DSP107 combines inhibition of CD47/SIRPα axis with activation of 4-1BB to trigger anticancer immunity.

BACKGROUND: Treatment of Diffuse Large B Cell Lymphoma (DLBCL) patients with rituximab and the CHOP treatment regimen is associated with frequent intrinsic and acquired resistance. However, treatment with a CD47 monoclonal antibody in combination with rituximab yielded high objective response rates in patients with relapsed/refractory DLBCL in a phase I trial. Here, we report on a new bispecific and fully human fusion protein comprising the extracellular domains of SIRPα and 4-1BBL, termed DSP107, for the treatment of DLBCL. DSP107 blocks the CD47:SIRPα 'don't eat me' signaling axis on phagocytes and promotes innate anticancer immunity. At the same time, CD47-specific binding of DSP107 enables activation of the costimulatory receptor 4-1BB on activated T cells, thereby, augmenting anticancer T cell immunity. METHODS: Using macrophages, polymorphonuclear neutrophils (PMNs), and T cells of healthy donors and DLBCL patients, DSP107-mediated reactivation of immune cells against B cell lymphoma cell lines and primary patient-derived blasts was studied with phagocytosis assays, T cell activation and cytotoxicity assays. DSP107 anticancer activity was further evaluated in a DLBCL xenograft mouse model and safety was evaluated in cynomolgus monkey. RESULTS: Treatment with DSP107 alone or in combination with rituximab significantly increased macrophage- and PMN-mediated phagocytosis and trogocytosis, respectively, of DLBCL cell lines and primary patient-derived blasts. Further, prolonged treatment of in vitro macrophage/cancer cell co-cultures with DSP107 and rituximab decreased cancer cell number by up to 85%. DSP107 treatment activated 4-1BB-mediated costimulatory signaling by HT1080.4-1BB reporter cells, which was strictly dependent on the SIRPα-mediated binding of DSP107 to CD47. In mixed cultures with CD47-expressing cancer cells, DSP107 augmented T cell cytotoxicity in vitro in an effector-to-target ratio-dependent manner. In mice with established SUDHL6 xenografts, the treatment with human PBMCs and DSP107 strongly reduced tumor size compared to treatment with PBMCs alone and increased the number of tumor-infiltrated T cells. Finally, DSP107 had an excellent safety profile in cynomolgus monkeys. CONCLUSIONS: DSP107 effectively (re)activated innate and adaptive anticancer immune responses and may be of therapeutic use alone and in combination with rituximab for the treatment of DLBCL patients.

Positions under positive selection--key for selectivity and potency of scorpion alpha-toxins.

Alpha-neurotoxins target voltage-gated sodium channels (Na(v)s) and constitute an important component in the venom of Buthidae scorpions. These toxins are short polypeptides highly conserved in sequence and three-dimensional structure, and yet they differ greatly in activity and preference for insect and various mammalian Na(v)s. Despite extensive studies of the structure-function relationship of these toxins, only little is known about their evolution and phylogeny. Using a broad data set based on published sequences and rigorous cloning, we reconstructed a reliable phylogenetic tree of scorpion alpha-toxins and estimated the evolutionary forces involved in the diversification of their genes using maximum likelihood-based methods. Although the toxins are largely conserved, four positions were found to evolve under positive selection, of which two (10 and 18; numbered according to LqhalphaIT and Lqh2 from the Israeli yellow scorpion Leiurus quinquestriatus hebraeus) have been previously shown to affect toxin activity. The putative role of the other two positions (39 and 41) was analyzed by mutagenesis of Lqh2 and LqhalphaIT. Whereas substitution P41K in Lqh2 did not alter its activity, substitution K41P in LqhalphaIT significantly decreased the activity at insect and mammalian Na(v)s. Surprisingly, not only that substitution A39L in both toxins increased their activity by 10-fold but also LqhalphaIT(A39L) was active at the mammalian brain channel rNa(v)1.2a, which otherwise is hardly affected by LqhalphaIT, and Lqh2(A39L) was active at the insect channel, DmNa(v)1, which is almost insensitive to Lqh2. Thus, position 39 is involved not only in activity but also in toxin selectivity. Overall, this study describes evolutionary forces involved in the diversification of scorpion alpha-toxins, highlights the key role of positions under positive selection for selectivity and potency, and raises new questions as to the toxin-channel face of interaction.

