Quantification and functional assessment of antigen(-) tumor cell variants

T-cell redirecting therapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies (bsAbs) creating an immunological synapse between target and effector cells are at the forefront of cancer immunotherapies. Selective pressure by these therapies can cause the enrichment of antigen negative (Ag)^low/^- tumor cell variants which are resistant to therapy and may drive clinical relapse, i.e. Ag escape. Ag^low/- cells generally exist in the tumor tissue prior to immunotherapies. Therefore, Ag escape is an integral part of tumor survival and poses a significant hurdle to overcome. Here we describe an approach used to quantify and functionally assess Ag^- tumor cells. This method enables high-throughput screening of single-cell biopsy or PBMC samples and allows for the assessment of their propensity to be targeted in a straightforward in vitro assay. Methods described here can be modulated and be incorporated to study samples derived from different anatomical locations and can be incorporated in a larger panel of flow cytometry for extensive phenotyping.