Catherine Prades

Two new genes from the human ATP-binding cassette transporter superfamily, ABCC11 and ABCC12, tandemly duplicated on chromosome 16q12.

Several years ago, we initiated a long-term project of cloning new human ATP-binding cassette (ABC) transporters and linking them to various disease phenotypes. As one of the results of this project, we present two new members of the human ABCC subfamily, ABCC11 and ABCC12. These two new human ABC transporters were fully characterized and mapped to the human chromosome 16q12. With the addition of these two genes, the complete human ABCC subfamily has 12 identified members (ABCC1-12), nine from the multidrug resistance-like subgroup, two from the sulfonylurea receptor subgroup, and the CFTR gene. Phylogenetic analysis determined that ABCC11 and ABCC12 are derived by duplication, and are most closely related to the ABCC5 gene. Genetic variation in some ABCC subfamily members is associated with human inherited diseases, including cystic fibrosis (CFTR/ABCC7), Dubin-Johnson syndrome (ABCC2), pseudoxanthoma elasticum (ABCC6) and familial persistent hyperinsulinemic hypoglycemia of infancy (ABCC8). Since ABCC11 and ABCC12 were mapped to a region harboring gene(s) for paroxysmal kinesigenic choreoathetosis, the two genes represent positional candidates for this disorder.

Identification and characterization of a novel ABCA subfamily member, ABCA12, located in the lamellar ichthyosis region on 2q34

The ABCA subfamily of ABC transporters includes ten members to date. In this study, we describe an additional gene, ABCA12. Four full-length cDNA sequences have been obtained from human placenta that contain two different polyadenylation sites and two splicing forms, coding for ABCA12 isoforms of 2,595 and 2,516 amino acid residues. Both isoforms are predicted to have two ATP-binding domains (nucleotide binding domain, NBD) and two transmembrane (TM) domains, features shared by all other ABCA subfamily proteins. ABCA12 is most closely related to ABCA1, with an amino acid similarity of 47%. Northern blot analysis demonstrates that a 9.5-kb transcript is mainly expressed in the stom- ach. ABCA12 was mapped to human chromosome 2q34. Two other genes from ABCA subfamily are associated with human inherited diseases, ABCA1 with the cholesterol transport disorders Tangier disease and familial hypoalphalipoproteinemia, and ABCA4 with several retinal degeneration disorders. The ABCA12 gene is located in a region of chromosome 2q34 that harbors the genes for lamellar ichthyosis, polymorphic congenital cataract, and insulin-dependent diabetes mellitus (IDDM13), and therefore is a positional candidate for these pathologies.

Comparative analysis of the promoter structure and genomic organization of the human and mouse ABCA7 gene encoding a novel ABCA transporter

We report here the genomic and transcriptional characterization in mouse and man of a novel transporter of the ABCA subclass, named ABCA7. As it is the case for other ABCA genes, the predicted protein encoded by ABCA7 is a full symmetric transporter, highly conserved across species. The ABCA7 gene maps to human chromosome 19 and to the homologous region at band B4-C1 on mouse chromosome 10. The preferential expression of ABCA7 in the spleen, thymus, and fetal liver is consistent with the finding, in both human and mouse promoter, of sites targeted by lymphomyeloid-specific transcription factors. This suggests that ABCA7 may play a pivotal role in the developmental specification of hematopoietic cell lineages.