Intron retention as a posttranscriptional regulatory mechanism of neurotoxin expression at early life stages of the starlet anemone Nematostella vectensis.

Sea anemones use an arsenal of peptide neurotoxins accumulated in special stinging cells (nematocytes) for defense and predation. Intriguingly, genomic analysis of Nematostella vectensis revealed only a single toxin, Nv1 (N. vectensis toxin 1), encoded by multiple extremely conserved genes. We examined the toxic potential of Nv1 and whether it is produced by the three developmental stages (embryo, planula, and polyp) of Nematostella. Nv1 was expressed in recombinant form and, similarly to Type I sea anemone toxins, inhibited the inactivation of voltage-gated sodium channels. However, in contrast to the other toxins, Nv1 revealed high specificity for insect over mammalian voltage-gated sodium channels. Transcript analysis indicated that multiple Nv1 loci are transcribed at all developmental stages of N. vectensis, whereas splicing of these transcripts is restricted to the polyp stage. This finding suggests that regulation of Nv1 synthesis is posttranscriptional and that the embryo and planula stages do not produce the Nv1 toxin. This rare phenomenon of intron retention at the early developmental stages is intriguing and raises the question as to the mechanism enabling such differential expression in sea anemones.

NMR analysis of interaction of LqhalphaIT scorpion toxin with a peptide corresponding to the D4/S3-S4 loop of insect para voltage-gated sodium channel.

Voltage-gated sodium channels (Navs) are large transmembrane proteins that initiate action potential in electrically excitable cells. This central role in the nervous system has made them a primary target for a large number of neurotoxins. Scorpion alpha-neurotoxins bind to Navs with high affinity and slow their inactivation, causing a prolonged action potential. Despite the similarity in their mode of action and three-dimensional structure, alpha-toxins exhibit great variations in selectivity toward insect and mammalian Navs, suggesting differences in the binding surfaces of the toxins and the channels. The scorpion alpha-toxin binding site, termed neurotoxin receptor site 3, has been shown to involve the extracellular S3-S4 loop in domain 4 of the alpha-subunit of voltage-gated sodium channels (D4/S3-S4). In this study, the binding site for peptides corresponding to the D4/S3-S4 loop of the para insect Nav was mapped on the highly insecticidal alpha-neurotoxin, LqhalphaIT, from the scorpion Leiurus quinquestriatus hebraeus, by following changes in the toxin amide 1H and 15N chemical shifts upon binding. This analysis suggests that the five-residue turn (residues LqK8-LqC12) of LqhalphaIT and those residues in its vicinity interact with the D4/S3-S4 loop of Nav. Residues LqR18, LqW38, and LqA39 could also form a patch contributing to the interaction with D4/S3-S4. Moreover, a new bioactive residue, LqV13, was identified as being important for Nav binding and specifically for the interaction with the D4/S3-S4 loop. The contribution of LqV13 to NaV binding was further verified by mutagenesis. Future studies involving other extracellular regions of Navs are required for further characterization of the structure of the LqhalphaIT-Navs binding site.

Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels.

Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Na(v)s (voltage-gated Na+ channels) like the structurally dissimilar scorpion alpha-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Na(v)s, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65+/-0.46 pmol/100 mg), to compete well with the site-3 toxin LqhalphaIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (K(i)=21.4+/-7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNa(v)1, but not that of mammalian Na(v)s expressed in Xenopus oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion beta-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNa(v)1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.

X-ray structure and mutagenesis of the scorpion depressant toxin LqhIT2 reveals key determinants crucial for activity and anti-insect selectivity.

Scorpion depressant beta-toxins show high preference for insect voltage-gated sodium channels (Na(v)s) and modulate their activation. Although their pharmacological and physiological effects were described, their three-dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatushebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 A resolution. The toxin molecule is composed of a conserved cysteine-stabilized alpha/beta-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na(v)s. The N-groove is flanked by Glu24 and Tyr28, which belong to the "pharmacophore" of scorpion beta-toxins, and by the side-chains of Trp53 and Asn58 that are important for receptor site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in "voltage-sensor trapping", the mode of action that typifies scorpion beta-toxins. The involvement of the N-groove in recognition of the receptor site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the receptor site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position.