Computational design of novel peptidomimetic inhibitors of cadherin homophilic interactions.

We report a first set of peptidomimetic ligands mimicking the adhesive interface identified by recent crystallographic structures of E- and N-cadherin. Compounds 2 and 3 inhibit adhesion of epithelial ovarian cancer (EOC) cells with improved efficacy compared to the ADH-1 peptide, a N-cadherin antagonist that is in early clinical trials in EOC patients.

Validity of Anti-PSMA ScFvD2B as a Theranostic Tool: A Narrative-Focused Review.

Prostate cancer (PCa) is the second leading cause of cancer among men, and its diagnosis and adequate staging are fundamental. Among the biomarkers identified in recent years for PCa management, prostate-specific-membrane-antigen (PSMA), physiologically expressed at a low level on healthy prostate and in other normal tissues and highly overexpressed in PCa, represents a reliable marker ideal for imaging and therapy. The development of anti-PSMA antibodies, such as D2B, demonstrated slow clearance of intact antibodies compared with fragments resulting in low tumor-to-blood ratios; however, the modular structural and functional nature of antibodies allowed the generation of smaller fragments, such as scFvs. In this review of the anti-PSMA antibody fragment scFvD2B, we combined further characterization of its biomolecular and tissue cross-reactivity characteristics with a comprehensive summary of what has already been performed in preclinical models to evaluate imaging and therapeutic activities. A molecular dynamics study was performed, and ScFvD2B occupied a limited conformational space, characterized by low-energy conformational basins, confirming the high stability of the protein structure. In the cross-reactivity study, the weak/absent immunoreactivity in non-tumor tissues was comparable to the PSMA expression reported in the literature. Biodistribution studies and therapeutic treatments were conducted in different animal models obtained by subcutaneous or locoregional injection of PSMA-positive-versus-negative xenografts. The maximum tumor uptake was observed for 123I(SPECT), 124I(PET), and optical imaging, which avoids kidney accumulation (compared with radiometals) and leads to an optimal tumor-to-kidney and tumor-to-background ratios. Regarding its possible use in therapy, experimental data suggested a strong and specific antitumor activity, in vitro and in vivo, obtained using CAR-T or NK-92/CAR cells expressing scFvD2B. Based on presented/reviewed data, we consider that scFvD2B, due to its versatility and robustness, seems to: (i) overcome some problems observed in other studied scFvs, very often relatively unstable and prone to form aggregates; (ii) have sufficient tumor-to-background ratios for targeting and imaging PSMA-expressing cancer; (iii) significantly redirect immune killing cells to PSMA-positive tumors when inserted in second-generation CAR-T or NK-92/CAR cells. These data suggest that our product can be considered the right reagent to fill the gap that still exists in PCa diagnosis and treatment.

SARS-CoV-2 Serology Monitoring of a Cancer Center Staff in the Pandemic Most Infected Italian Region.

Since the beginning of the COVID-19 outbreak, Cancer Centers adopted specific procedures both to protect patients and to monitor the possible spread of SARS-CoV-2 among healthcare personnel (HCP). In April 2020 at Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, one of the three oncologic hubs in Lombardy where the Health Regional Authorities referred all the cancer patients of the region, we implemented a prospective longitudinal study aimed at monitoring the serological response to SARS-Cov-2 in HCP. One hundred and ten HCP answered a questionnaire and were screened by nasopharyngeal swabs as well as for IgM/IgG levels; seropositive HCPs were further screened every 40-45 days using SARS-CoV-2-specific serology. We identified a fraction of HCP with long-term anti-SARS-CoV-2 antibody responses, though negative for viral RNA, and thus probably able to safely approach fragile cancer patients. Monitoring asymptomatic HCP might provide useful information to organize the healthcare service in a Cancer Center, while waiting for the effectiveness of the active immunization by SARS-CoV-2 vaccines, which will provide protection from infection.

Conversion of murine antibodies to human antibodies and their optimization for ovarian cancer therapy targeted to the folate receptor.

