Project 4
Development of NK cells as an alternative cell source for adoptive cell therapy
Prof. Dr. med. Sebastian Kobold
Abt. Klinische Pharmakologie, Klinikum der Universität München
Prof. Dr. med. Michael Hudecek
Medizinische Klinik II, UKW
Project Summary
Despite innovative combined therapeutic approaches consisting of chemoradiotherapy and immune checkpoint inhibitors, the successful treatment of advanced solid tumors continues to pose a major clinical challenge. There remains an urgent need to to develop new targeted immunotherapeutic approaches that target tumor-specific biomarkers and identify biomarkers that reliably predict which patients are most likely to respond to a particular therapy.
The Multhoff group has identified the stress-inducible molecular chaperone Hsp70 as a tumor-specific recognition structure on the cell surface of a large number of different, highly aggressive solid tumors. The group has also demosntrated that activation of NK cells with an Hsp70-derived peptide (TKD) in combination with low-dose IL-2 triggers cells to recognize and kill tumor cells expressing this membrane form of Hsp70. The safety and efficacy of this approach has been demonstrated in completed Phase I and Phase II clinical trials. In a pilot study, it has also been shown that a combination therapy consisting of membrane Hsp70 targeted NK cells and a second-line PD1 Immune Checkpoint Inhibitor therapy is well tolerated and significantly prolongs the survival.
Based on the sequence of a unique murine monoclonal antibody which recognizes membrane Hsp70, we have developed the first membrane Hsp70 CAR T cell which elicits cytolytic activity against membrane Hsp70 positive tumor cells in vitro. Using humanized sequences of this antibody, the proposed project will develop CAR expressing cytolytic NK and T cells targeting membrane Hsp70 using primary T and NK cells as well as GMP-compatible NK cell lines and test these in a range of in vitro and in vivo settings. The optimal CAR effector cell type will be further tested in combination with immune checkpoint inhibitors and developed for clinical use within the framework of this project.
It is known that the clinical translation of new therapies is more successful when there is a reliable biomarker which can identify and monitor potentially responsive patients. In this regard, tumor cells expressing membrane Hsp70 actively secrete microvesicle-associated Hsp70 into circulation, levels of which can be determined using the CE certified Hsp70-exo ELISA test developed by multimmune. Tumor patients have significantly increased levels of Hsp70 in the blood and it has been shown that the viable tumor mass correlates with the amount of Hsp70 in the blood. Levels can therfore be used as a surrogate measure of therapeutic response. For these reasons, the Hsp70-exo ELISA can be considered as a Companion Diagnostic for patient stratification and monitoring. The Hsp70-exo ELISA will be used to support the pre-clinical and clinical translational studies.
In summary, the innovative membrane Hsp70 targeted cell therapies that are to be developed in this project, combined with the availability of the Hsp70-exo Companion Diagnostic for patient startification and monitoring, have significant potential to deliver effective therapeutic options to the large proportion of patients having cancers expressing membrane Hsp70.
Publications
Yazdi M, Hasanzadeh Kafshgari M, Khademi Moghadam F, Zarezade V, Oellinger R, Khosravi M, Haas S, Hoch CC, Pockley AG, Wagner E, Wollenberg B, Multhoff G, Bashiri Dezfouli A. Crosstalk between NK cell receptors and tumor membrane Hsp70-derived peptide: A combined computational and experimental study. Adv Sci: 2305998, doi: 10.1002/advs.202305998, 2024. https://onlinelibrary.wiley.com/doi/full/10.1002/advs.202305998
Lennartz P, Thölke D, Bashiri Dezfouli A, Pilz M, Lobinger D, Messner V, Zanth H, Ainslie K, Kafshgari MH, Rammes G, Ballmann M, Schlegel M, Foulds GA, Pockley AG, Schmidt-Graf F, Multhoff G. Biomarkers in adult-type diffuse gliomas: Elevated levels of circulating vesicular heat shock protein 70 serve as a biomarker in grade 4 glioblastoma and increase nk cell frequencies in grade 3 glioma. Biomedicines 11(12): 3235, doi: 10.3390/biomedicines11123235, 2023. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10741018/
Xanthopoulos A, Samt AK, Guder C, Taylor N, Roberts E, Herf H, Messner V, Trill A, Holzmann KLK, Kiechle M, Seifert-Klauss V, Zschaeck S, Schatka I, Tauber R, Schmidt R, Enste K, Pockley AG, Lobinger D, Multhoff G. Hsp70-A universal biomarker for predicting therapeutic failure in human female cancers and a target for CTC isolation in advanced cancers. Biomedicines 11(8): 2276, doi: 10.3390/biomedicines 11082276, 2023. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10452093/
Safi S, Messner L, Kliebisch M, Eggert L, Ceylangil C, Lennartz P, Jefferies B, Klein H, Schirren M, Dommasch M, Lobinger D, Multhoff G. Circulating Hsp70 Levels and the immunophenotype of peripheral blood lymphocytes as potential biomarkers for advanced lung cancer and therapy failure after surgery. Biomolecules 13: 874, doi: 10.3390/biom13050874, 2023. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10216400/
Hantschel, M., K. Pfister, A. Jordan, R. Scholz, R. Andreesen, G. Schmitz, H. Schmetzer, W. Hiddemann, G. Multhoff. 2000. Hsp70 plasma membrane expression on primary tumor biopsy material and bone marrow of leukemic patients. Cell Stress Chaperones 5(5):438-42.
