Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16.

BACKGROUND: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and the evasion of host immune responses. Currently, debulking surgery and platinum-based chemotherapy are the treatment cornerstones, although recurrence is common. As the clinical efficacy of immune checkpoint blockade is low, new immunotherapeutic strategies are needed. Chimeric antigen receptor (CAR) T cell therapy empowers patients' own T cells to fight and eradicate cancer, and has been tested against various targets in OC. A promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC. CA125 itself has not been tested as a possible CAR target. In this study, we examined the suitability of the CA125 as a target for CAR T cell therapy. METHODS: We tested a series of antibodies raised against the CA125 extracellular repeat domain of MUC16 and adapted them to the CAR format. Comparisons between these candidates, and against an existing CAR targeting the MUC16 ectodomain, identified K101 as having high potency and specificity. The K101CAR was subjected to further biochemical and functional tests, including examination of the effect of soluble CA125 on its activity. Finally, we used cell lines and advanced orthotopic patient-derived xenograft (PDX) models to validate, in vivo, the efficiency of our K101CAR construct. RESULTS: We observed a high efficacy of K101CAR T cells against cell lines and patient-derived tumors, in vitro and in vivo. We also demonstrated that K101CAR functionality was not impaired by the soluble antigen. Finally, in direct comparisons, K101CAR, which targets the CA125 extracellular repeat domains, was shown to have similar efficacy to the previously validated 4H11CAR, which targets the MUC16 ectodomain. CONCLUSIONS: Our in vitro and in vivo results, including PDX studies, demonstrate that the CA125 domain of MUC16 represents an excellent target for treating MUC16-positive malignancies.

ALPL-1 is a target for chimeric antigen receptor therapy in osteosarcoma.

Osteosarcoma (OS) remains a dismal malignancy in children and young adults, with poor outcome for metastatic and recurrent disease. Immunotherapies in OS are not as promising as in some other cancer types due to intra-tumor heterogeneity and considerable off-target expression of the potentially targetable proteins. Here we show that chimeric antigen receptor (CAR) T cells could successfully target an isoform of alkaline phosphatase, ALPL-1, which is highly and specifically expressed in primary and metastatic OS. The target recognition element of the second-generation CAR construct is based on two antibodies, previously shown to react against OS. T cells transduced with these CAR constructs mediate efficient and effective cytotoxicity against ALPL-positive cells in in vitro settings and in state-of-the-art in vivo orthotopic models of primary and metastatic OS, without unexpected toxicities against hematopoietic stem cells or healthy tissues. In summary, CAR-T cells targeting ALPL-1 show efficiency and specificity in treating OS in preclinical models, paving the path for clinical translation.

SJI 2020 special issue: A catalogue of Ovarian Cancer targets for CAR therapy.

Ovarian Cancer (OC) is currently difficult to cure, mainly due to its late detection and the advanced state of the disease at the time of diagnosis. Therefore, conventional treatments such as debulking surgery and combination chemotherapy are rarely able to control progression of the tumour, and relapses are frequent. Alternative therapies are currently being evaluated, including immunotherapy and advanced T cell-based therapy. In the present review, we will focus on a description of those Chimeric Antigen Receptors (CARs) that have been validated in the laboratory or are being tested in the clinic. Numerous target antigens have been defined due to the identification of OC biomarkers, and many are being used as CAR targets. We provide an exhaustive list of these constructs and their current status. Despite being innovative and efficient, the OC-specific CARs face a barrier to their clinical efficacy: the tumour microenvironment (TME). Indeed, effector cells expressing CARs have been shown to be severely inhibited, rendering the CAR T cells useless once at the tumour site. Herein, we give a thorough description of the highly immunosuppressive OC TME and present recent studies and innovations that have enabled CAR T cells to counteract this negative environment and to destroy tumours.

Anti-PML-RARα shRNA sensitises promyelocytic leukaemia cells to all-trans retinoic acid.

The PML-RARα fusion gene disrupts the retinoic acid differentiation signal in a range of leukaemia types, promoting proliferation. We designed a shRNA to target the fusion mRNA, and the shRNA expression cassette was delivered via lentiviral transduction. Delivery of this shRNA significantly down regulated the target mRNA, with effects also evident at the protein level. When combined with ATRA administration, this down regulation of the fusion gene strongly inhibited proliferation in the NB4 PML cell line.

Outer egg coats of the marsupial conceptus: secretion and protein composition.

Little is known of the composition of the outer egg coats. We aimed to quantify secretion during embryonic development, identify precursor secreting cells and investigate protein composition. The study was based on 259 egg coats and 14 reproductive tracts of 104 T. vulpecula undergoing natural and induced cycles and 341 coats from 35 Sminthopsis macroura undergoing natural cycles. Following PAGE, Western blotting, and amino acid sequencing of egg coats, the short peptide sequences obtained from T. vulpecula and S. macroura coats were found to be dissimilar to each other and to any known protein. However, in T. vulpecula, S. macroura coat polyclonal antibody cross-reacted with coat precursors, suggesting similar epitopes, and showed mucoid precursors in secretory cells in oviduct epithelia and shell precursors in glands in the utero-tubal junction and uterus. Immuno-electron microscopy located shell coat precursors in various previously unidentified cell types, including pre-ovulatory apoptotic cells, early post-ovulatory holocrine cells, and milk-producing cells, found at blastocyst stages. Ultrastructural and quantitative volumetric analysis of the intact shell coat suggested a second wave of secretion at the blastocyst stages in T. vulpecula. Despite differences in protein composition, it was concluded that marsupial egg coats are homologous to each other because of similarities in ultrastructure and time and location of secretion.