1 - H of the “ huMAb1 _ 3 ” antibody . of MAbi/ huMAb1_ 1 /huMAb1 _ 2 / huMAb1_ 3 antibody SEO ID NO : 65 shows the light chain sequence of the 15 family based on residues identified as important for the negative control “ huMAb1 _ negA ” antibody with the muta - canonical structure and thus the binding of human LAMP1 tions 36A and 95A . using crystallography . SEQ ID NO : 66 shows the heavy chain sequence of the SEQ ID NO : 96 shows a consensus sequence for CDR3- H negative control " huMAb1 _ negA ” antibody with the muta of MAb1 /huMAb1 _ 1/ huMAb1 _ 2 /huMAb1 _ 3 antibody tion 101A . 20 family based on residues identified as important for the SEQ ID NO : 67 shows the heavy chain sequence of the canonical structure and thus the binding of human LAMP1 negative control " huMAb1 _ negB ” antibody with the muta using crystallography . tion 266A . SEQ ID NO : 97 shows the amino acid sequence corre SEQ ID NO : 68 shows the light chain sequence of the sponding to the amino acids 35 to 84 of SEQ ID NO : 24 . recombinant huMAb1_ 1 for crystallization . SEO ID NO : 98 shows the light chain sequence of the SEQ ID NO : 69 shows the heavy chain sequence of the “MAB4 ” antibody . recombinant huMAb1 _ 1 for crystallization comprising a SEQ ID NO : 99 shows the heavy chain sequence of the C -terminal His -tag . “ MAb4” antibody . SEQ ID NO : 70 shows the sequence of a human Loop1 - 2 region of LAMP1 with a cleavable thioredoxin (trx A ) tag , 30 EXAMPLES a His - Tag and a thrombin cleavage site . SEQ ID NO : 71 shows the sequence of the untagged Example 1 : Preparation of Patient- Derived Tumor hLAMP1- 29 - 195 . Xenografts ( PDX ) SEO ID NO : 72 shows the amino acid sequence corre sponding to the amino acids 101 to 110 of SEQ ID NO : 24 . 35 Example 1 . 1 : Preparation of CR - LRB - 010P , SEQ ID NO : 73 shows the amino acid sequence corre CR -LRB -003P , and CR - IGR -034P PDXs sponding to the amino acids 144 to 157 of SEO ID NO : 24 . SEQ ID NO : 74 shows the amino acid sequence corre A large collection of colorectal cancer models directly sponding to the amino acids 174 to 188 of SEQ ID NO : 24 . derived from tumor samples collected during patient surgery SEQ ID NO : 75 shows the amino acid sequence corre - 40 was develop . Patient- derived colorectal cancer tumor were sponding to the amino acids 29 to 41 of SEQ ID NO : 24 . collected , after patient' s informed consent, in 3 medical SEQ ID NO : 76 shows the amino acid sequence corre centers : Curie Institute (Paris , France ), Gustave Roussy sponding to the amino acids 68 to 80 of SEQ ID NO : 24 . Institute (Villejuif , France ), and Lariboisiere Hospital (Paris , SEQ ID NO : 77 shows the amino acid sequence corre - France ) . Immediately after surgery ( 1 hour after resection in sponding to the amino acids 29 to 100 of SEQ ID NO : 24 . 45 average ) , 2 fragments were transferred in culture medium SEQ ID NO : 78 shows the amino acid sequence corre - including DMEM with 10 mmol/ L HEPES , 4 . 5 g / L glucose , sponding to the amino acids 97 to 110 of SEQ ID NO : 24 . 1 mmol/ L pyruvate sodium , 200 U /mL penicillin , 200 SEQ ID NO : 79 shows the amino acid sequence corre - mg/mL streptomycin , 200 mg/mL gentamicin , 5 mg/mL sponding to the amino acids 173 to 189 of SEQ ID NO : 24 . ciprofloxacin , 20 mg/ mL metronidazole , 25 mg /mL vanco SEQ ID NO : 80 shows the amino acid sequence corre- 50 mycin , and 2 . 5 mg/mL fungizone orDMEM with Nanomy sponding to the amino acids 132 to 302 of SEQ ID NO : 70 . copulitine ( Abcys ) for engraftment. After 2 to 24 hours SEQ ID NO : 81 shows the sequence of the light chain of following the patient surgery , the tumor samples were the chimeric antibody “ chMAb3 VL _ R24 _ R93 ” . SEQ ID engrafted on 2 Swiss nude mice . Small fragments (50 mm ) NO : 82 shows the amino acid sequence corresponding to the were subcutaneously engrafted into the scapular area or on amino acids 360 to 375 of SEQ ID NO : 24 . 55 the flank of anesthetized mice . (xylazine /ketamine or iso SEQ ID NO : 83 - 85 show the sequences of the CDR1- H , flurane protocol) . Tumor growth was measured at least once CDR2 - H , CDR3 - H of the “ MAb4 ” antibody. a week and serial fragment grafts of each given tumor were SEQ ID NO : 86 and 87 show the sequences of the conducted on 3 to 5 Swiss nude or CB17 - SCID (after 3 CDR1- L and CDR3 - L of the " MAB4 ” antibody. passages) mice when the tumors reached a volume of 800 to SEQ ID NO : 88 shows the VH1 sequence of the antibody 60 1500 mm ”. ( Julien , S . 2012 , Clin . Cancer Res . 18 ( 19 ): 5314 “ MAD4” . 5328 . SEQ ID NO : 89 shows the VL1 sequence of the antibody “MAb4 ” . Example 1 .2 : Preparation of LUN -NIC - 0014 PDX SEQ ID NO : 90 shows the amino acid sequence corre and LUN -NIC -0070 PDXs sponding to the amino acids 47 to 61 of SEQ ID NO : 24 . 65 SEQ ID NO : 91 shows the amino acid sequence corre Non -small - cell lung carcinoma samples were collected , sponding to the amino acids 140 to 155 of SEQ ID NO : 24 . after patient ' s informed consent, in CHU Pasteur (Nice , US 9 ,809 ,653 B2 65 66 France ) . Immediately after surgery, a piece of the patient body binding was visualized by adding TMB - H , 0 , buffer tumor was transferred in AQIX medium and sent to Sanofi and read at a wavelength of 450 . Antibodies with the murine ( Vitry sur Seine, France ) . After 24 to 48 hours following the IgG , C kappa isotype were selected for further screening . patient surgery , the tumors samples were engrafted on 2 - 5 CB17 -SCID mice. Small fragments (50 mm ) were subcu - 5 Example 3 : Hybridoma Screening by taneously engrafted on the mice flank . Tumor growth was Immunohistochemistry (IHC ) followed at least once a week and serial fragment grafts of Individual hybridoma supernatants raised against tumor each given tumor were conducted on 5 to 10 CB17 - SCID tissue CR - LRB -010P were screened by IHC on a macroarray (after 3 passages ) mice when the tumor reached a volumeof slide containing frozen sections of immunizing tumor (CR 800 to 1500 mm . 10 LRB -010P ), human non - tumoral colon and human non tumoral skin . Frozen - OCT ( from Optimal Cutting Tempera Example 1 .3 : Preparation of BRE - IGR -0159 PDX ture ) specimens of non - tumoral colon and skin were obtained from surgical cases ( commercial sources such as Breast carcinoma samples were collected , after patient' s Asterand , US Biomax , Strasbourg Hospital ) . The automated informed consent, in Gustave Roussy Institute (Villejuif , 15 immunostaining was performed unsing Ventana Discovery France ) . Immediately after surgery ( 1 hour after resection in and Discovery XT automated systems (Ventana Medical average ) , 4 fragments were transferred in culture medium Systems, Inc , USA ) . including DMEM , penicillin , streptomycin and fungizone Frozen 10 um cryostat sections were incubated with IgG for engraftment. After a maximum of 12 hours following the culture supernatants as primary antibody (unknown concen patient surgery , the tumor samples ( fragments about 50 20 tration , dilution 11 in Phosphate Buffer Saline , PBS ) for 40 mm ) were engrafted on fat pad on 4 BALB nude mice . min at 37° C . Culture medium was used as negative control. Tumor growth was followed at least once a week and sent A postfixation step with glutaraldehyde ( 0 . 05 % in NaCl to Sanofi ( Vitry sur Seine ) . Serial fragment grafts of each 0 . 9 % w / v ) for 4 min was done. The secondary antibody given tumor were conducted on 3 to 5 BALB nude or Affinipure rabbit anti -mouse IgG (315 - 005 _ 008 , Jackson CB17 - SCID mice (after 3 passages ) when the tumors reach 25 Immunosearch Laboratories, Inc. USA ) was used at 4 . 8 a volume of 800 to 1500 mm " . ug/ mL and incubated for 12 min at 37° C . Immunostaining was done with UltraMap Red chromogenic detection Example 2 : Generation of Monoclonal Mouse Anti according to manufacturers recommendations for 8 min . LAMP1 Antibodies and First Screening Cryostat sections were subsequently couterstaining with 30 hematoxylin II (790 - 2208 , Ventana Medical Systems, Inc Immunizations, fusion and screening were performed USA ) and bluing for 4 min ( 760 -2037 ) . Stained slides were essentially as described previously using primary disaggre - dehydrated and coverslipped with Coverquick 2000 mount gated tumor CR -LRB -010P or CR -LRB - 003P or LUN -NIC - ing medium (Labonord , Ref 05547530 ) . 0014 mentioned in example 1 for immunization and P3X63 - Sections immunostainedimmunostaine with mAbs were analyzed by Ag8 .653 myeloma cells for fusion . Using the classical 35 microscope (Nikon Eclipse E400 ) . After the immunohisto method described by Wennerberg A . E et al. ( 1993 , Am . J . chemical screening clones of interest were identified as Pathol. 143 (4 ) : 1050 - 1054 ), 6 - 8 weeks old female BALBI those with reactivity with areas of tumoral colon cells but mice ( S082342 ; Charles River Labs, Bar Harbor, Me. ) each not normal epithelial cells of colon mucosa . MAb1 antibody received three rounds of immunization over a course of 41 showed evidence of tumor- associated reactivity and were days. Antigens were administered intraperitonealy to ventral 40 negative on epidermal human non - tumoral cells . site of mice . Three days after the last injection , mice were Similar results were obtained with MAb2 and MAb3 . sacrificed and spleens were isolated aseptically and washed Based on these IHC results , MAb1 MAb2 and MAb3 were with fresh RPMImedium . Lymphocytes were released from purified for further evaluation , including extensive IHC the spleens and single - cell suspension was washed twice characterization on non -tumoral and tumoral tissues for with RPMI medium before being fused with P3X63 - 45 MAb1 . AG8. 653 myeloma cells using polyethylene glycol. After fusion , the cell mixture was incubated in an incubator at 37° Example 4 : mAb Characterization C . for 16 - 24 hours . The resulting cells preparation was transferred into selective semi-solid medium and aseptically Antibodies MAb1 MAb2 and MAb3 were analysed for plated out into 100 mm Petri plates and incubated at 37° C . 50 cell surface binding on human primary disaggregated colon Ten days after initiation of selection , the plates were exam - tumor by FACS using Guava?easyCyteTM8HT Flow ined for hybridoma growth , and visible colonies were Cytometry System . picked -up and placed into 96 -well plates containing 200 uL The apparent affinity expressed as EC50 values was of growth medium . The 96 -well plates were kept in an estimated using BIOST @ T - SPEED software . incubator at 37° C . for 2 to 4 days . 