Evaluating Full Antitumor Activities of Monoclonal Antibody and Innate Immune Cells in Relapsed/Refractory Mantle Cell Lymphoma Using <i>Ex Vivo</i> Organoid Platform

Background MCL is a rare and incurable subtype of non-Hodgkin lymphoma. Resistance to therapies, including Bruton's tyrosine kinase inhibitor (BTKi), CD20-monoclonal antibody (rituximab), and CD19 CAR T cell-targeted therapies, is an urgent unmet clinical challenge in MCL. For relapsed/refractory (R/R) MCL, there is a few choices for effectively eradiating the MCL cells. Moreover, CAR T cell therapy is not suitable for some patients. Monoclonal antibody (mABb) is one of immunotherapies, the MCL cells with CD19 or CD20 positive, either anti-CD19 mAb Tafasitamab or Rituximab may be an effective therapy. NK cell-based immunotherapies become an attracting field of cancer treatment. Early clinical trials have shown promising outcomes with satisfactory product efficacy and safety. It is important to have a fast assay for validating NK cell activity before transplanting to patient. Patient-derived organoids (PDOs) possess the same gene mutational profile as the origin tumor tissue, and their accuracy in predicting drug sensitivity is typically greater than 80% when compared to other models. Due to the technical difficulty the development of PDOs of liquid tumors in particular MCL are very slow progress. We have established personalized MCL PDO culture system, which provides a simple way to measure, quantify, and monitor the kinetics of mAb mediated ADCC and ADCP antitumor activities as well as NK/macrophage killing activities in a mimicking in vivo microenvironment of MCL patient biopsies in real-time. Method For establishment of MCL PDO, the patient samples were resuspended in culture medium containing cytokine cocktails. The cell aggregates within 50% Matrigel were gently transferred into multiple-well plates as needed. The MCL PDOs were cultured in 3D Matrigel in the medium containing cytokine cocktail. The PBMCs or NK cells were labelled with green dye (CMFAD) prior to coculture with MCL PDOs. The treatment of the PDO/immune cells with mAbs routinely last for 72 hours. The cell viability and the cell populations in the PDO cocultures were determined using flow cytometry and automated cell counter. Results PDO model recapitulates at most the in vivo microenvironment of tumor cells, thereby we proposed that by adding normal immune cells such as NK cells/macrophages will reconstitute the immunocompetent MCL PDO platform which can be applied to rapidly validate anti-tumor activities of mAbs. To test this proposal, the PDOs were first generated from MCL patient apheresis. To distinguish immune cells from primary MCL cells the normal PBMCs which contain functional NK cells/macrophages or enriched NK cells were pre-labelled with CMFAD. These fluorescent immune cells were then cocultured with PDOs in the presence or absence of mAbs. The results indicated that addition of PBMCs alone significantly reduced the viable MCL cells in PDOs, indicating that the NK cells/macrophages in the PBMCs are activated by the cytokines such as IL-2 and IL-6 in the culture medium. In contrast, the single treatment of mAb against CD19 tafasitamab (Taf) resulted in mild decrease of viable MCL cells in the ex vivo assay. However, Combination of PBMCs and Taf greatly reduced the viable MCL cells, suggesting that antibody Fc-mediated activation of NK cells/macrophages in the PBMCs is attributed to the additional decrease in the viable MCL. In addition, we cocultured the purified NK cells with MCL cells (&amp;gt; 94% CD20+) in PDOs for 72 hours. The results demonstrated that NK cells alone did not significantly kill the MCL cells, but addition of monoclonal anti-CD20 antibody rituximab drastically lysed MCL cells in the PDO cocultures, indicating that the Fc domain of Rituximab activated NK cells leading to further reduction of viable MCL cells. Importantly, the ex vivo results of functional assays of mAbs are highly consistent with in vivo assessments using humanized mouse model. Our findings verified that MCL PDO platform can be used not only for evaluating antitumor activities of mAbs but also for assessing and predicting MCL patient's response of NK cell therapy in real time. Conclusion MCL PDO platform is a powerful and personized ex vivo assay for evaluating Fab neutralized activities of monoclonal antibody and its Fc-mediated ADCC /ADCP activities as well as for predicting response of R/R MCL patient to NK cell therapy.