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International audience; Nanoparticle-based biodetection commonly employs monoclonal antibodies (mAbs) for targeting. Although several types of conjugates have been used for biomarker labeling, the large size of mAbs limits the number of ligands per nanoparticle, impedes their intratumoral distribution, and limits intracellular penetration. Furthermore, the conditions of mAb conjugation using conventional techniques provide nanoprobes with irregular orientation of mAbs on the nanoparticle surface and often provoke mAb unfolding. Here, we have developed a protocol to engineer ultrasmall diagnostic nanoprobes through directional conjugation of semiconductor quantum dots (QDs) with 13-kDa single-domain antibodies (sdAbs) derived from llama immunoglobulin G (IgG). sdAbs are conjugated with QDs in a highly oriented manner via an additional cysteine residue specifically integrated into the sdAb C-terminus. The resultant nanoprobes are

International audience; The natural killer group 2 member D (NKG2D) receptor is a C-type lectin-like activating receptor mainly expressed by cytotoxic immune cells including NK, CD8 + T, γδ T and NKT cells and in some pathological conditions by a subset of CD4 + T cells. It binds a variety of ligands (NKG2DL) whose expressions is finely regulated by stress-related conditions. The NKG2DL/NKG2D axis plays a central and complex role in the regulation of immune responses against diverse cellular threats such as oncogene-mediated transformations or infections. We generated a panel of seven highly specific anti-human NKG2D single-domain antibodies targeting various epitopes. These single-domain antibodies were integrated into bivalent and bispecific antibodies using a versatile plug-and-play Fab-like format. Depending on the context, these Fab-like antibodies exhibited activating or inhibitory effects on the immune response mediated by the NKG2DL/NKG2D axis. In solution, the bivalent anti-NKG2D antibodies that compete with NKG2DL potently blocked the activation of NK cells seeded on immobilized MICA, thus constituting antagonizing candidates. Bispecific anti-NKG2DxHER2 antibodies that concomitantly engage HER2 on tumor cells and NKG2D on NK cells elicited cytotoxicity of unstimulated NK in a tumor-specific manner, regardless of their apparent affinities and epitopes. Importantly, the bispecific antibodies that do not compete with ligands binding retained their full cytotoxic activity in the presence of ligands, a valuable property to circumvent immunosuppressive effects induced by soluble ligands in the microenvironment.

Semiconductor quantum dots (QDs) are characterized by orders of magnitude higher multiphoton linear absorption cross-sections compared with conventional organic dyes. Combined with the QD photoluminescence quantum yield approaching 100%, this fact opens great prospects for the twophoton functional tumor imaging with QDs tagged with highly specific recognition molecules. Single-domain antibodies (sdAbs) or “nanobodies” derived from lamas are the smallest high-affinity recognition molecules, which may be tagged with the QDs thus permitting not only solid tumors multiphoton imaging but also rare disseminated cancer cells and micrometastases in the depth of the tissue to be detected. Additionally, unique photostability of QDs enables signal accumulation and significant enhancement of the sensitivity of routine biochemical and immunohistochemical assays to be obtained when the conjugates of QDs, instead of organic dyes, are used.

International audience; Mesothelin is a cell-surface glycoprotein restricted to mesothelial cells overexpressed in several types of cancer, including triple-negative breast cancer not responding to trastuzumab or hormone-based therapies. Mesothelin-targeting therapies are currently being developed. However, the identification of patients potentially eligible for such a therapeutic strategy remains challenging. The objective of this study was to perform the radiolabeling and preclinical evaluation of 99mTc-A1 and 99mTc-C6, two antimesothelin single-domain antibody (sdAb)-derived imaging agents. Methods: A1 and C6 were radiolabeled with 99mTc and evaluated in vitro on recombinant protein and cells, as well as in vivo in xenograft mouse models of the triple-negative breast cancer cell lines HCC70 (mesothelin-positive) and MDA-MB-231 (mesothelin-negative). Results: Both 99mTc-A1 and 99mTc-C6 bound mesothelin with high affinity in vitro, with 99mTc-A1 affinity being 2.4-fold higher than that of 99mTc-C6 (dissociation constant, 43.9 ± 4.0 vs. 107 ± 16 nM, P < 0.05). 99mTc-A1 and 99mTc-C6 remained stable in vivo in murine blood (>80% at 2 h) and ex vivo in human blood (>90% at 6 h). In vivo 99mTc-A1 uptake (percentage injected dose) in HCC70 tumors was 5-fold higher than in MDA-MB-231 tumors and 1.5-fold higher than that of 99mTc-C6 (2.34% ± 0.36% vs. 0.48% ± 0.18% and 1.56% ± 0.43%, respectively, P < 0.01) and resulted in elevated tumor-to-background ratios. In vivo competition experiments demonstrated the specificity of 99mTc-A1 uptake in HCC70 tumors. Conclusion: Mesothelin-positive tumors were successfully identified by SPECT using 99mTc-A1 and 99mTc-C6. Considering its superior characteristics, 99mTc-A1 was selected as the most suitable tool for further clinical translation.