55 Mouse hybridomas expressing antibody were produced Primary screening for IgG production was performed by into T500 flask and conditioned media collected after 7 days Enzyme- linked immunosorbent assay ( ELISA ) using a anti - of growth . Antibody was purified by passing the conditioned mouse kappa light chain antibody (Bethyl # A90 - 119A ) as media through a Protein - G column , washed and eluted with capturing antigen . Plates were coated with mouse kappa Glycine /HC1 100 mMPH 2 . 7 buffer . The eluate was dialyzed light chain antibody at 0 . 5 ug /well in PBS and 100 uL / well 60 against PBS before sterile filtration and stored at 4° C . of primary antibody was added to the plate . The plate was incubated at 37° C . for 1 h and washed five times with PBS Example 4 . 1 : Apparent Affinity of Antibodies containing 0 .05 % Tween - 20 (PBS - T ) . Then , 100 uL of a MAb1 and MAb2 to Human Primary Colon Tumor 1 :50 000 dilution of goat anti -mouse IgG (Fc ) conjugated PDX by Flow Cytometry with horseradish peroxidase ( Pierce # 31349 ) was added to 65 each well. Following incubation at 37° C . for 1 h in Advanced human primary colon tumor CR - IGR -034P darkness , plates were washed with PBS - T five times. Anti was obtained from Patient- derived xenograft in mice. Tumor US 9 ,809 , 653 B2 67 68 CR - IGR - 034P was enzymatically dissociated using collage TABLE 4 -continued nase Type IV ( Invitrogen ; # 17104 - 019 ) and deoxyribonu clease I (Invitrogen ; # 18047 -019 ) for 1 h at 4° C . Cell Antibody Binding Capacity by FACS on Patient - derived xenografts viability was estimated by Viacount application using Antibody Binding Guava? easyCyteTM 8HT Flow Cytometry System . For 5 Capacity (ABC ) apparent affinity estimation , CR - IGR -034P tumoral cells were coated at 40 , 000 cells /well on 96 -well High Bind plate MAD2 MAb1 (MSD L15XB -3 ) and 100 uL /well of antibody was added in Cell lines /origin 2 - fold serial dilutions starting at 20 ug /ml up to 12 dilutions Colo205 /colon 4 , 000 6 ,000 in assay diluent for 45 min at 4° C . and washed three times 10 SW480 / colon 1 , 700 2 ,500 with PBS 1 % BSA . 100 uL /well of goat anti -mouse IgG LS174T / colon 3 ,600 6 ,000 conjugated with Alexa647 ( Invitrogen ; # A2135 ) or goat anti- human IgG conjugated with Alexa488 ( Invitrogen ; The monoclonal antibodies MAb1 and MAb2 led to high # A11013 ) was added for 45 min at 4° C . and washed three 15 ABC in several PDXs of colorectal , ovary , stomach and lung times with PBS 1 % BSA . The antibody binding was evalu origin and lower ABC in cell lines than in PDXs of colon ated after centrifugation and resuspension of cells by adding origin . 200 ul /well PBS 1 % BSA and read using Guava® easy MAD3 Antibodies Bind to Multiple Cancer Cells CyteTM 8HT Flow Cytometry System . EC50 values were Advanced human primary tumors from colon (CR - IGR estimated using BIOST @ T -SPEED software . EC50 values 2 . 034P ), lung (LUN -NIC -014P and breast (BRE - IGR -0159 ) indications were obtained from patient - derived xenograft obtained with the advanced human primary colon tumor (PDX ) in mice as described in example 1 . PDXs were CR - IGR -034P are listet in Table 3. enzymatically dissociated using collagenase Type IV ( Invit rogen ; # 17104 -019 ) and deoxyribonuclease I ( Invitrogen , TABLE 3 # 18047 - 019 ) for 1 h at 4° C . Cell viability was estimated by ECso obtained with CR - IGR - 034P 25 Viacount application using Guava? easyCyteTM 8HT Flow Cytometry System . Tumoral cells were coated at 40 , 000 MAb1 MAD2 MAD3 cells /well on 96 - well High Bind plate (MSD L15XB -3 ) and 100 uL of antibody was added at 20 ug /mL for 45 min at 4° CR - IGR -034P 5 nM 14 nm 6nM C . and washed three times with PBS 1 % BSA . 100 uL of 30 goat anti - human IgG conjugated with Alexa488 ( Invitrogen ; Antibody binding capacity of antibody was determined # A11013 ) was added for 45 min at 4° C . and washed three using Mouse IgG Calibrator kit (Biocytex # 7208 ) or Human times with PBS 1 % BSA . The antibody binding was evalu IgG Calibrator Kit (Biocytex # CP010 ) according to the ated after centrifugation and resuspension of cells by adding manufacturer ' s instructions. Antibody binding capacityacity of 200 uL /well PBS 1 % BSA and read using Guava? easy 230 000 and 180 000 were measured for antibody MAb1 and 35 CyteTM 8HT Flow Cytometry System . The mean fluores MAb2 respectively on CR - IGR - 034P . cence was recorded and plotted in the graph shown in FIG . 12 to illustrate the expression profile of the three mAbs onto the three PDXs. Results presented in FIG . 12 show that Example 4 .2 : The Antibodies Bind to Multiple MAb3 binds to the different patient -derived xenografts from Cancer Cells 40 colon , lung and breast origin as Mab1 and Mab2 do . MAb1 and MAD2 Antibodies Bind to Multiple Cancer Example 4 . 3 : Internalization Score of MAbi, Cells and Determination of Antibody Binding Capacity MAb2 and MAb3 Following Binding to Colon Antibodies were found to be able of binding to multiple Colo205 Tumoral Cells Expressing LAMP1 by tumor cells by Flow Cytometry using the conditionswe A343 ImageStream Multispectral Imaging Flow described in example 4 .1 . The panel of tumor cells com Cytometer ( Amnis Corp . ) prises Patient- derived tumor xenografts from different ori Viable Colo205 cells (5x10 $ cells ) were seeded into wells gins and tumor cell lines . FIG . 2 illustrates the expression of 6 -well plates and incubated for 4 hours at 37° C . / 5 % CO2 profile and Table 4 summarizes the antibody binding capac 50 ( or 4° C . on ice for negative control) with 10 ug /ml of ity results . AlexaFluor488 - labeled antibody MAb1 or AlexaFluor488 labeled antibody M?b2 or AlexaFluor488 - labeled antibody TABLE 4 MAb3 . Cells were washed by centrifugation with PBS 1 % Antibody Binding Capacity by FACS on Patient -derived xenografts BSA at 400 g for 5 minutes . Cells were fixed and permea 55 bilized using 100 uL of Perm /Fix buffer on ice for 20 Antibody Binding minutes . Cells were washed by centrifugation with 1 mL of Capacity ( ABC ) Perm /Wash Cell buffer at 400 g for 5 minutes . MAD2 MAD1 To test whether internalized antibodies accumulate in lysosomes, simultaneous uptake of mAbs and Alex PDX /origin – 60 aFluor647 - labeled CD107a (a lysosomal marker) were car CR - LRB - 003P / colorectal 22 000 25 000 ried out. Labelled AlexaFluor647 anti -CD107a antibody at CR - LRB - 010P / colorectal 95 , 000 140 ,000 10 ug /mL was incubated on ice for 20 minutes . After CR - IGR - 034P / colorectal 180 ,000 230 ,000 incubation , 1 mL Perm /Wash Cell buffer was added to wash , OVA - IGR - 0022 / ovary 60 ,000 67 ,000 before centrifuging ( 400 g , 5 min ) . The supernatant was STO - IND - 006 / stomach 64, 000 90 , 000 LUN- NIC - 025 /lung 27 ,000 33 , 000 65 flicked from the plate before the cells were fixed with 200 uL LUN- NIC - 014/ lung 102 ,000 104 ,000 1 % formaldehyde on ice for 20 minutes . The fluorescence of cells was analyzed with the ImageStream multispectral US 9 ,809 , 653 B2 69 70 imaging flow cytometer ( Amnis corp .) using the Internal Our results show for the first time that LAMP1 can ization feature . Five thousand events were acquired for each function as a receptor mediating the internalization of anti experimental condition and the corresponding images were bodies very efficiently via receptor recycling to the cell surface and suggest that the availability of specific internal analyzed using the IDEAS image- analysis software . 5 izing antibodies should aid in developing novel therapeutic methods to target toxins, drugs or short- range isotopes to be TABLE 5 delivered specifically to the interior of the cancer cells , as Internalization score by Fluorescence- Based shown in Table 6 . ImageStream Imaging Flow Cytometer Internalization score (IS ) Internalization score ( IS ) TABLE 6 mAb 4° C . , 4 hr 37° C . , 4 hr Internalization measurements by Flow Cytometry MAb1 0 . 22 2 . 22 MAD2 0 . 19 2 . 24 4° C . 37° C . MAb3 0 . 11 1 . 56 mAb 4° C . Quencher 37° C . Quencher MFI, Alexa488 -MAbi 16 . 89 4 .46 172 . 14 167. 08 The monoclonal antibodies MAb1 MAb2 and MAb3 led MFI, Alexa488 - Transferrin 35. 78 8 .98 1228 1210 to high internalization scores in Colo205 cell line as shown in Table 5 . Example 4 .5 : Purification and Identification of the Example 4 . 4 : Quenching of Alexa488 by Use of 2020 MAb1, MAb2 and MAb3 Antibody Antigen Target the Anti - Alexa488 Antibody, Flow Cytometry and The antigen target of MAb1, MAb2 and MAb3 are Calculation of Internalized Fraction of MAb1 purified from a membrane fraction enriched by human primary colon tumor CR - LRB - 010P or CR - IGR -034P using Alexa488 - labelled MAb1 (66 nM ) was incubated with 25 Pierce Classic IP Kit ( # 26146 ) according to the manufac 6x10 $ Colo205 cells in complete medium for 4 h at 37° C . - turer 's instructions . or 4° C . The cells were washed twice in ice cold PBS in a Pulled - down proteins were separated by SDS - PAGE and cold centrifuge , and resuspended in 500 nM quenching proteins stained with silver nitrate . Stained bands were anti - Alexa488 antibody diluted in ice cold PBS . All tubes submitted to an in - gel tryptic digestion , and eluted peptides were incubated for 1 h on ice. Without washing, all cells 30 were analyzed by tandem MS (LC -MS / MS ) on an Orbitrap were fixed in two volumes of 2 % paraformaldehyde for 10 bentchtop mass spectrometer ( Thermo) . Raw MS /MS data min at room temperature . The paraformaldehyde was analysis with Mascot (Matrix Science ) database search removed by one wash in PBS, and the cells were resus engine, revealed LAMP1. pended in PBS and analyzed in a flow cytometer (Guava? This target was confirmed by ELISA with the recombi easyCyte 8HT Flow Cytometry System ). nant human LAMP1 as described in example 6 .2 (SEQ ID An internalization positive control experiment was done 35» NO : 28 ) . The obtained EC5o are listed in Table 7 and Table in parallel with Alexa488 - labelled Transferrin (600 nM ) . 11 . Mean fluorescence intensity (MFI ) values obtained from the flow cytometry reading of 5x104 cells per tube were used TABLE 7 for all calculations. Internalization was calculated as the MFI value of quenched cells (intracellular compartments EC5o determined by ELISA values on recombinant only ) divided by the MFI value of unquenched cells (both human LAMP1 ( 29 - 382 of SEO ID NO : 28 ) cell surface and intracellular compartments ) at 37° C . as Antibody EC50 described in the formula : MAb1 0 . 18 nM 45 MAD2 0 . 25 nM FL of quenched cells at 37° C . Percentage of iternalized fraction-: FL- of unquenched cells at 37° C* . *x^ 10010 Example 4 .6 : Specificity to LAMP1 The cells incubated with Alexa488 - labelled compounds at 50 LAMP2 is the closest member of the LAMP family with 4° C . were used as a control since internalization of anti - 35 % sequence identity to LAMP1. For evaluating specificity bodies does not take place significantly at 4° C . to LAMP1 ofMAb1 , MAD2 and MAb3 antibodies, 96 -well After 4 h at 37° C ., about 97 .0 % of the total cell plates were coated with recombinant human LAMP2 with a fluorescence from Alexa488 -MAb1 was intracellular. By C -terminal 10 His - tag (SEQ ID NO : 40 ) ( R & D Systems comparison , about 98 . 