Reduction of product composition variability using pooled microbiome ecosystem therapy and consequence in two infectious murine models.

Growing evidence demonstrates the key role of the gut microbiota in human health and disease. The recent success of microbiotherapy products to treat recurrent Clostridioides difficile infection has shed light on its potential in conditions associated with gut dysbiosis, such as acute graft-versus-host disease, intestinal bowel diseases, neurodegenerative diseases, or even cancer. However, the difficulty in defining a "good" donor as well as the intrinsic variability of donor-derived products' taxonomic composition limits the translatability and reproducibility of these studies. Thus, the pooling of donors' feces has been proposed to homogenize product composition and achieve higher taxonomic richness and diversity. In this study, we compared the metagenomic profile of pooled products to corresponding single donor-derived products. We demonstrated that pooled products are more homogeneous, diverse, and enriched in beneficial bacteria known to produce anti-inflammatory short chain fatty acids compared to single donor-derived products. We then evaluated pooled products' efficacy compared to corresponding single donor-derived products in Salmonella and C. difficile infectious mouse models. We were able to demonstrate that pooled products decreased pathogenicity by inducing a structural change in the intestinal microbiota composition. Single donor-derived product efficacy was variable, with some products failing to control disease progression. We further performed in vitro growth inhibition assays of two extremely drug-resistant bacteria, Enterococcus faecium vanA and Klebsiella pneumoniae oxa48, supporting the use of pooled microbiotherapies. Altogether, these results demonstrate that the heterogeneity of donor-derived products is corrected by pooled fecal microbiotherapies in several infectious preclinical models.IMPORTANCEGrowing evidence demonstrates the key role of the gut microbiota in human health and disease. Recent Food and Drug Administration approval of fecal microbiotherapy products to treat recurrent Clostridioides difficile infection has shed light on their potential to treat pathological conditions associated with gut dysbiosis. In this study, we combined metagenomic analysis with in vitro and in vivo studies to compare the efficacy of pooled microbiotherapy products to corresponding single donor-derived products. We demonstrate that pooled products are more homogeneous, diverse, and enriched in beneficial bacteria compared to single donor-derived products. We further reveal that pooled products decreased Salmonella and Clostridioides difficile pathogenicity in mice, while single donor-derived product efficacy was variable, with some products failing to control disease progression. Altogether, these findings support the development of pooled microbiotherapies to overcome donor-dependent treatment efficacy.

A novel antagonist anti-cMet antibody with antitumor activities targeting both ligand-dependent and ligand-independent c-Met receptors.

c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers.

Anti-c-Met monoclonal antibody ABT-700 breaks oncogene addiction in tumors with MET amplification.

BACKGROUND: c-Met is the receptor tyrosine kinase for hepatocyte growth factor (HGF) encoded by the MET proto-oncogene. Aberrant activation of c-Met resulting from MET amplification and c-Met overexpression is associated with poor clinical outcome in multiple malignancies underscoring the importance of c-Met signaling in cancer progression. Several c-Met inhibitors have advanced to the clinic; however, the development of inhibitory c-Met-directed therapeutic antibodies has been hampered by inherent agonistic activity. METHOD: We generated and tested a bivalent anti-c-Met monoclonal antibody ABT-700 in vitro for binding potency and antagonistic activity and in vivo for antitumor efficacy in human tumor xenografts. Human cancer cell lines and gastric cancer tissue microarrays were examined for MET amplification by fluorescence in situ hybridization (FISH). RESULTS: ABT-700 exhibits a distinctive ability to block both HGF-independent constitutive c-Met signaling and HGF-dependent activation of c-Met. Cancer cells addicted to the constitutively activated c-Met signaling driven by MET amplification undergo apoptosis upon exposure to ABT-700. ABT-700 induces tumor regression and tumor growth delay in preclinical tumor models of gastric and lung cancers harboring amplified MET. ABT-700 in combination with chemotherapeutics also shows additive antitumor effect. Amplification of MET in human cancer tissues can be identified by FISH. CONCLUSIONS: The preclinical attributes of ABT-700 in blocking c-Met signaling, inducing apoptosis and suppressing tumor growth in cancers with amplified MET provide rationale for examining its potential clinical utility for the treatment of cancers harboring MET amplification.

Innovative native MS methodologies for antibody drug conjugate characterization: High resolution native MS and IM-MS for average DAR and DAR distribution assessment.

