Tuning T-Cell Receptor Affinity to Optimize Clinical Risk-Benefit When Targeting Alpha-Fetoprotein-Positive Liver Cancer.

Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha-fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer-specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity-optimized T-cell receptor (TCR) with specificity to AFP/HLA-A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA-A*02-restricted AFP158-166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X-scan) and testing TCR-transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR-transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T-cell immunotherapy.

LMO4 is an essential cofactor in the Snail2-mediated epithelial-to-mesenchymal transition of neuroblastoma and neural crest cells.

Neuroblastoma is an embryonic tumor derived from cells of the neural crest. Taking advantage of a newly developed neural crest lineage tracer and based on the hypothesis that the molecular mechanisms that mediate neural crest delamination are also likely to be involved in the spread of neuroblastoma, we were able to identify genes that are active both in neural crest development and neuroblastoma tumor formation. A subsequent search of the neuroblastoma gene server for human orthologues of genes differentially expressed in the chick embryo neural crest screen retrieved the LIM domain only protein 4 (LMO4), which was expressed in both cell types analyzed. Functional experiments in these two model systems revealed that LMO4 activity is required for neuroblastoma cell invasion and neural crest delamination. Moreover, we identified LMO4 as an essential cofactor in Snail2-mediated cadherin repression and in the epithelial-to-mesenchymal transition of both neural crest and neuroblastoma cells. Together, our results suggest that the association of high levels of LMO4 with aggressive neuroblastomas is dependent on LMO4 regulation of cadherin expression and hence, tumor invasiveness.

Canonical BMP7 activity is required for the generation of discrete neuronal populations in the dorsal spinal cord.

BMP activity is essential for many steps of neural development, including the initial role in neural induction and the control of progenitor identities along the dorsal-ventral axis of the neural tube. Taking advantage of chick in ovo electroporation, we show a novel role for BMP7 at the time of neurogenesis initiation in the spinal cord. Using in vivo loss-of-function experiments, we show that BMP7 activity is required for the generation of three discrete subpopulations of dorsal interneurons: dI1-dI3-dI5. Analysis of the BMP7 mouse mutant shows the conservation of this activity in mammals. Furthermore, this BMP7 activity appears to be mediated by the canonical Smad pathway, as we demonstrate that Smad1 and Smad5 activities are similarly required for the generation of dI1-dI3-dI5. Moreover, we show that this role is independent of the patterned expression of progenitor proteins in the dorsal spinal cord, but depends on the BMP/Smad regulation of specific proneural proteins, thus narrowing this BMP7 activity to the time of neurogenesis. Together, these data establish a novel role for BMP7 in primary neurogenesis, the process by which a neural progenitor exits the cell cycle and enters the terminal differentiation pathway.