5 % of the total cell fluorescence from 55 6228 -LM ) using the same coating conditions described Alexa488 - Transferrin was intracellular . Transferrin is previously . Anti -LAMP1 antibodies were added to the plates known to be internalized very efficiently by Colo205 cells . and detected by using rabbit anti -mouse IgG conjugated After quenching , the fluorescence of Alexa488 -MAb1 with horseradish peroxidase (Sigma ; # A9044 ) . Antibody measured from cells labelled at 37° C . (both cell surface and binding was visualized by adding TMB - H , O , buffer and intracellular compartments ) was 10 - fold higher than that of 60 read at a wavelength of 450 nm . No binding to LAMP2 was cells labelled at 4° C . ( cell surface ). Because the fluores detected with MAb1 , MAb2 and MAb3 antibodies . cence of Alexa488 -MAb1 measured at cell surface at 4° C . is proportional to the antigen density , all the above results Example 4 .7 : Cross - Reactivity with Cynomolgus taken together indicate that each LAMP1 molecule is Monkey LAMP1 involved in several ( 10 on average ) internalization cycles via 65 recycling at cell membrane during the course of the experi Antibody MAb1 was assessed for its ability to bind ment. primate LAMP1 protein by ELISA . Extracellular domain of US 9 ,809 ,653 B2 71 72 LAMP1 of human ( Ala29 -Met382 of SEQ ID NO : 24 ) and ware . Antibody MAb1 binds to human and cynomolgus cynomolgus monkey LAMP1 ( Ala27 -Met380 of SEQ ID LAMP1 expressed at the cell surface of recombinant NO : 39 ) were prepared as described in example 6 . 2 . Plate HCT116 with similar affinity and EC50 of 4 . 9 and 5 . 5 nM was coated with cynomolgus monkey LAMP1 protein (SEQ respectively . ID NO : 29 ) , antibody M?bl was added to the plate and 5 Antibody M?b2 was assessed for its ability to bind detected with rabbit anti -mouse IgG conjugated with horse human LAMP1 and primate LAMP1 proteins expressed at radish peroxidase (Sigma ; # A9044 ) . The antibody binding the surface of recombinant HCT116 stable clones by FACS . Recombinant HCT116 cells were coated at 40 , 000 cells /well was visualized by adding TMB - H , 0 , buffer and read at a on 96 -well High Bind plate (MSD L15XB - 3 ) and 100 wavelength of 450 nm . Binding affinity was in the same UL /well of antibody M?b2 was added in 2 - fold serial range with both proteins as shown on FIG . 3 for MAbl. 10 dilutions starting at 20 ug /ml up to 12 dilutions in assay Antibody MAB1 was also assessed for its ability to bind diluent for 45 min at 4° C . and washed three times with PBS human LAMP1 and primate LAMP1 proteins expressed at 1 % BSA . 100 uL /well of goat anti -mouse IgG conjugated the surface of recombinant HEK293 cells by FACS. LAMP1 with Alexa647 ( Invitrogen ; # A2135 ) was added for 45 min Coding DNA Sequence, RefSeq NM _ 005561. 3 (SEQ ID at 4° C . and washed three times with PBS 1 % BSA . The NO : 23 ) was cloned internally . The CDS ofMacaca mulatta 15 antibody binding was evaluated after centrifugation and LAMP1, RefSeq XP _ 001087801 (SEQ ID NO : 27 ) was also re - suspension of cells by adding 200 ul/ well PBS 1 % BSA cloned internally . The predicted sequences of mature and read using Guava® easyCyteTM 8HT Flow Cytometry LAMP1 from Macaca mulatta and Macaca fascicularis are System . EC50 values were estimated using BIOST @ T identical to 99 % , said sequence differing by one additional SPEED software . Antibody M?b2 binds to human and Leucin at position 11 of Macaca mulatta (SEQ ID NO : 27 ) , 20 cynomolgus LAMP1 expressed at the cell surface of recom i . e . in the signal peptide . The mature LAMP1 proteins of binant HCT116 with similar affinity and EC of 6 . 3 and 6 .6 Macaca mulatta and Macaca fascicularis are identical. nM respectively for MAb2. Therefore the secreted LAMP1 used in the following Therefore MAb1 and MAb2 bind to LAMP1 of human example is referred to cynomolgus monkey . Both CDS were and cynomolgus origin with similar affinity . cloned into mammalian expression plasmids under CMV 25 Antibody M?b3 was assessed by flow cytometry for its ability to bind to human LAMP1 and primate LAMP1 enhancer/ promoter and SV40 polyA signals . HEK293 cells proteins expressed respectively at the surface of HCT116 or ( Invitrogen ; # K9000 - 10 . ) were transiently transfected with HEK293 stable clones . HCT116 stable clone was obtained human LAMP1 or cynomolgus LAMP1 plasmids using as described above . HEK293 cells were infected by a FreeStyleTM MAX 293 Expression System according to the lentiviral vector allowing stable integration of the human or manufacturer ' s instructions . Human LAMP1 transfected 30 the cynomolgus LAMP1 CDS in genomic DNA of cells . HEK293 cells and cynomolgus LAMP1 transfected Individual clones with different densities of cynomolgus HEK293 cells were coated at 40 ,000 cells /well on 96 -well LAMP1 cell surface localization were derived from a pool High Bind plate (MSD L15XB - 3 ) and 100 uL /well of of HEK293 infected cells . Protocol as described in example above . EC50 values were estimated using BIOST @ T antibody MAb1 was added in 2 - fold serial dilutions starting SPEED software . Antibody M?b3 binds to human and at 20 ug /ml up to 12 dilutions in assay diluent for 45 min at 35 cynomolgus LAMP1 expressed at the surface of HCT116 or 4° C . and washed three times with PBS 1 % BSA . 100 uL /well of goat anti -mouse IgG conjugated with Alexa647 respectivelyHEK293 with . similar affinity and EC50 of 7 .6 and 4. 0 nM ( Invitrogen ; # A2135 ) was added for 45 min at 4° C . and Therefore MAD1 , MAD2 and MAb3 bind to LAMP1 of washed three times with PBS 1 % BSA . The antibody human and cynomolgus origin with similar affinity . binding was evaluated after centrifugation and resuspension 40 of cells by adding 200 ul/ well PBS 1 % BSA and read using Example 4 . 8 : Binding Competition Between MAD Guava? easyCyteTM 8HT Flow Cytometry System . EC50 According to the Invention and / or Commercially values were estimated using BIOST @ T -SPEED software . Available Anti -LAMP1 H4A3 Binding affinity was in the same range with EC 50 of 14 and 44 nM to respectively human and cynomlogus monkey 45. The following examples present information on the com LAMP1 expressed transiently at the cell surface of HEK293 petition of the mAbs towards the epitope onto LAMP1 by ELISA . It confirmed data obtained on the epitope binding for MAb1. site as described in example 6 and allowed the comparison Antibody MAb1 was assessed for its ability to bind with a commercially available anti -LAMP1 mAb . human LAMP1 and primate LAMP1 proteins expressed at Binding Competition Between MAb1 and MAB2 the surface of recombinant HCT116 stable clones by FACS. 50 Competition between MAD2 (murine ) and MAb1 (chime HCT116 cells were infected by a lentiviral vector allowing ric ) for binding to LAMP1 was assayed by ELISA and is stable integration of the human or the cynomolgus LAMP1 illustrated on FIG . 4 . No competition was observed . CDS in genomic DNA of cells . Individual clones with Binding Competition Between MAbl and MAW2 or MAb3 different densities of human or cynomolgus LAMP1 cell Competition experiments between two anti -LAMP1 surface localization were derived from a pool of HCT116 55 mAbs were performed by ELISA with recombinant human infected cells . HCT116 cells expressing human or cynomol LAMP1 coated on plate (as described in example 6 . 2 ). gus LAMP1 were plated in 96 -well plates at 200 000 per Briefly , two mAbs were added simultaneously at concentra tions of 0 . 06 and 15 mg/ L , the concentration of 0 .06 mg /L well and MAbl was added in 2 - fold serial dilutions starting being close to the EC 50 . MAb format was chosen so that the at 40 ug /ml up to 12 dilutions in assay diluant for 1 h at 4° two mAbs had different Fc domains ( either human or C . and washed two times with PBS 1 % BSA . 100 uL / well 60 murine ) . Individual measurements of mAb binding could be of goat anti -human IgG conjugated with Alexa488 (Invitro performed specifically by their unique specific binding to Fc gen ; # A11013 ) was added for 1 h at 4° C . and washed two (with Peroxidase - AffiniPure Goat Anti -Human IgG Ab , Fcy times with PBS 1 % BSA . The antibody binding was evalu - Fragment SpecificSnecific ( Jacksonlackson 109 -- 035 - 098 )) or withwith Peroxi ated after centrifugation and resuspension of cells in 100 ul dase - AffiniPure Goat Anti -Mouse Igg Ab , Fcy Fragment fixing solution (paraformaldehyde at 4 % in PBS ) . Samples 65 Specific (Jackson 115 - 035 - 164 ) ) . Results were reported as a were read using Galaxy® Flow Cytometry System (Partec ) . percentage of the value obtained from the mAb alone at the EC50 values were estimated using BIOST @ T - SPEED soft same concentration , see Table 8 . US 9, 809, 653 B2 73 74 TABLE 8 Competition between chMAb1 and MAb2 or MAb3 Percentage of signal compared to Added mAb mAb control Sample ID mAbs concentration Secondary Ab alone chMAV1 + 0 .06 mg/ L Anti -Human IgG _ HRP 80 % MAB2 15 mg/ L chMAb1 + 0 .06 mg/ L Anti -Mouse IgG _ HRP 100 % MAD2 15 mg/ L chMAb1 0 .06 mg/ L Anti - Human IgG _ HRP 100 % chMAbi 0 .06 mg/ L Anti -Mouse IgG _ HRP 0 % MAD2 15 mg/ L Anti- Human IgG _ HRP 0 % MAD2 15 mg / L Anti -Mouse IgG _ HRP 100 % w chMAb1 + 0 .06 mg / L Anti - Human IgG _ HRP 80 % MAb3 15 mg / L A chMAbl + 0 .06 mg / L Anti -Mouse IgG _ HRP 90 % MAb3 15 mg/ L MAb3 15 mg/ L Anti - Human IgG _ HRP 0 % MAb3 15 mg/ L Anti -Mouse IgG _ HRP 100 % a chMAb1 + 0 .06 mg / L Anti- Human IgG _ HRP 10 % MAb1 15 mg/ L a chMAb1 + 0 .06 mg / L Anti -Mouse IgG _ HRP 90 % MAb1 15 mg/ L MAb1 15 mg / L Anti- Human IgG _ HRP 0 % MAb1 15 mg/ L Anti -Mouse IgG HRP 100 %

