What makes a good antibody-drug conjugate?

Introduction: Antibody-Drug Conjugates (ADCs) are becoming increasingly important weapons in the fight against cancer, as evidenced by the growing number of approved products. The complex nature of an ADC means that there is a vast array of choices to consider in the design of such drugs.Areas covered: We provide an overview of developments in each facet of ADC structure: the antibody, linker, and payload. Looking at the current clinical landscape, we discuss trends that have led to the evolution of ADC design.Expert opinion:Following a history of setbacks and high discontinuation rates, the understanding of the ADC field has grown. If developers can obtain a firm grasp of the structure-function relationship of their molecule, we expect the advances in ADC design to translate to improved clinical success. Moreover, the breadth of ADC applications will continue to expand to target new indications with novel targets and payloads.

Peptide profiling of Internet-obtained Cerebrolysin using high performance liquid chromatography - electrospray ionization ion trap and ultra high performance liquid chromatography - ion mobility - quadrupole time of flight mass spectrometry.

Cerebrolysin, a parenteral peptide preparation produced by controlled digestion of porcine brain proteins, is an approved nootropic medicine in some countries. However, it is also easily and globally available on the Internet. Nevertheless, until now, its exact chemical composition was unknown. Using high performance liquid chromatography (HPLC) coupled to ion trap and ultra high performance liquid chromatography (UHPLC) coupled to quadrupole-ion mobility-time-of-flight mass spectrometry (Q-IM-TOF MS), combined with UniProt pig protein database search and PEAKS de novo sequencing, we identified 638 unique peptides in an Internet-obtained Cerebrolysin sample. The main components in this sample originate from tubulin alpha- and beta-chain, actin, and myelin basic protein. No fragments of known neurotrophic factors like glial cell-derived neurotrophic factor (GDNF), neurotrophin nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) were found, suggesting that the activities reported in the literature are likely the result of new, hitherto unknown cryptic peptides with nootropic properties.

Characterisation of glycoproteins using a quadrupole time-of-flight mass spectrometer configured for electron transfer dissociation.

RATIONALE: Electron transfer dissociation (ETD) within ion trapping mass spectrometers has proven to be a useful tool for the characterisation of post-translational modifications. In this study, we describe the implementation of ETD upon a modified quadrupole time-of-flight (Q-ToF) system and methods for the analysis of glycoproteins. METHODS: Liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS) was performed using a hybrid quadrupole/ion mobility/oa-ToF mass spectrometer equipped with ETD functionality. 1,4-Dicyanobenzene reagent anions necessary for the ETD reaction were generated from a glow discharge region located within the ESI source block. ETD reactions occurred in the stacked ring travelling wave ion guide (located after the quadrupole mass filter and prior to the oa-ToF mass analyser). LC/ETD was performed upon 'super-charged' tryptic glycopeptide ions produced from the recombinant monoclonal antibody trastuzumab. LC/ETD was also performed on ions from the smaller glycopeptides obtained from erythropoietin. RESULTS: ETD performed upon the quadruply 'super-charged' N-linked glycopeptide ions of trastuzumab and the triply charged O-linked glycopeptide ions of erythropoietin provided both glycosylation site assignments and full sequence information, respectively. Tandem mass (MS/MS) spectra employing collision-induced dissociation (CID) were dominated by oxonium product ions hampering full peptide sequence characterisation. CONCLUSIONS: LC/ETD on the Q-ToF system proved effective at characterising a number of different N-linked glyco-forms of the tryptic peptide, EEQYNSTYR, from trastuzumab as well as glyco-forms from the O-linked tryptic peptide, EASIPPDAASAAPLR, from erythropoietin. The data demonstrates that the glycopeptide site heterogeneity of trastuzumab and erythropoietin can be accurately characterised. In addition, the post-column mixing of the super-charging reagent, m-NBA, is an effective method to increase the precursor ion charge state and to improve ETD reaction efficiency.

Shapes of supramolecular cages by ion mobility mass spectrometry.

Mass spectrometry and drift tube ion mobility mass spectrometry have been used to analyse several isobaric, multicomponent cages yielding information on three dimensional structure, interactions and dynamics of assembly in the gas phase.

