P-02.03 Artificial targets for functional characterization of CAR T cells

<h3>Background</h3> Immunotherapies using chimeric antigen receptor (CAR) T cells have proven efficacy against certain cancers. Ongoing research aims to extend their therapeutic potential to additional indications. Functional characterization is paramount throughout development. E.g. in pre-clinical phase, for selection of CAR constructs and manufacturing optimization; in clinical development, for product characterization; during clinical use, for quality control. Typically, CAR T cells are characterized functionally after co-culture with target cell lines. However, using target cell lines poses limitations to the establishment of standardized assays. These include variability due to cell fitness, metabolic state, or genetic drift. Moreover, it is work and time intensive, and introduce significant background due to allogeneic responses. <h3>Materials and Methods</h3> We generated a synthetic bead-based system (‘artificial target’) for activation of CAR T cells, which is composed of silica particles conjugated to anti-Biotin antibody via Michael addition. CAR T cell activation is achieved by simultaneously engaging CARs (via idiotype antibodies) and a costimulatory receptor, such as CD28, through the antibodies loaded on artificial targets. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture. As a cell free platform, artificial targets were tested in comparison to cellular targets during the co-culture with CAR T cells evaluating multiple parameters of CAR T cell functionality. <h3>Results</h3> Artificial targets were able to specifically induce CAR T cell effector functions comparable to cellular targets, as measured by intracellular cytokine expression, secretion of multiple effector molecules, and degranulation of cytotoxic mediators. Moreover, artificial targets specifically induced upregulation of activation markers and CAR T cell proliferation. <h3>Conclusions</h3> We propose artificial targets represent a significant improvement towards standardization of functional assays for screening, characterization and quality control of CAR T cell products, from early development to clinical products. <h3>References</h3> Wang X, Tokarew NJA, Borgelt N, Siemer R, Melo CC, Langer C, Kasampalidis I, Ogusuku IEY, Cathomen T, Gessner I, Dose C, Fauerbach JA, Richter A, Evaristo C. Artificial Targets: a versatile cell-free platform to characterize CAR T cell function in vitro. <i>Front. Immunol.</i> 2024;<b>15</b>:1254162. <b>Borgelt:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer Melo:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer Melo Langer:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer Melo Langer Kasampalidis:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer Melo Langer Kasampalidis Ogusuku:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer Melo Langer Kasampalidis Ogusuku Cathomen:</b> A. Employment (full or part-time); Significant; University Freiburg. <b>Borgelt Wang Tokarew Siemer Melo Langer Kasampalidis Ogusuku Cathomen Gessner:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer Melo Langer Kasampalidis Ogusuku Cathomen Gessner Dose:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer Melo Langer Kasampalidis Ogusuku Cathomen Gessner Dose Fauerbach:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer Melo Langer Kasampalidis Ogusuku Cathomen Gessner Dose Fauerbach Richter:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>Borgelt Wang Tokarew Siemer Melo Langer Kasampalidis Ogusuku Cathomen Gessner Dose Fauerbach Richter Evaristo:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG.