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Recombinant antibody fragments expressed in the cytoplasm of cells have considerable practical potential. However in the reducing environment of the cytoplasm, the intradomain disulphide bonds are not formed and the fragments are unstable and expressed in low yields. Here we attempted to overcome these limitations. We first isolated an antibody single chain Fv fragment that binds and activates an inactive mutant beta-galactosidase. We then subjected the gene encoding the scFv fragment to random mutation in vitro by error-prone polymerase chain reaction, and co-expressed the mutant beta-galactosidase and mutant antibody fragments in lac- bacteria. By plating on limiting lactose, we selected for antibody mutants with improved expression, and after four successive rounds of mutation and selection, isolated an antibody fragment that is expressed in the bacterial cytoplasm with yields of 0.5 g/l in a shaker flask (A600 nm of 5.5) and 3.1 g/l (A600 nm=33) in a fermentor. Analysis of the mutant antibody fragments revealed that the disulphide bonds are reduced in the cytoplasm, and that the fragments could be denatured and renatured efficiently under reducing conditions in vitro. This shows that with a suitable method of screening or selection, it is possible to make folded and functional antibody fragments in excellent yield in the cytoplasm.

OBJECTIVE: To compare the characteristics and clinical efficacy of low-molecular-weight heparins (LMWHs) and unfractionated heparin (UFH) in the treatment of deep-vein thrombosis (DVT). Adverse effects, dosing, and cost issues are also discussed. DATA SOURCES: A MEDLINE search (January 1984–October 1997) was used to identify pertinent French and English literature, including clinical trials and reviews on LMWHs and their use in DVT. STUDY SELECTION: Trials comparing dalteparin, enoxaparin, tinzaparin, and nadroparin with UFH were selected. As studies were numerous, only randomized trials including more than 50 patients were reviewed. Moreover, all patients studied had a first episode of symptomatic DVT confirmed by objective tests (i.e., venography, duplex ultrasonography, impedance plethysmography). Clinical efficacy and safety of LMWHs were assessed in these trials. DATA EXTRACTION: Results pertaining to venographic assessment, recurrent thromboembolism, total mortality, and bleeding complications were extracted from the selected studies. DATA SYNTHESIS: Compared with UFH, LMWHs have a longer plasma half-life, better subcutaneous bioavailability, more predictable anticoagulant response, and require less intense laboratory monitoring. Most trials demonstrate comparable effects on thrombus extension and incidence of recurrent thromboembolism. Compared with UFH, LMWHs do not alter total mortality. Although animal trials predict a lower hemorrhagic potential for LMWHs, the incidence of bleeding complications is generally similar to that observed with UFH. Outpatient management of DVT with LMWHs has shown comparable safety and efficacy with inpatient UFH use but a shorter hospital stay. CONCLUSIONS: Because LMWHs are as safe and as effective as UFH, and because of their more convenient method of administration, they can be considered valuable alternatives for the treatment of DVT. Savings generated by less intensive laboratory monitoring and the possibility of early hospital discharge and outpatient therapy may outweigh the higher acquisition cost of LMWHs.

Objective To report a case of clarithromycin-induced digoxin intoxication. Case Summary A 78-year-old white man with ischemic cardiomyopathy and chronic renal insufficiency was admitted 4 days after being prescribed clarithromycin for a suspected episode of bronchitis. He reported weakness, asthenia, and gastrointestinal symptoms; the digoxin serum concentration was measured at 3.89 ng/mL. The patient recovered uneventfully after digoxin and clarithromycin were discontinued. Discussion Erythromycin frequently interacts with other drugs that are also metabolized by the CYP3A4 isoenzyme. However, erythromycin is hypothesized to interact with digoxin by inhibiting Eubacterium lentum, which is a normal inhabitant of the human gut and is responsible for intestinal metabolism of digoxin in 10% of patients. Since clarithromycin shares a comparable antibacterial spectrum with erythromycin, the possibility of a drug interaction with digoxin remains. Only four cases of clarithromycin interacting with digoxin have been reported to date. Clinically, this interaction may have been more obvious because of our patient's moderate renal dysfunction and serum digoxin concentrations in the upper therapeutic range prior to clarithromycin initiation. Other causes for digoxin intoxication could not be identified. Conclusions Clarithromycin may inhibit the growth of E. lentum, which can lead to an increase in digoxin bioavailability and blood concentrations in patients in whom this intestinal metabolic pathway is present. Patients at risk include those with renal dysfunction, with serum concentrations in the upper therapeutic range, or with measured digoxin concentrations that are much lower than predicted by Pharmacokinetic calculations. For these patients, appropriate therapy includes the selection of an alternative, noninteracting antibiotic or, if this is not possible, a temporary reduction of digoxin dosage.

International audience; A set of permissive positions that tolerate insertions/deletions without major deleterious consequences for the binding activity of the protein was previously identified in the maltose-binding protein. The C3 epitope from poliovirus VP1 protein (93DNPASTTNKDK103) was inserted into eight of these positions and two nonpermissive control sites. NMR studies were performed on the MalE protein, the insertion/deletion mutants, and the C3MalE hybrids to selectively determine the flexible regions in these proteins. Comparison of the C3 epitope mobility in the different hybrid proteins indicates that, whatever its insertion site and independently from the specific sequences of its linkers, the epitope is mostly flexible. The vector protein was shown to unfold partially only in the two C3MalE hybrids that correspond to nonpermissive positions. For one of them (insertion at site 339), both sides of the insert are flexible, and at most one side for all the other hybrids. This result correlates with the antigenicity data on the inserted epitope (Martineau, P., Leclerc, C., and Hofnung, M. (1997) Mol. Immunol, in press.

