Triplebody Mediates Increased Anti-Leukemic Reactivity of IL-2 Activated Donor Natural Killer (NK) Cells and Impairs Viability of Their CD33-Expressing NK Subset

Natural killer cells (NK) are essential for the elimination of resistant acute myeloid and acute lymphoblastic leukemia (AML and ALL) cells. NK cell-based immunotherapies have already successfully entered for clinical trials, but limitations due to immune escape mechanisms were identified. Therefore, we extended our established NK cell protocol by integration of the previously investigated powerful trispecific immunoligand ULBP2-aCD19-aCD33 [the so-called triplebodies (TBs)] to improve the anti-leukemic specificity of activated NK cells. IL-2-driven expansion led to strongly elevated natural killer group 2 member D (NKG2D) expressions on donor NK cells which promote the binding to ULBP2+ TBs. Similarly, CD33 expression on these NK cells could be detected. Dual-specific targeting and elimination were investigated against the B-cell precursor leukemia cell line BV-173 and patient blasts, which were positive for myeloid marker CD33 and B lymphoid marker CD19 exclusively presented on biphenotypic B/myeloid leukemia’s. Cytotoxicity assays demonstrated improved killing properties of NK cells pre-coated with TBs compared to untreated controls. Specific NKG2D blocking on those NK cells in response to TBs diminished this killing activity. On the contrary, the observed upregulation of surface CD33 on about 28.0% of the NK cells decreased their viability in response to TBs during cytotoxic interaction of effector and target cells. Similar side effects were also detected against CD33+ T- and CD19+ B-cells. Very preliminary proof of principle results showed promising effects using NK cells and TBs against primary leukemic cells. In summary, we demonstrated a promising strategy for redirecting primary human NK cells in response to TBs against leukemia, which may lead to a future progress in NK cell-based immunotherapies.

[1]  C. Bokemeyer,et al.  Cetuximab Resistance in Head and Neck Cancer Is Mediated by EGFR-K521 Polymorphism. , 2017, Cancer research.

[2]  M. Hallek,et al.  Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia , 2016, Oncoimmunology.

[3]  J. Tolar,et al.  IL15 Trispecific Killer Engagers (TriKE) Make Natural Killer Cells Specific to CD33+ Targets While Also Inducing Persistence, In Vivo Expansion, and Enhanced Function , 2016, Clinical Cancer Research.

[4]  C. Kalberer,et al.  Advances in clinical NK cell studies: Donor selection, manufacturing and quality control , 2015, Oncoimmunology.

[5]  J. Koch,et al.  Cetuximab Reconstitutes Pro-Inflammatory Cytokine Secretions and Tumor-Infiltrating Capabilities of sMICA-Inhibited NK Cells in HNSCC Tumor Spheroids , 2015, Front. Immunol..

[6]  James C. Whisstock,et al.  Perforin and granzymes: function, dysfunction and human pathology , 2015, Nature Reviews Immunology.

[7]  A. Jauch,et al.  Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor. , 2015, Molecular therapy : the journal of the American Society of Gene Therapy.

[8]  H. Spits,et al.  The biology of innate lymphoid cells , 2015, Nature.

[9]  C. Klein,et al.  Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells , 2015, Journal of autoimmunity.

[10]  B. Suarez-Alvarez,et al.  Methylation of NKG2D ligands contributes to immune system evasion in acute myeloid leukemia , 2014, Genes and Immunity.

[11]  T. Robak.  Current and emerging monoclonal antibody treatments for chronic lymphocytic leukemia: state of the art , 2014, Expert review of hematology.

[12]  Bin Zhang,et al.  Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein. , 2014, Blood.

[13]  L. Weiner,et al.  CD16xCD33 bispecific killer cell engager (BiKE) activates NK cells against primary MDS and MDSC CD33+ targets. , 2014, Blood.

