CD3xCD19 DART molecule treatment induces non-apoptotic killing and is efficient against high-risk chemotherapy and venetoclax-resistant chronic lymphocytic leukemia cells

Background Bispecific antibodies are promising new therapeutics in B cell malignancies. Whether they lead to potent T cell activation despite described T cell dysfunction in chronic lymphocytic leukemia (CLL), and are able to effectively target high-risk or venetoclax-resistant samples, is currently unknown. Methods CD19+ cell lines or primary (high-risk) CLL were cocultured in vitro with healthy donor (HD) or CLL-derived T cells in the presence of a CD3xCD19 dual affinity retargeting molecule (CD3xCD19 DART). Cell cytotoxicity, T cell activation, proliferation and effector molecule production were analyzed using flow cytometry. Results Here, we report that a bispecific CD3xCD19 DART mediates efficient killing by HD T cells of CD19+ cell-lines and primary CLL cells, regardless of immunoglobulin heavy chain variable region (IGHV) mutational status TP53 status or chemotherapy, ibrutinib or venetoclax sensitivity. Whereas TCR stimulation of CLL-derived T cells resulted in dysfunctional T cell activation and proliferation, treatment with CD3xCD19 DART led to a similar activation profile in CLL-derived and HD-derived T cells. Consistently, co-culture of CLL derived T cells with JeKo-1 or CLL cells in the presence of CD3xCD19 DART resulted in significant cytotoxicity by both CD4+ and CD8+ T cells. On stimulation of CLL cells with CD40L, CLL cells become resistant to the specific inhibitor of anti-apoptotic Bcl-2 protein venetoclax, due to upregulation of Bcl-2 family members such as Bcl-XL. Nevertheless, CD40L stimulated CLL cells were as efficiently lysed on CD3xCD19 DART treatment as unstimulated CLL cells. Further examination of the mechanism of CD3xCD19 DART mediated killing showed that lysis was dependent on granules, but was independent of BAX/BAK or caspase activity, indicating non-apoptotic cell death. Conclusions These data show that CD3xCD19 DART in CLL leads to robust T cell activation and lysis of high-risk venetoclax resistant CLL cells through a non-apoptotic mechanism.

[1]  N. Worel,et al.  CAR T-cell therapy in diffuse large B-cell lymphoma , 2020, memo - Magazine of European Medical Oncology.

[2]  D. Baker,et al.  Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity , 2019, Science Immunology.

[3]  U. Jäger,et al.  CAR-T Cell Therapy in Diffuse Large B Cell Lymphoma: Hype and Hope , 2019, HemaSphere.

[4]  E. Thompson,et al.  Acquisition of the Recurrent Gly101Val Mutation in BCL2 Confers Resistance to Venetoclax in Patients with Progressive Chronic Lymphocytic Leukemia. , 2018, Cancer discovery.

[5]  D. Porter,et al.  Chronic lymphocytic leukemia cells impair mitochondrial fitness in CD8+ T cells and impede CAR T cell efficacy. , 2018, Blood.

[6]  E. M. Cook,et al.  A CD19/CD3 bispecific antibody for effective immunotherapy of chronic lymphocytic leukemia in the ibrutinib era. , 2018, Blood.

[7]  Hans Bitter,et al.  Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia , 2018, Nature Medicine.

[8]  Mithat Gonen,et al.  Long‐Term Follow‐up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia , 2018, The New England journal of medicine.

[9]  K. Young,et al.  PD-1 expression and clinical PD-1 blockade in B-cell lymphomas. , 2018, Blood.

[10]  Hao Liu,et al.  CD28 and 41BB Costimulation Enhances the Effector Function of CD19-Specific Engager T Cells , 2017, Cancer Immunology Research.

[11]  O. Elemento,et al.  Tailoring CD19xCD3-DART exposure enhances T-cells to eradication of B-cell neoplasms , 2017, Oncoimmunology.

