Highlights d IPH5201 and IPH5301 block cell-borne and soluble CD39 and CD73, respectively d IPH5201 maintains immunogenic extracellular ATP d When used in combination with chemotherapy, IPH5201 promotes antitumor immunity d Targeting CD39 and CD73 synergistically promotes cancer patient T cell activation

Highlights d NK cell engagers are multifunctional Abs targeting tumor antigens, NKp46 and CD16 d NKCEs bring tumor cells and NK cells together and trigger tumor-cell destruction d NKCEs can show killing potency superior to therapeutic Abs in vitro and in vivo d NKCEs may improve benefit-risk profile for cancer treatment compared to BiTEs

Asf1 is a conserved histone chaperone implicated in nucleosome assembly, transcriptional silencing, and the cellular response to DNA damage. We solved the NMR solution structure of the Nterminal functional domain of the human Asf1a isoform, and we identified by NMR chemical shift mapping a surface of Asf1a that binds the C-terminal helix of histone H3. This binding surface forms a highly conserved hydrophobic groove surrounded by charged residues. Mutations within this binding site decreased the affinity of Asf1a for the histone H3͞H4 complex in vitro, and the same mutations in the homologous yeast protein led to transcriptional silencing defects, DNA damage sensitivity, and thermosensitive growth. We have thus obtained direct experimental evidence of the mode of binding between a histone and one of its chaperones and genetic data suggesting that this interaction is important in both the DNA damage response and transcriptional silencing.Asf1 histone chaperone ͉ chromatin ͉ DNA damage ͉ NMR chemical shift mapping ͉ nucleosome assembly D NA in eukaryotic cells is packaged as nucleosome core particles containing Ϸ145 bp of DNA wrapped around an octamer comprised of two copies each of histones H2A, H2B, H3, and H4 (1). Assembly of histones into nucleosomes is a tightly orchestrated process (2, 3). Asf1 is a highly conserved histone chaperone that has been linked to both nucleosome assembly and disassembly (4-7). Asf1 interacts with two functional classes of protein: chromatin components, including histone H3 (8), the Hir proteins (9, 10), and the second subunit of CAF-I (5, 11, 12), and checkpoint kinases, including the Rad53 checkpoint kinase in budding yeast (13,14) and the Tousled-like kinases in metazoans (15). The function of most of these interactions has not been defined. However, a Hir binding region of Asf1 was implicated in telomeric silencing but not required for resistance to genotoxic stress (16). Further work is necessary to determine the functional role of the remaining interactions and, in particular, for defining which Asf1 partners are required for the DNA damage response and for optimal cell growth. In this work, we present the solution structure of the functional Nterminal domain of human Asf1a, and we identify its histone H3 binding site. We show that Asf1 mutants severely defective in histone H3͞H4 binding are incompetent in silencing and in providing resistance to DNA damage. MethodsProtein Production. pETM30 allowed the production of recombinant (His) 6 -GST-Tev site-fusion proteins in Escherichia coli strain BL21 gold (DE3). Unlabeled and uniformly labeled proteins were obtained as described in ref. 17. After Tev cleavage, the 15 N-labeled-H3 (122-135) peptide was further purified by reverse-phase chromatography. The NMR buffer was described in ref.17. An unlabeled peptide spanning the 122-133 sequence of histone H3 was obtained by chemical synthesis (Epytop, Nîmes, France). The protein concentrations were precisely measured by amino acid analysis.NMR Structure Determination and Binding Expe...