No abstract

Because of their favorable properties as macromolecular drugs, antibodies are a very successful therapeutic modality for interfering with disease-relevant targets in the extracellular space or at the cell membrane. However, a large number of diseases involve cytosolic targets and designing antibodies able to efficiently reach intracellular compartments would expand the antibody-tractable conditions. Here, we genetically fused cell penetrating peptides (CPPs) at various positions to an antibody targeting cancer cells, evaluated the developability features of the resulting antibody-peptide fusions and the ability of selected constructs to reach the cytosol. We first determined positions in the IgG structure that were permissive to CPP incorporation without destabilizing the antibody. Fusing CPPs to the C-terminus of the light chain and either before or after the hinge had the least effect on antibody developability features. These constructs were further evaluated for cell penetration efficiency. Two out of five tested CPPs significantly enhanced antibody penetration into the cytosol, in particular when fused before or after the hinge. Finally, we demonstrate that specific antibody binding to the cell surface target is necessary for efficient cell penetration of the CPP-antibody fusions. This study provides a solid basis for further exploration of therapeutic antibodies for intracellular targets.

Purpose: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a glycoprotein that has limited expression in normal adult tissues, but is overexpressed in carcinomas of the gastrointestinal tract, the genitourinary and respiratory systems, and breast cancer. As such, CEACAM5 is an attractive target for antibody-based therapies designed to selectively deliver cytotoxic drugs to certain epithelial tumors. Here, we describe preclinical data for a novel antibody–drug conjugate (ADC), SAR408701, which consists of an anti-CEACAM5 antibody (SAR408377) coupled to a maytansinoid agent DM4 via a cleavable linker. Experimental Design: The specificity and binding affinity of SAR408701 to human and cynomolgus monkey CEACAM5 were tested in vitro. The cytotoxic activity of SAR408701 was assessed in CEACAM5-expressing tumor cell lines and using patient-derived xenograft mouse models of CEACAM5-positive tumors. Pharmacokinetic-pharmacodynamic and pharmacokinetic-efficacy relationships were established. SAR408701 toxicity was evaluated in cynomolgus monkey. Results: SAR408701 bound selectively to human and cynomolgus monkey CEACAM5 with similar apparent Kd values (0.017 nmol/L and 0.024 nmol/L, respectively). Both in vitro and in vivo evaluations showed that SAR408701 has cytotoxic activity, leading to in vivo efficacy in single and repeated dosing. Single doses of SAR408701 induced significant increases in the tumor expression of phosphorylated histone H3, confirming the tubulin-targeting mechanism of action. The overall toxicity profile of SAR408701 in cynomolgus monkey was similar to that observed after intravenous administration of DM4 alone. Conclusions: On the basis of these preclinical data, the ADC SAR408701 is a promising candidate for development as a potential treatment for patients with CEACAM5-positive 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: Stéphanie Decary, Pierre-François Berne, Céline Nicolazzi, Anne-Marie Lefebvre, Tarik Dabdoubi, Beatrice Cameron, Catherine Devaud, Catherine Prades, Hervé Bouchard, Alhassan Cassé, Christophe Henry, Céline Amara, Paul Ferrari, Laetitia Maçon, Eric Lacoste, Cécile Combeau, Eric Beys, Souad Naimi, Francis Blanche, Carlos Garcia-Echeverria, Jean-François Mayaux, Véronique 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

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.