Jean-François Mayaux

High-level secretion of correctly processed recombinant human interleukin-1β in Kluyveromyces lactis

The lactose-assimilating yeast, Kluyveromyces lactis, has been developed as a microbial host for the synthesis and secretion of human proteins. Here, we report the use of multi-copy vectors based on the 2 mu-like plasmid pKD1 from Kluyveromyces drosophilarum [Chen et al., Nucleic Acids Res. 14 (1986) 4471-4481] for the secretion of recombinant human interleukin-1 beta (reIL-1 beta). High levels of reIL-1 beta were secreted into the growth medium when the structural gene was fused in-frame to a synthetic secretion signal derived from the pre-region of the K. lactis killer toxin. N-terminal sequencing of the excreted protein showed highly efficient (greater than 95%) maturation of the signal sequence. Synthesis as prepro-IL-1 beta, the pro-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1 beta. Cytoplasmic production of Met-IL-1 beta, without a secretion signal, was found to be toxic to K. lactis. As in Saccharomyces cerevisiae [Baldari et al., EMBO J. 6 (1987) 229-234], but unlike native human IL-1 beta, K. lactis reIL-1 beta is glycosylated. This glycosylation led to a 95% loss of its biological activity. Removal of the carbohydrate chains by endo-beta-N-acetyl-glucosamidase H treatment fully restored the biological activity. A modified form of IL-1 beta (Asn7----Gln7), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1 beta. The level of secretion of mature recombinant IL-1 beta ws glycosylation-independent.

Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase.

A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 mumol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrile hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSR1-derived shuttle vector.

Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa.

A Brevibacterium sp. R312 DNA fragment encoding the wide-spectrum amidase (EC 3.5.1.4) has been cloned and sequenced, using limited amino acid (aa) sequence information obtained from the purified enzyme. The deduced aa sequence showed more than 80% strict identity with the Pseudomonas aeruginosa aliphatic amidase, the product of the amiE gene, suggesting a horizontal transfer of the gene during evolution between Gram+ and Gram- bacteria.

Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1β

Expression plasmids carrying the coding sequence of mature human interleukin 1 beta (IL 1 beta) linked either to a Met start codon, or fused to different efficient Escherichia coli secretion signal sequences, have been constructed. In the latter case, we used signal peptides derived either from an outer membrane protein (OmpA) or from a periplasmic protein (PhoA). The synthesis of IL1 beta from these fusions was investigated in an otherwise strictly isogenic context using identical conditions of derepression and culture media. The Met-IL1 beta fusion produced a soluble cytoplasmic protein which could be released from the cells by osmotic shock whereas the OmpA and PhoA fusions were always insoluble. The extent of sOmpA-IL1 beta maturation was found to vary from 50 to 100%, mainly depending on the medium used, whereas no significant maturation of the signal peptide could be detected in the case of the sPhoA-IL1 beta fusion. Immuno-electron microscopy revealed that the sOmpA-IL1 beta fusion was targeted to the inner membrane, whereas the sPhoA-IL1 beta fusion remained within the cytoplasm and thus did not appear to enter the secretion pathway. Amplifying the E. coli signal peptidase lep gene on a multicopy plasmid did not improve signal peptide removal from sOmpA-IL1 beta. Moreover, these E. coli secretion vectors allowed us to produce, in high levels, IL1 beta fragments which otherwise could not be stably accumulated within the cytoplasmic compartment.

Biotin biosynthetic pathway in recombinant strains of Escherichia coli overexpressing bio genes: evidence for a limiting step upstream from KAPA

The effect of the overexpression of the bioABFCD operon on the biotin biosynthetic pathway was investigated in an Escherichia coli K12 bioR mutant with a chromosomal deletion for the biotin operon. When transformed with a multicopy number plasmid containing bioABFCD, this strain synthetized 10,000 times more biotin than a wild-type E. coli strain. In order to further increase biotin production, the bioA and bioB operons were subcloned into plasmids with stronger promoters and in some cases optimal ribosome binding sites. The new constructions led to the accumulation of large amounts of soluble Bio proteins (although not BioC) but did not improve biotin production. In all the constructed strains, BioA, BioD, and BioB activities were greatly amplified but these activitie did not correlate with the level of protein syntthesis. These strains accumulated only low levels of vitamers, auggesting that the major limiting step for higher biotin production occurs upstream from the first intermediate of the Bio pathway we assayed (7,keto-8-aminopelargonic acid). As BioC overproduction was shown to impair cell growth, we could not determine if this early step of pathway was limiting.

Rapid one-step automated sequencing reactions for 16 DNA samples using Taq polymerase and fluorescent primers

The optimization of fully automated sequencing reactions and analysis is a prerequisite for large scale sequencing programs. We describe here a very simple and reliable protocol which also solves all the evaporation problems encountered for sequencing reactions in microtiter plate. We used the property of the thermostable DNA polymerase from Thermophillus aquaticus, Taq, which is optimally active for DNA sequencing at high temperatures (60 to 80°C) in order to perform in a single heating step both primer-annealing and polymerization reactions. Hereafter is described a protocol using the BiomekTM1000 workstation equipped with the HCB-1000 thermocontroler (Beckman) which allows to perform simultaneous sequencing reactions with fluorescent primers on 16 different single-stranded DNAs in a microtiter plate. This protocol optimizes the full-speed utilization of the 370A automated DNA sequencer (Applied Biosystems). One of the most critical steps in successful DNA sequencing with fluorescent primers is the quality of DNA preparation. We strongly advise to wash out carefully any trace of PEG when using the protocol described by Messing et al. (1983). An amount of 3 ng of template generally gave excellent results even with high GC-percent DNAs (up to 66% tested). For instance, we can routinely obtain in that case accurate 370 bp sequences with more than 99% reliability. Taq DNA polymerase was purchased from Promega-Biotech. Deoxy and dideoxy-nucleotides were from Pharmacia-LKB, except for deaza-guanosine triphosphate (d-c7GTP), which was from Boehringer-Mannheim. M13 (—21) fluorescent-dye primers were from Applied Biosystems. Reactions are carried out in a V-bottom 96-wells vinyl microtiter plate (Dynatec, Polyabo, France) at + 4°C. Add in order:

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 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.