It was found thatMAb1 does not compete with MAb2 or MAD3 . Therefore the LAMP1 epitope binding site for MAb1 does not overlap with the epitope binding sites for MAW2 or MAD3 . Binding Competition Between H4A3 and MAbl or MAB230 or MAb3 and Between MAb2 and MAb3 Competition experiments between anti- LAMP1 H4A3 ( BioLegend 328602 ) and MAbi , Mab2 , or MAb3 and between MAb2 and MAb3 were performed as described in above Example B4 .81 Results were reported as a percentage 33 of the value obtained from the mAb alone at the same concentration , see Table 9 . TABLE 9 Competition between H4A3 and chMAbl or chMAW2 or chMAb3 Percentage of signal compared to mAb control Sample ID Added mAbs mAb concentration Secondary Ab alone H4A3 + 0 .06 mg/ L Anti- Human IgG _ HRP 96 % chMAb1 15 mg/ L H4A3 + 0 .06 mg/ L Anti- Mouse IgG _ HRP 98 % chMAb1 15 mg/ L chMAb1 15 mg/ L Anti - Human IgG _ HRP 100 % chMAb1 15 mg/ L Anti- Mouse IgG _ HRP 0 % H4A3 0 .06 mg/ L Anti -Human IgG _ HRP 0 % H4A3 0 .06 mg/ L Anti- Mouse IgG _ HRP 100 % MAb1 + 0 .06 mg/ L Anti- Human IgG _ HRP 96 % chMAb1 15 mg/ L MAbl + 0 .06 mg/ L Anti -Mouse IgG _HRP 28 % chMAb1 15 mg/ L MAb1 0 .06 mg/ L Anti- Human IgG _ HRP 0 % MAb1 0 .06 mg/ L Anti -Mouse IgG _ HRP 100 % 2 H4A3 + 0 .06 mg/ L Anti -Human IgG _ HRP 100 % chMAD2 15 mg/ L H4A3 + 0 .06 mg/ L Anti- Mouse IgG _ HRP 57 % chMAD2 15 mg/ L chMA62 15 mg/ L Anti- Human IgG _ HRP 100 % chMAD2 15 mg/ L Anti- Mouse IgG _ HRP 0 % MAD2 + 0 .06 mg/ L Anti - Human IgG _ HRP 100 % chMAD2 15 mg/ L US 9 ,809 , 653 B2 75 TABLE 9 -continued Competition between H4A3 and chMAbl or chMAW2 or chMAb3 Percentage of signal compared to mAb control Sample ID Added mAbs mAb concentration Secondary Ab alone MAD2 + 0 .06 mg/ L Anti- Mouse IgG _ HRP 9 % chMAD2 15 mg/ L MAD2 0 .06 mg/ L Anti -Human IgG _ HRP 0 % MA62 0 .06 mg/ L Anti -Mouse IgG _ HRP 100 % ??? H4A3 + 0 .06 mg/ L Anti - Human IgG _ HRP 100 % chMAb3 15 mg/ L H4A3 + 0 .06 mg/ L Anti- Mouse IgG _ HRP 11 % chMAb3 15 mg/ L chMAb3 15 mg/ L Anti - Human IgG _ HRP 100 % chMAb3 15 mg/ L Anti -Mouse IgG _ HRP 0 % MAb3 + 0 .06 mg/ L Anti- Human IgG _ HRP 100 % chMAb3 15 mg/ L MAb3 + 0 .06 mg/ L Anti- Mouse IgG _HRP 15 % chMAb3 15 mg/ L MAD3 0 .06 mg/ L Anti - Human IgG _ HRP 0 % MAD3 0 .06 mg/ L Anti -Mouse IgG _ HRP 100 % MAb3 + 0 .06 mg/ L Anti -Human IgG _ HRP 99 % chMAD2 15 mg/ L MAb3 + 0 .06 mg/ L Anti -Mouse IgG _HRP 58 % chMAD2 15 mg/ L MAb3 0 .06 mg/ L Anti - Human IgG _ HRP 0 % MAb3 0 .06 mg/ L Anti- Mouse IgG _ HRP 100 %

It was found that H4A3 competes with MAB3 , partially 30 were dehydrated and coverslipped with Coverquick 2000 competes with Mab2 and does not compete with MAb1 for mounting medium (Labonord , ref 05547530 ) . The negative binding to LAMP1 . controls used in this study consisted in omission of primary It was found that MAb2 and MAb3 partially compete for antibody and the use of IgG2a isotype ( final concentration 1 binding to LAMP1 . ug/ mL in PBS ). 35 Immunostaining on PFA Format Example 5 : Immunohistochemistry ( IHC ) Classical IHC was performed using Ventana automatic Characterization of Purified MAbl on Human instrument (Discovery XT, Ventana Medical Systems, Inc , Non - Tumoral and Tumoral Tissues USA ). Sections were dewaxed and antigen retrieval Cell Conditioning 1 ( CC1) buffer (ref 950 - 123 Ventana ) was The monoclonal antibody M?bl was purified for further 40 applied during 52 min . The sections were incubated with evaluation and antibody validation by extensive IHC char - avidin and biotin blocking reagent ( Endogenous Block , acterization on non - tumoral and tumoral tissues . Therefore a Ventana , 760 - 050 ) and Serum Block reagent (Ventana , 760 large panel of human non - tumoral and tumoral tissues from 4212 ) . The murine monoclonal antibody MAb1 was then commercial Tissue -Micro -Arrays or whole cryostat sections incubated at final concentration of 4 ug /mL during 1 hour at was tested for LAMP1 immunoreactivity either as Frozen - 45 37° C . A post- fixation step with glutaraldehyde ( 0 .05 % in OCTs (Optimal Cutting Temperature ) or Acetic Formalin NaCl 0 . 9 % w / v ) during 4 min was done. The secondary goat Alcohol (AFA ) or formalin patient- derived human xeno anti -mouse IgG2a -biotinilated was incubated for 12 min at grafts . The PDXs samples used were described in example 37° C . (Southern Biotech , Ref 1080 -08 , and dilution 1/ 200 in 1 . Ventana ' s diluent ) . Immunostaining was done with DAB Immunostaining on AFA Format 50 Map chromogenic detection kit according to manufacturer ' s Classical IHC was performed using Ventana automatic recommendations . A counterstaining step was applied to the instrument (Discovery XT, Ventana Medical Systems, Inc , cryostat sections with hematoxylin II (790 -2208 , Ventana USA ). Sections were dewaxed and incubated with avidin Medical Systems, Inc USA ) and bluing reagent was applied and biotin blocking reagent ( Endogenous Block , Ventana , for 4 min ( 760 - 2037 ) . Stained slides were dehydrated and 760 -050 ) followed by Serum Block incubation ( Ventana 55 coverslipped with Coverquick 2000 mounting medium ( La 760 - 4212 ) . The murine monoclonal antibody M?bl was bonord , ref 05547530 ) . The negative controls used in this then incubated at final concentration of 4 ug /mL during 1 study consisted in omission of primary antibody and the use hour at 37° C . A post - fixation step with glutaraldehyde of IgG2a isotype ( final concentration 1 ug /mL in PBS ) . ( 0 .05 % in NaCl 0 . 9 % w / v ) during 4 min was done . The Immunostaining on Frozen - OCT Format secondary goat anti- mouse IgG2a -biotinilated was incu - 60 After avidin and biotin blocking (Endogenous Block , bated for 12 min at 37° C . (Southern Biotech , Ref 1080 - 08 , Ventana, 760 - 050 ), frozen sections were incubated with and dilution 1/ 200 in Ventana ' s diluent ) . Immunostaining was murine monoclonal antibody MAb1 ( final concentration 1 done with DAB Map chromogenic detection kit according to ug /mL ( for human samples ) and 1 and 5 ug/ mL ( for monkey manufacturer ' s recommendations . A counterstaining step samples ) in Phosphate Buffer Saline, PBS ) for 32 min at 37° was applied to the cryostat sections with hematoxylin II 65 C . A postfixation step with glutaraldehyde ( 0 .05 % in NaCl (790 - 2208, Ventana Medical Systems, Inc USA ) and bluing 0 . 9 % w / v ) for 4 min was done . The secondary goat anti reagent was applied for 4 min ( 760 - 2037 ) . Stained slides mouse IgG2a -biotinylated was incubated for 12 min at 37° US 9 ,809 , 653 B2 77 78 C . ( Southern Biotech , Ref 1080 -08 , dilution 1 /200 in Venta Basic descriptive statistics were calculated with Microsoft na ' s diluent) . Immunostaining was done with DAB Map Excel 2003 . For each indication , number of cases, positive chromogenic detection kit according to manufacturers rec ommendations. A couterstaining step was applied to the cases number, prevalence , intensity score mean , frequency cryostat sections with hematoxylin II ( 790 - 2208 . Ventana 5 mean and Allred score were described . Medical Systems, Inc USA ) and bluing reagentwas applied Non - Tumoral Tissue Distribution for 4 min (760 - 2037 ). Stained slides were dehydrated and coverslipped with Coverquick 2000 mounting medium (La Globally , the experimental data show that the IHC pattern bonord , Ref 05547530 ) . of LAMP1 on cells of non - tumoral adult tissues is predomi The negative controls used in this study consisted in 10 nantly cytoplasmic . omission of primary antibody and the use of IgG2a isotype LAMP1 was expressed in the of a large panel ( final concentration 1 ug /mL in PBS) . of tissues , including vital organs, gastrointestinal, reproduc Data Analysis Sections immunostained with purified murine antibody tive , urinary, endocrine, lymphoid and others as skin , MAb1 were scanned and digitized at a magnification of x20 15 muscle , eye , spinal cord ) and no membrane staining was using Scan Scope XT system ( Aperio Technologies , Vista observed in main organs as heart , liver , pancreas , lung and Calif .) . Digitized images were then captured using Image kidney. Scope software ( v10 . 2 . 2 . 2319 Aperio , Technologies ). Staining evaluation included several parameters: histo - However, some LAMP1 expression at the membrane logic site of reactivity (cytoplasm . nuclei or membrane ), 20 occurred but was restricted to stomach epithelial cells , main type of reactive cell, staining intensity and cell staining oesophageal epithelial cells , breast epithelial cells , prostate frequency. The positive samples were scored with a scale of epithelial cells , testicular epithelial cells ( Table 10 ) . intensity from 1 to 3 . Ranges of intensities were described Nevertheless , prevalence and mean intensities for as negative ( 0 ) , weak ( 1 ), moderate ( 2 ) and strong ( 3 ) . Cell LAMP1 expression at the membrane of non - tumoral frequency was the percentage of immunostained cells and samples were lower than those found in tumours . TABLE 10 LAMP1 immunostaining in human non - tumoral samples - Membrane pattern Non tumoral tissues % Prev Intensity % + cells Prev % Prv Prev Memb Memb Memb Tissue Type N Cyto Cyto Memb (Mean ) (Mean ) (Mean ) Cell type Stomach 28 28 / 28 100 % 3 /28 11 % 16 Epithelial C . Esophagus 17 16 /17 94 % 2 / 17 12 % 5 Epithelial Basal C . Breast 17 17 / 17 100 % 6 / 17 35 % Epithelial C . Prostate 26 26 / 26 100 % 1 / 26 4 % Epithelial C . Testis 14 14 / 14 100 % 5 / 14 36 % Germinal + BE Euro Leyding was estimated by the histologist observation as a median by Tumoral Tissue Distribution sample . The cell frequency was ordered in 5 categories : 1. The immunohistochemical pattern using MAb1 or MAD2 ( 0 - 5 % ), 2 (6 -25 % ), 3 ( 26 - 50 % ) , 4 (51 -75 % ) and 5 ( 76 - in human tumoral tissues demonstrates that the antigen is 100 % ) . 45 located in the cytoplasm and /or membrane of tumoral tis A global expression was calculated according the Allred sues. Protein expression data for human tumoral samples Score ( AS ) description . AS was obtained by adding the displaying the membrane pattern show that LAMP1 antigen intensity and the proportion scores to obtain a total score that is not restricted to colon adenocarcinomas . A variety of other ranged from 0 - 8 . The AS was reported as a percent of the carcinomas, including gastrointestinal tumors ( small intes maximum global score and ranged in 5 categories: very low 50 tine, rectum , parotid gland ), vital organs tumors (lung , liver, ( 0 -25 % ) , weak ( 26 - 50 % ) , moderate (51 - 75 % ) and high stomach , pancreas and kidney ) , reproductive organ tumors (75 - 100 % ) . The prevalence was defined as the percent of (breast , ovary and prostate ) as well as skin , larynx and soft positive cases for the indication . tissue tumors ( Table 11 ) . TABLE 11 LAMP1 immunostaining in human tumoral samples : Membrane pattern TUMORAL TISSUES Intensity % + Cells Prev % Prev Memb Memb Alred 1 - 6 - 26 - 51- 76 Organ Tumor Type N Memb Memb (Mean ) (Mean ) Score Neg 5 % 25 % 50 % 75 % 100 % > 50 % Colon Adenocarcinoma 86 38 / 86 44 2 . 5 69 56 % 17 % 16 % 2 % 7 % 9 % Small Adenocarcinoma 1 11 / // 1 100 3 . . 0 0 30 75 100 % Intestine Rectum Adenocarcinoma 14 9 / 14 64 0 3. 2 1 63 36 % 21 % 14 % 21 % 7 % Parotid Adenocarcinoma 3 2 / 3 67 2. 0 18 50 33 % 33 % 33 % Gland US 9, 809, 653 B2 79 80 TABLE 11 -continued LAMP1 immunostaining in human tumoral samples : Membrane pattern TUMORAL TISSUES Intensity % + Cells Prev % Prev Memb Memb Alred 1 - 6 - 26 - 51 - 76 Organ Tumor Type N Memb Memb (Mean ) (Mean ) Score Neg 5 % 25 % 50 % 75 % 100 % > 50 % Lung Squamous Cell 29 6 / 29 21 2 . 5 31 69 79 % 3 % 10 % 3 % 3 % 6 % Carc Adenocarcinoma 12 4 / 12 33 2 .5 26 69 67 % 8 % 8 % 17 % Liver Hepatocellular 2 1 / 2 2 50250 38 50 % 50 % Carc Pancreas Adenocarcinoma 1 / 18 10 50 94 % 6 % Kidney Clear Cell Carc 9 1 / 9 11 89 % 11 % Breast InvDucCar 27 /70 MMM??? 61 % 13 % 9 % 7 % 7 % 14 % InvLobCar 2 / 3 ?= 8 33 % 33 % 33 % 33 % Ovary Adenocarcinoma 5 /21 15 76 % 5 % 14 % Serous Carcinoma 1 / 6 50 83 % 17 % Prostate Adenocarcinoma aw 4 / 16 43 75 75 % 19 % 6 % 6 % Stomach Adenocarcinoma wNa 8 / 32 45 NA 75 % 3 % 9 % 9 % 3 % 13 % Skin Squamous Cell 6 1/ / 6 17 M??owo 10 63 83 % 17 % Carc Malignant 4 11 / / 4 25 2. 0 75 % 25 % Melanoma Larynx Squamous Cell 1 / 5 2 . 0 u 38 80 % 20 % Carc Soft Giant cell tumor of 51 / 2 50 3 . 0 u 50 50 % 50 % Tissue thigh Na

