IgG light chain-independent secretion of heavy chain dimers: consequence for therapeutic antibody production and design

Rodent monoclonal antibodies with specificity towards important biological targets are developed for therapeutic use by a process of humanisation. This process involves the creation of molecules, which retain the specificity of the rodent antibody but contain predominantly human coding sequence. Here we show that some humanised heavy chains can fold, form dimers and be secreted even in the absence of light chain. Quality control of recombinant antibody assembly in vivo is thought to rely upon folding of the heavy chain CH1 domain. This domain acts as a switch for secretion, only folding upon interaction with the light chain CL domain. We show that the secreted heavy-chain dimers contain folded CH1 domains and contribute to the heterogeneity of antibody species secreted during the expression of therapeutic antibodies. This subversion of the normal quality control process is dependent upon the heavy chain variable domain, is prevalent with engineered antibodies and can occur when only the Fab fragments are expressed. This discovery will impact on the efficient production of both humanised antibodies as well as the design of novel antibody formats

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system

Increasing 3D Supramolecular Order by Decreasing Molecular Order. A Comparative Study of Helical Assemblies of Dendronized Nonchlorinated and Tetrachlorinated Perylene Bisimides

A nonplanar, twisted, and flexible tetrachlorinated perylene bisimide (Cl4PBI) was functionalized with two AB3 minidendrons containing hydrogenated or semifluorinated dodecyl groups. The hydrogenated dendron was attached to the imide groups of Cl4PBI via m = 0, 1, and 2 methylenic units, whereas the dendron containing semifluorinated groups was attached via m = 3 or a di(ethylene oxide) linker (m = 2EO). The supramolecular structures of these compounds, determined by a combination of differential scanning calorimetry, X-ray diffraction, and solid-state NMR, were compared with those of nonchlorinated planar and rigid PBI reported previously, which demonstrated the thermodynamically controlled formation of 2D periodic arrays at high temperatures and 3D arrays at low temperatures. The molecularly less ordered Cl4PBI containing hydrogenated dendrons self-organize into exclusively 3D crystalline periodic arrays under thermodynamic control for m = 0 and 2, while the more highly molecularly ordered PBI produced less stable and ordered 3D crystals and also 2D assemblies. This induction of a higher degree of 3D order in supramolecular assemblies of the less well-ordered molecular building blocks was unanticipated. The semifluorinated dendronized Cl4PBI with m = 3 formed a 2D columnar hexagonal array under kinetic control, whereas the compound with m = 2EO formed an unusual 2D honeycomb-like hexagonal phase under thermodynamic control. These Cl4PBI compounds provide a new route to stable crystalline assemblies via thermodynamic control at lower temperatures than previously obtained with PBI, thus generating 3D order in an accessible range of temperature of interest for structural analysis and for technological applications

Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D

Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53

Stability of biocontrol products carrying Candida sake CPA-1 in starch derivatives as a function of water activity

[EN] The preservation and shelf-life of formulations of the biocontrol agent Candida sake CPA-1 and starch derivatives as a function of water activity (aW) were studied in terms of the physical stability of the products and cell viability. Formulations of biocontrol products (BCPs), based on combinations of potato starch and pregelatinised potato starch (F1 and F2) or maltodextrines (MD) (F3) containing cell protectants, were obtained by fluidised-bed drying. The carriers and the formulated products were stored at 20°C under different aW conditions. The water sorption and water plasticization behaviour of the different products were analysed through the water sorption isotherms and glass transition temperatures (Tg). Likewise, the viability of C. sake over time was determined as a function of the aW. The solubility of the products was also assessed. Although formulations stored at 20°C and low aW (≤ 0.33) exhibited a better shelf-life, a significant decrease in cell survival ratio after 180 storage days was observed. Cold storage (5°C) was required to better maintain the cell viability, thus prolonging the shelf-life of BCPs. Formulations containing MD were the most effective at preserving cell viability and also exhibited the highest water solubility. All the formulations were physically stable at ambient temperature; therefore, the cell stability is the critical point at which to establish both the aW levels and temperature during storage. Packaging the product using high water vapour barrier material and under cold storage would be necessary to ensure a high number of viable cells and an effective and competitive BCPThe authors are grateful to the Spanish Government for the financial support from the national project RTA2012-00067-C02 (Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria, Spain and FEDER funds) and to the Conselleria d'Educacio of the Generalitat Valenciana, (Spain) for A. Marin's PhD grant.Marín-Gozalbo, A.; Atarés Huerta, LM.; Cháfer Nácher, MT.; Chiralt, A. (2017). Stability of biocontrol products carrying Candida sake CPA-1 in starch derivatives as a function of water activity. Biocontrol Science and Technology. 27(2):268-287. https://doi.org/10.1080/09583157.2017.1279587S26828727