In metastatic colorectal cancer (CRC) patients, chemotherapy in combination with targeted therapies such as Avastin, Erbitux, and Vectibix (monoclonal antibodies) has improved response rates with prolongation of progression free survival and overall survival. Despite these major therapeutic progresses, at least 30 to 50% of patients remain non-responders. Our project aimed to identify and validate new molecular targets of CRC. Comparison of gene expression profiles between normal mucosa and primary tumor of CRC patients followed by immunohistochemistry analysis identified claudin-1 (CLDN1) as potential therapeutic target for antibody-targeted therapy. CLDN1 is a major tight junction transmembrane protein, already found overexpressed in CRC in several studies and associated with tumor progression. We have generated monoclonal antibodies (mAb) directed against extracellular domains of CLDN1 and demonstrated their specificity against human CLDN1 in vitro and in vivo. Then, using mice xenografted with the CRC cell line SW620 we tested the therapeutic effect of the 6F6C3mAb. The mice were treated by i.p. injections with vehicle or 6F6C3mAb at two different doses. The results showed that 6F6C3mAb treated-groups had a significant (p= 0,018) reduced growth compared to the control group and this effect was dose-dependent. The in vitro effects of the binding of 6F6C3mAb to CLDN1 were also assessed by clonogenic assays. Treatment by 6F6C3mAb induced a significant (p< 0.05) reduction of the number of colony-forming cells for CLDN1high colorectal cancer cells (SW620, Caco2) but also for CLDN1high ovarian and pancreatic cancer cells (PANC1, BXPC3, SKOV3, IGROV1). Thus, 6F6C3mAb binding had a profound effect on cancer cell survival. In conclusion, CLDN1 seems to be a promising target for antibody-based therapy. The 6F6C3mAb is able to inhibit tumor growth and tumor survival by binding the extracellular domains of CLDN1. It can be used in the treatment of cancer diseases such as colorectal cancer as well in adjuvant as in metastatic setting. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B245. Citation Format: Adeline Ayrolles-Torro, Nadia Vezzio-Vie, Vincent Denis, Florence Boissiere-Michot, Véronique Garambois, Muriel Busson, Imade Ait Arsa, Caroline Mollevi, Martine Pugniere, Frédéric Bibeau, Pierre Martineau, Céline Gongora, Maguy Del Rio. Claudin-1 (CLDN1) as a new therapeutic target in colorectal cancer: Inhibition of cell growth and survival by an anti-CLDN1 monoclonal antibody. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B245.

Cetuximab is used in colorectal cancer (CRC), as targeted therapy against the Epithelial Growth Factor receptor (EGFR), in association with chemotherapy (5-FU and irinotecan). Its binding inhibits signalling pathways downstream to the receptor leading to a decrease in proliferation and surviving. In KRAS mutated CRC patients, cetuximab is ineffective, and half of KRAS Wild Type (WT) patients does not respond to cetuximab either. Thus, a better knowledge of cetuximab mechanism of action will help to improve response rate. We have previously shown that activation of the MAP Kinase p38 (p38MAPK) induces irinotecan resistance in vitro and in vivo. The aim of our project is to determine, in KRAS WT colorectal cells, if p38MAPK is involved as well in the cetuximab effect. Experiments were done on two KRAS WT CRC cell lines which respond differently to cetuximab: Caco2 cells (30% of survival inhibition, p53 mutated) and DiFi cells (80% of survival inhibition, p53 Wild Type). Cytotoxic experiments combining cetuximab treatment and inhibition of p38MAPK by a pharmacological inhibitor (SB202190) were performed. We assessed apoptosis and inhibition of proliferation by FACS analysis of cell cycle and DNA synthesis. In addition, Erk, Bim and p27 were analysed by Western Blot. Our results showed that inhibition of p38MAPK by SB202190 enhances cetuximab cytotoxic effect in Caco2 cells but impairs it in DiFi cells. We also observed that inhibition of p38MAPK decreases cetuximab induced apoptosis (from 40% of cells in subG1 to 15%) and anti-proliferative effect (from 10% EdU positive cells to 40%) in DiFi cells. The prevention of cell death by SB202190 in cetuximab treated DiFi cells could be explained by Erk pathway activation and the decrease of p27 and Bim respectively involved in cellular proliferation and mitochondrial apoptosis. We have shown that genetic inhibition of p53 also prevents cetuximab cytotoxicity in DiFi cells and we observed a synergistic effect with p38MAPK inhibition leading to higher impairment of cetuximab effect. Our results have shown the same inhibiting effect of SB202190 on the cytotoxic effect of two tyrosine-kinase inhibitors targeting EGFR (lapatinib and erlotinib) in DiFi cells indicating that p38MAPK implication is linked to inhibition of EGFR kinase activity not to cetuximab only. We have shown that p38MAPK is involved in response to the inhibition of EGFR activity. However, it seems to have different role according to the cell line used and genetic background (p53 status). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A62. Citation Format: Laetitia Marzi, Eve Combès, Nadia Vezzio-Vié, Gaelle Thomas, Christel Larbouret, Laetitia Linares, Clara Montagut, Mar Iglesias, Vincent Denis, Maguy Del Rio, Pierre Martineau, Céline Gongora. p38MAPK implication in cetuximab response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A62.

To identify genes implicated in metastatic colonization of the liver in colorectal cancer, we collected pairs of primary tumors and hepatic metastases before chemotherapy in 13 patients. We compared mRNA expression in the pairs of patients to identify genes deregulated during metastatic evolution. We then validated the identified genes using data obtained by different groups. The 33-gene signature was able to classify 87% of hepatic metastases, 98% of primary tumors, 97% of normal colon mucosa, and 95% of normal liver tissues in six datasets obtained using five different microarray platforms. The identified genes are specific to colon cancer and hepatic metastases since other metastatic locations and hepatic metastases originating from breast cancer were not classified by the signature. Gene Ontology term analysis showed that 50% of the genes are implicated in extracellular matrix remodeling, and more precisely in cell adhesion, extracellular matrix organization and angiogenesis. Because of the high efficiency of the signature to classify colon hepatic metastases, the identified genes represent promising targets to develop new therapies that will specifically affect hepatic metastasis microenvironment.

Background: Despite recent advances in the treatment of colorectal cancer, tumor resistance is a frequent cause of chemotherapy failure. Thus, new treatment options are needed to improve survival of patients with irinotecan-refractory colorectal cancers, particularly those bearing KRAS mutations that preclude the use of anti-EGFR therapies. In this study, we investigated whether sorafenib could reverse irinotecan resistance, thereby enhancing the therapeutic efficacy of routinely used irinotecan-based chemotherapy. Materials and methods: We used both in vitro (the HCT116, SW48, SW620 and HT29 colon adenocarcinoma cell lines and four SN-38 resistant HCT-116 and SW48 clones) and in vivo models (nude mice xenografted with SN-38 resistant HCT116 cells) to test the efficacy of sorafenib alone or in combination with irinotecan, or its active metabolite SN-38. Results: Sorafenib improved the anti-tumoral activity of irinotecan in vitro, in both parental and SN-38 resistant colon adenocarcinoma cell lines independently of their KRAS status, as well as in vivo, in xenografted mice. By inhibiting the drug-efflux pump ABCG2, sorafenib favors irinotecan intracellular accumulation and enhances its toxicity. Conclusion: Our results show that sorafenib can suppress resistance to irinotecan and suggest that sorafenib could be used to overcome resistance to irinotecan-based chemotherapies in colorectal cancer, particularly in KRAS mutated tumors. Citation Format: Thibault Mazard, Annick Causse, Joelle Simony, Wilhem Leconet, Marta Jarlier, Marc Ychou, Maguy Del Rio, Pierre Martineau, Bruno Robert, Céline Gongora. Sorafenib overcomes irinotecan resistance in colorectal cancer by inhibiting the ABCG2 drug-efflux pump. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 975. doi:10.1158/1538-7445.AM2013-975

In a previous study, a set of positions in the MalE protein from Escherichia coli were identified, which tolerated short insertions or deletions without compromising the maltose binding activity of the protein. It is now shown that these sites accommodate an insert of 13 amino acids and are, therefore, permissive. Eleven sites were used, including eight permissive sites, to display a linear neutralization B-cell epitope of poliovirus (C3 epitope) at different positions on the surface of MalE. The affinity of a monoclonal neutralizing anti-poliovirus antibody (anti-C3 mAb) for the hybrid proteins varied from undetectable, to more than 1000 times higher than for the synthetic peptide. Therefore, some MalEC3 proteins mimic interactions of the viral epitope with the monoclonal antibody more efficiently than the free peptide. The results are interpreted in terms of the mobility of the insert and its flanking regions. It was further shown that some of the purified hybrid proteins are able to induce high titer anti-C3-peptide antibodies in mice. A strong correlation exists between the capacity of a MalEC3 protein to induce anti-C3-peptide antibodies and the antigenicity of the inserted peptide, measured with a polyclonal serum raised against the synthetic peptide.

Despite recent advances in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. In the last decade, several studies have shown that cetuximab, an anti-EGFR monoclonal antibody, can overcome acquired resistance to irinotecan chemotherapy, but only in KRAS non mutant tumors. Thus, new treatment options are needed to improve survival in patients with irinotecan refractory and KRAS mutated colorectal cancer. In this study, we examined if treatment with sorafenib, a potent inhibitor of Raf kinase and VEGF receptor, could reverse the resistant phenotype in tumor, thereby enhancing the therapeutic efficacy of currently used irinotecan treatment. We used both in vitro and in vivo models to test the efficacy of sorafenib either as a single agent or in combination with irinotecan. We have first tested these different combinations on seven colorectal cancer cell lines displaying different molecular characteristics (mutated or not for KRAS, BRAF, p53 and PIK3CA). We have shown that sorafenib, as a single agent, showed antitumor properties and the effects were more pronounced when it was used in combination with the active metabolite of irinotecan, SN38. Then we have tested these treatments on SN38-resistant clones derived from the colon adenocarcinoma HCT-116 (KRAS mut) and SW48 (KRAS WT) cell lines, that show various levels (6 to 60 fold) of resistance to SN-38 as compared to the corresponding parental cells. Sorafenib has improved the anti-tumoral activity of SN-38 on all the SN38 resistant clones in vitro. Moreover, sorafenib sensitized tumor cells derived from both SN38-sensitive and -resistant HCT116 cells to irinotecan treatment in xenograft models. Signalling pathways involved in this effect have been identified on xenografts using the proteome profiler array. Altogether, our results show for the first time that sorafenib can inhibit resistance to irinotecan and suggest that sorafenib could be used to overcome resistance to irinotecan-based chemotherapies in colorectal cancer, particularly in KRAS mutated tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1902. doi:1538-7445.AM2012-1902