Tumor indications were ranked in terms of LAMP1 sylation sites . It is composed of two lumenal domains expression level based on the percentage of samples dis separated by a hinge . Each lumenal domain has two disul playing more than 50 % of membrane frequency ( positive 30 cells ) . phide bridges that define two loops. According to RefSeq Based on this parameter the first tumor indications were NP_ 005552. 3 (SEQ ID NO : 24 ) the different domains of colon , rectum , lung squamous cell carcinoma, breast inva LAMP1 have been mapped as shown in Table 1 . Based on sive ductal and lobular carcinoma, stomach adenocarcinoma and prostate adenocarcinoma . structural information and in particular beta -strands and Additionally , indications displaying 25 -50 % of positive amino acids differences between human and mouse LAMP1 cells at the membrane , could be also considered as relevant several hybrid LAMP1 molecules were designed . indications, including small intestine adenocarcinoma, parotid gland adenocarcinoma, lung adenocarcinoma, ovary 40 Example 6 .2 : Preparation of Recombinant adenocarcinoma, skin malignant melanoma and larynx Extracellular Domains of LAMP1 Proteins squamous cell carcinoma ( Table 11) . Moreover , LAMP1 immunostaining was not detected at The high level of glycosylation of the antigen required a the membrane in the following tumor indications : Lung specific approach to determine the binding site of the small cell carcinoma ( 0 / 3 ) , esophagus squamous cell carci- 45 noma ( 0 / 11 ) , cervix squamous cell carcinoma 0 / 3 ), endo anti -LAMP1 mAbs on LAMP1. The LAMP1 monoclonal metrium adenocarcinoma (0 / 3 ) , vulva squamous cell carci antibodies MAb1 and MAb2 do not show any binding to the noma ( 0 /6 ) , testis seminoma ( 0 / 4 ) , testis embryonal mouse LAMP1 protein . This absence of binding was used to carcinoma (0 / 1 ) , bladder transitional cell carcinoma (0 / 1 ) , design several chimeric LAMP1 proteins in which one or thyroid papillary adenocarcinoma ( 0 /3 ) and mullerian mixed 50 several of the LAMP1 domains (Loop1 - Loop4 ) in the tumor of the oral cavity ( 0 / 5 ) . human construct were replaced by the murine counterpart . The absence of binding once the binding site of the antibody Example 6 — Binding Site Identification was replaced by the murine counterpart allowed for identi In this example LAMP1 domains were defined and 55 fication of the antibody binding side . human -murine hybrid LAMP1 proteins were designed to Hence , the extracellular protein domains of LAMP1 from generate secreted as well as membrane -anchored LAMP1 human , cynomolgus monkey (c ) and murine ( m ) origin or proteins allowing the characterization of the binding site of hybrid between murine and human LAMP1 domains have the anti- LAMP1 mAbs towards LAMP1. been prepared by transient expression in human embryonic " kidney HEK293 cells with plasmids allowing expression of Example 6 . 1 : Definition of LAMP1 Domains the respective cDNA as outlined on Table 12. LAMP1 also named CD107a is the Lysosomal Associated Each expression plasmid was complexed with 293Fec tinTM ( Life Technologies ) and eight days post - transfection in Membrane Protein 1 . It is is a transmembrane type I protein 65 suspension - cultivated 293 -F cells (derived from HEK293 of around 120 kDa. The protein is a highly glycosylated cells ), the corresponding soluble protein was purified by monomer with eighteen N -glycosylation and six O -glyco IMAC (GE Healthcare ) to generate a protein batch . US 9 ,809 ,653 B2 81 TABLE 12 Description of the recombinant extracellular domains of LAMP1 proteins Protein name Description of protein domains Sequence ID . LAMP1: : histag human LAMP1 (29 -382 ) SEQ ID NO : 28 CLAMP1: : histag cynomolgus LAMP1 ( 27 - 380 ) SEQ ID NO : 29 mLAMP1 _ L1_ LAMP1 _ L234 : :histag Loopl : mouse LAMP1 (25 - 94 ) SEQ ID NO : 30 Loop2- 4 : human LAMP1 ( 101- 382) mLAMP1 L12 LAMP1 L34 : : histag Loop1 - 2 : mouse LAMP1 (25 - 189 ) SEQ ID NO : 31 Loop3 - 4 : human LAMP1 ( 196 - 382 ) LAMP1_ L12 _mLAMP1 _ L34 :: histag Loop1- 2 : human LAMP1 ( 29 - 195 ) SEQ ID NO : 32 Loop3 - 4 : mouse LAMP1 (190 - 369) LAMP1 _ L123 _mLAMP1 _ L4 : : histag Loop1 - 3 : human LAMP1 (29 -309 ) SEQ ID NO : 33 Loop4 : mouseLAMP1 (299 - 369 ) mLAMP1: : histag mouse LAMP1 (25 - 369 ) SEQ ID NO : 343

Example 6 . 3 : Determination of Binding Affinity and Epitope by ELISA Secreted LAMP1 proteins described in example 6 .2 were 20 used to identify the binding domain to anti -LAMP1 mAbs by ELISA . MAb1 recognizes loop 2 of LAMP1 and MAD2 recognizes loop 1 of LAMP1 with EC : n to LAMP1 of around 0 .2 and 0 .3 nM respectively . TABLE 13 ECs (nm ) obtained for murine or chimeric hybridoma mAbs Protein Loopl : Loop1 - 2 : Loop1 - 2 : Loop1- 3 : mLAMP1 mLAMP1 hLAMP1 hLAMP1 Loop2 - 4 : Loop3 - 4 : Loop3 - 4 : Loop4 : Antibody human LAMP1 Mouse LAMP1 hLAMP1 hLAMP1 mLAMP1 mLAMP1 MAb1 0 .18 No binding 0 .15 No binding 0 . 18 0 . 16 MA62 0 . 25 No binding No binding No binding 0 .25 0 . 25 chMAb1 0 . 12 No binding 0 . 11 No binding 0 . 11 0 . 11 chMAD3 0 . 11 No binding No binding No binding 0 .12 0 . 11

Example 6 . 4 : Expression of LAMP1 40 Mammalian plasmids had similar expression signals as Transmembrane Proteins plasmids used to produce recombinant LAMP1 described in Different LAMP1 proteins were expressed at the cell example 6 . 2 . Table 14 below lists all the plasmids that were membrane of HEK293 cells after transient expression from designed in order to confirm the results obtained with mammalian plasmids encoding the entire coding sequence 45 soluble LAMP1 protein by ELISA in example 6 . 3 and of LAMP1 deleted of the intracellular lysosome- targeting further characterize the binding domains of the anti- LAMP1 motif GYQTI and substituted by a 5 - Ala repeat sequence . mAbs . TABLE 14 Description of LAMP1 transmembrane proteins Short description of LAMP1 transmembrane protein / Plasmid Encoded Protein with amino acid positions according to SEQ ID NO : 24 PXL5626 hLAMP1_ AGYQTI human LAMP1 pXL5668 LAMP1 _mL1 _ hL234 _ AGYQTI Hybrid LAMP1 murine in L1 and human in L2 to L4 PXL5669 LAMP1 _ hL12 _mL34 _ AGYQTI Hybrid LAMP1 human in L1 and L2 murine in L3 and L4 pXL5719 hLAMP1_ AglycaninL1_ AGYQTI human LAMP1 with substitution ofN Q at positions 37 , 45 , 62 , 76 and 84 in Li pXL5720 hLAMP1_ AglycaninL2 _ AGYQTI human LAMP1 with substitution of N > Q at positions 103 , 107 , 121, 130 , 165 and 181 in L2 pXL5988 LAMP1 _mL1 _ hL2 _mL34 _ AGYQTI Hybrid LAMP1 murine in L1 , L3 and L4 human in L2 US 9 ,809 , 653 B2 83 84 TABLE 14 - continued Description of LAMP1 transmembrane proteins Short description of LAMP1 transmembrane protein / Plasmid Encoded Protein with amino acid positions according to SEQ ID NO : 24 pXL5997 LAMP1_ hL1 _mL2 _ hL34 _ AGYQTI Hybrid LAMP1 human in Li , L3 and L4 murine in L2 pXL5990 LAMP1_ mseq6 _ AGYQTI Hybrid LAMP1; human sequence except murine sequence at position 97 to 110 in L2 pXL5991 LAMP1 _ mseq7 _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 110 to 128 in L2 pXL5992 LAMP1 _mseq8 _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 128 to 144 in L2 PXL5993 LAMP1 _ mseq9 _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 144 to 157 in L2 pXL5994 LAMP1 _ mseq10 _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 157 to 173 in L2 pXL5995 LAMP1_ mseq11 _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 173 to 189 in L2 pXL5996 LAMP1 _ mseq12 _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 189 to 196 in L2 pXL6009 mLAMP1_ AGYQTI murine LAMP1 pXL6012 LAMP1_ mseql _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 29 to 41 in L1 pXL6013 LAMP1 _mseq2 _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 41 to 56 in L1 pXL6014 LAMP1_ mseq3_ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 56 to 68 in L1 pXL6015 LAMP1 _ mseq4 _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 68 to 80 in L1 pXL6017 LAMP1_ mseq5 _ AGYQTI Hybrid LAMP1 human sequence except murine sequence at position 80 to 97 in L1/ L2 pXL6041 mLAMP1 _ hseq6 - 11 _ AGYQTI Hybrid LAMP1 murine sequence except human sequence at position 91 to 104 and 167 to 183 in L2 pXL6047 mLAMP1 _ hseq6 _ AGYQTI Hybrid LAMP1 murine sequence except human sequence at position 91 to 104 in L2 pXL6048 mLAMP1_ hseq11 _ AGYQTI Hybrid LAMP1 murine sequence except human sequence at position 167 to 183 in L2 pXL6092 mLAMP1_ hseq6 - 9 - 11_ AGYQTI hybrid LAMP1 murine sequence except human sequence at position 91 to 104 and 138 to 151 and 167 to 183 in L2

Example 6 . 5 : Determination of Binding Affinity 50 cytometry (Guava® easyCyteTM 8HT) as mentioned in and Epitope by Flow Cytometry example 4 . 7 , and the mean fluorescence was recorded . This fluorescence represents a semi- quantitative assessment of Each expression plasmid described in example 6 .4 was binding . complexed with 293FectinTM in suspension - cultivated 293 - F The results obtained with plasmids pXL5626 , PXL5668 , cells as using the protocol outlined in example 6 . 2 . Two days 55 PXL5669, PXL5719 and PXL5720 are summarized in Table post transfection cells were processed , analyzed by flow 15 . TABLE 15 Binding of huMAb1 _ 1 , chMAbi , chMAb2 and chMAb3 onto LAMP1 proteins by flow cytometry (Mean fluorescence ) Plasmid pXL5626 pXL5668 pXL5669 Protein hLAMP1 _ AGYQTI LAMP1 _mLi _ hL234 _ AGYQTI LAMP1 _ hL12 _ mL34 _ AGYQTI huMAbl 1 864 1112 934 Binding Binding Binding US 9 ,809 ,653 B2 85 86 TABLE 15 -continued Binding of huMAb1_ 1 , chMAb1 , chMAb2 and chMAb3 onto LAMP1 proteins by flow cytometry (Mean fluorescence ) chMAb1 1047 1059 1484 Binding Binding Binding chMAD2 1025 6 814 Binding No binding Binding chMAb3 640 21 764 Binding No binding Binding Plasmid pXL5719 pXL5720 Protein hLAMP1_ AglycaninL1 _ AGYQTI HLAMP1_ AglycaninL2 _ AGYQTI huMAb1_ 1 1103 528 Binding Binding chMAb1 1113 1006 Binding Binding chMAb2 458 958 Binding Binding chMA3 706 765 Binding Binding

