Chimeric antigen receptors (CARs) link an antigen recognition domain to intracellular signaling domains to redirect T cell specificity and function. T cells expressing CARs with CD28/CD3ζ or 4-1BB/CD3ζ signaling domains are effective at treating refractory B cell malignancies but exhibit differences in effector function, clinical efficacy, and toxicity that are assumed to result from the activation of divergent signaling cascades. We analyzed stimulation-induced phosphorylation events in primary human CD8 CD28/CD3ζ and 4-1BB/CD3ζ CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3ζ CARs activated faster and larger-magnitude changes in protein phosphorylation, which correlated with an effector T cell-like phenotype and function. In contrast, 4-1BB/CD3ζ CAR T cells preferentially expressed T cell memory-associated genes and exhibited sustained antitumor activity against established tumors in vivo. Mutagenesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3ζ CAR signal intensity was partly related to constitutive association of Lck with this domain in CAR complexes. Our data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor and that signal strength is a key determinant of T cell fate. Thus, tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity.

Despite notably high response rates to B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells in multiple myeloma, few patients have a sustained, very good partial or complete response. This article presents a novel strategy to increase the efficacy of BCMA-directed CAR T-cell therapy and shows that γ-secretase inhibitors improve the efficacy of BCMA CAR T cells by increasing BCMA expression and reducing soluble BCMA.

Purpose This study examines cell-surface ROR1 expression in human tumors and normal tissues. ROR1 is considered a promising target for cancer therapy due to putative tumor-specific expression and multiple groups are developing antibodies and/or chimeric antigen receptor-modified T cells to target ROR1. On-target, off-tumor toxicity is a challenge for most non-mutated tumor antigens, however prior studies suggest that ROR1 is absent on most normal tissues. Experimental Design Our studies show that published antibodies lack sensitivity to detect endogenous levels of cell-surface ROR1 by immunohistochemistry (IHC) in FFPE tissues. We developed a ROR1-specific monoclonal antibody (mAb) targeting the carboxy-terminus of ROR1, and evaluated its specificity and sensitivity in IHC. Results The 6D4 mAb is a sensitive and specific reagent to detect cell-surface ROR1 by IHC. The data shows that ROR1 is homogenously expressed on a subset of ovarian cancer, triple negative breast cancer and lung adenocarcinomas. Contrary to previous findings, we found ROR1 is expressed on several normal tissues including parathyroid, pancreatic islets and regions of the esophagus, stomach and duodenum. The 6D4 mAb recognizes rhesus ROR1, and ROR1 expression was similar in human and macaque tissues suggesting that the macaque is a suitable model to evaluate safety of ROR1 targeted therapies. Conclusions ROR1 is a promising immunotherapeutic target in many epithelial tumors, however high cell-surface ROR1 expression in multiple normal tissues raises concerns for on-target off-tumor toxicities. Clinical translation of ROR1 targeted therapies warrants careful monitoring of toxicities to normal organs, and may require strategies to ensure patient safety.

Precise cell targeting is challenging because most mammalian cell types lack a single surface marker that distinguishes them from other cells. A solution would be to target cells based on specific combinations of proteins present on their surfaces. We design colocalization-dependent protein switches (Co-LOCKR) that perform AND, OR, and NOT Boolean logic operations. These switches activate through a conformational change only when all conditions are met, generating rapid, transcription-independent responses at single-cell resolution within complex cell populations. We implement AND gates to redirect T cell specificity against tumor cells expressing two surface antigens while avoiding off-target recognition of single-antigen cells, and 3-input switches that add NOT or OR logic to avoid or include cells expressing a third antigen. Thus, de novo designed proteins can perform computations on the surface of cells, integrating multiple distinct binding interactions into a single output.