Skip to main content

Advertisement

Log in

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells

  • Original Article
  • Published:
Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcγRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3ζ chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Maloney DG (2005) Immunotherapy for non-Hodgkin’s lymphoma: monoclonal antibodies and vaccines. J Clin Oncol 23:6421–6428

    Article  PubMed  CAS  Google Scholar 

  2. Cartron G, Watier H, Golay J, Solal-Celigny P (2004) From the bench to the bedside: ways to improve rituximab efficacy. Blood 104:2635–2642

    Article  PubMed  CAS  Google Scholar 

  3. Schilling V (2003) Immunotherapy with anti-CD20 compounds. Semin Cancer Biol 13:211–222

    Article  Google Scholar 

  4. Maloney DG, Liles TM, Czerwinski DK, Waldichuk C, Rosenberg J, Grillo-Lopez A, Levy R (1994) Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma. Blood 84:2457–2466

    PubMed  CAS  Google Scholar 

  5. Maloney DG, Grillo-Lopez AJ, White CA, Bodkin D, Schilder RJ, Neidhart JA, Janakiraman N, Foon KA, Liles TM, Dallaire BK, Wey K, Royston I, Davis T, Levy R (1997) IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood 90:2188–2195

    PubMed  CAS  Google Scholar 

  6. McLaughlin P, Grillo-Lopez AJ, Link BK, Levy R, Czuczman MS, Williams ME, Heyman MR, Bence-Bruckler I, White CA, Cabanillas F, Jain V, Ho AD, Lister J, Wey K, Shen D, Dallaire BK (1998) Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J Clin Oncol 16:2825–2833

    PubMed  CAS  Google Scholar 

  7. Vose JM, Link BK, Grossbard ML, Czuczman M, Grillo-Lopez A, Gilman P, Lowe A, Kunkel LA, Fisher RI (2001) Phase II study of rituximab in combination with chop chemotherapy in patients with previously untreated, aggressive non-Hodgkin’s lymphoma. J Clin Oncol 19:389–397

    PubMed  CAS  Google Scholar 

  8. Jazirehi AR, Bonavida B (2005) Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-CD20 mAb) in non-Hodgkin’s lymphoma: implications in chemosensitization and therapeutic intervention. Oncogene 24:2121–2143

    Article  PubMed  CAS  Google Scholar 

  9. Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, Watier H (2002) Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood 99:754–758

    Article  PubMed  CAS  Google Scholar 

  10. Weng WK, Levy R (2003) Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol 21:3940–3947

    Article  PubMed  CAS  Google Scholar 

  11. Hainsworth JD, Burris HA III, Morrissey LH, Litchy S, Scullin DC Jr, Bearden JD III, Richards P, Greco FA (2000) Rituximab monoclonal antibody as initial systemic therapy for patients with low-grade non-Hodgkin lymphoma. Blood 95:3052–3056

    PubMed  CAS  Google Scholar 

  12. Colombat P, Salles G, Brousse N, Eftekhari P, Soubeyran P, Delwail V, Deconinck E, Haioun C, Foussard C, Sebban C, Stamatoullas A, Milpied N, Boue F, Taillan B, Lederlin P, Najman A, Thieblemont C, Montestruc F, Mathieu-Boue A, Benzohra A, Solal-Celigny P (2001) Rituximab (anti-CD20 monoclonal antibody) as single first-line therapy for patients with follicular lymphoma with a low tumor burden: clinical and molecular evaluation. Blood 97:101–106

    Article  PubMed  CAS  Google Scholar 

  13. Davis TA, Grillo-Lopez AJ, White CA, McLaughlin P, Czuczman MS, Link BK, Maloney DG, Weaver RL, Rosenberg J, Levy R (2000) Rituximab anti-CD20 monoclonal antibody therapy in non-Hodgkin’s lymphoma: safety and efficacy of re-treatment. J Clin Oncol 18:3135–3143

    PubMed  CAS  Google Scholar 

  14. Uherek C, Tonn T, Uherek B, Becker S, Schnierle B, Klingemann HG, Wels W (2002) Retargeting of natural killer-cell cytolytic activity to ErbB2-expressing cancer cells results in efficient and selective tumor cell destruction. Blood 100:1265–1273