Interaction of human ß-defensin 2 (HBD2) with glycosaminoglycans.

Human ß-defensin 2 (HBD2) is a member of the defensin family of antimicrobial peptides that plays important roles in the innate and adaptive immune system of both vertebrates and invertebrates. In addition to their direct bactericidal action, defensins are also involved in chemotaxis and Toll-like receptor activation. In analogy to chemokine/glycosaminoglycan (GAG) interactions, GAG-defensin complexes are likely to play an important role in chemotaxis and in presenting defensins to their receptors. Using a gel mobility shift assay, we found that HBD2 bound to a range of GAGs including heparin/heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate. We used NMR spectroscopy of (15)N-labeled HBD2 to map the binding sites for two GAG model compounds, a heparin/HS pentasaccharide (fondaparinux sodium; FX) and enzymatically prepared DS hexasaccharide (DSdp6). We identified a number of basic amino acids that form a common ligand binding site, which indicated that these interactions are predominantly electrostatic. The dissociation constant of the [DSdp6-HBD2] complex was determined by NMR spectroscopy to be 5 ± 5 µM. Binding of FX could not be quantified because of slow exchange on the NMR chemical shift time scale. FX was found to induce HBD2 dimerization as evidenced by the analysis of diffusion coefficients, (15)N relaxation, and nESI-MS measurements. The formation of FX-bridged HBD2 dimers exhibited features of a cooperative binding mechanism. In contrast, the complex with DSdp6 was found to be mostly monomeric.

Peptide fragments of a beta-defensin derivative with potent bactericidal activity.

Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.

Conformational preferences of linear beta-defensins are revealed by ion mobility-mass spectrometry.

In recent times there has been an enormous rise in resistance to synthetic antibiotics as well as an increase in the virulence of bacteria, the so-called "superbugs". This problem has catalyzed a search for novel molecules to fight bacteria, which in turn relies on a better understanding of the molecular basis of the immune response. Beta-defensins are a class of small, cationic, cysteine-rich antimicrobial peptides expressed by humans and other animals to act against incoming pathogens. As well as their antimicrobial properties, beta-defensins also act as chemokines, recruiting cells to the sites of infection. Here the relationship between the tertiary structures of beta-defensin analogs and their chemotactic activities has been investigated using ion mobility-mass spectrometry (IM-MS) and biochemical assays. A panel of derivatives of the murine beta-defensin Defb14 has been formed and the ability of these peptides to chemoattract the receptor CCR6 has been assessed in vitro. The derivatives can be divided into two groups, those with chemotactic activity equal to that of the unmodified parent peptide, and those whose chemotactic activity has been lost upon modification. Analysis by ion mobility-mass spectrometry reveals the conformational preferences of these peptides upon ionization from different solvents. Under denaturing conditions, the chemotactic peptides adopt more compact conformations in the gas-phase at higher charge states than those which are inactive. While the conditions of these experiments are not akin to the environment around the receptor in vivo, this technique provides an in vacuo method for distinguishing between the different chemotactic activities of beta-defensin derivatives.

Isoleucine/leucine2 is essential for chemoattractant activity of beta-defensin Defb14 through chemokine receptor 6.

Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis.

Scale-up of batch kinetic models.

The scale-up of batch kinetic models was studied by examining the kinetic fitting results of batch esterification reactions completed in 75 mL and 5 L reactors. Different temperatures, amounts of catalysts, and amounts of initial starting reagents were used to completely characterize the reaction. A custom written Matlab toolbox called GUIPRO was used to fit first-principles kinetic models directly to in-line NIR and Raman spectroscopic data. Second-order kinetic models provided calibration-free estimates of kinetic and thermodynamic reaction parameters, time dependent concentration profiles, and pure component spectra of reagents and product. The estimated kinetic and thermodynamic parameters showed good agreement between small-scale and large-scale reactions. The accuracy of pure component spectra estimates was validated by comparison to collected NIR and Raman pure component spectra. The model estimated product concentrations were also validated by comparison to concentrations measured by off-line GC analysis. Based on the good agreement between kinetic and thermodynamic parameters and comparison between actual and estimated concentration and spectral profiles, it was concluded that the scale-up of batch kinetic models was successful.