The human ATP binding cassette gene ABCA13, located on chromosome 7p12.3, encodes a 5058 amino acid protein with an extracellular domain encoded in part by a 4.8-kb conserved exon

The ABCA subfamily of ATP-binding cassette (ABC) transporters includes eleven members to date. In this study, we describe a new, unusually large gene on chromosome 7p12.3, ABCA13. This gene spans over 450 kb and is split into 62 exons. The predicted ABCA13 protein consists of 5,058 ami- no acid residues making it the largest ABC protein described to date. Like the other ABCA subfamily members, ABCA13 contains a hydrophobic, predicted transmembrane segment at the N-terminus, followed by a large hydrophilic region. In the case of ABCA13, the hydrophilic region is unexpectedly large, more than 3,500 amino acids, encoded by 30 exons, two of which are 4.8 and 1.7 kb in length. These two large exons are adjacent to each other and are conserved in the mouse Abca13 gene. Tissue profiling of the major transcript reveals the highest expression in human trachea, testis, and bone marrow. The expression of the gene was also determined in 60 tumor cell lines and the highest expression was detected in the SR leukemia, SNB-19 CNS tumor and DU-145 prostate tumor cell lines. ABCA13 has high similarity with other ABCA subfamily genes which are associated with human inherited diseases: ABCA1 with the cholesterol transport disorders Tangier disease and familial hypoalphalipoproteinemia, and ABCA4 with several retinal degeneration disorders. The ABCA13 gene maps to chromosome 7p12.3, a region that contains an inherited disorder affecting the pancreas (Shwachman-Diamond syndrome) as well as a locus involved in T-cell tumor invasion and metastasis (INM7), and therefore is a positional candidate for these pathologies.

Abstract 1688: A novel anti-CEACAM5 maytansinoid-antibody-drug conjugate for the treatment of colorectal, lung and gastric tumors

Carcinoembryonic antigen cell adhesion molecule 5, CEACAM5 (CEA, CD66e) is a well known tumor marker, in particular in colorectal carcinomas, where circulating CEA is used to monitor response to chemotherapy. This GPI anchored glycoprotein belongs to the CEA-related cell adhesion molecule (CEACAM) family and shares domains identity to other members, like CEACAM6. CEACAM5 is expressed in non-human primate and also shares identity to different members, making it difficult to find an antibody both selective to human CEACAM5 and cross-reacting solely with monkey CEACAM5. CEACAM5 has been described in the literature as a poorly internalizing surface protein, but interestingly, antibodies with different uptake capacities have been found. CEACAM5 is highly expressed at the surface of tumor cells in several epithelial tumors, including CRC, lung and gastric tumors and displays a limited expression in normal tissue where it is found solely at the luminal surface of columnar absorptive cells. This prompted us to develop an anti-CEACAM5 antibody-drug conjugate (ADC) for the treatment of CEACAM5-positive tumors. We generated multiple anti-CEACAM5 antibodies by immunization of Balb/c mice with recombinant human and monkey CEACAM5 extracellular domain and CEACAM5-positive tumor cells. We selected a highly specific CEACAM5 antibody that cross-reacts with monkey. The Alexa488-labeled anti CEACAM5 antibody internalizes in tumor cells and is processed in lysosomes leading to free Alexa488 molecules. Although the internalization rate was shown to be moderate, the high number of CEACAM5 at the surface of tumor cells allowed to produce a high number of free Alexa488, suggesting that the antibody could be suited for conjugation to a cytotoxic molecule. Indeed, conjugation of the antibody to the cytotoxic maytansinoid, DM4, with the cleavable SPDB linker generated SAR408701, which kills different CEACAM5-positive tumor cells at sub-nM concentration. SAR408701 in vivo efficacy was evaluated in CRC, lung and gastric patient-derived xenografts (PDXs), following a single injection of ADC at low doses (2.5-5 mg/kg). The conjugate was able to elicit strong and specific antitumor efficacy in a number of PDX models representative of the CEACAM5-positive patient population. Repeating the administrations resulted in most cases in a more pronounced antitumor efficacy even at doses that were otherwise marginally active as single dose. SAR408701 was well tolerated in cynomologus monkey and displayed similar toxicity profile as other SPDB-DM4 ADCs. Based on preclinical efficacy data and the absence of target mediated toxicity in monkey, SAR408701 is expected to have anticancer activity with a favorable therapeutic index, warranting its evaluation in patients with CEACAM5-positive tumors. Citation Format: Stephanie Decary, Pierre-Francois Berne, Celine Nicolazzi, Anne-Marie Lefebvre, Tarik Dabdoubi, Beatrice Cameron, Catherine Devaud, Catherine Prades, Herve Bouchard, Alhassan Casse, Christophe Henry, Celine Amara, Paul Ferrari, Laetitia Macon, Eric Lacoste, Cecile Combeau, Eric Beys, Souad Naimi, Francis Blanche, Carlos Garcia-Echeverria, Jean-Francois Mayaux, Veronique Blanc. A novel anti-CEACAM5 maytansinoid-antibody-drug conjugate for the treatment of colorectal, lung and gastric tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1688. doi:10.1158/1538-7445.AM2015-1688