Semiconductor quantum dots (QDs) are characterized by orders of magnitude higher multiphoton linear absorption cross-sections compared with conventional organic dyes. Combined with the QD photoluminescence quantum yield approaching 100% and their rock-solid photostability, this fact opens great prospects for the two-photon functional tumor imaging with QDs tagged with highly specific recognition molecules. Single-domain antibodies (sdAbs) or “nanobodies” derived from lamas are the smallest high-affinity recognition molecules, which may be tagged with the QDs thus permitting not only solid tumors multiphoton imaging but also rare disseminated cancer cells and micrometastases in the depth of the tissue to be detected.

Supplemental Digital Content is Available in the Text., Background: Highly potent broadly neutralizing monoclonal antibodies (bNAbs) have been obtained from individuals infected by HIV-1 group M variants. We analyzed the cross-group neutralization potency of these bNAbs toward non-M primary isolates (PI). Material and Methods: The sensitivity to neutralization was analyzed in a neutralization assay using TZM-bl cells. Twenty-three bNAbs were used, including reagents targeting the CD4-binding site, the N160 glycan-V1/V2 site, the N332 glycan-V3 site, the membrane proximal external region of gp41, and complex epitopes spanning both env subunits. Two bispecific antibodies that combine the inhibitory activity of an anti-CD4 with that of PG9 or PG16 bNAbs were included in the study (PG9-iMab and PG16-iMab). Results: Cross-group neutralization was observed only with the bNAbs targeting the N160 glycan-V1/V2 site. Four group O PIs, 1 group N PI, and the group P PI were neutralized by PG9 and/or PG16 or PGT145 at low concentrations (0.04–9.39 μg/mL). None of the non-M PIs was neutralized by the bNAbs targeting other regions at the highest concentration tested, except 10E8 that neutralized weakly 2 group N PIs and 35O22 that neutralized 1 group O PI. The bispecific bNAbs neutralized very efficiently all the non-M PIs with IC50 below 1 μg/mL, except 2 group O strains. Conclusion: The N160 glycan-V1/V2 site is the most conserved neutralizing site within the 4 groups of HIV-1. This makes it an interesting target for the development of HIV vaccine immunogens. The corresponding bNAbs may be useful for immunotherapeutic strategies in patients infected by non-M variants.

International audience; The use of photoluminescence (PL) probes in immunodiagnostic procedures requires highly specific and precise labeling of biomarkers and he tags whose PL is easily differentiable from that of the autofluorescence of cells in clinical specimens. Nanoprobes consisting of core/shell quantum dots (QDs), notably CdSe/ZnS ones, and single-domain antibodies (sdAbs) with PL excited in the multiphoton mode meet both requirements. These diagnostic nanoprobes are

International audience; High-quality imaging for immunofluorescence diagnosis in medical practice requires a high sensitivity of labeling and discrimination between the fluorescence of immunostaining probes and tissue autofluorescence. Multiphoton microscopy with excitation in the near-infrared spectral region, far from the region of excitation of tissue autofluorescence, enables deep imaging of biological tissues. CdSe/ZnS quantum dots (QDs) conjugated to single-domain antibodies (sdAbs) are efficient diagnostic nanoprobes with diameters 40 times higher than that for one-photon excitation.

