Homogeneous antibody-drug conjugates : DAR 2 anti-HER2 obtained by conjugation on isolated light chain followed by mAb assembly

Despite advances in medical care, cancer remains a major threat to human health. Antibody-drug conjugates (ADCs) are a promising targeted therapy to overcome adverse side effects to normal tissues. In this field, the current challenge is obtaining homogeneous preparations of conjugates, where a defined number of drugs are conjugated to specific antibody sites. Site-directed cysteine-based conjugation is commonly used to obtain homogeneous ADC, but it is a time-consuming and expensive approach due to the need for extensive antibody engineering to identify the optimal conjugation sites and reduction - oxidation protocols are specific for each antibody. There is thus a need for ADC platforms that offer homogeneity and direct applicability to the already approved antibody therapeutics. Here we describe a novel approach to derive homogeneous ADCs with drug-to-antibody ratio of 2 from any human immunoglobulin 1 (IgG), using trastuzumab as a model. The method is based on the production of heavy chains (HC) and light chains (LC) in two recombinant HEK293 independent cultures, so the original amino acid sequence is not altered. Isolated LC was effectively conjugated to a single drug-linker (vcMMAE) construct and mixed to isolated HC dimers, in order to obtain a correctly folded ADC. The relevance of the work was validated in terms of ADC homogeneity (HIC-HPLC, MS), purity (SEC-HPLC), isolated antigen recognition (ELISA) and biological activity (HER2-positive breast cancer cells cytotoxicity assays)

A structural signature motif enlightens the origin and diversification of nuclear receptors

Nuclear receptors are ligand-activated transcription factors that modulate gene regulatory networks from embryonic development to adult physiology and thus represent major targets for clinical interventions in many diseases. Most nuclear receptors function either as homodimers or as heterodimers. The dimerization is crucial for gene regulation by nuclear receptors, by extending the repertoire of binding sites in the promoters or the enhancers of target genes via combinatorial interactions. Here, we focused our attention on an unusual structural variation of the alpha-helix, called pi-turn that is present in helix H7 of the ligand-binding domain of RXR and HNF4. By tracing back the complex evolutionary history of the pi-turn, we demonstrate that it was present ancestrally and then independently lost in several nuclear receptor lineages. Importantly, the evolutionary history of the pi-turn motif is parallel to the evolutionary diversification of the nuclear receptor dimerization ability from ancestral homodimers to derived heterodimers. We then carried out structural and biophysical analyses, in particular through point mutation studies of key RXR signature residues and showed that this motif plays a critical role in the network of interactions stabilizing homodimers. We further showed that the pi-turn was instrumental in allowing a flexible heterodimeric interface of RXR in order to accommodate multiple interfaces with numerous partners and critical for the emergence of high affinity receptors. Altogether, our work allows to identify a functional role for the pi-turn in oligomerization of nuclear receptors and reveals how this motif is linked to the emergence of a critical biological function. We conclude that the pi-turn can be viewed as a structural exaptation that has contributed to enlarging the functional repertoire of nuclear receptors

Facultative methanotrophy: false leads, true results, and suggestions for future research

Methanotrophs are a group of phylogenetically diverse microorganisms characterized by their ability to utilize methane as their sole source of carbon and energy. Early studies suggested that growth on methane could be stimulated with the addition of some small organic acids, but initial efforts to find facultative methanotrophs, i.e., methanotrophs able to utilize compounds with carbon–carbon bonds as sole growth substrates were inconclusive. Recently, however, facultative methanotrophs in the genera Methylocella , Methylocapsa , and Methylocystis have been reported that can grow on acetate, as well as on larger organic acids or ethanol for some species. All identified facultative methanotrophs group within the Alphaproteobacteria and utilize the serine cycle for carbon assimilation from formaldehyde. It is possible that facultative methanotrophs are able to convert acetate into intermediates of the serine cycle (e.g. malate and glyoxylate), because a variety of acetate assimilation pathways convert acetate into these compounds (e.g. the glyoxylate shunt of the tricarboxylic acid cycle, the ethylmalonyl‐CoA pathway, the citramalate cycle, and the methylaspartate cycle). In this review, we summarize the history of facultative methanotrophy, describe scenarios for the basis of facultative methanotrophy, and pose several topics for future research in this area.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86841/1/fml2315.pd