[12]  Deborah A. Bowen,et al.  Pembrolizumab in patients with CLL and Richter transformation or with relapsed CLL. , 2017, Blood.

[13]  M. Raffeld,et al.  Clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia. , 2017, Blood.

[14]  H. Putter,et al.  Long-term survival of patients with CLL after allogeneic transplantation: a report from the European Society for Blood and Marrow Transplantation , 2017, Bone Marrow Transplantation.

[15]  J. Gribben,et al.  Chronic lymphocytic leukaemia , 2017, Nature Reviews Disease Primers.

[16]  S. Kent,et al.  Cytotoxic CD4 T Cells—Friend or Foe during Viral Infection? , 2017, Front. Immunol..

[17]  R. Rosenquist,et al.  T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers , 2016, Haematologica.

[18]  P. Moore,et al.  MGD011, A CD19 x CD3 Dual-Affinity Retargeting Bi-specific Molecule Incorporating Extended Circulating Half-life for the Treatment of B-Cell Malignancies , 2016, Clinical Cancer Research.

[19]  S. Heimfeld,et al.  Immunotherapy of non-Hodgkin’s lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor–modified T cells , 2016, Science Translational Medicine.

[20]  Daniel Li,et al.  CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. , 2016, The Journal of clinical investigation.

[21]  Xu Luo,et al.  Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane , 2016, Genes & development.

[22]  S. Kaech,et al.  The multifaceted role of CD4+ T cells in CD8+ T cell memory , 2016, Nature Reviews Immunology.

[23]  K. Odunsi,et al.  Direct tumor recognition by a human CD4+ T-cell subset potently mediates tumor growth inhibition and orchestrates anti-tumor immune responses , 2015, Scientific Reports.

[24]  David L. Porter,et al.  Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia , 2015, Science Translational Medicine.

[25]  A. Kater,et al.  Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitors , 2015, Haematologica.

[26]  E. Kimby,et al.  Managing high-risk CLL during transition to a new treatment era: stem cell transplantation or novel agents? , 2014, Blood.

[27]  E. Remmerswaal,et al.  CMV-specific CD8+ T-cell function is not impaired in chronic lymphocytic leukemia. , 2014, Blood.

[28]  C. Fegan,et al.  Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells , 2013, Haematologica.

[29]  Lieping Chen,et al.  Molecular mechanisms of T cell co-stimulation and co-inhibition , 2013, Nature Reviews Immunology.

[30]  J. Gribben,et al.  T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production. , 2013, Blood.

[31]  H. Einsele,et al.  Long-term follow-up of hematologic relapse-free survival in a phase 2 study of blinatumomab in patients with MRD in B-lineage ALL. , 2012, Blood.

[32]  P. Kufer,et al.  Blinatumomab: a historical perspective. , 2012, Pharmacology & therapeutics.

[33]  Michel Sadelain,et al.  Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. , 2011, Blood.

[34]  J. Timmer,et al.  Definition and characterization of the systemic T-cell dysregulation in untreated indolent B-cell lymphoma and very early CLL. , 2011, Blood.

[35]  N. Schmitz,et al.  Allogeneic stem cell transplantation provides durable disease control in poor-risk chronic lymphocytic leukemia: long-term clinical and MRD results of the German CLL Study Group CLL3X trial. , 2010, Blood.

[36]  R. Blasberg,et al.  Tumor-reactive CD4+ T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts , 2010, The Journal of experimental medicine.

[37]  D. Green,et al.  Immunogenic and tolerogenic cell death , 2009, Nature Reviews Immunology.

[38]  A. Kater,et al.  c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches. , 2008, Blood.

[39]  J. Byrd,et al.  Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug. , 2008, The Journal of clinical investigation.

[40]  T. Hamblin,et al.  Chronic lymphocytic leukaemia , 2008, The Lancet.

