Céline Nicolazzi

Anionic polyethyleneglycol lipids added to cationic lipoplexes increase their plasmatic circulation time

Cationic liposomes have been widely sensed as good DNA compacting delivery agents. Although their use generally met with encouraging results in vitro, the results in vivo were rather disappointing, as they strongly interact with the blood components before they can reach the therapeutic target. Polyethyleneglycol (PEG) shielding has been proposed as a way to alleviate this effect, but was still found unsatisfactory in most instances for systemic administration. We demonstrate here that the insertion of anionic functions between the lipid part and the PEG, at a correct distance to favor electrostatic interactions with the outer cationic layer of the lipoplexes, provides not only a decrease in the mean peripheral charge of the lipoplex (ζ potential), but also a greater colloidal stability of the particles in the presence of serum. Transfection in the lung is also decreased with negatively charged PEG shielding, although no significant changes are observed in the tumor. This encouraging new approach should consequently be combined with active extra-cellular receptor targeting to achieve the desired delivery of the therapeutic DNA to tumor tissues.

Translational Model-Based Strategy to Guide the Choice of Clinical Doses for Antibody–Drug Conjugates

This work proposes a model‐based approach to help select the phase 1 dosing regimen for the antibody–drug conjugate (ADC) SAR408701 leveraging the available data for 2 other ADCs of the same construct: SAR3419 and SAR566658. First, monkey and human pharmacokinetic (PK) data of SAR566658 and SAR3419 were used to establish the appropriate allometric approach to be applied to SAR408701 monkey PK data. Second, a population pharmacokinetics‐pharmacodynamics (PK‐PD) model was developed to describe tumor volume evolution following SAR408701 injection in mice. Third, allometric approaches identified for SAR566658 and SAR3419 were applied to SAR408701 monkey PK data to predict the human PK profile. Both SAR566658 and SAR3419 human and monkey PK were best described by a 2‐compartment linear model. The relative difference was less than 10% between predicted and observed clearance using allometric exponents of 0.75 and 1, respectively. Tumor volume evolution following SAR408701 injection was best described by a full Simeoni model with a plasma concentration threshold of 4.6 μg/mL for eradication in mice. Both allometric exponents were used to predict SAR408701 PK in human from PK in monkey and to identify the potential effective dosing regimens. This translational strategy may be a valuable tool to design future clinical studies for ADCs, to support selection of the most appropriate dosing regimen, and to estimate the minimal dose required to assure antitumor activity, according to the schedule used.

Abstract 1197: Outstanding preclinical efficacy of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 in patient-derived xenograft solid tumors

Lysosome-associated membrane protein 1 (LAMP1) is involved in the maintenance of lysosome membrane integrity and phagolysosome formation. LAMP1 shows very limited expression at the cell surface in normal tissues while, a moderate to high expression was found in a number of solid tumors. We generated SAR428926, an antibody drug conjugate (ADC) comprising an anti-LAMP1 humanized monoclonal antibody conjugated via a cleavable SPDB linker, to the maytansinoid derivative DM4, a tubulin interfering molecule. Here we report the annotation of LAMP1 expression in a large panel of human tumors and correlate it to the in vivo efficacy of SAR428926 in a panel of over 50 human patient-derived xenograft (PDX) solid tumors. Indications of interest were identified by immunohistochemistry (IHC) conducted on a large panel of frozen tumor samples using the murine version of the therapeutic anti-human LAMP1 antibody. The prevalence of tumor samples with positive membrane staining was 49% in breast invasive lobular or ductal carcinoma, and in particular in a TNBC subset (87%), 25% in gastric adenocarcinoma, 52% in colon/rectum adenocarcinoma, 22% in lung adenocarcinoma, 20% in lung squamous cell carcinoma, 25% in prostate adenocarcinoma and 31% in ovary adenocarcinoma. In vivo efficacy of SAR428926 was evaluated in PDX models engrafted subcutaneously into immunocompromised SCID mice. PDX models retain the molecular diversity, cellular heterogeneity, and histology typically seen in patient tumors and offer a distinct advantage over cell line models. The PDX models used in the study, which include colon, breast, lung, prostate, gastric and ovarian cancer, were selected by IHC based on LAMP1 expression levels distribution and the frequency of LAMP1 membrane positive cells, which varied between 5% and 100% of the tumor cells, allowing coverage of a wide variety of cases. Outstanding efficacy with complete regressions was observed at 5 mg/kg iv single administration, a dose that gives an exposure tolerated in toxicology species. Efficacy was also observed at lower doses of 2.5 mg/kg and 1.25 mg/kg after single administration. Our results show that while the presence of the antigen is required as there is no efficacy in LAMP1-negative PDX, there is no linear correlation between the level of antigen expression and the antitumor activity across the panel of PDX tested. In addition to membrane LAMP1 expression, a key driver for efficacy was the intrinsic sensitivity of the PDX models to DM4. These results highlight the potential of ADCs that target human cancers expressing membrane LAMP1 at the surface of tumor cells. These encouraging preclinical data have prompted us to initiate IND-enabling studies with the goal to progress anti LAMP1-ADC to the clinic in patients with solid tumors expressing LAMP1 at the cell surface. The phase I study started in October 2015. Citation Format: Loreley Calvet, Anne-Marie Lefebvre, Celine Nicolazzi, Lydia Blot, Corinne Thomas, Yves Baudat, Beatrice Cameron, Carlos Garcia-Echeverria, Jean-Francois Mayaux, Veronique Blanc, Cecile Combeau, Souad Naimi, Sukhvinder Sidhu. Outstanding preclinical efficacy of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 in patient-derived xenograft solid tumors. [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 1197.