This first set of affinity data ( Table 15 ) with membrane anchored LAMP1 proteins are in agreement with the ELISA data reported on Example 6 . 3 with the secreted LAMP1 proteins . MAb1 binds to hLAMP1 in L2 positions 101 to 25 195 of hLAMP1 ( SEQ ID No : 24 ) , MAb2 and MAb3 bind to hLAMP1 in L1 positions 29 to 100 of hLAMP1. These data also showed that none of the three anti -LAMP1 bind to a glycotope since MAb1 binds to LAMP1 for which L2 was engineered to have no N - glycosylation site and MAb2 and 2 MAb3 bind to LAMP1 for which L1 was engineered to have 30 no N - glycosylation site . The results obtained with plasmids pXL5626 , PXL5988 , PXL5669, PXL5990 to PXL 5997 are summarized in Table 16 . TABLE 16 Binding of huMAb1_ 1 and chMAb2 onto LAMP1 proteins by flow cytometry (mean fluorescence )

Plasmid Protein huMAbl 1 chMAb2 pXL5626 HLAMP1 _ AGYQTI 1412 1498 Binding Binding pXL5988 LAMP1_ mL1_ hL2 _mL34 _ AGYQTI 1180 10 Binding No binding pXL5997 LAMP1_ hL1 _mL2 _ hL34 _ AGYQTI 25 1167 No Binding Binding pXL5990 LAMP1_ mseq6 _ AGYQTI 11 1721 No Binding Binding PXL5991 LAMP1_ mseq7 _ AGYQTI 1400 1412 Binding Binding pXL5992 LAMP1_ mseq8 _ AGYQTI 1440 1688 Binding Binding pXL5993 LAMP1_ mseq9 _ AGYQTI 545 1461 Binding Binding pXL5994 LAMP1_ mseq10 _ AGYQTI 1414 1555 Binding Binding pXL5995 LAMP1 _mseq11 _ AGYQTI 16 1378 No Binding Binding pXL5996 LAMP1_ mseq12 _ AGYQTI 1303 1365 Binding Binding ballast no LAMP1 No binding No binding US 9 ,809 ,653 B2 87 88 The results obtained with plasmids pXL5626 , PXL6041 , From these sets of affinity data ( Tables 15 , 16 , 17 and 18 ) pXL6047 , pXL6048 , and PXL6009 are summarized in Table obtained by flow cytometry with membrane - anchored 17 . LAMP1 proteins and the ELISA results with the secreted TABLE 17 Binding of MAb1 and MAb2 onto LAMP1 proteins by flow cytometry mean( fluorescence Experiment n°1 Experiment n°2 Plasmid Protein MAb1 MAb2 MAb1 pXL5626 HLAMP1_ AGYQTI 1748 1183 551 Binding binding Full binding pXL6041 mLAMP1 _ hseq6 - 11 _ AGYQTI 680 28 211 Binding No binding binding pXL6047 mLAMP1_ hseq6 _ AGYQTI 7 25 No binding No binding No binding pXL6048 mLAMP1_ hseq11_ AGYQTI 6 28 No binding No binding No binding pXL6092 mLAMP1_ hseq6 -9 -11 _ AGYQTI Not done Not done 499 Full binding pXL6009 mLAMP1 _ AGYQTI 4 No binding No binding No binding

The results obtained with plasmids pXL5626 , PXL6012 LAMP1 proteins described in Example 6 . 3 , the following to PXL6015 , PXL6017 and pXL6009 are summarized in 25 conclusions could be derived : Table 18 . MAb1 binds to L2 positions 101 to 195 of LAMP1, MAb2 and MAb3 bind to Ll positions 29 to 100 of LAMP1. TABLE 18 None of the three anti -LAMP1 bind to a glycotope since MAb1 binds to LAMP1 for which L2 was engineered to Binding of MAb1 , MAb2 and MAb3 onto LAMP1 proteins 30 have no N - glycosylation site and MAD2 and MAD3 bind to by flow cytometry (mean fluorescence LAMP1 for which L1 was engineered to have no N - glyco Plasmid Protein MAb1 MA62 MAb3 sylation site . pXL5626 hLAMP1_ AGYQTI 914 757 749 MAbl does not bind to hybrid LAMP1 protein where Binding Binding Binding human LAMP1 residues from positions 97 to 110 (SEQ ID pXL6012 LAMP1 _mseq1 _ AGYQTI 1027 105 2. 8 35 NO : 78 ) or from positions 173 to 189 (SEQ ID NO : 79 ) have Low binding No been substituted by murine LAMP1 residues . But it binds to binding hybrid LAMP1 protein where human LAMP1 residues from pXL6013 LAMP1_ mseq2 _AGYQTI Binding990 Binding694 Binding803 positions 110 to 173 or from positions 189 to 196 in L2 have pXL6014 LAMP1_ mseq3 _ AGYQTI 888 694 674 been substituted by murine LAMP1 residues . pXL6015 LAMP1_ mseq4 _ AGYQTI Binding89127 1Binding Binding 40 MAb1 interacts with amino acids located within L2 and Binding No binding No more specifically MAb1 interacts with amino acids located mabinding within sequences from positions 101 to 110 (SEQ ID NO : pXL6017 LAMP1_ mseq5 _ AGYQTI 846 629 721 72 ) and /or from positions 174 to 188 (SEQ ID NO : 74 ) and lolo:121Binding 1Binding Binding pXL6009 mLAMP1 _ AGYQTI 13 27 to some extent to sequence from positions 144 to 157 (SEQ No No binding No 45 ID NO : 73 ) . Therefore, we can infer from these results and binding binding from amino acid differences between murine and human sequences that human LAMP1 residues among R146 , D150 , The affinity data described in Table 16 with membrane K152, R106 , A108 , N181, S182 , S183 , R186 and G187 are anchored LAMP1 proteins demonstrated that MAb1 binds to likely to interact with MAb1 . LAMP1 in L2 positions 101 to 195 of hLAMP1 (SEQ ID 50 MA62 interacts with amino acids located within L1 and NO : 24 ) (pXL5626 , pXL5988 and pXL5997) . More spe more specifically MAb2 interacts with amino acids located cifically MAb1 does not bind to hybrid LAMP1 protein within sequences from positions 68 to 80 ( SEQ ID NO : 76 ) where human LAMP1 residues from positions 97 to 110 or and to some extent within sequences from positions 29 to 41 from positions 173 to 189 have been substituted by murine (SEQ ID NO : 75 ) . Therefore , we can infer from these results LAMP1 residues, but it binds to hybrid LAMPi protein 55 and from amino acid differences between murine and human where human LAMP1 residues from positions 110 to 173 or sequences that human LAMP1 residues among A29 , M30 , from positions 189 to 196 in L2 have been substituted by M32 , G36 , A40 , S69, D70 , 172 , 174 , 175 , and R77 are murine LAMP1 residues . Of note some binding is also lost likely to interact with MAb2. when human LAMP1 residues from position 144 to 157 are MAb3 interacts with amino acids located within L1 and replaced by the murine LAMP1 residues . In addition data 60 more specifically MAb3 interacts with amino acids located reported in Table 17 Experiment No 1 showed that residues within sequences from positions 29 to 41 (SEQ ID NO : 75 ) from positions 97 to 110 and from positions 173 to 189 are and /or from positions 68 to 80 ( SEQ ID NO : 76 ) . Therefore , simultaneously needed to restore some of the binding . Data we can infer from these results and from amino acid differ reported in Table 17 experiment No 2 showed that residues ences between murine and human sequences that human from positions 91 to 104 and 138 to 151 and 167 to 183 in 65 LAMP1 residues among A29 , M30 , M32 , G36 , A40 , 569 , Loop2 are simultaneously needed to restore full binding of D70 , 172 , 174 , 175 , and R77 are likely to interact with MAb1. MAb3 . US 9 ,809 , 653 B2 89 90 Example 6 . 6 : Determination of Individual Amino located at the 5 ' - end of the cDNA encoding the variable Acid Involved in Epitope Binding by Ala Scan regions and the constant domains. High Resolution Mass Spectrometry of Hybridoma Individual residues identified in example 6 . 5 and not (HRMS ) : involved in B - strand structure have been individually 5 Mass spectra were obtained on a Waters Synapt G2 TOF replaced by an alanine residue in the LAMP1 sequence system in electrospray positive mode (ES + ). Chromato derived from hLAMP1_ AGYQTI and encoded in plasmid graphic conditions are the following : column: UPLC pXL5626 ( example 6 . 4 ) . A total of 21 plasmids were engi MassPrep 20 um 2 . 1X5 mm ; solvents : A : H20 + 0 . 1 % formic neered from pXL5626 ( see Table 19 ) and used to assay acid : B ; CH3CN + 0 . 1 % formic acid ; column temperature : LAMP1 expression at the cell membrane of HEK293 cells 10 80° C .; flow rate 0 . 2 mL /min ; gradient elution ( 10 min ) : 10 % after transient transfection . Two days post transfection cells B for 30 sec ; from 10 to 50 % of B in 7 min 10 sec ; 8 were processed , analyzed by flow cytometry (Guava® easy min : 90 % B ; 8 min 30 sec: 10 % B ; 10 min : 10 % B . Samples CyteTM 8HT) as mentioned in example 4 . 7 , and the mean were reduced 30 min at 37° C . in Gdn .HCL 6M /DTT 1M fluorescence was recorded . This fluorescence represents a before LC /MS analysis. semi- quantitative assessment of binding . Loss of binding is The derived amino acid sequences provided information reported on Table 19 when there is a decrease of more than in agreement with the data obtained on purified mAbs 50 % of the mean fluorescence compared to the control derived from the hybridoma by N - terminal sequencing and protein encoded from PXL5626 . mass spectrometry (LC /MS ) of the heavy and light chains (LC , HC ) Table 20 . No identical sequences were found in TABLE 19 the patented sequences from GenomeQuest. Loss of binding to anti- LAMP1 mAb with Alascan LAMP1 transmembrane proteins TABLE 20 Position of mutation Mass spectrometry analysis of anti- LAMP1 mAbs from hybridoma in Binding to Binding to 25 Plasmid hLAMP1 _ AGYQTI MAb1 MAb3 Mass ( Da ) pXL5626 none in silico value retrieved pXL6058 G36A Clone ID Chain by LC /MS from batch from sequence pXL6065 N37A pXL6059 G38A Binding loss 30 MAb1 LC 23587 23587 pXL6060 L67A HC (GOF ) 50700 50700 pXL6066 P68A MAD2 LC 23911 23911 pXL6067 S69A HC (GOF ) 50702 50704 pXL6069 D70A Binding loss MAb3 LC 23725 + higher masses 23723 pXL6072 N107A due to N - glycans pXL6073 A108T 5 HC (GOF ) 5085250852 50848 pXL6080 T109A pXL6070 1149A Binding loss pXL6085 D150A Binding loss The nucleic acid sequences of the variable domains VH pXL6071 K151A and VL were cloned into expression vectors in fusion with pXL6079 Y178A pXL6074 L179A the human IgG1 or the human Ckappa constant domain pXL6075 S180A 40 coding sequences, respectively , to then generate batches of pXL6076 N181A chimeric mAbs by transient expression in 293 - F cells as PXL6077 F184A described in Example 6 . 2 . Batches were purified by protein pXL6081 R186A Binding loss A affinity chromatography (MabSelect , GE Heathcare ) . The pXL6082 6187A eluate was dialyzed against PBS before sterile filtration and 45 storage at 4° C . Loss of binding to MAbl at positions 1149, D150 and Affinity to LAMP1 remained similar for murine and R186 due to Ala substitution in LAMP1 protein indicates chimeric mAbs illustrated by the EC so obtained by ELISA that these positions are important for MAb1 binding to with LAMP1 in Table 21 . LAMP1. Loss of binding to MA63 at positions G38 and D70 due 50 TABLE 21 to Ala substitution in LAMP1 protein indicate that these EC50 (nM ) obtained with LAMP1 for murine hybridoma and positions are important for MAb2 binding to LAMP1. corresponding chimeric mAbs Example 7: Determination of mAb Sequences EC5o obtained for murine 55 hybridoma mAbs Example 7 . 1 : Determination of mAb Sequences EC - o obtained for chimeric mAbs and Generation of Chimeric mAbs clone ID hLAMP1 clone ID hLAMP1 The sequences of the variable domains of the mAb were MAb1 0 . 18 nM chMAb1 0 . 12 nM ( assay A ) retrieved from the hybridoma and cloned into an expression 60 M 0 . 12 nM (assay B ) vector to ensure that the cloned mAbs had the same char- MAb2 0 . 25 nM chMAb2 0 .12 nM ( assay A ) acteristics as the initial murine mAbs . chMAb2 can 0 . 