Antibody drug conjugates (ADCs) are macromolecules composed of cytotoxic drugs covalently attached via a conditionally stable linker to monoclonal antibodies (mAbs). ADCs are among the most promising next generation of empowered mAbs foreseen to treat cancers. Compared to naked mAbs, ADCs have an increased level of complexity as the heterogeneity of conjugation cumulates with the inherent microvariability of the biomolecule. An increasing need underlying ADC's development and optimization is to improve its analytical and bioanalytical characterization by assessing three main ADC quality attributes: drug distribution, amount of naked antibody, and average drug to antibody ratio (DAR). Here, the analytical potential of native mass spectrometry (MS) and native ion mobility MS (IM-MS) is compared to hydrophobic interaction chromatography (HIC), the reference method for quality control of interchain cysteinyl-linked ADCs. Brentuximab vedotin, first in class and gold standard, was chosen for a proof of principle. High resolution native MS provided accurate mass measurement (<30 ppm) of intact ADCs together with average DAR and drug distribution, confirming the unique ability of native MS for simultaneous detection of mixtures of covalent and noncovalent products. Native IM-MS was next used for the first time to characterize an ADC. IM-MS evidenced ADC multiple drug loading, collisional cross sections measurement of each payload species attesting slight conformational changes. A semiquantitative interpretation of IM-MS data was developed to directly extrapolate average DAR and DAR distribution. Additionally, HIC fractions were collected and analyzed by native MS and IM-MS, assessing the interpretation of each HIC peak. Altogether, our results illustrate how native MS and IM-MS can rapidly assess ADC structural heterogeneity and how easily these methods can be implemented into MS workflows for in-depth ADC analytical characterization.

Melanoma and microbiota: Current understanding and future directions.

Over the last decade, the composition of the gut microbiota has been found to correlate with the outcomes of cancer patients treated with immunotherapy. Accumulating evidence points to the various mechanisms by which intestinal bacteria act on distal tumors and how to harness this complex ecosystem to circumvent primary resistance to immune checkpoint inhibitors. Here, we review the state of the microbiota field in the context of melanoma, the recent breakthroughs in defining microbial modes of action, and how to modulate the microbiota to enhance response to cancer immunotherapy. The host-microbe interaction may be deciphered by the use of "omics" technologies, and will guide patient stratification and the development of microbiota-centered interventions. Efforts needed to advance the field and current gaps of knowledge are also discussed.

A novel role for junctional adhesion molecule-A in tumor proliferation: modulation by an anti-JAM-A monoclonal antibody.

To identify new potential targets in oncology, functional approaches were developed using tumor cells as immunogens to select monoclonal antibodies targeting membrane receptors involved in cell proliferation. For that purpose cancer cells were injected into mice and resulting hybridomas were screened for their ability to inhibit cell proliferation in vitro. Based on this functional approach coupled to proteomic analysis, a monoclonal antibody specifically recognizing the human junctional adhesion molecule-A (JAM-A) was defined. Interestingly, compared to both normal and tumor tissues, we observed that JAM-A was mainly overexpressed on breast, lung and kidney tumor tissues. In vivo experiments demonstrated that injections of anti-JAM-A antibody resulted in a significant tumor growth inhibition of xenograft human tumors. Treatment with monoclonal antibody induced a decrease of the Ki67 expression and downregulated JAM-A levels. All together, our results show for the first time that JAM-A can interfere with tumor proliferation and suggest that JAM-A is a potential novel target in oncology. The results also demonstrate that a functional approach coupled to a robust proteomic analysis can be successful to identify new antibody target molecules that lead to promising new antibody-based therapies against cancers.

Time resolved native ion-mobility mass spectrometry to monitor dynamics of IgG4 Fab arm exchange and "bispecific" monoclonal antibody formation.

Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half molecules (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissociate and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Molecule-A (JAM-A) and alpha4 integrin, respectively. In addition, native MS analysis of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen molecules, confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb production follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixtures, and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies.

A JAM-A-tetraspanin-αvß5 integrin complex regulates contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvß5 integrin. JAM-A binds Csk and inhibits the activity of αvß5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell-cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell-matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvß5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.

Tetraspanin CD151 as a target for antibody-based cancer immunotherapy.

