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P06.06 Enhancing trafficking and resistance to immunosuppression of synthetic agonistic receptor-transduced T cells in solid tumor models
  1. M Schwerdtfeger1,2,
  2. M Benmebarek1,
  3. F Märkl1,
  4. CH Karches1,
  5. A Öner1,
  6. M Geiger3,
  7. B Cadilha1,
  8. S Endres1,
  9. V Desiderio2,
  10. C Klein3 and
  11. S Kobold1,4,5
  1. 1Center of Integrated Protein Science Munich (CIPS-M) and Division of Clinical Pharmacology, Department of Medicine IV, Klinikum der Universität München, LMU Munich, Member of the German Center for Lung Research (DZL), Munich, Germany
  2. 2Department of Experimental Medicine, University of Campania ‘Luigi Vanvitelli’, Naples, Italy
  3. 3Roche Innovation Center Zurich, Schlieren, Switzerland
  4. 4German Center for Translational Cancer Research (DKTK), partner site Munich, Munich, Germany
  5. 5Einheit für Klinische Pharmakologie (EKLiP), Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), Munich, Germany


Background Chimeric antigen receptor therapy – although very efficacious in B cell malignancies – is facing many challenges which limit its success in solid tumors, e.g. on-target off-tumor toxicities, antigen heterogeneity, lack of T cell migration into tumors and an immunosuppressive tumor microenvironment. To better control on-target off-tumor effects and address antigen heterogeneity we developed a modular approach where we equipped T cells with a synthetic agonistic receptor (SAR). The SAR is only activated in the presence of a bispecific antibody (BiAb) cross-linking the receptor with a tumor-associated antigen. While we could show efficacy of the SAR platform in different models, limited infiltration and immune suppression still hamper its function. We could previously demonstrate that T cell infiltration can be enhanced by transduction with carefully chosen chemokine receptors like CXCR6, CCR4 and CCR8. At the same time, gene silencing of checkpoint molecules like PD-1 can make T cells more resistant to immunosuppression, thus we assumed that combining these approaches might generate a desired T cell product.

Materials and Methods All constructs had been generated previously by overlap-extension cloning. The EGFRvIII (E3) SAR consists of extracellular EGFRvIII, transmembrane CD28 and intracellular CD28 and CD3ζ. Human CXCR6-GFP, CCR4-GFP and CCR8-GFP are composed of the chemokine receptors fused to GFP via a 2A sequence. Primary human T cells were retrovirally transduced to stably express the SAR and chemokine receptors. We analyzed migration, cytotoxicity and activation of the single and double (E3 SAR and chemokine receptor) transduced T cells. In addition, PD-1 was knocked out using CRISPR-Cas9 and killing kinetics of target cells and T cell activation were assessed.

Results Co-transduction with chemokine receptors significantly increased migration of E3 SAR T cells to their respective ligand while lysis of target-expressing tumor cell and T cell activation in the presence of BiAb were not affected in vitro. Additionally knocking out PD-1 enhanced killing kinetics and activation of E3 SAR and E3 SAR + CXCR6-GFP transduced T cells compared to corresponding mock electroporated T cells.

Conclusions Using the controllable and modular SAR – BiAb platform SAR T cell activation can be limited by stopping BiAb dosing if adverse events occur. In addition, SAR T cells can be redirected to an alternative tumor-associated antigen by exchanging the BiAb in the case of antigen escape. Here we present add-ons to this approach for increased tumor infiltration and resistance to immunosuppression. Since migration is enhanced upon co-transduction with chemokine receptors and target cell lysis is accelerated upon PD-1 knockout in vitro these two additional modifications seem very promising options to further improve tumor control in vivo.

Disclosure Information M. Schwerdtfeger: None. M. Benmebarek: None. F. Märkl: None. C.H. Karches: A. Employment (full or part-time); Significant; Daiichi Sankyo Deutschland GmbH. A. Öner: None. M. Geiger: A. Employment (full or part-time); Significant; Roche. B. Cadilha: None. S. Endres: None. V. Desiderio: None. C. Klein: A. Employment (full or part-time); Significant; Roche. S. Kobold: None.

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