Biology subjects

Villa, A.

Publications and source records attributed to Villa, A..

24 records · Page 2Linked to original sources

bioRxiv2019

The immunocytokine L19-TNF eradicates Sarcomas in combination with Chemotherapy or with immune check-point inhibitors

Antibody-cytokine fusion proteins (also called "immunocytokines") represent an emerging class of biopharmaceutical products, which are being considered for cancer immunotherapy. When used as single agents, pro-inflammatory immunocytokines are rarely capable of induce complete and durable cancer regression in mouse models and in patients. However, the combination treatment with conventional chemotherapy or with other immune-stimulatory agents typically increases the therapeutic efficacy of immunocytokines. In this article, we describe combination treatments of a tumor-targeting antibody-cytokine fusion protein based on the L19 antibody (specific to a splice isoform of fibronectin) fused to murine tumor necrosis factor (TNF) with standard chemotherapy (dacarbazine, trabectedin or melphalan) or with an immune check-point inhibitor (anti-PD-1) in a BALB/c derived immunocompetent murine model of sarcoma (WEHI-164). All combination treatments led to improved tumor remission compared to single agent treatments, suggesting that these combination partners may be suitable for further clinical development in sarcoma patients.

cancer biology↗

bioRxiv2019

Localized axolemma deformations suggest mechanoporation as axonal injury trigger

Traumatic brain injuries are a leading cause of morbidity and mortality worldwide. With almost 50% of traumatic brain injuries being related to axonal damage, understanding the nature of cellular level impairment is crucial. Experimental observations have so far led to the formulation of conflicting theories regarding the cellular primary injury mechanism. Disruption of the axolemma, or alternatively cytoskeletal damage has been suggested mainly as injury trigger. However, mechanoporation thresholds of generic membranes seem not to overlap with the axonal injury deformation range and microtubules appear too stiff and too weakly connected to undergo mechanical breaking. Here, we aim to shed a light on the mechanism of primary axonal injury, bridging finite element and molecular dynamics simulations. Despite the necessary level of approximation, our models can accurately describe the mechanical behavior of the unmyelinated axon and its membrane. More importantly, they give access to quantities that would be inaccessible with an experimental approach. We show that in a typical injury scenario, the axonal cortex sustains deformations large enough to entail pore formation in the adjoining lipid bilayer. The observed axonal deformation of 10-12% agree well with the thresholds proposed in the literature for axonal injury and, above all, allow us to provide quantitative evidences that do not exclude pore formation in the membrane as a result of trauma. Our findings bring to an increased knowledge of axonal injury mechanism that will have positive implications for the prevention and treatment of brain injuries.

bioengineering↗

bioRxiv2019

A novel fully-human potency-matched dual cytokine-antibody fusion protein targets carbonic anhydrase IX in renal cell carcinomas

Certain cytokines synergize in activating anti-cancer immunity at the site of disease and it may be desirable to generate biopharmaceutical agents, capable of simultaneous delivery of cytokine pairs to the tumor. In this article, we have described the cloning, expression and characterization of IL2-XE114-TNFmut, a dual-cytokine biopharmaceutical featuring the sequential fusion of interleukin-2 (IL2) with the XE114 antibody in scFv format and a tumor necrosis factor mutant (TNFmut). The fusion protein recognized the cognate antigen (carbonic anhydrase IX, a marker of hypoxia and of renal cell carcinoma) with high affinity and specificity. IL2-XE114-TNFmut formed a stable non-covalent homotrimeric structure, displayed cytokine activity in in vitro tests and preferentially localized to solid tumors in vivo. The product exhibited a partial growth inhibition of murine CT26 tumors transfected for carbonic anhydrase IX. When administered to Cynomolgus monkey as intravenous injection, IL2-XE114-TNFmut showed the expected plasma concentration of ~1500 ng/ml at early time points, indicating the absence of any in vivo trapping events, and a half-life of ~2 hours. IL2-XE114-TNFmut may thus be considered as a promising biopharmaceutical for the treatment of metastatic clear-cell renal cell carcinoma, since these tumors are known to be sensitive to IL2 and to TNF.\n\nContribution to the fieldThere is a growing interest in the antibody-based targeted delivery of pro-inflammatory cytokines for tumor therapy, which may be complementary or alternative to immune checkpoint inhibitors for immunotherapeutic applications. In this article, we have described a novel fusion protein, featuring antibody moieties specific to carbonic anhydrase IX, as well as interleukin-2 and tumor necrosis factor as pro-inflammatory cytokines, which combines the ability to recognize a tumor-associated antigen on the surface of renal cell carcinomas with the simultaneous display of two potent therapeutic payloads. The newly developed product (termed IL2-XE114-TNFmut) exhibited favorable biochemical characteristics, the ability to preferentially localize at the tumor site, a cancer growth inhibition activity and a suitable pharmacokinetic profile in Cynomolgus monkey. IL2-XE114-TNFmut may therefore represent an attractive candidate for the immunotherapy of renal cell carcinoma.