12 nM ( assay A ) The cDNA encoding the variable domains of the mono chMAb3 0 . 12 nM ( assay B ) clonal antibodies were obtained as follows. cDNA has been chMAb3VL _ R24 _ R93 0 . 11 nM ( assay B ) retrieved and sequenced by RT- PCR ( transcriptase Super- 65 Script III from Invitrogen and polymerase Phusion from Based on the data described above , the amino acid Finnzymes) from 100 hybridoma cells and oligonucleotides sequences of the HC and the LC were validated . US 9 ,809 , 653 B2 91 92 The LC and HC sequences ofMAb1 are shown in SEQ ID a lower binding efficiency than Mabi , Mab 2 and MAD 3 NO : 35 and SEQ ID NO : 36 , respectively . with a Kd of around 100 nM . The LC and HC sequences ofMAb2 are shown in SEQ ID NO : 37 and SEQ ID NO : 38 , respectively. TABLE 22 The sequences for the CDR regions were deduced from 5 the protein sequence using the IMGT nomenclature. Binding kinetics to LAMP1 for the murine , chimer or humanized mAbs The LC and HC sequences of chMAb1 are shown in SEQ ID NO : 18 and SEQ ID NO : 17 , respectively , and the LC and Mab kg ( M -15 - 15 kg ( s -1 ) Kp (nM ) HC sequences of chMAb2 are shown in SEO ID NO : 20 and Mab1 14 . 8E + 04 0 . 71E - 03 4 . 8 huMAbl 1 19 . 1E + 04 1 . 57E - 03 8 . 2 SEQ ID NO : 19 , respectively . The LC and HC sequences of 10 chMAb2can 7 .21E + 04 4 . 96E - 03 68 . 8 chMAb3 are shown in SEQ ID NO : 49 and SEQ ID NO : 50 , Mab2 6 . 33E + 04 4 . 02E - 03 63. 5 respectively. chMAb3VL R24 R93 17. 3E + 04 1 . 25E - 03 7. 2 Of note , canonical residues have been introduced into MAb3 24 . 2E + 04 1 .13E - 03 4 . 7 clone MAb2 at positions A9, L51, L58 , G72 and L108 on Murine H4A3 5 . 80E + 04 6 . 09E - 03 105 VL and at position T116 on VH sequence, to generate 15 (3286027 BioLegend MAb2. com . The corresponding amino acid sequences of the VH and the VL of MAb2can are SEQ ID NO : 15 and SEQ ID NO : 16 , respectively . The HC and LC sequences of chMAb2 can are shown in SEQ ID NO : 21 and 22, respec Example 7 . 2 : Obtention and Characterisation of tively . Humanized Variants Derived from MAb1 A batch of clone chMAb2. can was generated in the same conditions as the batch corresponding to clone chMAb2 . In this example , humanized variants of parental murine This highlights that point mutations in the FR can be made IgG MAb1 have been designed in silico . The resulting without any impact on binding butmore importantly provide huMAbl variants were produced and provided similar char an alternative to the production process . 25 acteristics as the chimer chMAb1. A batch of clone chMAb3 _ VLR24 -R93 was generated in the same conditions as the batch corresponding to clone Example 7 . 2 . 1 Design of the Humanized chMAb3. This highlights that point mutations in the CDR Anti -LAMP1 huMAb1 can be made without any impact on binding . Affinity to LAMP1 by SPR : 30 Humanization Based on CDR Grafting The binding kinetics of the murine, chimer or humanized This approach consists in the transplantation of CDRs of anti -LAMP1 mAbs were determined by surface plasmon the parental murine MAbl into relevant human FRs. The resonance assay using a BIAcore 2000 (BIAcore Inc ., variable light and heavy regions of murine MAbl were Uppsala , N . J . ) . Briefly , a CM5 BIAcore biosensor chip was compared to human germline sequences from IMGT Infor docked into the instrument and activated with 70 uL of 1 : 1 35 mation system (Lefranc et al. Nucl. Acids . Res. 2009 , NHS / EDC at room temperature . A mouse anti- ahuman Fc 37 :D1006 -D1012 ) to select the human light and heavy IgG1 (BIAcore # BR - 1008 - 39 ) and rabbit anti -amurine Fc variable sequences that would serve as the basis of the IgG1 (BIAcore # BR - 1008 - 38 ) ( 50 ug /mL in 1 M acetate humanized MAb1 regions (huMAB1 ). buffer, pH5 ) were immobilized on the activated chips in all The mouse light chain variable region displayed 68 . 8 % flow cells . The immobilization was carried out at a flow rate 40 identity over the V region and 74 . 7 % identity within the FRs of 10 uL /min up to saturation . The chip was then blocked by alone to the human germline kappa light chain IGKV1 -27 . injection of 70 uL of ethanolamine -HC1 , pH 8 . 5 , followed by For the joining region , mouse J region displayed 90 % one wash with 3 M MgCl2 for anti - ahuman Fc IgG1 and one identity to human germline IGKJ4 . Consequently human V wash with 10 mM Glycine -HCl pH 1 . 7 for anti -amurine Fc region IGKV1- 27 combined to human J region IGKJ4 given IgG1. To measure the binding of anti- LAMP1 mAbs to 45 a global germinality index ( identity calculated on FRs only ) LAMP1, antibodies were used at 1 - 5 ug /mL in BIAcore of 76 . 4 % have been selected as human acceptor sequences running buffer (HBS - EP ) . The antigen (SEQ ID NO : 28 for humanization of the mouse MAb1 light chain . This then protein produced as described in example 6 . 2 ) was injected became the basis of the humanized variant of the anti from 1 to 256 nM . Following completion of the injection LAMP1 MAb1 light chain , which comprised the CDRs of phase , dissociation was monitored in a BIAcore running 50 the murine MAbi Vk region and the FRs of the human buffer at the same flow rate for 600 sec . The surface was IGKV1 - 27 _ IGKJ4 regions . regenerated between injections using 2x5 uL 3 M MgCl2 The mouse heavy chain variable region displayed 65 . 3 % (2x30 s) or anti - ahuman Fc IgG1 and 1x30 UL 10 mM identity over the V region and 70 .0 % identity within the FRs Glycine -HC1 pH 1 . 7 for anti - amurine Fc IgG1 ( 180 s ) . alone to the human heavy variable germline IGHV1 -69 . For Individual sensorgrams were analyzed using BIAevaluation 55 the joining region , mouse J region displayed 78 % identity to software . human heavy joining germline IGHJ4 . Consequently human Affinity to LAMP1 for the murine, chimer or humanized germline V region IGHV1 -69 combined to human germline mAbs is reported on Table 22 . It was found to be indepen J region IGHJ4 given a global germinality index ( identity dent of the MAb format. calculated on FRs only ) of 71. 4 % have been selected as MAb1 binds to LAMP1 with K , ranging from 4 . 8 to 8 .2 60 human acceptor sequences for humanization of the murine nM MAb1 heavy region . This then became the basis of the MAD2 binds to LAMP1 with K , ranging from 63 . 5 to humanized variant of the anti -LAMP1 MAb1 heavy chain , 68 . 8 nM which comprised the CDRs of the murine MAb1 Vh region MAb3 binds to LAMP1 with Ky ranging from 4 .7 to 7 .2 and the FRs of the human IGHV1- 69 _ IGHJ4 regions . nM 65 However, some FRs residues are also important for the The commercially available anti -LAMP1 mAb ( H4A3 biological activity of the antibody since they can impact ( BioLegend 328602 ) has a significantly kigher K , and thus CDRs conformation and thus antigen binding . Back muta US 9 , 809, 653 B2 93 94 tions to murine amino acid may be introduced at selected These 49 human models have been built by systematically positions of FRs grafted antibody in order to retain the combining a representative panel of 7 human light chains binding specificity and affinity of the parent antibody . Thus, (namely vk1, vk2 , vk3 , Vk4 , vlambdal, vlambda2, the next step in the design process was to study the protein vlambda3 ) with a representative panel of 7 human heavy sequences of the humanized variant to determine if any of 5 chains ( namely vhla , vhlb , vh2 , vh3 , vh4, vh5 , vh6 ) . these amino acid residues were likely to alter the confor - The vk1 -. vh1bvh1 h combination showed the highest AD4D simisimi. mation or orientation of the CDRs loops . A 3D homology larity of its flexible amino acids compared to the flexible model of the variable regions of both the murine and the humanized antibodies were built using model antibody amino acids of the murine MAb1 antibody ; this model was framework protocol of Discovery studio 3. 1 from Accelrys 10 therefore used to humanize the MAb1 antibody , focusing on Software Inc . the flexible amino acids . For the pairwise amino acid asso The VL and VH sequences of the murine MA1 were ciation between the murine MAb1 and vk1 - vhlb amino compared to the protein database (PDB ) (Berman et al . acids, the 2 sequences were aligned based on the optimal 3D Nucleic Acids Research , 2000 , 28 :235 - 242 ). superposition of the alpha carbons of the 2 corresponding The structure model of the antithrombotic monoclonal 15 homology models . antibody 82D6A3 with the PDB identity number 2ADF was In addition , to improve the stability of the resulting used as template for the light chain ( 96 . 6 % identity on light humanized MAbl antibody, the amino acids of the light and chain framework ) and the structure model of of IL -23 in heavy chains with low frequency of occurrence vs their complex with neutralizing FAB with the PDB identity respective canonical sequences, excluding the CDRs, are number 3D85 was used as template for the heavychain 20 originally proposed to be mutated into the most frequently (83 . 5 % identity on heavy chain framework ) . found amino acids ( AAGth > 0 . 5 kcal/ mol ; (Monsellier et al . In the same way, the VL and VH sequences of the J . Mol. Biol. 2006 , 362 , 580 -593 ) . A first list of consensus humanized variant (human FRs and murine CDR ) were mutations for the LC and for the HC has been restricted to compared to the protein database (PDB ) (Berman et al. the amino acids found in the closest human model ( i . e . Nucleic Acids Research , 2000 , 28 : 235 - 242 ) . The model with 25 vk1 - vhlb ) . None of these mutations are located in the the PDB identity number 3AAZ was used as template for the “ Vernier” zone (Foote et al. , J . Mol. Biol. 1992, 224 , light chain (86 .6 % identity on light chain framework ) and 487 - 499 ) . Other criteria are taken into account to consider the model with the PDB identity number 3KDM was used as these consensus mutations for potentially stabilizing the template for the heavy chain ( 84 . 3 % identity on heavy chain anti -LAMP1 MAb1 antibody. These criteria are a favourable framework ) ( All PDB references refer to the PDB identity 30 change of hydropathy at the surface or a molecular mechan number as available on Nov . 