[14]  M. Hallek,et al.  Natural ligands and antibody-based fusion proteins: harnessing the immune system against cancer. , 2014, Trends in molecular medicine.

[15]  M. Hallek,et al.  The bispecific immunoligand ULBP2‐aCEA redirects natural killer cells to tumor cells and reveals potent anti‐tumor activity against colon carcinoma , 2013, International journal of cancer.

[16]  O. Janssen,et al.  Shedding of endogenous MHC class I‐related chain molecules A and B from different human tumor entities: Heterogeneous involvement of the “a disintegrin and metalloproteases” 10 and 17 , 2013, International journal of cancer.

[17]  C. Rossig.  Extending the chimeric receptor-based T-cell targeting strategy to solid tumors , 2013, Oncoimmunology.

[18]  J. Passweg,et al.  Clinical Grade Purification and Expansion of NK Cell Products for an Optimized Manufacturing Protocol , 2013, Front. Oncol..

[19]  J. Kwekkeboom,et al.  Defining Early Human NK Cell Developmental Stages in Primary and Secondary Lymphoid Tissues , 2012, PloS one.

[20]  G. Fey,et al.  Heterodimeric bispecific antibody-derivatives against CD19 and CD16 induce effective antibody-dependent cellular cytotoxicity against B-lymphoid tumor cells. , 2011, Cancer letters.

[21]  D. Saul,et al.  A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting , 2011, mAbs.

[22]  J. Koch,et al.  IL‐2‐activated haploidentical NK cells restore NKG2D‐mediated NK‐cell cytotoxicity in neuroblastoma patients by scavenging of plasma MICA , 2010, European journal of immunology.

[23]  D. Saul,et al.  A recombinant trispecific single‐chain Fv derivative directed against CD123 and CD33 mediates effective elimination of acute myeloid leukaemia cells by dual targeting , 2010, British journal of haematology.

[24]  M. Keating,et al.  Alemtuzumab by continuous intravenous infusion followed by subcutaneous injection plus rituximab in the treatment of patients with chronic lymphocytic leukemia recurrence , 2010, Cancer.

[25]  J. Passweg,et al.  IL-2−driven Regulation of NK Cell Receptors With Regard to the Distribution of CD16+ and CD16− Subpopulations and In Vivo Influence After Haploidentical NK Cell Infusion , 2010, Journal of immunotherapy.

[26]  J. Skepper,et al.  Differential Mechanisms of Shedding of the Glycosylphosphatidylinositol (GPI)-anchored NKG2D Ligands* , 2010, The Journal of Biological Chemistry.

[27]  J. Trowsdale,et al.  ULBP6/RAET1L is an additional human NKG2D ligand , 2009, European journal of immunology.

[28]  G. Ball,et al.  NKG2D Ligand Expression in Human Colorectal Cancer Reveals Associations with Prognosis and Evidence for Immunoediting , 2009, Clinical Cancer Research.

[29]  A. Nesterova,et al.  Anti-leukemic activity of Lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia , 2009, mAbs.

[30]  H. Ljunggren,et al.  Tumor cell recognition by the NK cell activating receptor NKG2D , 2008, European journal of immunology.

[31]  B. Suarez-Alvarez,et al.  NKG2D ligands: key targets of the immune response. , 2008, Trends in immunology.

[32]  W. Wels,et al.  A novel five-colour flow cytometric assay to determine NK cell cytotoxicity against neuroblastoma and other adherent tumour cells. , 2007, Journal of immunological methods.

[33]  W. Held,et al.  The role of the NKG2D receptor for tumor immunity. , 2006, Seminars in cancer biology.

[34]  H. Salih,et al.  Soluble MICB in malignant diseases: analysis of diagnostic significance and correlation with soluble MICA , 2006, Cancer Immunology, Immunotherapy.

[35]  M. Hallek,et al.  A novel bispecific protein (ULBP2-BB4) targeting the NKG2D receptor on natural killer (NK) cells and CD138 activates NK cells and has potent antitumor activity against human multiple myeloma in vitro and in vivo. , 2006, Blood.