International audience

International audience; Mesothelin, a cancer biomarker overexpressed in tumors of epithelial origin, is a target for nanotechnology-based diagnostic, therapeutic, and prognostic applications. The currently available anti-mesothelin antibodies present limitations, including low penetration due to large size and/or lack of in vivo stability. Single domain antibodies (sdAbs) or nanobodies (Nbs) provide powerful solutions to these specific problems. We generated a phage-display library of Nbs that were amplified from B cells of a llama that was immunized with human recombinant mesothelin. Two nanobodies (Nb A1 and Nb C6) were selected on the basis of affinity (K(D) = 15 and 30 nM, respectively). Nb A1 was further modified by adding either a cysteine to permit maleimide-based bioconjugations or a sequence for the site-specific metabolic addition of a biotin in vivo. Both systems of conjugation (thiol-maleimide and streptavidin/biotin) were used to characterize and validate Nb A1 and to functionalize nanoparticles. We showed that anti-mesothelin Nb A1 could detect native and denatured mesothelin in various diagnostic applications, including flow cytometry, western blotting, immunofluorescence, and optical imaging. In conclusion, anti-mesothelin Nbs are novel, cost-effective, small, and single domain reagents with high affinity and specificity for the tumor-associated antigen mesothelin, which can be simply bioengineered for attachment to nanoparticles or modified surfaces using multiple bioconjugation strategies. These anti-mesothelin Nbs can be useful in both conventional and nanotechnology-based diagnostic, therapeutic and prognostic biomedical applications.

International audience; The targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system.

An ideal multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible two-photon absorption cross section (TPACS) and the smallest highly specific recognition molecules bound in an oriented manner. CdSe/ZnS quantum dots (QDs) conjugated to 13-kDa single-domain antibodies (sdAbs) derived from camelid IgG or streptavidin have been used as efficient two-photon excitation (TPE) probes for carcinoembryonic antigen (CEA) imaging on normal human appendix and colon carcinoma tissue. The TPACS for some conjugates was higher than 49,000 GM (Goeppert-Mayer units), considerably exceeding that of organic dyes being close to the theoretical value of 50,000 GM calculated for CdSe QDs. The ratio of sdAb-QD emission to the autofluorescence for 800 nm TPE was 40 times higher than that for 457.9 nm one-photon excitation. TPE ensures a clear discrimination of CEA-overexpressing tumor areas from normal tissue. Oriented sdAb-QD conjugates are bright specific labels for detecting low concentrations of antigens using multiphoton microscopy. From the Clinical Editor This study demonstrates carcinoembryonic antigen imaging on normal human appendix and colon carcinoma tissue utilizing CdSe/ZnS quantum dots conjugated to streptavidin or to 13-kDa single-domain antibodies as efficient two-photon excitation probes.

International audience; Trastuzumab is established as treatment of HER2high metastatic breast cancers but many limitations impair its efficacy. Here, we report the design of a Fab-like bispecific antibody (HER2bsFab) that displays a moderate affinity for HER2 and a unique, specific and high affinity for FcγRIII. In vitro characterization showed that ADCC was the major mechanism of action of HER2bsFab as no significant HER2-driven effect was observed. HER2bsFab mediated ADCC at picomolar concentration against HER2high, HER2low as well as trastuzumab-refractive cell lines. In vivo HER2bsFab potently inhibited HER2high tumor growth by recruitment of mouse FcγRIII and IV-positive resident effector cells and more importantly, exhibited a net superiority over trastuzumab at inhibiting HER2low tumor growth. Moreover, FcγRIIIA-engagement by HER2bsFab was independent of V/F158 polymorphism and induced a stronger NK cells activation in response to target cell recognition. Thus, taking advantage of its epitope specificity and affinity for HER2 and FcγRIIIA, HER2bsFab exhibits potent anti-tumor activity against HER2low tumors while evading most of trastuzumab Fc-linked limitations thereby potentially enlarging the number of patients eligible for breast cancer immunotherapy.

The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif. In vitro and in vivo characterization of these Fab-like bispecific molecules revealed favorable features for further development as a therapeutic molecule. They are easy to produce in Escherichia coli, very stable, and elicit potent lysis of tumor cells by human natural killer cells at picomolar concentrations. Unlike conventional antibodies, they do not engage inhibitory FcγRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism. As opposed to anti-CD3 bispecific antitumor antibodies, they do not engage regulatory T cells as these latter cells do not express FcγRIII. Studies in nonobese diabetic/severe combined immunodeficient gamma mice xenografted with carcinoembryonic antigen–positive tumor cells showed that Fab-like bispecific molecules in the presence of human peripheral blood mononuclear cells significantly slow down tumor growth. This new compact, linker-free bispecific antibody format offers a promising approach for optimizing antibody-based therapies. Mol Cancer Ther; 12(8); 1481–91. ©2013 AACR.

