Project

Utilization of Multiplex Fluorescent Immunohistochemistry to Assess CAR Efficacy in B-Cell Malignancies

Diffuse large B-cell lymphoma (DLBCL) is the most common type of Non-Hodgkin’s lymphoma. In DLBCL, immunologically ignorant and immature B-cells are overproduced, resulting in lymphoma. The primary method of treatment for DLBCL is R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone regimen). However, approximately 30 to 50% of patients will not respond to this treatment and experience relapsed/refractory DLBCL (r/r-DLBCL). Kymriah is a chimeric antigen receptor T-cell (CAR-T) therapy that has been approved for these patients if they do not respond to other secondary treatments. In Kymriah, patient T-cells are isolated from blood and transduced to contain a chimeric antigen receptor (CAR) that allows these T-cells to specifically target, bind, and eliminate CD19-expressing cells, a protein highly expressed on B-cells. Although Kymriah is approved for use by the FDA, it is still critical to determine the expression of persistent CAR-T in these patients. There are several testing platforms commonly used to assess CAR-T expression including flow cytometry and chromogenic immunohistochemistry (cIHC). While flow cytometry involves the use of fluorescently labelled cells in patient blood samples, cIHC is able to determine the expression of CAR-T cells on solid tumor sites. However, cIHC is limited in distinguishing multiple markers on the same cells. Multiplex fluorescent immunohistochemistry (mFIHC) serves as a platform to overcome the limitation of cIHC and is capable of performing staining with multiple markers. mFIHC enables the visualization of spatial interaction of multiple markers on a single slide. At Navigate Biopharma Services, a CAR-T/CD3/CD19 mFIHC assay was developed and validated for use on patient tumor biopsies in CAR-T related clinical trials. The assay will help gauge the status of tumor burden, and the effect of CAR-T by measuring CAR-T and CD19 interactions at the tumor site. The CAR-T/ CD3/CD19 mFIHC assay met the assay validation acceptance criteria and therefore is ready for use on clinical patient samples and patient selection. This assay will help determine the stability of patients treated with CAR-T therapy and offer unique insight as to how the transduced CAR-T interacts with DLBCL.

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