Foreign polypeptides can be expressed as genetic inserts in several permissive sites of MalE and LamB, two Escherichia coli envelope proteins. Several viral B and T-cell epitopes have been inserted in these proteins and we analyzed the role of the molecular environment on the immunogenicity of the foreign epitopes. These studies demonstrated that the antigenicity and immunogenicity of B-cell epitopes depend on their site of insertion in the carrier protein. Using bacteria expressing B-cell epitopes either at the cell surface or in the periplasm, it was also shown that the cellular location of a foreign B-cell epitope expressed by recombinant bacteria determines its T-cell dependent or independent characteristics. Analysis of in vivo immunogenicity of purified LamB or MalE hybrid proteins expressing two different T-cell epitopes established that the immunogenicity of recombinant T-cell epitopes may be strongly affected by both the insertion site and inserted adjacent residues. The in vitro analysis of specific T-cell hybridoma response to hybrid MalE proteins also showed that the molecular context of a T-cell determinant alters the diversity of its T-cell recognition.

Despite recent advances in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. To better elucidate the molecular mechanisms involved in resistance to irinotecan (and its active metabolite SN38), we established SN38-resistant clones derived from the colon adenocarcinoma HCT-116 and SW48 cell lines. These clones show various levels (6 to 60 fold) of resistance to SN-38 and display enhanced levels of activated MAPK p38 as compared to the corresponding parental cells. Because four different isoforms of p38 have been described, we then studied the effect of p38 overexpression or downregulation of each isoform on cell sensivity to SN38 and found that both alpha and beta isoforms are involved in the development of resistance to SN38. In this line, we show that cell treatment with SB202190, which specifically inhibits p38 alpha and beta, enhanced the cytotoxic activity of SN38, but not of 5-FU or oxaliplatin. Moreover, p38 inhibition sensitized tumor cells derived from both SN38-sensitive and -resistant HCT116 cells to irinotecan treatment in xenograft models. Finally, we detected less phosphorylated p38 in primary colon cancer of patients sensitive to irinotecan-based treatment, compared to non-responder patients. This indicates that enhanced level of expression of phosphorylated p38 could predict the absence of clinical response to irinotecan. Altogether, our results show for the first time that the p38 MAPK pathway is involved in irinotecan sensitivity and suggest that phosphorylated p38 expression level could be used as a marker of clinical resistance to irinotecan. They further suggest that targeting the p38 pathway may be a potential strategy to overcome resistance to irinotecan-based chemotherapies in colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2011-1639

In a previous study [(1987) J. Mol. Biol. 194, 663-673], we isolated ten insertion/deletion mutants (indels) of the maltose binding protein for which the maltose binding constant was only a little or not at all affected. In this paper, we have localized these mutations in the recently solved three-dimensional structure. Contrary to the general expectation, most of the insertion/deletion modifications occurred within elements of secondary structure. An analysis of the inserted residues for three indels found within α helices allowed an interpretation regarding protein structure accommodation to such modifications.

The Escherichia coli maltose-binding protein is a highly versatile carrier protein allowing the construction of genetically engineered hybrid proteins. It accepts large fusions to both C- and N-termini as well as the insertion of shorter peptides at 'permissive sites' within the continuity of the protein. We have genetically inserted immunogenic peptides corresponding to defined viral B- and T-cell epitopes into two permissive sites: one at amino acid site 133, the other at site 303. The hybrid proteins are easily purifiable and immunogenic, inducing peptide-specific B- and T-cell responses. When delivered by live bacteria (E. coli K12 and aroA Salmonella typhimurium) antibody responses can be induced against both the MalE carrier and the inserted B-cell epitope. We discuss the induction of T-cell responses by bacterial delivery systems.

Despite the recent advances achieved in the treatment of colon cancer, tumour resistance is a frequent cause of chemotherapy failure. Our work was aimed to determine the molecular mechanisms involved in the resistance to irinotecan (SN38), an anticancer agent widely used in colorectal cancer treatment. To this end, we have established an SN38-resistant cellular model from the colon adenocarcinoma cell line HCT-116 and we have obtained 5 resistant clones. In all these five clones we have observed an activation of the MAPK p38 compared to the sensitive cell line called HCT116-s. We have shown in the HCT116-SN6 clone that the p38 pathway is responsible for the resistance to SN38. The family of MAPK p38 counting four isoformes α, β, γ and δ, we have generated cells lines overexpressing each isoforms or inhibiting each isoform expression, and we have demonstrate that the α and β forms are involved in SN38 resistance. In a xenograft model we have shown that the HCT116-SN6 cell line is also resistant to Campto treatment in vivo, and that the α and β isoforms are still responsible for drug resistance. Finally, we have also detected phosphorylated p38 in colon cancer cells of patients resistant to Irinotecan-based chemotherapy. These results show for the first time that the p38 pathway is implicated in the drug sensitivity of colon cancer cells and that targeting p38 could be a good alternative to decrease drug resistance. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2565.

Lactational insufficiency is one of the major problems in swine production, and the consequences on the growth of the litter must be emphasized. Pathophysiology involves neuroendocrinologic interactions and the role of target organs such as the mammary glands, uterus, bladder, and gut. The complexity of these interactions and the fact that all interactions are not well understood leads to a broad approach. Some risk factors and their associations with lactational insufficiency are described to help the practitioner in the clinical approach to such a problem.

