Targeting of low ALK antigen density neuroblastoma using AND logic-gate engineered CAR-T cells.

BACKGROUND AIMS: The targeting of solid cancers with chimeric antigen receptor (CAR) T cells faces many technological hurdles, including selection of optimal target antigens. Promising pre-clinical and clinical data of CAR T-cell activity have emerged from targeting surface antigens such as GD2 and B7H3 in childhood cancer neuroblastoma. Anaplastic lymphoma kinase (ALK) is expressed in a majority of neuroblastomas at low antigen density but is largely absent from healthy tissues. METHODS: To explore an alternate target antigen for neuroblastoma CAR T-cell therapy, the authors generated and screened a single-chain variable fragment library targeting ALK extracellular domain to make a panel of new anti-ALK CAR T-cell constructs. RESULTS: A lead novel CAR T-cell construct was capable of specific cytotoxicity against neuroblastoma cells expressing low levels of ALK, but with only weak cytokine and proliferative T-cell responses. To explore strategies for amplifying ALK CAR T cells, the authors generated a co-CAR approach in which T cells received signal 1 from a first-generation ALK construct and signal 2 from anti-B7H3 or GD2 chimeric co-stimulatory receptors. The co-CAR approach successfully demonstrated the ability to avoid targeting single-antigen-positive targets as a strategy for mitigating on-target off-tumor toxicity. CONCLUSIONS: These data provide further proof of concept for ALK as a neuroblastoma CAR T-cell target.

Identification of plasma complement C3 as a potential biomarker for neuroblastoma using a quantitative proteomic approach.

The majority of patients diagnosed with neuroblastoma present with aggressive disease. Improved detection of neuroblastoma cancer cells following initial therapy may help in stratifying patient outcome and monitoring for relapse. To identify potential plasma biomarkers, we utilised a liquid chromatography-tandem mass spectrometry-based proteomics approach to detect differentially-expressed proteins in serum from TH-MYCN mice. TH-MYCN mice carry multiple copies of the human MYCN oncogene in the germline and homozygous mice for the transgene develop neuroblastoma in a manner resembling the human disease. The abundance of plasma proteins was measured over the course of disease initiation and progression. A list of 86 candidate plasma biomarkers was generated. Pathway analysis identified significant association of these proteins with genes involved in the complement system. One candidate, complement C3 protein, was significantly enriched in the plasma of TH-MYCN(+/+) mice at both 4 and 6weeks of age, and was found to be elevated in a cohort of human neuroblastoma plasma samples, compared to healthy subjects. In conclusion, we have demonstrated the suitability of the TH-MYCN(+/+) mouse model of neuroblastoma for identification of novel disease biomarkers in humans, and have identified Complement C3 as a candidate plasma biomarker for measuring disease state in neuroblastoma patients. BIOLOGICAL SIGNIFICANCE: This study has utilised a unique murine model which develops neuroblastoma tumours that are biologically indistinguishable from human neuroblastoma. This animal model has effectively allowed the identification of plasma proteins which may serve as potential biomarkers of neuroblastoma. Furthermore, the label-free ion count quantitation technique which was used displays significant benefits as it is less labour intensive, feasible and accurate. We have been able to successfully validate this approach by confirming the differential abundance of two different plasma proteins. In addition, we have been able to confirm that the candidate biomarker Complement C3, is more abundant in the plasma of human neuroblastoma patient plasma samples when compared to healthy counterparts. Overall we have demonstrated that this approach can be potentially useful in the identification of biomarker candidates, and that further validation of the candidates may lead to the discovery of novel, clinically useful diagnostic tools in the detection of sub-clinical neuroblastoma.