bioengineering↗

bioRxiv2019

Targeted enhancement of the therapeutic window of L19-TNF by transient and selective inhibition of RIPK1-signaling cascade

IntroductionCytokine-based products are gaining importance for cancer immunotherapy. L19-TNF is a clinical-stage antibody-cytokine fusion protein that selectively accumulates to tumors and displays potent anticancer activity in preclinical models. Here, we describe an innovative approach to transiently inhibit off-target toxicity of L19-TNF, while maintaining antitumor activity.\n\nMethodsGSK963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice for its ability to reduce acute toxicity associated with TNF signaling. The biological effects of L19-TNF on tumor cells, lymphocytes and tumor vessels were investigated with the aim to enable the administration of TNF doses, which would otherwise be lethal.\n\nResultsTransient inhibition of RIPK1 allowed to increase the maximal tolerated dose of L19-TNF. The protective effect of GSK963 did not affect the selective localization of the immunocytokine to tumors as evidenced by quantitative biodistribution analysis and allowed to reach high local TNF concentrations around tumor blood vessels, causing diffused vascular shutdown and hemorrhagic necrosis within the neoplastic mass.\n\nConclusionsThe selective inhibition of RIPK1 with small molecule inhibitors can be used as a pharmaceutical tool to transiently mask TNF activity and improve the therapeutic window of TNF-based biopharmaceuticals. Similar approaches may be applicable to other pro-inflammatory cytokines.

pharmacology and toxicology↗

bioRxiv2019

The antibody-based delivery of interleukin-12 to solid tumors boosts NK and CD8+ T cell activity and synergizes with immune check-point inhibitors

We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 anti-body in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively-spliced EDB domain of fibronectin), retained the activity of the parental cyto-kine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent anti-tumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with check-point blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anti-cancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.\n\nNOVELTY AND IMPACT STATEMENTIn this study, we describe the generation of a novel fusion protein consisting of murine inter-leukin-12 fused to the L19 antibody (specific to the EDB domain of fibronectin). L19-mIL12 revealed favourable tumor to organ ratios 24h after intravenous administration and it was able to cure 60% of CT26 tumor-bearing mice. From a clinical perspective, the rapid clearance from circulation should ease the administration to patients as infusions could be stopped upon onset of side-effects.

cancer biology↗

bioRxiv2019

Molecular basis of egg coat cross-linking sheds light on ZP1-associated female infertility

Interaction between sperm and the egg zona pellucida (ZP) is the first step of mammalian fertilization, and ZP component ZP1 is important for fertility by covalently cross-linking ZP filaments into a matrix. Like ZP4, a structurally-related subunit absent in the mouse, ZP1 is predicted to contain an N-terminal ZP-N domain of unknown function. Characterization of ZP1 proteins carrying mutations from infertile patients suggests that, unlike in the mouse, filament cross-linking by ZP1 is crucial for human ZP assembly. We map the function of ZP1 to its ZP-N1 domain and determine crystal structures of ZP-N1 homodimers from a chicken homolog of ZP1. These reveal that ZP filament cross-linking is highly plastic and can be modulated by ZP1 fucosylation and, potentially, zinc sparks. Moreover, we show that ZP4 ZP-N1 forms non-covalent homodimers in chicken but not human. Together, these data identify human ZP1 cross-links as a promising target for non-hormonal contraception.

biophysics↗