A census of metals and baryons in stars in the local Universe

We combine stellar metallicity and stellar mass estimates for a large sample of galaxies drawn from the SDSS DR2 spanning wide ranges in physical properties, in order to derive an inventory of the total mass of metals and baryons locked up in stars today. Physical parameter estimates are derived from galaxy spectra with high S/N (>20). Coadded spectra of galaxies with similar velocity dispersions, absolute r-band magnitudes and 4000\AA-break values are used for those regions of parameter space where individual spectra have lower S/N. We estimate the total density of metals and of baryons in stars and, from these two quantities, we obtain a mass- and volume-averaged stellar metallicity of =1.04+-0.14 Z_sun, i.e. consistent with solar. We also study how metals are distributed in galaxies according to their mass, morphology and age, and we then compare these distributions with the corresponding distributions of stellar mass. We find that the bulk of metals locked up in stars in the local Universe reside in massive, bulge-dominated galaxies, with red colours and high 4000\AA-break values corresponding to old stellar populations. Bulge-dominated and disc-dominated galaxies contribute similar amounts to the total stellar mass density, but have different fractional contributions to the mass density of metals in stars, in agreement with the mass-metallicity relation. Bulge-dominated galaxies contain roughly 40% of the total amount of metals in stars, while disc-dominated galaxies less than 25%. Finally, at a given galaxy stellar mass, we define two characteristic ages as the median of the distributions of mass and metals as a function of age. These characteristic ages decrease progressively from high-mass to low-mass galaxies, consistent with the high formation epochs of stars in massive galaxies.Comment: replaced with accepted version, minor changes, references adde

A critical look at the mass-metallicity-star formation rate relation in the local universe. I. An improved analysis framework and confounding systematics

It has been proposed that the mass-metallicity relation of galaxies exhibits a secondary dependence on star formation rate (SFR), and that the resulting M-Z-SFR relation may be redshift-invariant, i.e., "fundamental." However, conflicting results on the character of the SFR dependence, and whether it exists, have been reported. To gain insight into the origins of the conflicting results, we (a) devise a non-parametric, astrophysically motivated analysis framework based on the offset from the star-forming ("main") sequence at a given stellar mass (relative specific SFR), (b) apply this methodology and perform a comprehensive re-analysis of the local M-Z-SFR relation, based on SDSS, GALEX, and WISE data, and (c) study the impact of sample selection, and of using different metallicity and SFR indicators. We show that metallicity is anti-correlated with specific SFR regardless of the indicators used. We do not find that the relation is spurious due to correlations arising from biased metallicity measurements, or fiber aperture effects. We emphasize that the dependence is weak/absent for massive galaxies ($\log M_*>10.5$), and that the overall scatter in the M-Z-SFR relation does not greatly decrease from the M-Z relation. We find that the dependence is stronger for the highest SSFR galaxies above the star-forming sequence. This two-mode behavior can be described with a broken linear fit in 12+log(O/H) vs. log (SFR$/M_*$), at a given $M_*$. Previous parameterizations used for comparative analysis with higher redshift samples that do not account for the more detailed behavior of the local M-Z-SFR relation may incorrectly lead to the conclusion that those samples follow a different relationship.Comment: ApJ. Several minor correction

The COS Legacy Archive Spectroscopy SurveY (CLASSY) Treasury Atlas

Far-ultraviolet (FUV; ~1200-2000 angstroms) spectra are fundamental to our understanding of star-forming galaxies, providing a unique window on massive stellar populations, chemical evolution, feedback processes, and reionization. The launch of JWST will soon usher in a new era, pushing the UV spectroscopic frontier to higher redshifts than ever before, however, its success hinges on a comprehensive understanding of the massive star populations and gas conditions that power the observed UV spectral features. This requires a level of detail that is only possible with a combination of ample wavelength coverage, signal-to-noise, spectral-resolution, and sample diversity that has not yet been achieved by any FUV spectral database. We present the COS Legacy Spectroscopic SurveY (CLASSY) treasury and its first high level science product, the CLASSY atlas. CLASSY builds on the HST archive to construct the first high-quality (S/N_1500 >~ 5/resel), high-resolution (R~15,000) FUV spectral database of 45 nearby (0.002 < z < 0.182) star-forming galaxies. The CLASSY atlas, available to the public via the CLASSY website, is the result of optimally extracting and coadding 170 archival+new spectra from 312 orbits of HST observations. The CLASSY sample covers a broad range of properties including stellar mass (6.2 < logM_star(M_sol) < 10.1), star formation rate (-2.0 < log SFR (M_sol/yr) < +1.6), direct gas-phase metallicity (7.0 < 12+log(O/H) < 8.8), ionization (0.5 < O_32 < 38.0), reddening (0.02 < E(B-V < 0.67), and nebular density (10 < n_e (cm^-3) < 1120). CLASSY is biased to UV-bright star-forming galaxies, resulting in a sample that is consistent with z~0 mass-metallicity relationship, but is offset to higher SFRs by roughly 2 dex, similar to z >~2 galaxies. This unique set of properties makes the CLASSY atlas the benchmark training set for star-forming galaxies across cosmic time.Comment: Accepted for publication in Ap