We previously developed murine and chimeric antibodies against a specific epithelial ovarian carcinoma (EOC) marker, named folate receptor (FR), and promising results were obtained in phase II trials. More recently, we successfully generated a completely human Fab fragment, C4, by conversion of one of the murine anti-FR antibodies to human antibody using phage display and guided selection. However, subsequent efforts to obtain C4 in a dimer format, which seems especially desirable for EOC locoregional treatment, resulted in a highly heterogeneous product upon natural dimerization and in a very poor production yield upon chemical dimerization by a non-hydrolyzable linker to a di-Fab-maleimide (DFM). We therefore designed, constructed and characterized a large Fab dual combinatorial human antibody phage display library obtained from EOC patients and potentially biased toward an anti-tumor response in an effort to obtain new anti-FR human antibodies suitable for therapy. Using this library and guiding the selection on FR-expressing cells with murine/human antibody chains, we generated four new human anti-FR antibody (AFRA) Fab fragments, one of which was genetically and chemically manipulated to obtain a chemical dimer, designated AFRA-DFM5.3, with high yield production and the capability for purification scaled-up to clinical grade. Overall affinity of AFRA-DFM5.3 was in the 2-digit nanomolar range, and immunohistochemistry indicated that the reagent recognized the FR expressed on EOC samples. (131)I-AFRA-DFM5.3 showed high immunoreactivity, in vitro stability and integrity, and specifically accumulated only in FR-expressing tumors in subcutaneous preclinical in vivo models. Overall, our studies demonstrate the successful conversion of murine to completely human anti-FR antibodies through the combined use of antibody phage display libraries biased toward an anti-tumor response, guided selection and chain shuffling, and point to the suitability of AFRA5.3 for future clinical application in ovarian cancer.

Sensitization of p53-mutated epithelial ovarian cancer to CD95-mediated apoptosis is synergistically induced by cisplatin pretreatment.

Epithelial ovarian carcinoma (EOC) remains a highly lethal malignancy. Despite the progress in surgical and therapeutic strategies, resistance to chemotherapy is still a major concern. Cytotoxic therapies mediate killing of cancer cells by activating the intrinsic mitochondrial apoptotic pathway, and p53 status is a key factor in determining the efficacy of apoptotic signaling. The extrinsic (CD95) death receptor-dependent signaling pathway also contributes to the efficacy of cancer therapy. We previously showed that EOC are generally resistant to CD95-dependent apoptosis. In p53 wild-type EOC tumors, CD95-mediated apoptosis is impaired at the receptor level by the long form of cellular FLICE-inhibitory protein, whereas this mechanism does not account for resistance in tumors with mutated p53 (p53mu). In the present study, we examined both intrinsic and death receptor-dependent apoptotic signaling in p53mu OVCAR3 EOC cell line, showing that these cells are less susceptible to cisplatin treatment as compared with p53 wild-type EOC cells and also resist CD95-mediated apoptosis due to inefficient formation of the death-inducing signaling complex and weak mitochondrial signal amplification. However, pretreatment of OVCAR3 cells with clinically relevant cisplatin concentrations significantly improved receptor-dependent apoptotic signaling by up-modulating CD95 receptor expression and increasing death-inducing signaling complex formation efficiency. The synergy of cisplatin pretreatment and CD95 triggering in inducing cell death was also shown in p53mu tumor cells derived from ascitic fluid of advanced-stage EOC patients. These findings support the effectiveness of a combined therapeutic treatment able to sensitize cancer cells to apoptosis even when p53 is functionally inactivated.

Full preclinical validation of the 123I-labeled anti-PSMA antibody fragment ScFvD2B for prostate cancer imaging.