The construction of recombinant proteins by genetic engineering has opened new avenues in basic research (studies on protein organization, protein folding, immunogenicity of proteins, etc) and in the development of applications (vaccines, diagnosis, targeting of polypeptides, combination of activities, etc). Recombinant proteins which retain properties of both parental proteins are especially interesting. For example, if one protein (the vector protein) is targeted to a given cellular compartment, the other protein (the passenger) may be identically targeted. Also, if the vector protein can be purified by a simple affinity chromatographic procedure, this property may be extended to the passenger. However, recombinant proteins constructed without precaution are often unstable, toxic, insoluble or have lost some of the useful properties of the “parent” proteins.

We present a simple and flexible procedure to elicit and assay anti-peptide antibodies without peptide synthesis. It consists of expressing the peptide of interest in the form of a genetic insert within two different "recipient" bacterial proteins. One hybrid protein is used as immunogen for the induction of antibodies against the inserted peptide and the other as antigen for monitoring the anti-peptide antibodies raised. The two "recipient" proteins used are the MalE and the LamB proteins from E. coli. The MalE hybrid proteins can be affinity purified on an amylose column using mild nondenaturing conditions and can be crystalized for structural studies; LamB hybrid proteins express the inserted peptide on the cell surface so that intact bacteria can be used as a reagent. We chose, as a model peptide, a B-cell epitope from the pre-S(2) region of Hepatitis B virus. With both MalE and LamB hybrid proteins, high titres of anti-preS antibodies, able to react with native HBsAg particles, were induced in mice. The anti-peptide antibody titres recorded by ELISA were comparable to those obtained when either a synthetic peptide, or the hybrid proteins, were used as immobilized antigen.

The periplasmic maltose-binding protein (MBP or MalE protein) of Escherichia coli is an essential element in the transport of maltose and maltodextrins and in the chemotaxis towards these sugars. On the basis of previous results suggesting their possible role in the activity and fluorescence of MBP, we have changed independently to alanine each of the eight tryptophan residues as well as asparagine 294, which is conserved among four periplasmic sugar-binding proteins. Five of the tryptophan mutations affected activity. In four cases (substitution of Trp62, Trp230, Trp232 and Trp340), there was a decrease in MBP affinity towards maltose correlated with modifications in transport and chemotaxis. According to the present state of the 2.3 A three-dimensional structure of MBP, all four residues are in the binding site. Residues Trp62 and Trp340 are in the immediate vicinity of the bound substrate and appear to have direct contacts with maltose; this is in agreement with the drastic increases in Kd values (respectively 67 and 300-fold) upon their substitution by alanine residues. The modest increase in Kd (12-fold) observed upon mutation of Trp230 would be compatible with the lesser degree of interaction this residue has with the bound substrate and the idea that it plays an indirect role, presumably by keeping other residues involved directly in binding in their proper orientation. Substitution of Trp232 resulted in a small increase in Kd value (2-fold) in spite of the fact that this residue is the closest to the ligand of the tryptophan residues according to the three-dimensional model. In the fifth case, replacement of Trp158, which is distant from the binding site, strongly reduced the chemotactic response towards maltose without affecting the transport parameters or the sugar-binding activities of the mutant protein. Trp158 may therefore be specifically implicated in the interaction of MBP with the chemotransducer Tar, but this effect is likely to be indirect, since Trp158 is buried in the structure of MBP. Of course, some structural rearrangements could be responsible in part for the effects of these mutations. The remaining four mutations were silent. The corresponding residues (Trp10, Trp94, Trp129 and Asn294) are all distant from the sugar-binding site on the crystallographic model of MBP, which is in agreement with their lack of effect on binding. In addition, our results show that they play no role in the interactions with the other proteins of the maltose transport (MalF, MalG or MalK) or chemotaxis (Tar) systems.(ABSTRACT TRUNCATED AT 400 WORDS)

The periplasmic maltose binding protein (MBP) is required for the high affinity transport of maltose and maltodextrins and for chemotaxis towards these sugars. In these functions, MBP interacts with proteins of the cytoplasmic membrane: MalF and MalG for transport, Tar for chemotaxis. A large number of MBP mutations have been isolated by us and other laboratories. We grouped these mutations into classes depending on the interactions affected and we represented the corresponding residues on the 3-D model for MBP so as to further identify the sites of MBP interacting with the MalF-MalG complex and with the Tar protein. MBP (like the other binding proteins) is composed of 2 lobes enclosing a cleft where the substrate binds. The face of the protein opposite the cleft seems to interact neither with MalF-MalG nor with Tar. The other face, corresponding to the cleft, contains sites for interactions with MalF-MalG and Tar. These sites appear to cover both sides of the cleft and may overlap in part. The present definition of the interaction sites suggests further that MBP has different in vivo orientations when it interacts with MalF-MalG or with Tar. This work constitutes an additional step in combining the use of genetic and structural analysis to define the interaction sites on MBP. Because of the structural similarities between periplasmic binding proteins, the regions of interaction defined could be relevant for other members of this family.

Background Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli.