Abstract 1198: Characterization of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 expressed at the surface of tumor cells

Lysosome-associated membrane protein 1 (LAMP1), one of the most abundant protein expressed at the membrane of lysosomes, is a type I transmembrane protein involved in the maintenance of lysosome membrane integrity and phagolysosome formation. In activated lymphocytes LAMP1 is described as a marker of degranulation. Unexpectedly, by immunizing mice with a colon patient-derived xenograft (PDX) followed by a screening of monoclonal antibodies (mAb) by immunohistochemistry (IHC) for selection of antibodies that specifically stain tumor plasma membrane and de-orphaning by Immunoprecipitation-Mass spectrometry, we identified LAMP1 as the target of several antibodies. One of them, Ab-1, showed binding to the luminal domain of human LAMP1 with nM affinity. Crystal structure of its Fab with LAMP1 extracellular domain, showed that the epitope was non-linear, not a glycotope and spanned between position 29 to195. LAMP1 expression was further documented by IHC with Ab-1, showing limited cell surface expression in normal tissues while moderate to high plasma membrane expression was found in a number of breast, including TNBC, colorectal, gastric, prostate, lung and ovary tumors. The humanized Ab-1 mAb, humAb-1, was shown to display rapid cycling after binding to LAMP1 at the surface of colo205 cell line, allowing internalization and processing of a number of LAMP1/antibody complex 10 folds higher than the number of LAMP1 molecules at the cell surface. humAb-1 was conjugated to DM4 maytansinoid derivative using an SPDB cleavable linker to generate a new antibody-drug conjugate, SAR428926, for the treatment of patients with cell surface LAMP1-positive tumors. Conjugated and naked antibody displayed similar affinities for LAMP1. SAR428926 killed tumor cell lines (engineered to express cell surface LAMP1) in the sub-nM range. In contrast, no target-mediated cytotoxicity was observed when SAR428926 was incubated with normal cells, including resting or activated lymphocytes. PDX models reflecting the pattern and level of LAMP1 expression at the surface of human tumors were selected to evaluate SAR428926 in vivo efficacy. Outstanding in vivo activity was observed in different indications, including TNBC, lung and colon PDXs, with complete regressions after a single administration at 5 mg/kg. These encouraging preclinical data have prompted the initiation of IND-enabling studies with the goal to progress humAb-1-ADC to the clinic in patients with tumors expressing LAMP1 at the cell surface. The First-In-Human trial has been initiated in October 2015. Citation Format: Yves Baudat, Beatrice Cameron, Tarik Dabdoubi, Anne-Marie Lefebvre, Ana Merino-Trigo, Corinne Thomas, Veronique Pecheux, Bruno Genet, Loreley Calvet, Lydia Blot, Magali Mathieu, Laurence Gauzy, Laurence Berthou-Soulie, Catherine Prades, Celine Amara, Manoel Nunes, Christophe Henry, Cecile Combeau, Francis Blanche, Jean-Francois Mayaux, Carlos Garcia-Echeverria, Souad Naimi, Veronique Blanc. Characterization of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 expressed at the surface of tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1198.