Middle Level IM-MS and CIU Experiments for Improved Therapeutic Immunoglobulin Subclass Fingerprinting.

Most of the current FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are based on humanized or human IgG1, 2, or 4 subclasses and engineered variants. On the structural side, these subclasses are characterized by specific interchain disulfide bridge connections. Different analytical techniques have been reported to assess intact IgGs subclasses, with recently special interest in native ion mobility (IM) and collision induced unfolding (CIU) mass spectrometry (MS).

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.

A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants.

The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nondenaturing conditions have proven to be valuable for mAb-related compound characterization.

A New Anti-CXCR4 Antibody That Blocks the CXCR4/SDF-1 Axis and Mobilizes Effector Cells.

The type IV C-X-C-motif chemokine receptor (CXCR4) is expressed in a large variety of human cancers, including hematologic malignancies, and this receptor and its ligand, stromal cell-derived factor-1 (SDF-1), play a crucial role in cancer progression. We generated a humanized immunoglobulin G1 mAb, hz515H7, which binds human CXCR4, efficiently competes for SDF-1 binding, and induces a conformational change in CXCR4 homodimers. Furthermore, it inhibits both CXCR4 receptor-mediated G-protein activation and β-arrestin-2 recruitment following CXCR4 activation.

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.

A sensitive multidimensional method for the detection, characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection.

Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window.

Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion.

Here we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA )-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms,electropherograms and mass spectra to those reported for hinge Cys-linked ADCs.

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.

The respiratory syncytial virus G protein conserved domain induces a persistent and protective antibody response in rodents.

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130-230).

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.

The next generation of antibody-drug conjugates comes of age.

Monoclonal antibodies (mAbs) and derivatives are currently the fastest growing class of therapeutic molecules. More than 30 G-type immunoglobulins (IgG) and related agents have been approved over the past 25 years mainly for cancers and inflammatory diseases. In oncology, mAbs are often combined with cytotoxic drugs to enhance their therapeutic efficacy.

[Immunoconjugates, drug-armed antibodies to fight against cancer].

Monoclonal antibodies constitute a growing class of therapeutic agents. They are classically used in combination with chemotherapeutic drugs for cancer treatment. The concept of coupling a cytotoxic agent to an antibody can be viewed as a means to confer a selectivity for tumoral cells to highly cytotoxic drugs which cannot be used in human, or a higher power to antibodies which have a low anti-tumoral activity on their own.

Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins.

Monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. More than 20 MAbs have been approved and several hundreds are in clinical trials in various therapeutic indications including oncology, inflammatory diseases, organ transplantation, cardiology, viral infection, allergy, and tissue growth and repair. Most of the current therapeutic antibodies are humanized or human Immunoglobulins (IgGs) and are produced as recombinant glycoproteins in eukaryotic cells.

Glycoproteomics and glycomics investigation of membrane N-glycosylproteins from human colon carcinoma cells.

Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT-29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin-affinity chromatography, SDS-PAGE and MS analysis, allowed the identification of membrane N-glycoproteins from Triton X-100-solubilized proteins from membrane preparation.

Scaled-down purification protocol to access proteomic analysis of 20S proteasome from human tissue samples: comparison of normal and tumor colorectal cells.

The proteasome is a proteolytic complex that constitutes the main pathway for degradation of intracellular proteins in eukaryotic cells. It regulates many physiological processes and its dysfunction can lead to several pathologies like cancer. To study the 20S proteasome structure/activity relationship in cells that derive from human biopsy samples, we optimized an immuno-purification protocol for the analysis of samples containing a small number of cells using magnetic beads.

Peptides as tools and drugs for immunotherapies.

Peptides are essential tools for discovery and pre-clinical and pharmaceutical development of viral and cancer vaccines ('active immunotherapies') as well as for therapeutic antibodies ('passive immunotherapies'). They help to trigger and analyze immune responses at a molecular level (B-cell, T-helper and CTL epitopes). They contribute largely to the design of new vaccine candidates and to the generation of monoclonal antibodies.

Separation and quantitative analysis of alkyl sulfobetaine-type detergents by high-performance liquid chromatography and light-scattering detection.