L’ideologie securitaire et le raisonnement paradoxal a propos des risques sont les reflets d’un « malaise dans la Cite ». Celui-ci entretient une souffrance sociale des adolescents qui reveille leurs failles narcissiques et les incertitudes sexuelles et generationnelles. La disqualification des referents identificatoires et des garants de l’autorite aggravent leur sentiment d’exilation. En resultent l’ennui, les prises de risques mortiferes ou des actes destructeurs que tentent de pallier les interventions medico-psycho-socio-judiciaires trop souvent centrees sur les actes et non les personnes. Nous evoquons ici des initiatives citoyennes en vue de developper des « mi-lieux » praticables et des formes de mediation sociale capables de rallier ces jeunes en perte de culture.

We previously determined a number of ‘permissive’ sites in the periplasmic maltose-binding protein (MaIE) from Escherichia coli . These sites accept the insertion of heterologous peptides without major deleterious consequences for the activities, structure and cellular location of the protein. This study explores the versatility of two such permissive sites for the synthesis of foreign peptides, and examines the antigenicity and the immunogenicity of the inserts. One site is located after amino acid 133 (aa 133 ) of MalE, and the other after aa 303 . Both sites tolerate inserts of up to at least 70 aa and accept sequences of different natures. Hydrophobic aa sequences are accepted, although strongly hydrophobic sequences, such as the Sendai virus F protein membrane anchor, affected export. We compared the antigenic and the immunogenic properties of peptides derived from the coat proteins of HBV and poliovirus which contain well defined B-cell epitopes. Specific monoclonal antibodies show that the antigenic properties of the inserted B-cell epitopes were different at the two sites. Despite these differences, the inserted peptides elicited strong and comparable antibody responses in mice against the corresponding synthetic peptides. In this case, and with these criteria, the molecular context of the peptides did not affect the immunogenicity of B-cell epitopes. We show for the first time that when a foreign peptide carrying a T-cell epitope was inserted in MalE, the hybrid proteins can elicit a T-cell response against the foreign peptide both in vivo and in vitro. Furthermore, the MalE hybrid was as efficient as free peptide in stimulating T-cell hybridomas in vitro. The MalE vectors provide a powerful genetic system to study how the position and the conformation of a peptide within a protein affect the B-cell and T-cell responses.

IMPACT: 4.894; International audience; Antibody molecules are able to recognize any antigen with high affinity and specificity. To get insight into the molecular diversity at the source of this functional diversity, we compiled and analyzed a non-redundant aligned collection of 227 structures of antibody-antigen complexes. Free energy of binding of all the residue side-chains was quantified by computational alanine scanning, allowing the first large-scale quantitative description of antibody paratopes. This demonstrated that as few as 8 residues among 30 key positions are sufficient to explain 80% of the binding free energy in most complexes. At these positions, the residue distribution is not only different from that of other surface residues, but also dependent on the role played by the side chain in the interaction, residues participating in the binding energy being mainly aromatic residues, and Gly or Ser otherwise. To question the generality of these binding characteristics, we isolated an antibody fragment by phage-display using a biased synthetic repertoire with only two diversified complementary determining regions (CDRs) and solved its structure in complex with its antigen. Despite this restricted diversity, the structure demonstrated that all CDRs were involved in the interaction with the antigen and that the rules derived from the natural antibody repertoire apply to this synthetic binder, thus demonstrating the robustness and universality of our results.

Competition for binding to MHC class II molecules between processed peptides derived from a single protein Ag is considered an important parameter leading to the presentation of a limited set of peptides by APCs. We tested the relevance of this competition process in a model Ag, the MalE protein, by deleting T cell epitopes or by introducing a competitor T cell peptide. We identified in DBA/1 (I-Aq) mice six immunodominant T cell determinants in the MalE sequence, 89–95, 116–123, 198–205, 211–219, 274–281, and 335–341. Synthetic peptides carrying these determinants were classified in three groups as weak, intermediate, or strong I-Aq binders in competition experiments with the PreS:T peptide of hepatitis B surface Ag. In vivo, synthetic MalE peptides with weak and intermediate MHC binding capacity were inhibited in their capacity to stimulate proliferative response in the presence of the PreS:T competitor peptide, whereas the strongest MHC binder was not. Strikingly, the insertion of the potent competitor PreS:T peptide into the MalE sequence, as a single copy or as four copies, did not inhibit the proliferative response to the six immunodominant peptides of the recipient protein. Moreover, deletion in the protein sequence disrupting either the weak (198–205) or strong (335–341) MHC binding determinant of MalE did not modify the proliferative response to the remaining T cell determinants as compared with wild-type MalE protein. Altogether, these results show that peptide competition for MHC binding may not represent the most important event in processes leading to immunodominance.

Background Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. In this work our aim was to study the role of the phosphoserine aminotransferase PSAT1 in colorectal cancer development. Results We first observed that PSAT1 is overexpressed in colon tumors. In addition, we showed that after drug treatment, PSAT1 expression level in hepatic metastases increased in non responder and decreased in responder patients. In experiments using human cell lines, we showed that ectopic PSAT1 overexpression in colon carcinoma SW480 cell line resulted in an increase in its growth rate and survival. In addition, SW480-PSAT1 cells presented a higher tumorigenic potential than SW480 control cells in xenografted mice. Moreover, the SW480-PSAT1 cell line was more resistant to oxaliplatin treatment than the non-transfected SW480 cell line. This resistance resulted from a decrease in the apoptotic response and in the mitotic catastrophes induced by the drug treatment. Conclusion These results show that an enzyme playing a role in the L-serine biosynthesis could be implicated in colon cancer progression and chemoresistance and indicate that PSAT1 represents a new interesting target for CRC therapy.