    PubMed  CAS  Google Scholar 

  15. Daldrup-Link HE, Meier R, Rudelius M, Piontek G, Piert M, Metz S, Settles M, Uherek C, Wels W, Schlegel J, Rummeny EJ (2005) In vivo tracking of genetically engineered, anti-HER2/neu directed natural killer cells to HER2/neu positive mammary tumors with magnetic resonance imaging. Eur Radiol 15:4–13

    Article  PubMed  Google Scholar 

  16. Imai C, Iwamoto S, Campana D (2005) Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 106:376–383

    Article  PubMed  CAS  Google Scholar 

  17. Klingemann HG, Wong E, Maki G (1996) A cytotoxic NK-cell line (NK-92) for ex vivo purging of leukemia from blood. Biol Blood Marrow Transplant 2:68–75

    PubMed  CAS  Google Scholar 

  18. Yan Y, Steinherz P, Klingemann HG, Dennig D, Childs BH, McGuirk J, O’Reilly RJ (1998) Antileukemia activity of a natural killer cell line against human leukemias. Clin Cancer Res 4:2859–2868

    PubMed  CAS  Google Scholar 

  19. Tam YK, Miyagawa B, Ho VC, Klingemann HG (1999) Immunotherapy of malignant melanoma in a SCID mouse model using the highly cytotoxic natural killer cell line NK-92. J Hematother 8:281–290

    Article  PubMed  CAS  Google Scholar 

  20. Tonn T, Becker S, Esser R, Schwabe D, Seifried E (2001) Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92. J Hematother Stem Cell Res 10:535–544

    Article  PubMed  CAS  Google Scholar 

  21. Gong JH, Maki G, Klingemann HG (1994) Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 8:652–658

    PubMed  CAS  Google Scholar 

  22. Burshtyn DN, Scharenberg AM, Wagtmann N, Rajagopalan S, Berrada K, Yi T, Kinet JP, Long EO (1996) Recruitment of tyrosine phosphatase HCP by the killer cell inhibitor receptor. Immunity 4:77–85

    Article  PubMed  CAS  Google Scholar 

  23. Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, Biassoni R, Moretta L (2001) Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol 19:197–223

    Article  PubMed  CAS  Google Scholar 

  24. Romanski A, Bug G, Becker S, Kampfmann M, Seifried E, Hoelzer D, Ottmann OG, Tonn T (2005) Mechanisms of resistance to natural killer cell-mediated cytotoxicity in acute lymphoblastic leukemia. Exp Hematol 33:344–352

    Article  PubMed  CAS  Google Scholar 

  25. Wu AM, Tan GJ, Sherman MA, Clarke P, Olafsen T, Forman SJ, Raubitschek AA (2001) Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange. Protein Eng 14:1025–1033

    Article  PubMed  CAS  Google Scholar 

  26. Chow KU, Sommerlad WD, Boehrer S, Schneider B, Seipelt G, Rummel MJ, Hoelzer D, Mitrou PS, Weidmann E (2002) Anti-CD20 antibody (IDEC-C2B8, rituximab) enhances efficacy of cytotoxic drugs on neoplastic lymphocytes in vitro: role of cytokines, complement, and caspases. Haematologica 87:33–43

    PubMed  CAS  Google Scholar 

  27. Hoogenboom HR, Griffiths AD, Johnson KS, Chiswell DJ, Hudson P, Winter G (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19:4133–4137

    Article  PubMed  CAS  Google Scholar 

  28. Altenschmidt U, Kahl R, Moritz D, Schnierle BS, Gerstmayer B, Wels W, Groner B (1996) Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes. Clin Cancer Res 2:1001–1008

    PubMed  CAS  Google Scholar 

  29. Rohrbach F, Gerstmayer B, Biburger M, Wels W (2000) Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain and single chain antibody fragments for tumor targeting. Clin Cancer Res 6:4314–4322