[36]  A. B. Pérez-Oliva,et al.  A study of CD33 (SIGLEC‐3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing , 2006, Journal of leukocyte biology.

[37]  D. Scheinberg,et al.  Phase III randomized multicenter study of a humanized anti-CD33 monoclonal antibody, lintuzumab, in combination with chemotherapy, versus chemotherapy alone in patients with refractory or first-relapsed acute myeloid leukemia. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[38]  C. Kalberer,et al.  Ligands for natural killer cell-activating receptors are expressed upon the maturation of normal myelomonocytic cells but at low levels in acute myeloid leukemias. , 2005, Blood.

[39]  C. Le,et al.  Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. , 2005, Blood.

[40]  E. Lanino,et al.  Analysis of the receptor-ligand interactions in the natural killer-mediated lysis of freshly isolated myeloid or lymphoblastic leukemias: evidence for the involvement of the Poliovirus receptor (CD155) and Nectin-2 (CD112). , 2005, Blood.

[41]  H. Rammensee,et al.  Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. , 2003, Blood.

[42]  C. Yee,et al.  Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation , 2002, Nature.

[43]  Katia Perruccio,et al.  Effectiveness of Donor Natural Killer Cell Alloreactivity in Mismatched Hematopoietic Transplants , 2002, Science.

[44]  M. Martelli,et al.  Cellular therapy: exploiting NK cell alloreactivity in transplantation , 2001, Current opinion in hematology.

[45]  Jeffrey S. Miller,et al.  The biology of natural killer cells in cancer, infection, and pregnancy. , 2001, Experimental hematology.

[46]  D. Olive,et al.  Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: Engagement of CD33 induces apoptosis of leukemic cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[47]  K. Nakachi,et al.  Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population , 2000, The Lancet.

[48]  T. Whiteside,et al.  Human tumor antigen-specific T lymphocytes and interleukin-2-activated natural killer cells: comparisons of antitumor effects in vitro and in vivo. , 1998, Clinical cancer research : an official journal of the American Association for Cancer Research.

[49]  G. Trinchieri.  Natural killer cells wear different hats: effector cells of innate resistance and regulatory cells of adaptive immunity and of hematopoiesis. , 1995, Seminars in immunology.

[50]  R. Handgretinger,et al.  Expression of an early myelopoietic antigen (CD33) on a subset of human umbilical cord blood-derived natural killer cells. , 1993, Immunology letters.

[51]  G. Trinchieri,et al.  Biology of Natural Killer Cells , 1989, Advances in Immunology.

[52]  A. Tomita.  Genetic and Epigenetic Modulation of CD20 Expression in B-Cell Malignancies: Molecular Mechanisms and Significance to Rituximab Resistance. , 2016, Journal of clinical and experimental hematopathology : JCEH.

[53]  Jeffrey S. Miller,et al.  NK cells in therapy of cancer. , 2014, Critical reviews in oncogenesis.

[54]  D. Maloney,et al.  Rituximab resistance. , 2011, Best practice & research. Clinical haematology.

[55]  Hyunkeun Song,et al.  Soluble ULBP suppresses natural killer cell activity via down-regulating NKG2D expression. , 2006, Cellular immunology.

[56]  S. Zimmermann,et al.  A novel four-colour flow cytometric assay to determine natural killer cell or T-cell-mediated cellular cytotoxicity against leukaemic cells in peripheral or bone marrow specimens containing greater than 20% of normal cells. , 2005, Journal of immunological methods.

[57]  J. Luhm,et al.  NK cells: a lesson from mismatched hematopoietic transplantation. , 2003, Trends in immunology.

[58]  T. Whiteside,et al.  Natural killer cells and tumor therapy. , 1998, Current topics in microbiology and immunology.

[59]  Markus Voelter,et al.  State of the Art , 1997, Pediatric Research.