[41]  C. Borner,et al.  Granzyme B-induced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs , 2008, Cell Death and Differentiation.

[42]  I. Goping,et al.  Cytotoxic T lymphocytes overcome Bcl-2 inhibition: target cells contribute to their own demise. , 2008, Blood.

[43]  N. Hellings,et al.  CD4+CD28null T cells in autoimmune disease: pathogenic features and decreased susceptibility to immunoregulation. , 2007, Journal of immunology.

[44]  N. Hellings,et al.  CD4+CD28null T Cells in Autoimmune Disease: Pathogenic Features and Decreased Susceptibility to Immunoregulation1 , 2007, The Journal of Immunology.

[45]  J. Gribben,et al.  Autologous and allogeneic stem cell transplantations for poor-risk chronic lymphocytic leukemia. , 2005, Blood.

[46]  J. Gribben,et al.  Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells. , 2005, The Journal of clinical investigation.

[47]  A. Morales,et al.  High expression of Bfl‐1 contributes to the apoptosis resistant phenotype in B‐cell chronic lymphocytic leukemia , 2005, International journal of cancer.

[48]  E. Remmerswaal,et al.  CD40 stimulation of B‐cell chronic lymphocytic leukaemia cells enhances the anti‐apoptotic profile, but also Bid expression and cells remain susceptible to autologous cytotoxic T‐lymphocyte attack , 2004, British journal of haematology.

[49]  E. Remmerswaal,et al.  Emergence of a CD4+CD28− Granzyme B+, Cytomegalovirus-Specific T Cell Subset after Recovery of Primary Cytomegalovirus Infection , 2004, The Journal of Immunology.

[50]  S. Lens,et al.  Apoptosis via the B cell antigen receptor requires Bax translocation and involves mitochondrial depolarization, cytochrome C release, and caspase‐9 activation , 2004, European journal of immunology.

[51]  M. V. van Oers,et al.  Expansion of CMV-specific CD8+CD45RA+CD27- T cells in B-cell chronic lymphocytic leukemia. , 2003, Blood.

[52]  T. McDonnell,et al.  Characterization of 4 mantle cell lymphoma cell lines. , 2003, Archives of pathology & laboratory medicine.

[53]  Yong J. Lee,et al.  Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis , 2003, The Journal of cell biology.

[54]  S. Lederman,et al.  CD20-induced B cell death can bypass mitochondria and caspase activation , 2002, Leukemia.

[55]  J. Roliński,et al.  T type 1/type 2 subsets balance in B-cell chronic lymphocytic leukemia--the three-color flow cytometry analysis. , 2002, Leukemia research.

[56]  S. Korsmeyer,et al.  Granzyme B can cause mitochondrial depolarization and cell death in the absence of BID, BAX, and BAK , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[57]  D. Green,et al.  Granzyme B Short-Circuits the Need for Caspase 8 Activity during Granule-Mediated Cytotoxic T-Lymphocyte Killing by Directly Cleaving Bid , 2000, Molecular and Cellular Biology.

[58]  Michael Loran Dustin,et al.  Costimulation: Building an Immunological Synapse , 1999, Science.

[59]  D. Green,et al.  Granzyme B Mimics Apical Caspases , 1998, The Journal of Biological Chemistry.

[60]  S. Yonehara,et al.  Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. , 1996, Journal of immunology.

[61]  E. Kimby,et al.  T lymphocyte subpopulations in chronic lymphocytic leukemia of B cell type in relation to immunoglobulin isotype(s) on the leukemic clone and to clinical features , 1987, European journal of haematology.

[62]  R. Larson,et al.  Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia: a multicentre, single-arm, phase 2 study. , 2015, The Lancet. Oncology.

[63]  N. Schmitz,et al.  Allogeneic stem cell transplantation provides durable disease control in poor-risk chronic lymphocytic leukemia : long-term clinical and MRD results of the German CLL Study Group CLL 3 X trial , 2010 .