CD151 is a plasma membrane protein belonging to the tetraspanin superfamily which is expressed on normal cells such as endothelial cells and platelets and frequently overexpressed on cancer cells. It is known to be functionally linked to cancer metastasis. In humans, increased expression of CD151 is indicative of a poor prognosis in different cancer types. Whereas its mechanism of action remains obscure, CD151 was shown to regulate cell motility and adhesion through association with laminin-binding integrins such as α3ß1 or α6ß4. Several anti-CD151 mAbs (monoclonal antibodies) have been shown to display anti-metastatic activity in vivo. Inhibition of metastasis was not attributed to any effect of these mAbs on tumour cell growth, but was essentially attributed to inhibition of cell motility. We have generated anti-CD151 mAbs which can inhibit the tumoral growth in different xenograft cancer models. As expected, these mAbs were also able to inhibit metastasis in orthotopic cancer models. These data suggest that CD151 could function at multiple cancer stages, including not only metastasis cascade steps, but also earlier steps of primary tumour growth, thus reinforcing the interest of this innovative target in oncology. mAbs targeting CD151 may be of significant interest for cancer biotherapy.

Effect of microtubule-targeting drugs on cell-cell and cell-matrix junctions in tumor epithelial cells.

In this study, we investigated the effects of microtubule-targeting drugs, which either destabilize (the Vinca alkaloid vincristine) or stabilize (the taxane derivative docetaxel) microtubules, on the cell-cell and cell-matrix adhesive junctions of Caco-2 tumor epithelial cells, using fluorescence imaging and functional assays. We found that, in sub-confluent (but not confluent) cells, vincristine (but not docetaxel) affected cell-cell junction morphology. Furthermore, docetaxel (but not vincristine) attenuated the formation of the peri-junctional actomyosin ring and enhanced the internalization of junctional adhesion molecule-A. However, these effects of vincristine and docetaxel did not translate into appreciable functional changes during the opening and resealing of the cell-cell junctions. We also found that vincristine caused enlargement of focal adhesions (the major cell-matrix junctions) without affecting cell adhesion onto the matrix. Thus, we conclude that the microtubule-targeting drugs interfere to variable degrees with the morphology and/or function of the cell-cell and cell-matrix adhesive junctions. In addition, the results highlight the importance of considering the cellular context and dynamics (e.g. cell confluence and junction opening, respectively), when determining the final effects of microtubule manipulation on cell adhesiveness.

Efficacy of the Antibody-Drug Conjugate W0101 in Preclinical Models of IGF-1 Receptor Overexpressing Solid Tumors.

The insulin-like growth factor type 1 receptor (IGF-1R) is important in tumorigenesis, and its overexpression occurs in numerous tumor tissues. To date, therapeutic approaches based on mAbs and tyrosine kinase inhibitors targeting IGF-1R have only shown clinical benefit in specific patient populations. We report a unique IGF-1R-targeted antibody-drug conjugate (ADC), W0101, designed to deliver a highly potent cytotoxic auristatin derivative selectively to IGF-1R overexpressing tumor cells. The mAb (hz208F2-4) used to prepare the ADC was selected for its specific binding properties to IGF-1R compared with the insulin receptor, and for its internalization properties. Conjugation of a novel auristatin derivative drug linker to hz208F2-4 did not alter its binding and internalization properties. W0101 induced receptor-dependent cell cytotoxicity in vitro when applied to various cell lines overexpressing IGF-1R, but it did not affect normal cells. Efficacy studies were conducted in several mouse models expressing different levels of IGF-1R to determine the sensitivity of the tumors to W0101. W0101 induced potent tumor regression in certain mouse models. Interestingly, the potency of W0101 correlated with the expression level of IGF-1R evaluated by IHC. In an MCF-7 breast cancer model with high-level IGF-1R expression, a single injection of W0101 3 mg/kg led to strong inhibition of tumor growth. W0101 provides a potential new therapeutic option for patients overexpressing IGF-1R. A first-in-human trial of W0101 is currently ongoing to address clinical safety.

Molecular mechanisms involved in activity of h7C10, a humanized monoclonal antibody, to IGF-1 receptor.