PURPOSE: In the context of prostate cancer (PCa) imaging, the aim of this study was to optimize (in vitro) the specificity and assess preclinically (in vivo) the tumor targeting properties of the 123I-scFvD2B antibody specific for prostate-specific membrane antigen (PSMA). EXPERIMENTAL DESIGN: The 123I-labeling conditions of the antibody fragment scFvD2B, produced in an eukaryotic system under GMP-compliant conditions, were optimized and assessed for purity and immunoreactivity. The specificity and potency of tumor uptake were tested in three preclinical in vivo models of subcutaneously xenografted human tumors expressing different levels of PSMA (LNCaP, naturally expressing PSMA; PC3-PIP and LS174T-PSMA, transfected with PSMA) or PC3 and LS174T, as negative controls, to assess the clearance, biodistribution and imaging potential of 123I-scFvD2B. RESULTS: The set conditions of production and radiolabeling yielded a reagent suitable for human delivery thanks to the purity of the formulation and the high immunoreactivity. In all preclinical models 123I-scFvD2B showed specific targeting only to PSMA-positive tumors with the final specific activity ranging up to 1500 MBq/mg. Despite different levels of PSMA expression, biodistribution analyses and SPECT/CT imaging demonstrated similar results and maximal signal-to-background ratios 24 hours after injection. CONCLUSIONS: Due to its in vitro and in vivo properties, 123I-scFvD2B could be a promising tool for the early diagnosis of PCa, and may represent a molecular imaging option to monitor disease progression and assist in the clinical management of PCa patients.

CD95-mediated apoptosis is impaired at receptor level by cellular FLICE-inhibitory protein (long form) in wild-type p53 human ovarian carcinoma.

PURPOSE: Ovarian carcinoma is a highly lethal malignancy that often becomes resistant to chemotherapy. Alterations in apoptotic signals and p53 status contribute to drug resistance, and CD95-mediated apoptosis is also deficient in resistant cells. We analyzed the mechanism of resistance to CD95-mediated apoptosis in ovarian carcinoma cell lines differing in p53 status. EXPERIMENTAL DESIGN: CD95-mediated apoptosis was induced by agonistic anti-CD95 antibody, and the apoptotic cascade was monitored with biochemical and functional assays. RESULTS: CD95-mediated apoptosis was blocked in human ovarian cancer cells. In cell lines with wild-type p53, treatment with the protein synthesis inhibitor cycloheximide (CHX) together with anti-CD95 overcame the resistance, suggesting the presence of a labile inhibiting protein. Indeed, the labile protein cellular FLICE-inhibitory protein long form (c-FLIP(L)) was found to block caspase-8 recruitment to the death-inducing signaling complex (DISC), and sensitization of cells by CHX was due to c-FLIP(L) down-modulation at the DISC level. Down-regulation of c-FLIP(L) with antisense oligonucleotides increased CD95-mediated apoptosis as in cells sensitized by CHX, demonstrating the direct involvement of c-FLIP(L) in apoptosis resistance. Removal of c-FLIP(L) block at DISC level allowed full activation of the mitochondrial pathway and, eventually, apoptosis in wild-type p53 cells, whereas in cells with mutated p53, c-FLIP(L) involvement in CD95-mediated apoptosis resistance appeared to be irrelevant. Immunohistochemical analysis of an ovarian tumor tissue array revealed c-FLIP(L) expression in samples with no p53 accumulation (P = 0.034), and a significant (P = 0.037) inverse relationship between c-FLIP(L) and p53 expression levels was also observed in 27 epithelial ovarian cancer specimens with known p53 status. CONCLUSION: The inhibitory protein c-FLIP(L) is involved in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild-type p53.

Effect of radiochemical modification on biodistribution of scFvD2B antibody fragment recognising prostate specific membrane antigen.

