PurposeFollowing two cases of neutralizing antibodies to epoetin alfa in an investigational clinical study, a small number of individual syringes of two drug product batches were found to contain unusually high levels of aggregation at the end of the clinical trial.MethodsWe undertook an extensive analytical approach to determine the root-cause of the increased aggregation in the affected batches.ResultsSoluble tungsten was found in the syringes, most likely derived from the pins used to manufacture the syringes. Spiking of epoetin alfa with sodium polytungstate or an extract of tungsten pins used to manufacture the syringes induced the formation of aggregates, both dimers that appeared to be covalently linked by disulphide bonds as well as higher-order aggregates. Sodium polytungstate had also a strong denaturing effect on the protein.ConclusionsWe propose tungsten-mediated unfolding and aggregation of epoetin alfa in pre-filled syringes as a potential root cause for increased immunogenicity. This finding may be more broadly applicable to this and other classes of therapeutic proteins.

Background Women have a markedly increased lifetime risk for cardiovascular morbidity and mortality following hypertensive disorders of pregnancy. Arterial stiffness is regarded as a target parameter for reducing cardiovascular risk and can be modified by lifestyle changes. Methods In a prospective, randomised, controlled interventional study, starting 6 weeks postpartum, the effect of nutritional intervention combined with an intensive 6-month cardiovascular exercise programme on arterial stiffness was investigated by means of pulse wave velocity (PWV) in 38 women with severe hypertensive disorder of pregnancy (preeclampsia with or without pre-existing hypertension and/or HELLP syndrome). A reference group was formed with postpartum women without pregnancy complications or known cardiovascular risk and the arterial stiffness was studied by means of PWV at the time of delivery. The PWV was measured in the intervention and control groups within a week after delivery and after 32 weeks (6 weeks + 6 months). A feasibility analysis was performed in addition. Results 29 of 38 women with severe hypertensive disorder of pregnancy and 38 postpartum women in the reference group were included in the analysis (intervention group n = 14; control group n = 15; reference group n = 38). Adherence to a) the nutritional counselling and b) the intensive cardiovascular exercise programme was 73% and 79% respectively. A clinically significant difference (d = 0.65) in pulse wave velocity between the intervention and control groups was found after 6 months (6.36 ± 0.76 vs. 7.33 ± 2.25 m/s; group × time: p = 0.632). The PWV of the intervention group corresponded to that of the reference group at the end of the study (6.36 ± 0.76 m/s vs. 6.5 ± 0.70; d = 0.19), while the results in the control group differed markedly from this (7.33 ± 2.25 m/s; d = 0.56). Conclusion The study documents the feasibility of lifestyle intervention with physical exercise after delivery (starting 6 weeks postpartum). The intervention showed a significant clinical effect by reducing arterial stiffness to the level of the reference group. Before this intervention can be included in the standard of care and prevention, follow-up studies must confirm these results and the medium-term effects on cardiovascular risk.

SummaryTemperature-dependent activation of bacterial virulence factors at 37°C is well investigated. The molecular mechanism underlying the expression of toxicity determinants at environmental temperatures, however, has not been characterized. The insecticidal activity of Yersinia enterocolitica strain W22703 requires the toxin complex subunit A (TcaA) encoded on the pathogenicity island Tc-PAIYe. Genes tcaA and tcaB encoding this subunit are maximally produced at low temperatures (10-20°C), but repressed at body temperature. Two further insecticidal genes, tcaC (subunit B) and tccC1 (subunit C), are silent at both temperatures. A novel LysR-type transcriptional regulator (LTTR), TcaR2, revealed to be autoregulated and essential for tcaA and tcaB expression in W22703. Expression of tcaR2 is negatively controlled by a second LTTR-like regulator, TcaR1. Gel mobility shift assays confirmed the interaction of TcaR2 with the tcaR2, tcaA and tcaB promoters. The activity of the tcaA promoter in heterologous hosts in the presence of TcaR2 excludes the requirement of additional, Yersinia-specific (co)factors for toxin gene expression. Overproduced TcaR2 protein is shown to be unstable at 37°C, whereas the mRNA of tcaA and tcaR2 is equally stable at low and high temperature. Thus, TcaR2 is a key player in the induction of insecticidal genes in Y. enterocolitica at low temperatures.

Purpose: While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1–positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti–4-1BB mAbs. Experimental Design: We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1–expressing murine MC38 cells to assess in vivo antitumoral activity. Results: PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti–PD-L1–resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. Conclusions: The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB–mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development. See related commentary by Shu et al., p. 3182

The central step in endothelin biosynthesis is site-specific cleavage of big endothelins by endothelin-converting enzymes (ECEs). ECE-1 is a membrane-bound metalloprotease, predominantly but not exclusively expressed in endothelial cells. ECE-1 is expressed in two mRNA isoforms, termed alpha and beta, which differ only in the 5'-terminal regions but are functionally very similar when expressed in vitro. The structure of the human ECE-1 gene suggests either alternative splicing or alternative promoters as underlying mechanisms of mRNA isoform expression. We have previously shown that the alpha-upstream region exerts promoter activity in endothelial cells. To clarify whether the 5'-untranslated region upstream of exon 3, which contains the beta-specific sequence, acts as an alternative transcriptional promoter, we sequenced and cloned 1,206 bp upstream of the beta-specific translation initiation codon in a luciferase reporter vector. After transfection, we detected strong promoter activity in primary cultured endothelial cells (HU-VECs, BAECs) but only marginal activity in the endothelial cell line ECV304 and in CHO cells. Maximal promoter activity was observed with the full-length construct, 1206 (136% of the SV40 promoter activity in BAECs). Transfection of serial deletion mutants indicated at least three major regulatory regions within the promoter. Our results are consistent with cell type-restricted action of the beta-promoter and, in conjunction with the previously reported transcriptional start sites, clearly prove the existence of an alternative beta-specific promoter located in intron 2 of the human ECE-1 gene.