Selection of a PD-1 blocking antibody from a novel fully human phage display library.

Programmed cell death protein 1 (PD-1) is an immunoregulatory target which is recognized by different monoclonal antibodies, approved for the therapy of multiple types of cancer. Different anti-PD-1 antibodies display different therapeutic properties and there is a pharmaceutical interest to generate and characterize novel anti-PD-1 antibodies. We screened multiple human antibody phage display libraries to target novel epitopes on the PD-1 surface and we discovered a unique and previously undescribed binding specificity (termed D12) from a new antibody library (termed AMG). The library featured antibody fragments in single-chain fragment variable (scFv) format, based on the IGHV3-23*03 (VH ) and IGKV1-39*01 (Vκ) genes. The D12 antibody was characterized by surface plasmon resonance (SPR), cross-reacted with the Cynomolgus monkey antigen and bound to primary human T cells, as shown by flow cytometry. The antibody blocked the PD-1/PD-L1 interaction in vitro with an EC50 value which was comparable to the one of nivolumab, a clinically approved antibody. The fine details of the interaction between D12 and PD-1 were elucidated by x-ray crystallography of the complex at a 3.5 Å resolution, revealing an unprecedented conformational change at the N-terminus of PD-1 following D12 binding, as well as partial overlap with the binding site for the cognate PD-L1 and PD-L2 ligands which prevents their binding. The results of the study suggest that the expansion of antibody library repertoires may facilitate the discovery of novel binding specificities with unique properties that hold promises for the modulation of PD-1 activity in vitro and in vivo.

ABCA6 affects the malignancy of Ewing sarcoma cells via cholesterol-guided inhibition of the IGF1R/AKT/MDM2 axis.

PURPOSE: The relevance of the subfamily A members of ATP-binding cassette (ABCA) transporters as biomarkers of risk and response is emerging in different tumors, but their mechanisms of action have only been partially defined. In this work, we investigated their role in Ewing sarcoma (EWS), a pediatric cancer with unmet clinical issues. METHODS: The expression of ABC members was evaluated by RT-qPCR in patients with localized EWS. The correlation with clinical outcome was established in different datasets using univariate and multivariate statistical methods. Functional studies were conducted in cell lines from patient-derived xenografts (PDXs) using gain- or loss-of-function approaches. The impact of intracellular cholesterol levels and cholesterol lowering drugs on malignant parameters was considered. RESULTS: We found that ABCA6, which is usually poorly expressed in EWS, when upregulated became a prognostic factor of a favorable outcome in patients. Mechanistically, high expression of ABCA6 impaired cell migration and increased cell chemosensitivity by diminishing the intracellular levels of cholesterol and by constitutive IGF1R/AKT/mTOR expression/activation. Accordingly, while exposure of cells to exogenous cholesterol increased AKT/mTOR activation, the cholesterol lowering drug simvastatin inhibited IGF1R/AKT/mTOR signaling and prevented Ser166 phosphorylation of MDM2. This, in turn, favored p53 activation and enhanced pro-apoptotic effects of doxorubicin. CONCLUSIONS: Our study reveals that ABCA6 acts as tumor suppressor in EWS cells via cholesterol-mediated inhibition of IGF1R/AKT/MDM2 signaling, which promotes the pro-apoptotic effects of doxorubicin and reduces cell migration. Our findings also support a role of ABCA6 as biomarker of EWS progression and sustains its assessment for a more rational use of statins as adjuvant drugs.

Serial Profiling of Circulating Tumor DNA Identifies Dynamic Evolution of Clinically Actionable Genomic Alterations in High-Risk Neuroblastoma.

Neuroblastoma evolution, heterogeneity, and resistance remain inadequately defined, suggesting a role for circulating tumor DNA (ctDNA) sequencing. To define the utility of ctDNA profiling in neuroblastoma, 167 blood samples from 48 high-risk patients were evaluated for ctDNA using comprehensive genomic profiling. At least one pathogenic genomic alteration was identified in 56% of samples and 73% of evaluable patients, including clinically actionable ALK and RAS-MAPK pathway variants. Fifteen patients received ALK inhibition (ALKi), and ctDNA data revealed dynamic genomic evolution under ALKi therapeutic pressure. Serial ctDNA profiling detected disease evolution in 15 of 16 patients with a recurrently identified variant-in some cases confirming disease progression prior to standard surveillance methods. Finally, ctDNA-defined ERRFI1 loss-of-function variants were validated in neuroblastoma cellular models, with the mutant proteins exhibiting loss of wild-type ERRFI1's tumor-suppressive functions. Taken together, ctDNA is prevalent in children with high-risk neuroblastoma and should be followed throughout neuroblastoma treatment. SIGNIFICANCE: ctDNA is prevalent in children with neuroblastoma. Serial ctDNA profiling in patients with neuroblastoma improves the detection of potentially clinically actionable and functionally relevant variants in cancer driver genes and delineates dynamic tumor evolution and disease progression beyond that of standard tumor sequencing and clinical surveillance practices. See related commentary by Deubzer et al., p. 2727. This article is highlighted in the In This Issue feature, p. 2711.

The ABC subfamily A transporters: Multifaceted players with incipient potentialities in cancer.

Overexpression of ATP-binding cassette (ABC) transporters is a cause of drug resistance in a plethora of tumors. More recent evidence indicates additional contribution of these transporters to other processes, such as tumor cell dissemination and metastasis, thereby extending their possible roles in tumor progression. While the role of some ABC transporters, such as ABCB1, ABCC1 and ABCG2, in multidrug resistance is well documented, the mechanisms by which ABC transporters affect the proliferation, differentiation, migration and invasion of cancer cells are still poorly defined and are frequently controversial. This review, summarizes recent advances that highlight the role of subfamily A members in cancer. Emerging evidence highlights the potential value of ABCA members as biomarkers of risk and response in different tumors, but information is disperse and very little is known about their possible mechanisms of action. The only clear evidence is that ABCA members are involved in lipid metabolism and homeostasis. In particular, the relationship between ABCA1 and cholesterol is becoming evident in different fields of biology, including cancer. In parallel, emerging findings indicate that cholesterol, the main component of cell membranes, can influence many physiological and pathological processes, including cell migration, cancer progression and metastasis. This review aims to link the dispersed knowledge regarding the relationship of ABCA members with lipid metabolism and cancer in an effort to stimulate and guide readers to areas that the writers consider to have significant impact and relevant potentialities.

Construction of Human Naïve Antibody Gene Libraries.

Size and variability often represent an obstacle in generating an effective antibody gene library for the detection of an abundant repertoire of antigens. Therefore, optimizing the construction of a large library is essential for the selection of high-affinity reactive fragments. Here, we report a highly efficient method for the construction of a human naïve antibody gene library for the selection of antibodies as single-chain variable fragments. This protocol is based on many different sets of oligonucleotide primers and multistep electroporation and ligation reactions.This advanced method can be adopted by any molecular biology laboratory to generate a naïve library for use in isolating single-chain fragment variables against specific targets.