Engineering CAR-T cells against neuroblastoma with synthetic notch receptors to increase tumor-specificity and endogenous immune activation

Research Area

CAR-T cell therapy

Project Summary

Adoptive T cell therapies are one of the most promising therapeutic approaches to cure cancer, but targeting solid cancers with chimeric antigen receptor (CAR-) T cells remains challenging. A lot of effort goes into lymphocyte engineering with the goal to improve CAR-T cell therapy by increasing CAR-T cell effector function and persistence in an immunosuppressive tumor microenvironment. With enhanced efficacy, the risk increases for on-target, off-tumor toxicity and tumor escape via antigen loss or downregulation. CRISPR/Cas9 geneediting technology makes elaborate lymphocyte engineering possible, but the risk rises that the recipient’s immune system could reject CAR-T cells the more elaborately they are engineered.

Furthermore, the large numbers of starting cells often required can be difficult or even impossible to obtain for pediatric patients and the time required to manufacture autologous CAR-T cell products is often too long to fight fast growing tumors. The common and devastating solid childhood tumor, neuroblastoma, will be used as a model cancer entity for our three-step approach to improve therapy without raising toxicity. Project A06 aims to (1) allow the use of potent CAR constructs by limiting on-target activity to only tumor cells expressing the neuroblastoma-specific antigens GD2 and L1CAM, (2) reduce risk for rejection and manufacture failure by developing an allogenic CAR-T cell product for these heavily pretreated children suffering from neuroblastoma and (3) prevent tumor escape by armoring CAR-T cells with cytokines to modulate the neuroblastoma tumor microenvironment. In Aim 1, we will use logic-gated CAR-T cells to enhance on-target activity against neuroblastoma cells and reduce toxicity to bystander tissues also expressing targeted antigens. The CAR initiating the cytotoxic re- sponse against the tumor cell and binding a second tumor-specific antigen is only produced when the synthetic Notch (synNotch) receptor binds to the first tumor-specific antigen. In Aim 2, we will generate CAR-T cells from allogeneic instead of autologous T cells to reduce the risk of donor T cell rejection, manufacture failure for severely pretreated children and overly long product generation times. In a step-wise approach, multiple gene- editing steps will start by knocking out B2M to reduce immunogenicity and end by inserting the CAR into the constant region of the TCR alpha chain (TRAC), thus silencing TCR expression and allowing the use of al- logeneic T cells without risking graft-versus-host disease. Aim 3 will armor L1CAM-specific CAR-T cells with synNotch-inducible IL18/IL12 expression to improve CAR-T cell fitness. What we learn about how to improve neuroblastoma-targeting CAR-T cells will render important steps forwarding CAR-T cell therapy for solid tu- mors in general and provide valuable information for the CRC and beyond.

Project-Related Publications

Künkele, A., Johnson, A.J., Rolczynski, L.S., Chang, C.A., Hoglund, V., Kelly-Spratt, K.S., Jensen, M.C et al: Functional Tuning of CARs Reveals Signaling Threshold above Which CD8+ CTL Antitumor Potency Is Attenu- ated due to Cell Fas-FasL-Dependent AICD. Cancer Immunol Res 2015; 3, 368-379

Künkele, A., Taraseviciute, A., Finn, L.S., Johnson, A.J., Berger, C., Finney, O., Chang, C.A., Rolczynski, L.S., Brown, C., Mgebroff, S., Berger, M., Park, J.R., Jensen, M.C.: Preclinical Assessment of CD171-Directed CAR T-cell Adoptive Therapy for Childhood Neuroblastoma: CE7 Epitope Target Safety and Product Manufacturing Feasibility. Clin Cancer Res 2017; 23, 466-477

Künkele, A., Brown, C., Beebe, A., Mgebroff, S., Johnson, A.J., Taraseviciute, A., Rolczynski, L.S., Chang, C.A., Finney, O., Park, J.R., Jensen, M.C.: Manufacture of chimeric antigen receptor T cells from mobilized cryo- preserved peripheral blood stem cell units depends on monocyte depletion. Biol Blood Marrow Transplant 2019; 25:223-232

Andersch, L., Radke, J., Klaus, A., Schwiebert, S., Winkler, A., Schumann, E., Grunewald, L., Zirngibl, F., Flem- mig, C., Jensen, M.C., Rossig, C., Joussen, A., Henssen, A., Eggert, A., Schulte, J.H., Künkele A: CD171- and GD2-specific CAR T cells potently target retinoblastoma cells in preclinical in vitro testing. BMC Cancer 2019; 19, 895

Ali, S., Toews, K., Schwiebert, S., Klaus, A., Winkler, A., Grunewald, L., Oevermann, L., Deubzer, H.E., Tüns, A., Jensen, M.C., Henssen, A.G., Eggert, A., Schulte, J.H., Schwich, E., Rebmann, V., Schramm, A., Künkele A: Tumor-derived extracellular vesicles impair CD171-specific CD4+ CAR T cell efficacy. Front Immunol 2020; 11:531

Toews, K., Grunewald, L., Schwiebert, S., Klaus, A., Winkler, A., Ali, S., Zirngibl, F., Astrahantseff, K., Wagner, D.L., Henssen, A.G., Deubzer, H.E., Schulte, J.H., Ochsenreither, S., Eggert, A., Künkele A: Central memory phenotype drives success of checkpoint inhibition in combination with CAR T cells. Mol Carcinog 2020; 59:p.724- 735