Antibody-based reagents represent a promising strategy as clinical diagnostic tools. Prostate cancer (PCa) is the second-leading cause of death in males in the Western population. There is a presently unmet need for accurate diagnostic tool to localize and define the extent of both primary PCa and occult recurrent disease. One of the most suitable targets for PCa is the prostate-specific membrane antigen (PSMA) recognised by the monoclonal antibody D2B that we re-shaped into the single chain Fv (scFv format). Aim of this study was to evaluate in preclinical in vivo models the target specificity of scFvD2B after labelling with different radionuclides. (111)In radiolabelling was performed via the chelator Bz-NOTA, and (131)I radioiodination was performed using iodogen. The potential for molecular imaging and the biological behaviour of the radiolabelled scFvD2B were evaluated in mice bearing two subcutaneous PCa isogenic cell lines that differed only in PSMA expression. Biodistribution studies were performed at 3, 9, 15 and 24h after injection to determine the optimal imaging time point. A significant kidney accumulation, as percentage of injected dose of tissue (%ID/g), was observed for (111)In-scFvD2B at 3h after injection (45%ID/g) and it was maintained up to 24h (26%ID/g). By contrast, kidney accumulation of (131)I-scFvD2B was only marginally (0.3%ID/g at 24h). At the optimal time point defined between 15h and 24h, regardless of the radionuclide used, the scFvD2B was able to localize significantly better in the PSMA expressing tumours compared to the negative control; with (131)I-scFvD2B yielding a significantly better target/background ratio compared to (111)In-scFvD2B. These data suggest that, besides antigen specificity, chemical modification may affect antibody fragment biodistribution.

Targeting FR-expressing cells in ovarian cancer with Fab-functionalized nanoparticles: a full study to provide the proof of principle from in vitro to in vivo.

Efficient targeting in tumor therapies is still an open issue: systemic biodistribution and poor specific accumulation of drugs weaken efficacy of treatments. Engineered nanoparticles are expected to bring benefits by allowing specific delivery of drug to the tumor or acting themselves as localized therapeutic agents. In this study we have targeted epithelial ovarian cancer with inorganic nanoparticles conjugated to a human antibody fragment against the folate receptor over-expressed on cancer cells. The conjugation approach is generally applicable. Indeed several types of nanoparticles (either magnetic or fluorescent) were engineered with the fragment, and their biological activity was preserved as demonstrated by biochemical methods in vitro. In vivo studies with mice bearing orthotopic and subcutaneous tumors were performed. Elemental and histological analyses showed that the conjugated magnetic nanoparticles accumulated specifically and were retained at tumor sites longer than the non-conjugated nanoparticles.

Role of tyrosyl-DNA phosphodiesterase 1 and inter-players in regulation of tumor cell sensitivity to topoisomerase I inhibition.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) plays a unique function as it catalyzes the repair of topoisomerase I-mediated DNA damage. Thus, ovarian carcinoma cell lines exhibiting increased TDP1 levels and resistance to the topoisomerase I poisons campthotecins were used to clarify the role of this enzyme. The camptothecin gimatecan was employed as a tool to inhibit topoisomerase I because it produces a persistent damage. The resistant sublines displayed an increased capability to repair drug-induced single-strand breaks and a reduced amount of drug-induced double-strand breaks, which was enhanced following TDP1 silencing. In loss of function studies using U2-OS cells, we found that TDP1 knockdown did not produce a change in sensitivity to camptothecin, whereas co-silencing of other pathways cooperating with TDP1 in cell response to topoisomerase I poisons indicated that XRCC1 and BRCA1 were major regulators of sensitivity. No change in cellular sensitivity was observed when TDP1 was silenced concomitantly to RAD17, which participates in the stabilization of collapsed replication forks. The expression of dominant-negative PARP1 in cells with reduced expression of TDP1 due to a constitutively expressed TDP1 targeting microRNA did not modulate cell sensitivity to camptothecin. Mild resistance to gimatecan was observed in cells over-expressing TDP1, a feature associated with decreased levels of drug-induced single-strand breaks. In conclusion, since TDP1 alone can account for mild levels of camptothecin resistance, repair of topoisomerase I-mediated DNA damage likely occurs through redundant pathways mainly implicating BRCA1 and XRCC1, but not RAD17 and PARP1. These findings may be relevant to define novel therapeutic strategies.