Since 2000, monoclonal antibodies (mAb) have become essential and routine drugs in therapy and particularly in oncology. The field continues to grow very quickly and given the abundance of molecules available, it is increasingly important to bring innovative molecules with a high added value for therapy.Two main approaches are used to select these mAbs: hybridoma technology using normal or humanized mice; display systems such as phage-display. The major interests of phage display are the speed of mAb development, the facilities offered by E. coli and the easy access to protein engineering techniques. Typically, antibodies are first selected on their ability to bind to the antigen, and then tested for their functional efficiency in cellular models. However, only a part of the activity of antibodies is explained by their binding to the antigen, and the therapeutic activity also depends strongly on their ability to recruit the immune system (ADCC) and activate the complement cascade (CDC).This thesis project consists in the development of a new recombinant antibody library format combining the power of phage display selection with functional screening in a whole IgG format produced in eukaryotic cells. This new system is based on hybrid promoter and signal peptide regions allowing expression both in prokaryotic and eukaryotic cells, and a site-specific recombination event that exchanges the Fab between the display vector and the chromosome of an especially developed mammalian cell line resulting in the secretion of a monoclonal human antibody by the cell. The usual approach of recloning one by one from E.Coli vector to an IgG format is no more needed since it is done directly by transfection. This new system makes possible to couple selection by phage display with a direct functional screening of a large population of human monoclonal clones.; Les anticorps monoclonaux (mAb) sont depuis les années 2000 devenus des médicaments incontournables et de routine en thérapie et notamment en cancérologie. Le domaine continue à croître très rapidement et devant l’abondance des molécules disponibles, il est de plus en plus important d’apporter des molécules innovantes à haute valeur ajoutée pour la thérapie. Deux grandes approches sont utilisées pour sélectionner ces mAbs : l’hybridation lymphocytaire à partir de souris normales ou humanisées ; Les systèmes de display comme le phage-display. Les intérêts majeurs du phage display sont la rapidité de développement des mAbs, la facilité de manipulation chez E. coli, l’accès aux techniques de "protein engineering". Classiquement, les anticorps sont d’abord sélectionnés sur leur capacité de liaison à l’antigène puis ensuite testés pour leur efficacité fonctionnelle dans des modèles cellulaires. Cependant, seulement une partie de l'activité des anticorps est expliquée par leur liaison à l’antigène et l’activité thérapeutique dépend aussi fortement de leur capacité à recruter le système immunitaire (ADCC) et à activer la cascade du complément (CDC).Ce projet de thèse consiste à développer un nouveau format de banque d'anticorps recombinants combinant la puissance de la sélection par phage display à un criblage fonctionnel au format IgG entières produites en cellules eucaryotes. Ce nouveau système est basé sur des régions initiatrices hybrides contenant à la fois des promoteurs et des séquences signal procaryotes et eucaryotes permettant l’expression dans ces deux systèmes cellulaires, et des évènements de recombinaisons sites-spécifiques transférant le fragment Fab du vecteur de display vers le chromosome d’une lignée cellulaire de mammifère spécialement développée pour aboutir à la sécrétion d’un anticorps humain monoclonal par la cellule. L’approche habituelle de reclonage un par un du vecteur E. Coli au format IgG n’est plus nécessaire puisqu’il se fait directement par transfection. Ce nouveau système rend possible le couplage d’une sélection par phage display à un criblage fonctionnel direct sur une large population de clones monoclonaux humains.

Since 2000, monoclonal antibodies (mAb) have become essential and routine drugs in therapy and particularly in oncology. The field continues to grow very quickly and given the abundance of molecules available, it is increasingly important to bring innovative molecules with a high added value for therapy.Two main approaches are used to select these mAbs: hybridoma technology using normal or humanized mice; display systems such as phage-display. The major interests of phage display are the speed of mAb development, the facilities offered by E. coli and the easy access to protein engineering techniques. Typically, antibodies are first selected on their ability to bind to the antigen, and then tested for their functional efficiency in cellular models. However, only a part of the activity of antibodies is explained by their binding to the antigen, and the therapeutic activity also depends strongly on their ability to recruit the immune system (ADCC) and activate the complement cascade (CDC).This thesis project consists in the development of a new recombinant antibody library format combining the power of phage display selection with functional screening in a whole IgG format produced in eukaryotic cells. This new system is based on hybrid promoter and signal peptide regions allowing expression both in prokaryotic and eukaryotic cells, and a site-specific recombination event that exchanges the Fab between the display vector and the chromosome of an especially developed mammalian cell line resulting in the secretion of a monoclonal human antibody by the cell. The usual approach of recloning one by one from E.Coli vector to an IgG format is no more needed since it is done directly by transfection. This new system makes possible to couple selection by phage display with a direct functional screening of a large population of human monoclonal clones., Les anticorps monoclonaux (mAb) sont depuis les années 2000 devenus des médicaments incontournables et de routine en thérapie et notamment en cancérologie. Le domaine continue à croître très rapidement et devant l’abondance des molécules disponibles, il est de plus en plus important d’apporter des molécules innovantes à haute valeur ajoutée pour la thérapie. Deux grandes approches sont utilisées pour sélectionner ces mAbs : l’hybridation lymphocytaire à partir de souris normales ou humanisées ; Les systèmes de display comme le phage-display. Les intérêts majeurs du phage display sont la rapidité de développement des mAbs, la facilité de manipulation chez E. coli, l’accès aux techniques de "protein engineering". Classiquement, les anticorps sont d’abord sélectionnés sur leur capacité de liaison à l’antigène puis ensuite testés pour leur efficacité fonctionnelle dans des modèles cellulaires. Cependant, seulement une partie de l'activité des anticorps est expliquée par leur liaison à l’antigène et l’activité thérapeutique dépend aussi fortement de leur capacité à recruter le système immunitaire (ADCC) et à activer la cascade du complément (CDC).Ce projet de thèse consiste à développer un nouveau format de banque d'anticorps recombinants combinant la puissance de la sélection par phage display à un criblage fonctionnel au format IgG entières produites en cellules eucaryotes. Ce nouveau système est basé sur des régions initiatrices hybrides contenant à la fois des promoteurs et des séquences signal procaryotes et eucaryotes permettant l’expression dans ces deux systèmes cellulaires, et des évènements de recombinaisons sites-spécifiques transférant le fragment Fab du vecteur de display vers le chromosome d’une lignée cellulaire de mammifère spécialement développée pour aboutir à la sécrétion d’un anticorps humain monoclonal par la cellule. L’approche habituelle de reclonage un par un du vecteur E. Coli au format IgG n’est plus nécessaire puisqu’il se fait directement par transfection. Ce nouveau système rend possible le couplage d’une sélection par phage display à un criblage fonctionnel direct sur une large population de clones monoclonaux humains.