The Cosmic Origins Spectrograph

The Cosmic Origins Spectrograph (COS) is a moderate-resolution spectrograph with unprecedented sensitivity that was installed into the Hubble Space Telescope (HST) in May 2009, during HST Servicing Mission 4 (STS-125). We present the design philosophy and summarize the key characteristics of the instrument that will be of interest to potential observers. For faint targets, with flux F_lambda ~ 1.0E10-14 ergs/s/cm2/Angstrom, COS can achieve comparable signal to noise (when compared to STIS echelle modes) in 1-2% of the observing time. This has led to a significant increase in the total data volume and data quality available to the community. For example, in the first 20 months of science operation (September 2009 - June 2011) the cumulative redshift pathlength of extragalactic sight lines sampled by COS is 9 times that sampled at moderate resolution in 19 previous years of Hubble observations. COS programs have observed 214 distinct lines of sight suitable for study of the intergalactic medium as of June 2011. COS has measured, for the first time with high reliability, broad Lya absorbers and Ne VIII in the intergalactic medium, and observed the HeII reionization epoch along multiple sightlines. COS has detected the first CO emission and absorption in the UV spectra of low-mass circumstellar disks at the epoch of giant planet formation, and detected multiple ionization states of metals in extra-solar planetary atmospheres. In the coming years, COS will continue its census of intergalactic gas, probe galactic and cosmic structure, and explore physics in our solar system and Galaxy.Comment: 17 pages, 15 figure

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. Methodology/Principal Findings The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid).Conclusion/Significance These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.Organismic and Evolutionary Biolog

A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate Using Middle-Down Mass Spectrometry

International audienceMiddle-down mass spectrometry (MD MS) has emerged as a promising alternative to classical bottom-up approaches for protein characterization. Middle-level experiments after enzymatic digestion are routinely used for subunit analysis of monoclonal antibody (mAb)-related compounds, providing information on drug load distribution and average drug-to-antibody ratio (DAR). However, peptide mapping is still the gold standard for primary amino acid sequence assessment, post-translational modifications (PTM), and drug conjugation identification and localization. However, peptide mapping strategies can be challenging when dealing with more complex and heterogeneous mAb formats, like antibody-drug conjugates (ADCs). We report here, for the first time, MD MS analysis of a third-generation site-specific DAR4 ADC using different fragmentation techniques, including higher-energy collisional- (HCD), electron-transfer (ETD) dissociation and 213 nm ultraviolet photodissociation (UVPD). UVPD used as a standalone technique for ADC subunit analysis afforded, within the same liquid chromatography-MS/MS run, enhanced performance in terms of primary sequence coverage compared to HCD- or ETD-based MD approaches, and generated substantially more MS/MS fragments containing either drug conjugation or glycosylation site information, leading to confident drug/glycosylation site identification. In addition, our results highlight the complementarity of ETD and UVPD for both primary sequence validation and drug conjugation/glycosylation site assessment. Altogether, our results highlight the potential of UVPD for ADC MD MS analysis for drug conjugation/glycosylation site assessment, and indicate that MD MS strategies can improve structural characterization of empowered next-generation mAb-based formats, especially for PTMs and drug conjugation sites validation

Insight on the Impact of the Reduction Step on the Site‐Directed Conjugation of an Anti‐HER2 Cysteine‐Engineered Antibody

International audienceCysteine‐based conjugation is one of the main antibody conjugation strategies for generating both heterogeneous and homogeneous Antibody Drug Conjugates (ADCs), being the reduction of the antibody a crucial step in these processes. In this work we have analysed the reduction conditions for the site‐directed conjugation of an anti‐HER2 (Human Epidermal Receptor 2) Trastuzumab_cys114 antibody to the cytotoxic drug vcMMAE (valine‐citrulline monomethyl auristatin E), with an aimed average DAR (Drug‐Antibody Ratio) of 2. Initial reduction was found to have a direct impact on the availability of free thiols for the conjugation: increasing of reducing agent concentration (until a molar excess of 50x) in the reduction step resulted in a lower proportion of naked antibody in the final ADC product and allowed us to obtain an ADC close to the DAR of interest. This work shows that reduction conditions must be adjusted in order to obtain the desired homogeneous ADC product