IGF-1 receptor (IGF-1R) plays a key role in the development of numerous tumors. Blockade of IGF-1R axis using monoclonal antibodies constitutes an interesting approach to inhibit tumor growth. We have previously shown that h7C10, a humanized anti-IGF-1R Mab, exhibited potent antitumor activity in vivo. However, mechanisms of action of h7C10 are still unknown. Here, we showed that h7C10 inhibited IGF-1-induced IGF-1R phosphorylation in a dose-dependent manner. Also, h7C10 abolished IGF-1-induced activation of PI3K/AKT and MAPK pathways. Cell cycle progression and colony formation were affected in the presence of h7C10 probably because of the inhibition of IGF-1-induced cyclin D1 and E expression. In addition, we demonstrated that h7C10 induced a rapid IGF-1R internalization leading to an accumulation into cytoplasm resulting in receptor degradation. Using lysosome and proteasome inhibitors, we observed that the IGF-1R alpha- and beta-chains could follow different degradation routes. Thus, we demonstrated that antitumoral properties of h7C10 are the result of IGF-1-induced cell signaling inhibition and down-regulation of IGF-1R level suggesting that h7C10 could be a candidate for therapeutic applications.

Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS.

Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen molecules with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.

Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody.

Despite technological advances, detection of deamidation in large proteins remains a challenge and the use of orthogonal methods is needed for unequivocal assignment. By a combination of cation-exchange separation, papain digestion, and a panel of mass spectrometry techniques we identified asparagine deamidation in light chain complementarity determining region 1 (CDR1) of a humanized IgG1 monoclonal antibody. The reaction yields both Asp and isoAsp, which were assigned by Edman degradation and by isoAsp detection using protein isoaspartate methyltransferase. The deamidated antibody variants were less potent in antigen binding compared to the nondegraded antibody. Changes in near-UV CD spectra, susceptibility to papain cleavage in an adjacent CDR2 loop, and the tendency of the newly formed isoAsp to undergo isomerization suggest local perturbations in the structure of the isoAsp-containing antibody.

[Therapeutic antibodies and related products: choosing the right structure for success]. / Anticorps thérapeutiques et dérivés : une palette de structures pour une pléthore d'indications. Quel format et quelle glycosylation choisir ? Pour quelles applications ?

Monoclonal antibodies (mAb) and related-products represent the fastest growing class of therapeutics in the biotechnological and pharmaceutical industry. In just as short as 20 years, more than 30 immunoglobulins (IgG) and derivatives have been approved in a wide range of indications (oncology, inflammation and auto-immunity, transplantation, angioplasty, hematology, ophthalmology, viral infections, allergy). The mAb structure toolbox contains mouse, chimeric, humanized and human antibodies from different isotypes (IgG1, 2 and 4), as well as IgG-related products (immunoconjugates, radio-immunoconjugates, Fab fragments, Fc-fusion proteins and peptides, bispecifics). Furthermore from a structural point of view, mAb glycosylation is linked to their production systems and may impact on their effector functions and immunogenicity. Based on the current knowledge, choosing the right antibody format, isotype and glycosylation profile are some of the key issues to address early during the lead selection.

[Protein scaffolds as alternatives to whole antibodies: from discovery research to clinical development]. / Ingénierie de charpentes protéiques comme alternative aux anticorps : de la recherche au développement clinique.

Recent advances in combinatorial protein engineering have made it possible to develop non-Ig protein scaffolds that can potentially substitute for most whole antibody-associated properties. These protein scaffolds display most of the binding properties associated with the variable domain of antibodies. In theory, many different natural human protein backbones are suitable to be used as recombinant templates for engineering ; in practice however, only a few have yielded the necessary properties to be translated into << druggable biologicals >>. Amongst these properties, potential broad specificities towards any kind of target, ease of production, small size, good tolerability and low immunogenicity are essential. Intellectual property is another key issue. In this review, a particular emphasis will be given to the most validated non-Ig scaffolds that have reached the clinical development phase.

[The potential of monoclonal antibodies in cancer : established trastuzumab and cetuximab and promising targets IGF-1R and c-MET]. / Anticorps et tumeurs solides : cibles établies et pistes prometteuses.

Recent biotechnological advances allowed the development of a novel class of anti-cancer drugs called monoclonal antibodies (mAb). To illustrate the potential of these new agents, two mAbs used in daily practice (i.e., trastuzumab and cetuximab) and two promising targets (i.e., IGF-1R and c-Met) for which mAbs should be available in a near future are discussed here. Trastuzumab and cetuximab deeply changed treatment strategies for breast, colon, and head and neck cancers. However their efficacy is observed in a fraction of patients only and is often time limited. Thus, current challenges are to better understand the mechanisms of action of mAbs, to identify mechanisms of resistance, to exploit the synergy between mAbs and chemotherapy drugs, and to better select patients with a potential benefit. Resolving these issues should pave the way for tailored treatment according to tumor and patient characteristics.

