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Immuno-oncology has traditionally focused on conventional MHC-restricted αβ T cells. Yet, unconventional γδ T cells, which kill tumor cells in an MHC-unrestricted manner, display characteristics of effector activity and stemness without exhaustion and are nearly universally observed in human gynecologic malignancies, correlating with improved outcomes. These cells do not have a clear counterpart in mice but are also found in the healthy female reproductive tract. Interventions that modulate their in vivo activity, or cellular therapies utilizing γδ T cells as an allogeneic, “off-the-shelf” platform (e.g., for chimeric antigen receptor expression) hold significant potential against challenging tumors like ovarian cancer, which has been stubbornly resistant to the immune checkpoint inhibitors that change the landscape of other human tumors. Here, we discuss recent discoveries on the specific populations of γδ T cells that infiltrate human gynecologic cancers, their anti-tumor activity, and the prospect of redirecting their effector function against tumor cells to develop a new generation of immunotherapies that extends beyond the traditional αβ T cell-centric view of the field.

Summary: Understanding the heterogenicity of tumor-infiltraing lymphocyte (TIL) populations and the immunobiology in human cancer is a key to establish efficient immunotherapies. Here, we have established a protocol for the characterization of CD8+ TILs in tumors by single-cell RNA-seq paired to VDJ profiling and chromatin structure including dissociation of tumor biopsies. We have also provided guidance for subsequent fluorescence-activated cell sorting (FACS), single-cell encapsulation, bioinformatics analysis, and troubleshooting.For complete details on the use and execution of this protocol, please refer to Anadon et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Objectives: To develop chimeric antigen receptor (CAR) T cells targeting the olfactory receptor family 5 subfamily V member 1 (OR5V1) for the treatment of ovarian cancer. Methods: OR5V1 expression was quantitated by real-time quantitative polymerase chain reaction (RT-QPCR) and western blot analysis of 17 normal tissues and 75 ovarian cancers of various histologies. The sequence of an antibody fragment that recognizes the extracellular domain of OR5V1 was predicted from the amino acid sequence of OR5V1. Focusing on this antibody fragment, potential linear B cell epitopes were predicted. The peptides were biotinylated and tetramerized using fluorescently labeled streptavidin. Immortalized B cells derived from 10 human high-grade serous ovarian cancers reacting with the tetramer were fluorescence-activated cell sorting (FACS) sorted and subjected to 10x single cell B cell receptor sequencing. The sequences of the variable heavy and variable light chains of a dominant clone representing 54% of the population were identified and used to predict the sequence of a single-chain variable fragment (scFv) interacting with OR5V1. We designed the CAR construct using the olfactory receptor signal peptide, followed by the OR5V1 scFv, linked by a glycine/serine spacer, followed by a CD8a hinge, and transmembrane domains, and intracellular domains of co-stimulatory 4-1BB and CD3ζ. The construct was cloned into a retroviral vector, and human T cells were retrovirally transduced to express the OR5V1 CAR. The OR5V1 -specific cytotoxic killing of HeLa cells with endogenous expression of OR5V1 was demonstrated in vitro using luciferase assays and in vivo using an immunodeficient mouse model. Differences between the means of experimental groups were calculated using a two-tailed unpaired Student's t-test or two-way ANOVA with multiple comparisons. A p-value of <0.05 was considered statistically significant. Results: OR5V1 is expressed in ovarian cancers of multiple histologies, with expression in normal human tissues limited to the testis. There was no cytotoxic killing by OR5V1 CAR T cells of healthy human adipocytes, hepatocytes, or neurons. OR5V1 CAR T cells demonstrated cytotoxicity against HeLa cells, with nearly 100% specific killing in a dose-dependent manner, compared to mock transduced T cells (p=0.0002). Administration of OR5V1 CAR T cells in HeLa tumor-bearing mice significantly abrogated tumor growth, while mock transduced T cells were unable to prevent accelerated malignant progression (p<0.0001). Tumors treated with OR5V1 CAR T cells demonstrated not only reduced tumor volumes (p<0.0001) and weights (p=0.0023) but also significant central necrosis compared to those treated with mock transduced T cells. Conclusions: This study characterizes the expression pattern of OR5V1 in normal tissues and ovarian tumors of multiple histologies. We also demonstrate the therapeutic efficacy of targeting OR5V1- expressing tumors with CAR T cells re-directed against OR5V1, with a likely admissible toxicity profile. [ABSTRACT FROM AUTHOR]