26 , 2013 ) . ics based predicted stabilization of the mutant. Stabilizing Both 3D homology models , the murine MAb1 and the mutations reported to be successful in the literature humanized version were compared and each amino acid (Bedouelle , H . J. Mol. Biol. 2006 , 362 , 580 -593 ; Steipe B . substitution from mouse to human version were carefully J . Mol. Biol. 1994 , 240 , 188 - 192 ) were considered . looked . When the substitution of a mouse to a human residue 35 Resulting Humanized VL and VH Regions was done at a position that could influence the conformation Based on both approached , the CDRs grafting and the 4D of the CDRs , a back mutation to the murine residue was protocols , three versions for the variable light chain (VL1 , done . VL2 and VL3 ) and three versions for the variable heavy Humanization Based on Molecular Dynamic Trajectories chain (VH1 , VH2 and VH3 ) are proposed . The particular (4D Humanization Protocol) 40 combination of amino acid residues mutated in each human A molecular dynamics (MD ) simulation of the 3D homol- ized MAD1 VL and VH variants are set forth in Table 23 and ogy model of the murine MAb1 (as described in section Table 24 respectively . The complete amino acid sequences above on grafting protocol) was subsequently performed , of the humanized VH and VL domains are set forth in Table with constraints on the protein backbone at 500 K tempera - 25 . ture for 1. 1 nanoseconds (ns ) in Generalized Born implicit 45 For the Variable Light Region : solvent. 10 diverse conformations were extracted from this The humanized VL1 variant with SEQ ID NO : 56 dis first MD run every 100 picoseconds (ps ) for the last 1 ns. plays a total of 12 mutations compared to mouse sequence These diverse conformations were then each submitted to a with SEQ ID NO : 5 . This variant derives from frameworks MD simulation , with no constraints on the protein backbone of human germline IGKV1- 27 _ IGKJ4 sequences with 6 and at 300 K temperature , for 2 . 3 ns. For each of the 10 MD 50 back mutations done because they were suspected to have runs , the last 2 ,000 snapshots, one every ps , from the MD negative impact on mAb structure , CDRs conformation and trajectory were then used to calculate , for each murine therefore, on binding to its target . In addition , for amino MAb1 amino acid , its root mean square deviations ( rmsd ) acids at position 43 and 83 , mutation in a more frequent compared to a reference medoid position . By comparing the amino acid present in IGKV1 germlines ( A43 and F83 ) was average rmsd on the 10 separate MD runs of a given amino 55 preferred . acid to the overall average rmsd of all MAb1 murine amino The humanized VL2 variant with SEQ ID NO : 57 dis acids, one decides if the amino acid is flexible enough , as plays 2 mutations which derive from the direct comparison seen during the MD to be considered as likely to interact between the non -CDR most flexible amino acids of the with B - cell receptors and responsible for activation of the murine MAb1 light chain and the vk1 human light chain immune response . 28 amino acids were identified as flexible 60 sequence . in the murine MAb1 antibody , excluding the CDRs and its The humanized VL3 variant with SEQ ID NO : 58 derives immediate 5 Å vicinity . from VL2 and includes 6 new mutations that are consensus The motion of the 60 most flexible murine MAbl amino ( vkl sequence) and potentially stabilizing . acids , during the 20 ns ( 10x2 ns) of molecular dynamic The particular combination of amino acid residues simulation , were then compared to the motion of the corre - 65 mutated in the individual humanized light chains of sponding flexible amino acids of 49 human 3D homology huMAb1 thus VL of huMab1 _ 1 , huMab1 _ 2 and huMab1 _ 3 models , for each of which were run the same simulations. are set forth in Table 23 . US 9, 809, 653 B2 95 96 TABLE 23 The particular combination of amino acid residues mutated in the individual humanized light chains of Mutations of the VL variants of the anti -LAMP1 MAb1 antibody huMAb1 thus VH ofhuMab1 _ 1 , huMab1 _ 2 and huMab1_ 3 Mouse HuMab1 _ 1 HUMAbl 2 HUMAb1 _ 3 are set forth in Table 24 . MAb1 VL (VL1 ) (VL2 ) (VL3 ) 5 P9 S9 $ 9 TABLE 24 L15 V15 V15 V15 G17 D17 Mutations of the VH variants of the anti - LAMP1 MAb1 antibody K18 R18 D38 Q38 10 Mouse MAb1 HuMabi 1 HuMab1 _ 2 HuMab1 _ 3 G43 A43 VH ( VH1 ) ( VH2 ) ( VH3) R45 K45 K45 P56 S56 Q5 V5 V5 V5 158 V58 V58 L11 V11 S72 T72 V12 K12 F73 L73 L73 S74 T74 T74 A16 S16 N77 S77 M20 V20 183 F83 K38 R38 L103 V103 K39 Q39 S40 A40 20 S61 A61 For the Variable Heavy Region : K65 Q65 Q65 Q65 The VH1 variant (SEQ ID NO : 53) displays a total of a D66 666 G66 total of 15 residues substitution compared to the mouse K67 R67 R67 sequence (SEQ ID NO : 1 ). This variant derives from frame K74 T74 T74 T74 works of human germline IGHV1 -69 _ IGHJ4 sequences S76 176 with 9 back mutations done because they were expected to 25 082 E82 E82 E82 have negative impact on mAb structure , CDRs conformation R85 S85 585 585 and therefore , on binding to its target. In addition , K74 of T87 R87 SEQ ID NO : 1 in vicinity of CDRs was mutated into T to 591 327318T91 T91 anticipate a potential problem if targeted by the conjugation S115 T115 T115 process . A118 S118 S118 S118 The VH2 variant displays 7 mutations : 6 mutations deriv ing from the direct comparison between the non - CDR most flexible amino acids of the murine MAb1 heavy chain and The resulting humanized sequences were blasted for the vhlb human heavy chain sequence , plus mutation of K74 sequence similarity against the Immune Epitope Data Base of SEQ ID NO : 1 into T to anticipate a potential problem if 3535 (IEDB ) database ( (PLos Biol (2005 ) 3 (3 )?91 ) on the world targeted by the conjugation process . wide web at immuneepitope . org ; ) to ensure that none of the The VH3 variant derives from VH2 and includes 7 new sequences contain any known B - or T- cell epitope listed in . mutations that are consensus ( vhlb sequence ) and poten The complete amino acid sequences of the humanized VH tially stabilizing . and VL domains are set forth in Table 25 . TABLE 25 VH and VL amino acid sequences of humanized anti - LAMP1 antibodies . VH or VL variant Sequence SEQ ID NO . humAb1 _ 1 VH1 QVOLVQSGAEVKKPGSSVKVSCKASGYIFTN SEQ ID NO : 53 YNIHWVKKSPGOGLEWIGAIYPGNGDAPYSO KFOGKATLTADTSTSTTYMELSSLRSEDTAVY YCVRANWDVAFAYWGQGTLVTVSS humAb1 _ 2 VH2 QVOLVOSGAELVKPGASVKMSCKASGYIFTN SEQ ID NO : 54 YNIHWVKKSPGQGLEWIGAIYPGNGDAPYSQ KFODRATLTADTSSSTTYMELSSLTSEDSAVY YCVRANWDVAFAYWGQGTLVSVSS humAb1 3 VH3 QVOLVQSGAELVKPGASVKMSCKASGYIFTN SEQ ID NO : 55 YNIHWVROAPGOGLEWIGAIYPGNGDAPYAO KFOGRATLTADTSSSTTYMELSSLTSEDTAVY YCVRANWDVAFAYWGQGTLVTVSS huMab1 _ 1 VL1 DIQMTQSPSSLSASVGDRVTITCKASQDIDRY SEQ ID NO : 56 MAWYQDKPGKAPRLLIHDTSTLQSGVPSRFS GSGSGRDYTLTISNLEPEDFATYYCLQYDNLW TFGGGTKVEIK humAb1_ 2 VL2 DIOMTQSPPSLSASVGGKVTITCKASQDIDRY SEQ ID NO : 57 MAWYQDKPGKGPKLLIHDTSTLQPGIPSRFS GSGSGRDYSFSISNLEPEDIATYYCLQYDNLW TFGGGTKLEIK US 9 ,809 , 653 B2 97 98 TABLE 25 - continued VH and VL amino acid sequences of humanized anti - LAMP1 antibodies . VH or VL variant Sequence SEQ ID NO . huMab1 _ 3 VL3 DIQMTQSPSSLSASVGGKVTITCKASQDIDRY SEQ ID NO : 58 MAWYQOKPGKGPKLLIHDTSTLQPGVPSRFS GSGSGRDYSLTISSLEPEDIATYYCLQYDNLW TFGGGTKLEIK

Example 7 . 2 . 2 : Production and Characterization of TABLE 26 - continued Three Humanized Anti- LAMP1 huMAb1 Variants Mass spectrometry analysis of humanized anti- LAMP1 mAbs The corresponding nucleic acid sequences encoding thethe 15 humanized variable VH and VL domains described in Mass (Da ) example 7 . 2 . 1 were synthesized at Geneart and cloned into expression vectors in fusion with the human IgG1 or the by LC /MS in silico value retrieved human Ckappa constant domain coding sequences , respec - 20 mAh ID Chain from batch from sequence tively , to then generate batches of humanized mAbs by transient expression in 293 - F cells as described in Example huMab1 _ 2 LC2 23375 Da 23376 Da 6 . 2 . The three mAbs were referred to — huMAb1_ 1 that HC2 (GOF ) 50209 Da 50209 Da contains LC1 (VL1 - huCk ) (SEQ ID NO : 59 ) and HCl huMAb1 _ 3 LC3 23318 Da 23318 Da ( VH1- hulgG1) (SEQ ID NO : 60 ) , 25 huMAb1 _ 2 that contains LC2 ( VL2- huCk ) (SEQ ID NO : HC3 (GOF ) 50176 Da 50175 Da 61 ) and HC2 (VH2 -hulgG1 ) (SEQ ID NO : 62 ), huMAbl_ nega LCnega 23277 Da 23277 Da huMAb1 _ 3 that contains LC3 (VL3 - huCk ) ( SEQ ID NO : HCnega 50103 Da 50104 Da 63) and HC3 (VH3 -hulgG1 ) ( SEQ ID NO : 64 ) . (GOF ) A negative control was generated and referred to as 30 huMAb1 _ negB LC1 23484 Da 23484 Da huMAb1 _ negA . It contains LCneGA ( VL1_ 36A - 95A -huCk ) HCnegB 50175 Da 50175 Da (SEQ ID NO : 65 ) and HCneGA ( VH1_ 101A -hulgG1 ) (SEQ (GOF ) ID NO : 67 ) . Another control was generated and referred to as huMAb1 negB . It contains LC1 (VL1 - huCk ) (SEQ ID NO : 35 Secreted human LAMP1 protein described in example 6 . 2 59 ) and HCnegB (VH1 -hulgG1 _ 266A ) ( SEQ ID NO : 68 ) . was used to determine the binding domain to the humanized The mutation 266A in the hulgG1 corresponds to the D265A anti -LAMP1 mAbs by ELISA . Affinity to LAMP1 remained mutation according to the nomenclature described by Kabat similar for chimer and humanized mAbs as illustrated by the et al. , Sequences of Proteins of Immunological Interest , 5th EC obtained by ELISA with LAMP1 in Table 27 . No edition , National Institute of Health , Bethesda , Md. , 1991. It 40 bin was reported to significantly decrease binding to FcyRs and 40 binding is detected with huMAb1 _ negA . ADCC ( Lund et al. , J . Immunol. , 157 :4963 - 4969 , 1996 ; Shields et al. , J . Biol. Chem ., 276 ( 1 ): 6591- 6604, 2001 ) . TABLE 27 Significant decrease in binding to FcYRI, II and III was ECso ( nM ) obtained with LAMP1 for chimer and humanized mAbs also verified by ELISA with recombinant proteins (recom - 45 binant human FcYRI/ CD64 reference 1257 -FC - 050 , recom mAb ID HLAMP1 binant human FcYRIIA /CD32a reference 1330 -CD - 050 /CF , chMAb1 0 . 09 nM recombinant human FcyRilla /CD16a , reference 4325 - FC huMAbl 1 0 . 11 nM 050 , all obtained from R & D System ) . huMAb1 2 0 . 11 nM huMAbi 3 0 .12 nM Batches were purified by protein A affinity chromatogra - 50 huMAbl negA No binding detected phy (MabSelect , GE Heathcare ) . The eluate was dialyzed huMAbi _ negB 0 . 07 nM against PBS before sterile filtration and storage at 4° C . Batches were analysed by High Resolution Mass Spectrom etry as described in Example 7 . Data were in agreement with the in silico value retrieved from amino acid sequences , 55 Example 7 . 2 .3 : Cross Reactivity of humAb1 _ 1 ; Table 26 . huMAb1 _ 2 and huMAb1_ 3 with Cynomolgus Monkey LAMP1 TABLE 26 Mass spectrometry analysis of humanized anti -LAMP1 mAbs - 60 HUMAb1 _ 1 , huMAb1 _ 2 and huMAb1 _ 3 antibodies were assessed by flow cytometry for their ability to bind to human Mass ( Da ) LAMP1 or cynomolgus LAMP1 proteins expressed respec by LC /MS in silico value retrieved tively at the surface of HCT116 or HEK293 stable clones . mAb ID Chain from batch from sequence HCT116 stable clone was obtained as described in example huMAbi 1 LC1 23483 Da 23484 Da 65 4 . 7. HEK293 stable clone was obtained according to the HC1 (GOF ) 50219 Da 50219 Da protocol described in example 4 . 7 . EC50S , estimated using BIOST @ T - SPEED software , are listed in Table 28 .