An improved high-performance liquid chromatographic (HPLC) method for the separation of zwitterionic detergents is described. It is based on a reversed-phase liquid chromatography with evaporative light-scattering detection (ELSD). The method was shown to be highly specific, allowing the separation of three detergents of the alkyl sulfobetaine family: 3-(N-dodecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB12), 3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB14) and 3-(N-hexadecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB16).

Evaluation of a novel Vi conjugate vaccine in a murine model of salmonellosis.

Immunisation of BALB/c mice with a vaccine containing Vi polysaccharide conjugated to the Klebsiella pneumoniae outer membrane 40 kDa protein (rP40), in combination with Escherichia coli heat-labile toxin adjuvant (LT), elicited anti-Vi IgG antibodies after administration using different routes. Testing of the immune serum in opsonisation assays demonstrated the specific enhancement of Vi-positive bacterial uptake by cultured murine bone marrow derived macrophages. Intra-peritoneal challenge of mice immunised with the Vi-based vaccine elicited a degree of protection against virulent Vi+ Salmonella enterica serovar typhimurium (S.

Complexity and complementarity of outer membrane protein A recognition by cellular and humoral innate immunity receptors.

Outer membrane protein A (OmpA) is a conserved major component of the outer membrane of Enterobacteriaceae. Here, we report that OmpA from Klebsiella pneumoniae (KpOmpA) activates macrophages and dendritic cells (DCs) in a TLR2-dependent way. However, TLR2 does not account for binding of KpOmpA to innate immune cells.

Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells.

7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI-TOF, ES-TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain).

Protective levels of polysaccharide-specific maternal antibodies may enhance the immune response elicited by pneumococcal conjugates in neonatal and infant mice.

Maternal antibodies (MatAbs) may protect the offspring against infections but may also interfere with their immune responses to vaccination. We have previously shown that maternal immunization with pneumococcal polysaccharides (PPS) conjugated to tetanus protein (Pnc-TT) protected the offspring against infections caused by three important pediatric serotypes. To study the influence of MatAb on the immune response to Pnc-TT early in life, adult female mice were immunized twice with Pnc-TT of serotype 1 (Pnc1-TT), and their offspring received Pnc1-TT subcutaneously three times at 3-week intervals starting at 1 week (neonatal) or 3 weeks (infant) of age.

A recombinant humanized anti-insulin-like growth factor receptor type I antibody (h7C10) enhances the antitumor activity of vinorelbine and anti-epidermal growth factor receptor therapy against human cancer xenografts.

Interaction of insulin-like growth factor receptor I (IGF-IR) with its ligands has been reported to induce cell proliferation, transformation and blockade of cell apoptotic functions. IGF-IR is overexpressed on numerous tumor cell types and its blockade could be of importance for anti-cancer therapy. We have generated a humanized anti-IGF-IR antibody h7C10 that blocks in vitro IGF-I and IGF-II-induced cell proliferation of MCF-7 breast cancer cells.

Immunization of female mice with glycoconjugates protects their offspring against encapsulated bacteria.

The immune system of the newborn is immature, and therefore it is difficult to induce protective immunity by vaccination in the neonatal period. Immunization of mothers during pregnancy against infections caused by encapsulated bacteria could thus be particularly attractive, as infants do not respond to polysaccharide (PS) antigens. Transmission of maternal vaccine-specific antibodies and protection of offspring against pneumococcal bacteremia and/or lung infection were studied in a neonatal murine model of pneumococcal immunization and infections.

The outer membrane protein X from Escherichia coli exhibits immune properties.

Outer membrane proteins (OMP) are expressed in Gram-negative bacterial cell wall. OmpA from Klebsiella pneumoniae (KpOmpA) has been shown to bind and to activate selectively antigen presenting cells (APCs), eliciting protective CTL responses. In this study, we investigated whether OmpX, another member of the OMP family and structurally related to OmpA, exhibits the same immune properties.

Identification of B- and T-cell epitopes of BB, a carrier protein derived from the G protein of Streptococcus strain G148.

Most conventional vaccines consist of killed organisms or purified antigenic proteins. Such molecules are generally poorly immunogenic and need to be coupled to carrier proteins. We have identified a new carrier molecule, BB, derived from the G protein of Streptococcus strain G148.