Few broadly neutralizing antibodies targeting determinants of the HIV-1 surface envelope glycoprotein (gp120) involved in sequential binding to host CD4 and chemokine receptors have been characterized. While these epitopes show low diversity among various isolates, HIV-1 employs many strategies to evade humoral immune response toward these sensitive sites, including a carbohydrate shield, low accessibility to these buried cavities, and conformational masking. Using trimeric gp140, free or bound to a CD4 mimic, as immunogens in llamas, we selected a panel of broadly neutralizing single-domain antibodies (sdAbs) that bind to either the CD4 or the coreceptor binding site (CD4BS and CoRBS, respectively). When analyzed as monomers or as homo- or heteromultimers, the best sdAb candidates could not only neutralize viruses carrying subtype B envelopes, corresponding to the Env molecule used for immunization and selection, but were also efficient in neutralizing a broad panel of envelopes from subtypes A, C, G, CRF01_AE, and CRF02_AG, including tier 3 viruses. Interestingly, sdAb multimers exhibited a broader neutralizing activity spectrum than the parental sdAb monomers. The extreme stability and high recombinant production yield combined with their broad neutralization capacity make these sdAbs new potential microbicide candidates for HIV-1 transmission prevention.

Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms.The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.

International audience; Ideal diagnostic nanoprobes should not exceed 15 nm in size and should contain high-affinity homogeneously oriented capture molecules on their surface. An advanced procedure for antibody (Ab) reduction was used to cleave each Ab into two functional half-Abs, 75-kDa heavy–light chain fragments, each containing an intact antigen-binding site. Affinity purification of half-Abs followed by their linkage to quantum dots (QDs) yielded oriented QD–Ab conjugates whose functionality was considerably improved compared to those obtained using the standard protocols. Ultrasmall diagnostic nanoprobes were engineered through oriented conjugation of QDs with 13-kDa single-domain Abs (sdAbs) derived from llama IgG. sdAbs were tagged with QDs via an additional cysteine residue specifically integrated into the C-terminal region of sdAb using genetic engineering. This approach made it possible to obtain sdAb–QD nanoprobes

International audience; Antibody microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnoses and proteomic analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and expression yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecules for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs, produced in crude bacterial lysates, to generate a proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of "multi-tagged" sdAbs via anti-tag antibodies and a direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers.

International audience; The channel formed by colicin A in planar lipid bilayers has an outsized selectivity for protons compared to any other ion, even though it allows large ions, such as tetraethylammonium, to permeate readily. A mechanism to account for this discrepancy remains obscure. We considered that protons may traverse a separate pathway but were unable to find any evidence for one. Manipulations that interfere with ionic conduction, such as replacing some of the water in the pore with a nonelectrolyte, reduce the proton current along with the ionic current. Lipids have been proposed to play a structural role in the channel, but we found that the proton selectivity was unaffected by various gross changes in the lipid composition of the bilayer, effectively ruling out any specific effect of lipids in the selectivity and offering no support for their role in structure. The 10-helix channel-forming domains of colicins Ia and E1 are structurally homologous to that of colicin A but do not select so remarkably for protons; thus we were able to use them to probe for the regions responsible for the high selectivity. Using hybrids made by helix swapping among these proteins, we found that the anomalous selectivity could be localized to the five C-terminal helices of colicin A.

International audience; Pancreatic cancer is a devastating disease with the worst mortality rate and an overall 5-year survival rate lower than 5%. In the U.S., this disease is the fourth leading cause of death and represents 6% of all cancer-related deaths. Gemcitabine, the current standard first-line treatment, offers marginal benefits to patients in terms of symptom control and prolongation of life. Since 1996, about 20 randomized phase III trials have been performed to improve the efficacy of gemcitabine, with little success regarding a significant improvement in survival outcomes. The need for novel therapeutic strategies, such as target therapy, is obvious. Monoclonal antibodies have finally come of age as therapeutics and several molecules are now approved for cancer therapies. This review aims to give a general view on the clinical results obtained so far by antibodies for the treatment of pancreatic cancer and describes the most promising avenues toward a significant improvement in the treatment of this frustrating disease.