Tumor Cells Hijack Macrophage-Produced Complement C1q to Promote Tumor Growth.

Clear-cell renal cell carcinoma (ccRCC) possesses an unmet medical need, particularly at the metastatic stage, when surgery is ineffective. Complement is a key factor in tissue inflammation, favoring cancer progression through the production of complement component 5a (C5a). However, the activation pathways that generate C5a in tumors remain obscure. By data mining, we identified ccRCC as a cancer type expressing concomitantly high expression of the components that are part of the classical complement pathway. To understand how the complement cascade is activated in ccRCC and impacts patients' clinical outcome, primary tumors from three patient cohorts (n = 106, 154, and 43), ccRCC cell lines, and tumor models in complement-deficient mice were used. High densities of cells producing classical complement pathway components C1q and C4 and the presence of C4 activation fragment deposits in primary tumors correlated with poor prognosis. The in situ orchestrated production of C1q by tumor-associated macrophages (TAM) and C1r, C1s, C4, and C3 by tumor cells associated with IgG deposits, led to C1 complex assembly, and complement activation. Accordingly, mice deficient in C1q, C4, or C3 displayed decreased tumor growth. However, the ccRCC tumors infiltrated with high densities of C1q-producing TAMs exhibited an immunosuppressed microenvironment, characterized by high expression of immune checkpoints (i.e., PD-1, Lag-3, PD-L1, and PD-L2). Our data have identified the classical complement pathway as a key inflammatory mechanism activated by the cooperation between tumor cells and TAMs, favoring cancer progression, and highlight potential therapeutic targets to restore an efficient immune reaction to cancer.

The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies.

Glycosylation which plays a crucial role in the pharmacological properties of therapeutic monoclonal antibodies (MAbs) is influenced by several factors like production systems, selected clonal population and manufacturing processes. Efficient analytical methods are therefore required in order to characterize glycosylation at different stages of MAbs discovery and production. Three mass spectrometry (MS)-based strategies were compared to analyze N-glycosylation of MAbs either expressed in murine myeloma (NS0) or Chinese Hamster Ovary (CHO) cell lines, the two current main production systems used for therapeutic MAbs. First a top-down approach was used on intact and reduced MAbs by liquid chromatography coupled to an electrospray ionization-time of flight mass spectrometer (LC-ESI-TOF), which provided fast and accurate profiles of MAbs glycosylation patterns for routine controls. Secondly, after digestion of the antibody with the peptide N-glycosidase F (PNGase F) enzyme, released N-linked glycans were directly analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) without any prior derivatization, which gave precise details on the structure of the most abundant glycoforms. Finally, a bottom-up approach on tryptic glycopeptides using a nanoLC-Chip-MS/MS ion trap (IT) system equipped with a graphitized carbon column was investigated. Data were compared to those obtained with a more classical C18 reversed phase column showing that this last method is well suited to detect low abundant glycoforms and to provide in one shot information regarding both the oligosaccharide structure and the amino acid sequence of its peptide moiety.

Functional responses and in vivo anti-tumour activity of h7C10: a humanised monoclonal antibody with neutralising activity against the insulin-like growth factor-1 (IGF-1) receptor and insulin/IGF-1 hybrid receptors.

A novel humanised monoclonal antibody (Mab, h7C10) was raised against the human insulin-like growth factor-1 receptor (IGF-1R); it exhibited potent inhibition of tumour growth in animal models. Further evaluation of its inhibitory activity at hybrid receptors (Hybrid-Rs) composed of the association between IGF-1R and insulin receptor (IR) was performed. Selective, potent and efficacious inhibition of [(125)I]IGF-1 binding as well as IGF-1- and IGF-2-mediated receptor phosphorylation was demonstrated at both IGF-1R and Hybrid-Rs, without activity at IR. Ligand-independent down-regulation of both IGF-1R and Hybrid-Rs was obtained upon long-term association with h7C10. In vivo evaluation was performed in a MDA-MB-231 xenograft mouse model, showing a 14-fold higher level of Hybrid-Rs as compared to IGF-1R. A more potent anti-tumoural response was obtained for h7C10 as compared to Mabs targeting solely IGF-1R or Hybrid-Rs. The herewith described neutralising properties of h7C10 as potent inhibitor of both IGF-1R and Hybrid-Rs are likely to participate in its anti-tumoural activities and maybe of interest for therapeutic applications.