T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

Direct quantification of biomolecular interaction by single-molecule force spectroscopy has evolved into a powerful tool for materials and life sciences. We introduce an approach in which the unbinding forces required to break intermolecular bonds are measured in a differential format by comparison with a known reference bond (here, a short DNA duplex). In addition to a marked increase in sensitivity and force resolution, which enabled us to resolve single-base pair mismatches, this concept allows for highly specific parallel assays. This option was exploited to overcome cross-reactions of antibodies in a protein biochip application.

Small ligands and their receptors are widely used noncovalent couplers in various biotech applications. One prominent example, the digoxigenin-antibody complex, was often used to immobilize samples for single molecule force measurements by optical trap or AFM. Here, we employed dynamic AFM spectroscopy to demonstrate that a single digoxigenin-antibody bond is likely to fail even under moderate loading rates. This effect potentially could lower the yield of measurements or even obscure the unbinding data of the sample by the rupture events of the coupler. Immobilization by multiple antibody-antigen bonds, therefore, is highly recommended. The analysis of our data revealed a pronounced loading rate dependence of the rupture force, which we analyzed based on the well-established BellEvans-model with two subsequent unbinding barriers. We could show that the first barrier has a width of Dx 1 = 1.15 nm and a spontaneous rate of k off1 = 0.015 s À1 and the second has a width of Dx 2 = 0.35 nm and a spontaneous rate of k off2 = 4.56 s À1. In the crossover region between the two regimes, we found a marked discrepancy between the predicted bond rupture probability density and the measured rupture force histograms, which we discuss as non-Markovian contribution to the unbinding process.

3 + complexes, making the scavenged iron available to the cells. We show that inactivation of the fes gene causes iron limitation on rich medium plates and a parallel SpoT-dependent increase of the ppGpp pool, as judged by the induction of the iron-regulated fiu :: lacZ fusion and the repression of the stringently controlled P1 rrnB :: lacZ fusion respectively. We further show, by direct ppGpp assays, that iron starvation in liquid medium produces a SpoT-dependent increase of the ppGpp pool, strongly suggesting a role for iron in the balance of the two activities of SpoT, synthesis and hydrolysis of (p)ppGpp. Finally, we present evidence that ppGpp exerts direct or indirect positive control on iron uptake, suggesting a simple homeostatic regulatory circuit: iron limitation leads to an increased ppGpp pool, which increases the expression of iron uptake genes, thereby alleviating the limitation.

In this paper, we measure the single chain elasticity of an oligomer single-stranded DNA (ssDNA) in both aqueous and nonaqueous, apolar liquid environments by AFM-based single molecule force spectroscopy. We find a marked deviation between the force-extension relations recorded for the two conditions. This difference is attributed to the additional energy required to break the H-bond-directed water bridges around the ssDNA chain in aqueous solutions, which are nonexistent in organic solvents. The results obtained in 8 M guanidine-HCl solution provide more evidence that water bridges around ssDNA originate the observed deviation. On the basis of the results obtained by an ab initio quantum mechanics calculation, a parameter-free freely rotating chain model is proposed. We find that this model is in perfect agreement with the experimental force-extension curve obtained in organic solvents, which further corroborates our assumption. On the basis of the experimental results, it is suggested that the weak H-bonding between ssDNA and water molecules may be a precondition for stable double-stranded DNA to exist in water.

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The significantly improved results on 3 outcome measures after 10 years suggest that MACT represents a suitable option in the treatment of local cartilage defects in the knee.

Patients treated with a MACI implant demonstrated significant clinical improvement and good quality repair tissue 5 years after surgery. The MACI procedure was shown to be a safe and effective treatment for symptomatic, traumatic chondral knee defects in this study.