International audience; With 23 approvals in the US and other countries and four approvals outside US, antibodies are now widely recognized as therapeutic molecules. The therapeutic and commercial successes met by rituximab, trastuzumab, cetuximab and other mAbs have inspired antibody engineers to improve the efficacy of these molecules. Consequently, a new wave of antibodies with engineered Fc leading to much higher effector functions such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity is being evaluated in the clinic, and several approvals are expected soon. In addition, research on a different class of antibody therapeutics, bispecific antibodies, has recently led to outstanding clinical results, and the first approval of the bispecific antibody catumaxomab, a T cell retargeting agent that was approved in the European Union in April 2009. This review describes the most recent advances and clinical study results in the field of bispecific antibodies, a new class of molecules that might outshine conventional mAbs as cancer immunotherapeutics in a near future.

International audience; Single-domain antibodies (sdAbs), which occur naturally in camelids, are endowed with many characteristics that make them attractive candidates as building blocks to create new antibody-related therapeutic molecules. In this study, we isolated from an immunized llama several high-affinity sdAbs directed against human carcinoembryonic antigen (CEA), a heavily glycosylated tumor-associated molecule expressed in a variety of cancers. These llama sdAbs bind a different epitope from those defined by current murine mAbs, as shown by binding competition experiments using immunofluorescence and surface plasmon resonance. Flow cytometry analysis shows that they bind strongly to CEA-positive tumor cells but show no cross-reaction toward nonspecific cross-reacting antigen, a highly CEA-related molecule expressed on human granulocytes. When injected into mice xenografted with a human CEA-positive tumor, up to 2% of the injected dose of one of these sdAbs was found in the tumor, despite rapid clearance of this 15 kDa protein, demonstrating its high potential as a targeting moiety. The single-domain nature of these new anti-CEA IgG fragments should facilitate the design of new molecules for immunotherapy or diagnosis of CEA-positive tumors.

Understanding cellular systems requires identification and analysis of their multiple components and determination of how they act together and are regulated. Microarray technology is one of the few tools that is able to solve such problems. It is based on high-throughput recognition of a target to the probe and has the potential to simultaneously measure the presence of numerous molecules in multiplexed tests, all contained in a small drop of test fluid. Microarrays allow the parallel analysis of genomic or proteomic content in healthy versus disease-affected or altered tissues or cells. The signal read-out from the microarrays is done with organic dyes which often suffer of photobleaching, low brightness and background fluorescence. Recent data show that the use of fluorescent nanocrystals named "quantum dots" (QDs) allows to push these limits away. QDs are sufficiently bright to be detected as individual particles, extremely resistant against photobleaching and provide unique possibilities for multiplexing, thus supplying the microarray technology with a novel read-out option enabling the sensitivity of detection to reach the single-molecule level. This paper reviews QDs applications to microarray-based detection and demonstrates how the combination of microarray and QDs technologies may increase sensitivity and highly parallel capacities of multiplexed microarrays. Such a combination should provide the breakthrough results in drug discovery, cancer diagnosis and establish new therapeutic approaches through the identification of binding target molecules and better understanding of cell signalling pathways.

FcgammaRIII (CD16) plays an important role in the anti-tumor effects of therapeutic antibodies. Bi-specific antibodies (bsAbs) targeting FcgammaRIII represent a powerful alternative to the recruitment of the receptor via the Fc fragment, but are not efficiently produced. Single-domain antibodies (sdAbs) endowed with many valuable structural features might help to bypass this problem. In the present work, we have isolated anti-FcgammaRIII sdAbs (C21 and C28) from a phage library generated from a llama immunized with FcgammaRIIIB extra-cellular domains. These sdAbs bind FcgammaRIIIA(+) NK cells and FcgammaRIIIB(+) polymorphonuclear cells, but not FcgammaRI(+) or FcgammaRII(+) cells, as detected by indirect immunofluorescence. Competition experiments showed that C21 and C28 sdAbs bind different FcgammaRIII epitopes, with C21 recognizing a linear and C28 a conformational epitope of the receptor. Surface plasmon resonance experiments showed that C21 and C28 sdAbs bind FcgammaRIII with a K(D) in the 10 and 80 nM range, respectively. Importantly, the engagement by both molecules of FcgammaRIIIA expressed by transfected Jurkat T cells or by NK cells derived from peripheral blood induced a strong IL-2 and IFN-gamma production, respectively. These anti-FcgammaRIII sdAbs represent versatile tools for generating bsAbs under various formats, able to recruit FcgammaRIII killer cells to target and destroy tumor cells.

Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L. A single band around 16,000Da corresponding to VHH fragments was found after analysis by SDS-PAGE and Western blotting, while competition ELISA demonstrated selective binding to soluble MTX. Surface plasmon resonance (SPR) analysis showed that anti-MTX VHH domains had affinities in the nanomolar range (29-515nM) to MTX-serum albumin conjugates. The genes encoding anti-MTX VHH were found by IMGT/V-QUEST to be similar to the previously reported llama and human IGHV germline genes. The V-D and D-J junction rearrangements in the seven anti-MTX CDR3 sequences indicate that they were originated from three distinct progenitor B cells. Our results demonstrate that camelid single-domain antibodies are capable of high affinity binding to low molecular weight hydrosoluble haptens. Furthermore, these anti-MTX VHH give new insights on how the antigen binding repertoire of llama single-domain antibody can provide combining sites to haptens in the absence of a VL. This type of single-domain antibodies offers advantages compared to murine recombinant antibodies in terms of production rate and sequence similarity to the human IGHV3 subgroup genes.

The voltage-dependent gating of the colicin channel involves a substantial structural rearrangement that results in the transfer of about 35% of the 200 residues in its pore-forming domain across the membrane. This transfer appears to represent an unusual type of protein translocation that does not depend on a large, multimeric, protein pore. To investigate the ability of this system to transport arbitrary proteins, we made use of a pair of strongly interacting proteins, either of which could serve as a translocated cargo or as a probe to detect the other. Here we show that both an 86-residue and a 134-residue hydrophilic protein inserted into the translocated segment of colicin A are themselves translocated and are functional on the trans side of the bilayer. The disparate features of these proteins suggest that the colicin channel has a general protein translocation mechanism.

International audience; A mouse monoclonal anti-7-(O carboxymethyl)oximinoestradiol antibody 9D3, raised against the same immunogen as that employed for generating the reported anti-estradiol antibody 15H11 [Rousselot, P., et al. (1997) Biochemistry 36, 7860-7868], was found to exhibit an opposite specificity profile with a much stronger recognition of the D-ring than of the A-ring extremity of the steroid, but a similar lack of specificity for both 6- and 7-positions of the B-ring. This antibody was photoaffinity-labeled with five (5-azido-2-nitrobenzoyl)amido (ANBA) derivatives of [17 alpha-H-3]estradiol, synthesized from 3-aminoethyloxy, 3-(aniinoethylamido)carboxymethyloxy, 6 alpha- and 6 beta -amino, and 7-[O-(aminoethylamido)carboxymethyl]oximino precursors. After tryptic digestion, the radioactive peptides on L and H chains were immunopurified with the immobilized antibody 9D3, separated by reversed-phase liquid chromatography, sequenced, and characterized by mass spectrometry, including post-source decay-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The long 3-(ANBA-ethylamido)carboxymethyl ether photoreagent was found to label TyrL-32 (on CDR L1), whereas no labeling was observed with the shorter 3-derivative, a result in agreement with a binding pocket large enough to explain the high crossreactivity with estradiol 3-conjugates. The two 6 alpha- and 6 beta -ANBA-estradiol isomers, as well as the 7-[O-(ANBA-ethylamido)carboxymethyl]oximinoestradioI photoreagent derived from the steroid hapten, labeled the same TyrL-32 residue. The 6 beta -ANBA epimer also labeled TyrH-50 (at the basis of CDR H2). These experiments indicate that TyrL-32 is freely accessible from the three C3, C6, and C7 positions, all presumed to be exposed to solvent, while TyrH-50 is probably located on the beta -face of estradiol. These results, obtained in solution, provide experimental data useful for molecular modeling of the steroid-antibody complex.

The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA). This was inserted into the Escherichia coli inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai). We constructed a series of fusion proteins in which various numbers of sp-pfColA α-helices were fused to alkaline phosphatase (AP). We showed that a fusion protein made up of the hydrophobic α-helices 8 and 9 of sp-pfColA fused to AP was specifically coimmunoprecipitated with EpCai produced in the same cells. This is the first biochemical evidence that Cai recognizes and interacts with the colicin A hydrophobic helical hairpin.