Abstract LB-053: Towards new cryptophycins as promising payloads for ADC

Cryptophycins are a class of macrocyclic depsipeptides produced as secondary metabolites by cyanobacteria of the genus Nostoc and were found to bind to microtubules at the vinca site. The natural product C-1 isolated in 1990 and the synthetic derivative C-52 displayed potent in vitro and in vivo antitumor activity in preclinical models. Cryptophycin C-52 (LY355703) produced marginal antitumor activity at MTD in two phase II lung cancer trials and was therefore discontinued. Considering its higher potency versus other tubulin binders such as maytansine and auristatin, this chemical series was selected for an ADC approach. Several conjugates were evaluated in vitro and in vivo based on the conjugation of C52 derivatized at the para-benzylic position of the macrocycle. As described in this study, this optimization led to the discovery of cryptophycin ADC which displayed potent antitumor activity in vivo. However, these conjugates were found unstable in mice plasma while being stable in the plasma of non-rodent species. This species-dependent instability was shown to be the result of a mice plasmatic metabolization of C52 macrocycle once conjugated to the antibody. We therefore designed and synthesized new cryptophycin ADC which exhibited improved plasmatic stability when tested in mice and enhanced therapeutic index in comparison with C52 ADC. In conclusion, these data demonstrated the potent in vitro and in vivo antitumor activity of these new cryptophycin ADC and warrant further development of this cytotoxic payload for an ADC approach. Citation Format: Marie-Priscille Brun, Herve Bouchard, Francois Clerc, Jidong Zhang, Pierre-Yves Abecassis, Celine Amara, Eric Beys, Florence Efremenko, Celine Nicolazzi, Marie-Helene Pascual, Olivier Pasquier, Alain Krick, Pierre-Francois Berne, Laurence Gauzy, Nathalie Karst, Sylvain Huille, Christophe Henry, Anne-Marie Lefebvre, Ingrid Sassoon. Towards new cryptophycins as promising payloads for ADC. [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 LB-053.

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 B145: PK/PD evaluation of an anti-CEACAM5 antibody drug conjugate, in a colon patient-derived-xenografted mice model

CEACAM5 is an oncofetal protein that is expressed in a number of tumors of epithelial origin such as colorectal cancer (CRC), lung and gastric cancer. Differential expression of CEACAM5 protein in tumor tissues compared to normal tissues make this target attractive for targeting with an antibody-drug conjugate (ADC). The ADC that is characterized in this study contains a specific anti-CEACAM5 antibody and the maytansinoid derivative DM4, a potent antimitotic agent that inhibits microtubule assembly. DM4 is covalently bound to the humanized IgG1 (hIgG) antibody through a N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB) linker that is stable in plasma and cleavable inside cells. After binding and internalization into CEACAM5-positive cancer cells, this ADC is degraded, releasing cytotoxic DM4 metabolites. In the present study the Pharmacokinetic (PK), Pharmacodynamic (PD) and efficacy relationships of this ADC were investigated against subcutaneous colon Patient-Derived-Xenograft (PDX) in severe combined immunodeficient (SCID) mice. The modulation of several biomarkers after a single administration of this compound at 3 dose levels were quantified in the tumor by immunoassay (phosphorylated Histone H3 (pHH3), a marker of cells arrested in mitosis and free human CEACAM5 expression). The spatial localization in the tumor of the biomarkers (pHH3, free human CEACAM5, DM4 and hIgG) was analyzed by immunohistochemistry (IHC). A population pharmacokinetic analysis of the ADC was performed first, followed by population PK/PD analysis for pHH3 induction and tumor growth inhibition (TGI). This study has shown both by immunoassay and IHC that a single administration of this anti-CEACAM5 ADC induced in the xenografted tumor a dose dependent increase of pHH3 expression compared to control, with a maximal induction observed at 24-48 hours. The levels of DM4 and hIgG detected by IHC in the tumor were dose dependent and progressively decreased with time. DM4 and hIgG part of the ADC were essentially immunodetected at the membrane level of tumor cells and sub-localization was observed in vesicular intracellular compartments at later time point which confirms ADC tumor cell penetration. The PK/PD relationship analysis indicated that compound concentration producing 50% of the maximum pHH3 stimulation was 2.3 μg/mL and the threshold concentration for tumor eradication was 8 μg/mL in this colon PDX model. Both immunoassay and IHC assessment confirmed that single administration of the ADC had no impact on CEACAM5 expression at the tumor cell membrane. Overall these data have confirmed mechanism of action of this anti-CEACAM5 ADC and its attractiveness as a potent compound to induce dose dependent CEACAM5 expressing tumor cell death. This compound is now being explored in a phase 1 clinical trial in cancer patients. Citation Format: Christophe Henry, Celine Nicolazzi, Celine Amara, Anne-Marie Lefebvre, Christelle Larois, Florence Attenot, Claire Brillac, Marion Bouillon-Pichault, Nathalie Fagniez, Stephanie Decary, Cecile Combeau, Pierre-Francois Berne, Souâd Naimi, Marielle Chiron, Veronique Blanc. PK/PD evaluation of an anti-CEACAM5 antibody drug conjugate, in a colon patient-derived-xenografted mice model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B145.