1University Hospital Frankfurt, Department for Children and Adolescents, Frankfurt, Germany, 2Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany
Background: Pediatric patients with high-risk alveolar rhabdomyosarcoma (aRMS) above the age of 10 years cannot be cured by conventional therapies. Immune cells targeting ErbB2 with a chimeric antigen receptor (CAR) were recently considered for these patients. Cytokine-induced killer (CIK) cells already capable of natural killer (NK)-like anti-tumor capacity additionally redirected with an ErbB2 CAR may provide overall disease control in these high-risk tumors.
Methods: ErbB2-CAR modified CIK cells were generated from conventional CIK cells (WT-CIK) by lentiviral gene transduction on day 4 of culture. The codon-optimized CAR sequence consists of an IgG heavy-chain signal peptide, an ErbB2-specific antibody fragment scFv (FRP5) and a modified CD8α hinge region, as well as CD28 transmembrane and intracellular domains and a CD3ζ intracellular domain. 1x105 luciferase gene-transduced RH30 (aRMS) cells were engrafted in immunodeficient NOD/SCID/γc-(NSG) mice. Mice were randomly selected into 5 different treatment groups (DBPS on day +1, 2.5x106 WT-CIK or ErbB2 CAR-CIK cells on days +1 and +36, 2.5x106 WT-CIK or ErbB2 CAR-CIK cells on days +22 and +57). Mice were monitored by bioluminescence imaging (BLI) until day +100. Tumor engraftment and immune cell homing at tumor sites were analyzed by FACS, chimerism and immunohistochemistry analyses.
Results: Human RMS xenografts were established in all mice treated with DBPS only. Control-mice showed a median survival of 62 days. Human RMS was identified in all analyzed organs, with the highest tumor burden seen in livers of DBPS-treated mice.
Mice injected with WT or ErbB2-CAR CIK cells on days +1 and +36 showed a significant improved (p < 0.014 and p < 0.01) disease-free survival, respectively. Furthermore, no signs of tumor engraftment were shown by BLI in ErbB2 CAR-CIK cell treated mice while some of the mice treated with WT-CIK cells developed positive tumor signals between weeks 7 and 10. In 4 out of 6 (64%) WT- and in all (8 of 8, 100%) CAR-CIK cells treated mice no residual tumor cells were identified by PCR-based analysis. In contrast, tumor cells were detectable in all mice with delayed anti-tumor treatment applied on day +22 and +57. However, tumor growth was lower in these groups. Correspondingly, BLI showed delayed tumor engraftment in mice with WT- and even more with CAR-CIK cell treatment given on day 22. Treatment on day 22 resulted in a significantly improved survival of ErbB2-CAR CIK cell treated mice (p < 0.01), while survival was not improved after WT-CIK cell infusion (p > 0.07). Within all treatment groups, immune cells were detected by chimerism and FACS analyses. FACS analyses showed a significant increase of NK-like T cells (p < 0.01 and < 0.05, WT- and ErbB2-CAR CIK cells). Additionally, a higher, but not significant, amount of effector memory and stem cell memory T cells were detected.
Conclusions: These pre-clinical in vivo results indicate that ErbB2- CAR redirection of CIK cells improves both homing and NK-like cytotoxicity of CIK cells in the presence of ErbB2-positive tumors, implying that this therapy may represent a step forward in the treatment of patients with resistant, relapsed and advanced RMS.
Disclosure: Michael Merker, Juliane Wagner, Vida Meyer, Thomas Klingebiel, Winfried S. Wels and Eva Rettinger have nothing to declare. Peter Bader declares the following potential conflicts of interest: Novartis (consultancy: included expert testimony, speaker bureau, Honoraria), Medac (Research Funding, Patents and Royalties), Riemser (Research Funding), Neovii (Research Funding), Amgen (Honoraria).