Multhoff, G., K. Pfister, M. Gehrmann, M. Hantschel, C. Gross, M. Hafner, W. Hiddemann. 2001. A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity. Cell Stress Chaperones 6(4):337-44.
Stangl, S., M. Gehrmann, J. Riegger, K. Kuhs, I. Riederer, W. Sievert, K. Hube, R. Mocikat, R. Dressel, E. Kremmer, A. G. Pockley, L. Friedrich, L. Vigh, A. Skerra, G. Multhoff. 2011. Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc Natl Acad Sci U S A 108(2):733-8.
Bashiri Dezfouli, A., M. Yazdi, M. R. Benmebarek, M. Schwab, S. Michaelides, A. Micciche, D. Geerts, S. Stangl, A. Klapproth, E. Wagner, S. Kobold, G. Multhoff. 2022. CAR T Targeting membrane-bound Hsp70 on tumor cells mimic Hsp70-primed NK cells. Front Immunol 13(883694).
Bashiri Dezfouli, A., M. Yazdi, A. G. Pockley, M. Khosravi, S. Kobold, E. Wagner, G. Multhoff. 2021. NK cells armed with Chimeric Antigen Receptors (CAR): Roadblocks to successful development. Cells 10(12):3390.
Krause, S. W., R. Gastpar, R. Andreesen, C. Gross, H. Ullrich, G. Thonigs, K. Pfister, and G. Multhoff. 2004. Treatment of colon and lung cancer patients with ex vivo heat shock protein 70-peptide- activated, autologous natural killer cells: a clinical phase I trial. Clin Cancer Res 10(11):3699-707.
Milani, V., S. Stangl, R. Issels, M. Gehrmann, B. Wagner, K. Hube, D. Mayr, W. Hiddemann, M. Molls, G. Multhoff. 2009. Anti-tumor activity of patient-derived NK cells after cell-based immunotherapy - a case report. J Transl Med 7:50.
Kokowski, K., S. Stangl, S. Seier, M. Hildebrandt, P. Vaupel, G. Multhoff. 2019. Radiochemotherapy combined with NK cell transfer followed by second-line PD-1 inhibition in a patient with NSCLC stage IIIb inducing long-term tumor control: a case study. Strahlenther Onkol 195(4):352-61.
Multhoff, G., S. Seier, S. Stangl, W. Sievert, M. Shevtsov, C. Werner, A. G. Pockley, C. Blankenstein, M. Hildebrandt, R. Offner, N. Ahrens, K. Kokowski, M. Hautmann, C. Rödel, R. Fietkau, D. Lubgan, R. Huber, H. Hautmann, T. Duell, M. Molls, H. Specht, B. Haller, M. Devecka, A. Sauter, S. E. Combs. 2020. Targeted natural killer cell-based adoptive immunotherapy for the treatment of patients with NSCLC after radiochemotherapy: A randomized phase II clinical trial. Clin Cancer Res 26(20):5368-79.
Shevtsov, M., E. Pitkin, A. Ischenko, S. Stangl, W. Khachatryan, O. Galibin, S. Edmond, D. Lobinger, G. Multhoff. 2019. Ex vivo hsp70-activated NK cells in combination with PD-1 inhibition significantly increase overall survival in preclinical models of glioblastoma and lung cancer. Front Immunol 10:454.
Breuninger, S., J. Ertl, C. Bayer, C. Knape, S. Günther, I. Regel, F. Rödel, U. Gaipl, J. Thorsteinsdottir, L. Giannitrapani, A. Dickinson, G. Multhoff. 2015. Quantitative analysis of liposomal heat shock protein 70 in the blood of tumor patients using a novel lipHsp70 ELISA. J Clin Cellular Immunol 5:4- 10.
Gunther, S., C. Ostheimer, S. Stangl, H. M. Specht, P. Mozes, M. Jesinghaus, D. Vordermark, S. E. Combs, F. Peltz, M. P. Jung, G. Multhoff. 2015. Correlation of Hsp70 serum levels with gross tumor volume and composition of lymphocyte subpopulations in patients with squamous cell and adeno non-small cell lung cancer. Front Immunol 6:556.
Werner, C., S. Stangl, L. Salvermoser, M. Schwab, M. Shevtsov, A. Xanthopoulos, F. Wang, A. Bashiri Dezfouli, D. Tholke, C. Ostheimer, D. Medenwald, M. Windberg, M. Bache, M. Schlapschy, A. Skerra, G. Multhoff. 2021. Hsp70 in liquid biopsies - A tumor-specific biomarker for detection and response monitoring in cancer. Cancers (Basel) 13(15):3706.
Seier S, A. Bashiri Dezfouli, P. Lennartz, A.G. Pockley, H. Klein, G. Multhoff. 2022. Elevated levels of circulating Hsp70 and an increased prevalence of CD94+/CD69+ NK cells is predictive for advanced stage Non-Small Cell Lung Cancer. Cancers (Basel). Nov 21;14(22):5701.
Xanthopoulos A, AK Samt, C. Guder, N. Taylor, E Roberts, H. Herf, V. Messner, A. Trill, K.L.K. Holzmann, M. Kiechle, V. Seifert-Klauss, S. Zschaeck, I. Schatka, R. Tauber, R. Schmidt, K. Enste, A.G. Pockley, D. Lobinger, G. Multhoff. 2023. Hsp70-A universal biomarker for predicting therapeutic failure in human female cancers and a target for CTC isolation in advanced cancers. Biomedicines. ;11(8):2276.