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of Escherichia coli from the periplasmic side and forms a functional channel. The soluble structure of pfColA consists of a ten-helix bundle containing a hydrophobic helical hairpin. Here, we generated a series of mutants in which an increasing number of sp-pfColA α-helices was deleted. These peptides were tested for their ability to form ion channels in vivo and in vitro. We found that the shortest sp-pfColA mutant protein that killed Escherichia coli was composed of the five last α-helices of sp-pfColA, whereas the shortest peptide that formed a channel in planar lipid bilayer membranes similar to that of intact pfColA was the protein composed of the last six α-helices. The peptide composed of the last five α-helices of pfColA generated a voltage-independent conductance in planar lipid bilayer with properties very different from that of intact pfColA. Thus, helices 1 to 4 are unnecessary for channel formation, while helix 5, or some part of it, is important but not absolutely necessary. Voltage-dependence of colicin is evidently controlled by the first four α-helices of pfColA.

International audience; Four colicin A double-cysteine mutants possessing a disulfide bond in their pore-forming domain were constructed to study the translocation and the pore formation of colicin A. The disulfide bonds connected alpha-helices 1 and 2, 2 and 10, 3 and 9, or 3 and 10 of the pore-forming domain. The disulfide bonds did not prevent the colicin A translocation through the Escherichia coli envelope. However, the mutated colicins were able to exert their in vivo channel activity only after reduction of their disulfide bonds. In vitro studies with brominated phospholipid vesicles and planar lipid bilayers revealed that the disulfide bond that connects the alpha-helices 2 and 10 prevented the colicin A membrane insertion, whereas the other double-cysteine mutants inserted into lipid vesicles. The disulfide bonds that connect either the alpha-helices 1 and 2 or 3 and 10 were unable to prevent the formation of a conducting channel in presence of membrane potential. These results indicate that alpha-helices 1, 2, 3, and 10 remain at the membrane surface after application of a membrane potential.

International audience; Four disulfide bonds were engineered into the pore-forming domain of colicin A to probe the conformational changes associated with its membrane insertion and channel formation. The soluble pore-forming domain consists of 10 alpha-helices with two outer layers (helices 1, 2, and 3-7, respectively) sandwiching a middle layer of three helices (8-10). Helices 8 and 9 form a hairpin which is completely buried and consists of hydrophobic and neutral residues only. This helical hairpin has been hypothesized to be the membrane anchor. Each double-cysteine mutant possessing an individual disulfide bond, cross-linking either helices 1 to 9 (H1/H9), 5 to 6 (H5/H6), 7 to 8 (H7/H8), or 9 to 10 (H9/H10), respectively, is unable to promote K+ efflux from sensitive Escherichia coli cells. Activity can be restored by addition of a reducing agent. In vitro studies with brominated lipid vesicles and planar lipid bilayers show that the disulfide bond which connects the helices 1 to 9 prevents colicin A membrane insertion, whereas the other disulfide bond mutants insert readily into lipid vesicles. All of the engineered bridges prevented the formation of a conducting channel in the presence of a membrane potential. This novel approach indicates that membrane insertion and channel formation are two separate steps. Moreover, the effects of the distance constraints introduced by the different disulfide bonds on colicin A activity indicate that the helical pair 1 and 2 moves away from the other helices upon membrane insertion. Helices 3-10 remain associated together. As a consequence, the results imply that the helical hairpin lies parallel to the membrane surface. In contrast, induction of the colicin channel by the membrane potential requires a profound reorganization of the helices association. These results are discussed in light of several proposed models of the membrane-bound colicin and channel structures.

International audience; The bacterial toxin colicin A binds spontaneously to the surfaces of negatively charged membranes. The surface-bound toxin must subsequently, however, become an acidic 'molten globule' before it can fully insert into the lipid bilayer. Clearly, electrostatic interactions must play a significant role in both events. The electrostatic field around the toxin in solution was calculated using the finite-difference Poisson-Boltzmann method of the Delphi programme and the known X-ray structure. A large positively charged surface was identified which could be involved in the binding of colicin to negatively charged membranes. The applicability of the result was tested by also calculating the fields around modelled structures of the closely related colicins B and N. Surprisingly, colicin N showed a similar charge distribution in spite of its isoelectric point of pI 10.20 (colicin A has pI 5.44). One reason for this is the strong conservation of certain negative charges in all colicins. There is a single highly conserved aspartate residue (Asp78) on the positively charged face which provides a small but discrete region of negative charge. This residue, Asp78, was replaced by asparagine in the mutant D78N. D78N binds faster to negatively charged vesicles but inserts only half as fast as the wild-type protein into the membrane core. This indicates that, first, the initial membrane binding has a significant electrostatic component and, second, that the isolated charge on Asp78 plays a role in the formation of the insertion intermediate.