Concomitant CIS on TURBT does not impact oncological outcomes in patients treated with neoadjuvant or induction chemotherapy followed by radical cystectomy

© Springer-Verlag GmbH Germany, part of Springer Nature 2018Background: Cisplatin-based neoadjuvant chemotherapy (NAC) for muscle invasive bladder cancer improves all-cause and cancer specific survival. We aimed to evaluate whether the detection of carcinoma in situ (CIS) at the time of initial transurethral resection of bladder tumor (TURBT) has an oncological impact on the response to NAC prior to radical cystectomy. Patients and methods: Patients were identified retrospectively from 19 centers who received at least three cycles of NAC or induction chemotherapy for cT2-T4aN0-3M0 urothelial carcinoma of the bladder followed by radical cystectomy between 2000 and 2013. The primary and secondary outcomes were pathological response and overall survival, respectively. Multivariable analysis was performed to determine the independent predictive value of CIS on these outcomes. Results: Of 1213 patients included in the analysis, 21.8% had concomitant CIS. Baseline clinical and pathologic characteristics of the ‘CIS’ versus ‘no-CIS’ groups were similar. The pathological response did not differ between the two arms when response was defined as pT0N0 (17.9% with CIS vs 21.9% without CIS; p = 0.16) which may indicate that patients with CIS may be less sensitive to NAC or ≤ pT1N0 (42.8% with CIS vs 37.8% without CIS; p = 0.15). On Cox regression model for overall survival for the cN0 cohort, the presence of CIS was not associated with survival (HR 0.86 (95% CI 0.63–1.18; p = 0.35). The presence of LVI (HR 1.41, 95% CI 1.01–1.96; p = 0.04), hydronephrosis (HR 1.63, 95% CI 1.23–2.16; p = 0.001) and use of chemotherapy other than ddMVAC (HR 0.57, 95% CI 0.34–0.94; p = 0.03) were associated with shorter overall survival. For the whole cohort, the presence of CIS was also not associated with survival (HR 1.05 (95% CI 0.82–1.35; p = 0.70). Conclusion: In this multicenter, real-world cohort, CIS status at TURBT did not affect pathologic response to neoadjuvant or induction chemotherapy. This study is limited by its retrospective nature as well as variability in chemotherapy regimens and surveillance regimens.Peer reviewedFinal Accepted Versio

Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8(+) T cells

Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8(+) T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4(+)CD25(+) regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8(+) T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The γ (C) cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of γ (C) cytokine–responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8(+) T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells

Liver-Targeting of Interferon-Alpha with Tissue-Specific Domain Antibodies

PMCID: PMC3581439This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Molecular mode-coupling theory applied to a liquid of diatomic molecules

We study the molecular mode coupling theory for a liquid of diatomic molecules. The equations for the critical tensorial nonergodicity parameters ${\bf F}_{ll'}^m(q)$ and the critical amplitudes of the $\beta$ - relaxation ${\bf H}_{ll'}^m(q)$ are solved up to a cut off $l_{co}$ = 2 without any further approximations. Here $l,m$ are indices of spherical harmonics. Contrary to previous studies, where additional approximations were applied, we find in agreement with simulations, that all molecular degrees of freedom vitrify at a single temperature $T_c$. The theoretical results for the non ergodicity parameters and the critical amplitudes are compared with those from simulations. The qualitative agreement is good for all molecular degrees of freedom. To study the influence of the cut off on the non ergodicity parameter, we also calculate the non ergodicity parameters for an upper cut off $l_{co}=4$. In addition we also propose a new method for the calculation of the critical nonergodicity parameterComment: 27 pages, 17 figure