ABC transporters of the A subfamily: potential prognostic markers and candidates for antibody selection from phage-display libraries for therapeutic use in Ewing sarcoma

The prognostic value of ABC transporters in Ewing sarcoma is still poorly explored and controversial. We described for the first time the impact of various ABCs on Ewing sarcoma prognosis by assessment of their gene expression in two independent cohorts of patients. Unexpected associations with favourable outcomes were observed for two ABCs of the A-subfamily, ABCA6 and ABCA7, whereas no associations with the canonical multidrug ABC transporters were identified. The ABCs of the A-subfamily are involved in cholesterol/phospholipids transportation and efflux from cells. Our clinical data support the drug-efflux independent contribution to cancer progression of the ABCAs, which has been confirmed in PDX-derived cell lines. The impact of these ABCA transporters on tumor progression seems to be mediated by lowering intracellular cholesterol, supporting the role of these proteins in lipid transport. In addition, the gene expression of ABCA6 and ABCA7 is regulated by transcription factors which control lipid metabolism: ABCA6 was induced by the binding of FoxO1/FoxO3a to its promoter and repressed by IGF1R/Akt signaling, whereas the expression of ABCA7 was regulated by p53. The data point to ABCA6 and ABCA7 as potential prognostic markers in Ewing sarcoma and suggest the IGF1/ABCA/lipid axis as an intriguing therapeutic target. Agonist monoclonal antibodies towards ABCA6/7 or inhibitors of cholesterol biosynthesis, such as statins or aminobiphoshonates, may be investigated as therapeutic options in combination with chemotherapy. Considering that no monoclonal antibodies selectively targeting extracellular domains of ABCA6/7 are available, the second part of the project has been dedicated to the generation of human antibody phage-display libraries as tools for selecting monoclonal antibodies. A novel synthetic human antibody phage-display library has been designed, cloned and characterized. The library takes advantages of the high variability of a designed naïve repertoire to be a useful tool for isolating antibodies towards all potential antigens, including the ABCAs.

Neonatal outcomes following maternal SARS-CoV-2 infection in pregnancy - longitudinal follow-up and assessment of transplacental antibody transfer

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in pregnancy has been associated with multiple adverse pregnancy outcomes, including the risk of in utero mother-to-child transmission. Short- and long-term outcomes of SARS-CoV-2 exposed neonates and the extent to which maternal SARS-CoV-2 antibodies are transferred to neonates are still unclear. METHODS: Prospective observational study enrolling neonates born to mothers with SARS-CoV-2 infection in pregnancy, between April 2020-April 2021. Neonates were evaluated at birth and enrolled in a 12-month follow-up. SARS-CoV-2 IgG transplacental transfer ratio was assessed in mother-neonate dyads at birth. Maternal derived IgG were followed in infants until negativizing. RESULTS: Of 2745 neonates, 106 (3.9%) were delivered by mothers with SARS-CoV-2 infection in pregnancy. Seventy-six of 106 (71.7%) mothers were symptomatic. Median gestational age and mean birth weight were 39 weeks (range 25+5-41+4) and 3305 grams (SD 468). Six of 106 (6%) neonates were born preterm, without significant differences between asymptomatic and symptomatic mothers (P=0.67). No confirmed cases of in utero infection were detected. All infants had normal cerebral ultrasound and clinical evaluation at birth and during follow-up, until a median age of 7 months (range 5-12). All mothers and 96/106 (90.5%) neonates had detectable SARS-CoV-2 IgG at birth. Transplacental transfer ratio was higher following second trimester maternal infections (mean 0.940.46 versus 1.070.64 versus 0.750.44, P=0.039), but was not significantly different between asymptomatic and symptomatic women (P=0.20). IgG level in infants progressively decreased after birth: at 3 months 53% (51/96) and at four months 68% (63/96) had lost maternal antibodies respectively. The durability of maternal antibodies was positively correlated to the IgG level at birth (r=0.66; P<0.00001). CONCLUSIONS: Maternal SARS-CoV-2 infection was not associated with increased neonatal or long-term morbidity. No cases of confirmed in utero infection were detected. Efficient transplacental IgG transfer was found following second trimester maternal infections.

Research of predictive biomarkers to anti-CD22 antibody-drug conjugate treatment in B-cell acute lymphoblastic leukemia

Background: The treatment of B-cell acute lymphoblastic leukemia (B-ALL) has been enriched by novel agents targeting surface markers CD19 and CD22. Inotuzumab ozogamicin (INO) is a CD22-calicheamicin conjugated monoclonal antibody approved in the setting of relapse/refractory (R/R) B-ALL able to induce a high rate of deep responses, not durable over time. Aims: This study aims to identify predictive biomarkers to INO treatment in B- ALL by flow cytometric analysis of CD22 expression and gene expression profile. Materials and methods: Firstly, the impact on patient outcome in 30 R/R B-ALL patients of baseline CD22 expression in terms of CD22 blast percentage and CD22 fluorescent intensity (CD22-FI) was explored. Secondly, baseline gene expression profile of 18 R/R B-ALL patient samples was analyzed. For statistical analysis of differentially expressed genes (DEGs) patients were divided in non-responders (NR), defined as either INO-refractory or with duration of response (DoR) < 3 months, and responders (R). Gene expression results were analyzed with Ingenuity pathway analysis (IPA). Results: In our patient set higher CD22-FI, defined as higher quartiles (Q2-Q4), correlated with better patient outcome in terms of CR rate, OS and DoR, compared to lower CD22-FI (Q1). CD22 blast percentage was less able to discriminate patients’ outcome, although a trend for better outcome in patients with CD22 ≥ 90% could be appreciated. Concerning gene expression profile, 32 genes with corrected p value <0.05 and absolute FC ≥2 were differentially expressed in NR as compared to R. IPA upstream regulator and regulator effect analysis individuated the inhibition of tumor suppressor HIPK2 as causal upstream condition of the downregulation of 6 DEGs. Conclusions: CD22-FI integrates CD22-percentage on leukemic blasts for a more comprehensive target pre-treatment evaluation. Moreover, a unique pattern of gene expression signature based on HIPK2 downregulation was identified, providing important insights in mechanisms of resistance to INO.