International audience; Three double cysteine mutants, each possessing a disulfide bond in its pore-forming domain, were used to study the translocation of colicin A through the Escherichia coli envelope. These mutated colicins were able to exert their in vivo channel activity only after their disulfide bonds had been reduced by dithiothreitol. In solution, the reduction of the disulfide bonds by dithiothreitol was a slow process whose kinetics depended on the position of the disulfide bond (t1/2 varying between 35 and 100 s). This t1/2 was strongly decreased (t1/2 = 8-9 s) upon predenaturation of the mutated colicins with urea. The t1/2 values of reduction of the mutants bound to E. coli-sensitive cells were similar to those of predenatured colicins. This suggested that the interaction of the oxidized double cysteine mutants with the E. coli envelope triggered their unfolding. The disulfide bonds did not prevent but delayed the translocation of the colicins. The amplitude of the delay and the time at which it occurred during translocation depended on the position of the disulfide bond. We could discriminate between the delays accumulated during binding to the receptor and those during the translocation via OmpF and the Tol proteins.

International audience; Pore-forming colicins constitute a large family of homologous bacteriocins that are of interest for a variety of problems related to membranes. Topics such as toxin action, polypeptide translocation across and into membranes, voltage-gated channels, receptor function, protein-protein interactions in membranes will benefit from the simple model system provided by the colicins. Mostly colicins A and El have been studied, however, only the three-dimensional structure of the pore-forming C-terminal domain of colicin A has been solved at 2.4 Å resolution. This domain can exist both in a water soluble as well as in a membrane form. Colicins bind to a receptor in the outer membrane and are then translocated across the cell envelope to the inner membrane. Import of colicins involves a cascade of steps which use specific proteins in the outer and inner membranes that are probably required for the acquisition and stabilization of a translocation-competent intermediate state. A membrane potential-dependent movement of a large region of the channel peptide into the membrane bilayer is required for channel opening. Pore-forming colicins span the whole cell envelope even after channel opening. Thus, they probably remain at putative translocation contact sites between the outer and inner membranes. Site-directed mutagenesis studies are now used to probe models concerning the dynamics of conformational changes of the C-terminal domain upon pore-formation. The protective action of immunity proteins against pore-forming colicins is a challenging problem. This action is specifically directed against the C-terminal domain of colicins. Studies using homologous recombination between the genes for colicins A and B provided hybrid colicins which allowed us to demonstrate that the specificity determinant in the pore-forming domain is constituted by the two hydrophobic helices of the structure. Despite extensive studies on the topography and identification of critical regions in the immunity polypeptide which spans the inner membrane four times, it is not yet known whether the formation of the channel or its opening is prevented by the immunity protein.

International audience; Colicin action is initiated by adsorption of the bacteriocin onto a specific receptor protein located in the outer membrane of susceptible cells. The presence or absence of such receptor proteins is a critical factor in defining the activity spectrum of a particular colicin (Graham & Stocker, 1977). The presence of an outer membrane receptor however is not, in itself, sufficient to bring about the lethal events. Following binding, the colicin has to be transported to its ultimate target. Mutant strains, possessing functional receptor, but which are not killed by a particular colicin have been isolated and described. Such mutants are usually denoted as “tolerant”.

International audience; All bacterial organisms have an envelope which has the primary role to constitute a physical barrier between the cytoplasm and the extracellular medium thereby protecting cells from harmful compounds from this medium. However, exchanges with the latter are required since cells must take up nutrients useful for growth.

International audience; Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and E1. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results, one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin E1 is located in the N-terminal domain of colicin E1; (4) the 183 N-terminal amino acid residues of colicin E1 are sufficient to promote E1AA uptake and thus probably colicin E1 uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin E1 by their respective immunity proteins.