Anti-tumoral monoclonal antibodies are currently produced in eukaryotic cells. For cost and time reasons, a limited number of potential candidates are selected after in vitro tests. They are produced at large scale and then tested in vivo. To test more antibodies and more rapidly, we chose to produce single chain variable fragments (scFv) in bacteria, and to couple them to the eukaryotic constant fragment (Fc) thanks to click chemistry to reconstitute immunoglobulin-like compounds. For a given cost, this enables to produce and test in vivo a larger number of clones. This independent production of fragments is also a flexible tool allowing the combination of different Fc isotypes/allotypes with different scFvs.Click chemistry is based on a specific and high-yield reaction between and azide and a cyclooctyne. Therefore, antibody fragments were functionalised on specific residues (tags) by chemical linker so that each part will contain one of these chemical moieties at their extremity. The first step consisted in introducing tags into the anti-HER2 scFv 4D5 C-terminus and human IgG1 Fc N-termini sequences. The scFvs were produced with yields higher than 100 mg/L in the E. coli cytoplasm and in vitro oxidized with copper sulfate. The Fc fragment was classically produced in human cells. Five chemical or enzymatical reactions were optimised and compared in terms of specificity and yield. The coupling between an amine and a glutamine tag catalysed by microbial transglutaminase gave the best results. The scFv fragment was thus functionalised with an azadibenzocyclooctyne and the Fc fragment with an azide at 60-70%. When mixed together, these fragments formed a (scFv)2-Fc and a scFv-Fc with global yields respectively of 10-20% and 20-30% after optimisation.After the click reaction, the scFv + Fc mix binds to the HER2 receptor on the same way as the eukaryotic (scFv)2-Fc in terms of HER2-binding and proliferation inhibition capacity. Now, it must be demonstrated that their proliferation inhibition of a HER2-positive cell line is similar. The final aim is to get a similar tumour growth inhibition on murine xenografts.; Les anticorps monoclonaux anti-tumoraux sont produits en cellules eucaryotes. Pour des raisons de temps et de coût, peu de candidats sont sélectionnés après des tests in vitro pour être produits à large échelle et testés in vivo. Pour tester plus d’anticorps plus rapidement, nous souhaitons produire des fragments variables simple chaine (scFv) chez E. coli. et les coupler à un fragment constant Fc par chimie click pour reconstituer des mimes d’immunoglobulines naturelles. Cette production indépendante des fragments est aussi un outil modulaire permettant de combiner rapidement un grand nombre scFv et de Fc différents.La chimie click est basée sur une réaction spécifique et de haut rendement entre un azide et un cyclooctyne. Les fragments ont donc été fonctionnalisés sur des résidus spécifiques (tags) par des composés chimiques pour introduire ces fonctions à leur extrémité. La première étape a consisté à introduire des tags en C-terminal du scFv anti-HER2 4D5 et en N-terminal du Fc d’IgG1 humaine. Les scFv ont été produits en cytoplasme d’E. coli à hauteur d’au moins 100 mg/L puis oxydés in vitro au sulfate de cuivre. Le fragment Fc a été produit classiquement en cellules humaines. Cinq réactions chimiques ou enzymatiques ont été optimisées et comparées en termes de spécificité et de rendement. La conjugaison d’une amine sur un tag glutamine catalysée par la transglutaminase microbienne a donné les meilleurs résultats. Le scFv a ainsi été dérivé par l’azadibenzocyclooctyne et le Fc par l’azide à hauteur de 60-70%. Lorsqu’ils sont mélangés, ces fragments forment un (scFv)2-Fc et un scFv-Fc avec des rendements globals respectifs de 10-20% et 20-30% après optimisation.Les mélanges scFv + Fc après réaction de chimie click se fixent de la même façon que le (scFv)2-Fc eucaryote au récepteur HER2. Il reste désormais à montrer que leur capacité à inhiber la prolifération d’une lignée exprimant ce récepteur est similaire. L’objectif final est d’obtenir une inhibition de croissance tumorale similaire sur des xénogreffes.