    PubMed  CAS  Google Scholar 

  30. Evan GI, Lewis GK, Ramsay G, Bishop JM (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616

    PubMed  CAS  Google Scholar 

  31. Gerstmayer B, Groner B, Wels W, Schnierle BS (1999) Stable expression of the ecotropic retrovirus receptor in amphotropic packaging cells facilitates the transfer of recombinant vectors and enhances the yield of retroviral particles. J Virol Methods 81:71–75

    Article  PubMed  CAS  Google Scholar 

  32. Hobbs S, Jitrapakdee S, Wallace JC (1998) Development of a bicistronic vector driven by the human polypeptide chain elongation factor 1alpha promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins. Biochem Biophys Res Commun 252:368–372

    Article  PubMed  CAS  Google Scholar 

  33. Moritz D, Wels W, Mattern J, Groner B (1994) Cytotoxic T lymphocytes with a grafted recognition specificity for ERBB2-expressing tumor cells. Proc Natl Acad Sci USA 91:4318–4322

    Article  PubMed  CAS  Google Scholar 

  34. Bitton N, Debre P, Eshhar Z, Gorochov G (2001) T-bodies as antiviral agents. Curr Top Microbiol Immunol 260:271–300

    PubMed  CAS  Google Scholar 

  35. Uherek C, Groner B, Wels W (2001) Chimeric antigen receptors for the retargeting of cytotoxic effector cells. J Hematother Stem Cell Res 10:523–534

    Article  PubMed  CAS  Google Scholar 

  36. Abken H, Hombach A, Heuser C, Kronfeld K, Seliger B (2002) Tuning tumor-specific T-cell activation: a matter of costimulation? Trends Immunol 23:240–245

    Article  PubMed  CAS  Google Scholar 

  37. Kershaw MH, Teng MW, Smyth MJ, Darcy PK (2005) Supernatural T cells: genetic modification of T cells for cancer therapy. Nat Rev Immunol 5:928–940

    Article  PubMed  CAS  Google Scholar 

  38. Bach N, Waks T, Eshhar Z (1995) Specific lysis of tumor cells by an NK-like cell line transfected with chimeric receptor genes. Tumor Target 1:203–209

    Google Scholar 

  39. Tran AC, Zhang D, Byrn R, Roberts MR (1995) Chimeric zeta-receptors direct human natural killer (NK) effector function to permit killing of NK-resistant tumor cells and HIV-infected T lymphocytes. J Immunol 155:1000–1009

    PubMed  CAS  Google Scholar 

  40. Whiteside TL, Vujanovic NL, Herberman RB (1998) Natural killer cells and tumor therapy. Curr Top Microbiol Immunol 230:221–244

    PubMed  CAS  Google Scholar 

  41. Smyth MJ, Hayakawa Y, Takeda K, Yagita H (2002) New aspects of natural-killer-cell surveillance and therapy of cancer. Nat Rev Cancer 2:850–861

    Article  PubMed  CAS  Google Scholar 

  42. Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri MA (2002) Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood 100:1935–1947

    Article  PubMed  CAS  Google Scholar 

  43. Mahle NH, Radcliff G, Sevilla CL, Kornbluth J, Callewaert DM (1989) Kinetics of cellular cytotoxicity mediated by a cloned human natural killer cell line. Immunobiology 179:230–243

    PubMed  CAS  Google Scholar 

  44. Dälken B, Giesübel U, Knauer SK, Wels WS (2006) Targeted induction of apoptosis by chimeric granzyme B fusion proteins carrying antibody and growth factor domains for cell recognition. Cell Death Differ 13:576–585

    Article  PubMed  Google Scholar 

  45. Mahrus S, Craik CS (2005) Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells. Chem Biol 12:567–577

    Article  PubMed  CAS  Google Scholar 

  46. Sedlmayr P, Rabinowich H, Elder EM, Ernstoff MS, Kirkwood JM, Herberman RB, Whiteside TL (1991) Depressed ability of patients with melanoma or renal cell carcinoma to generate adherent lymphokine-activated killer cells. J Immunother 10:336–346

    Article  PubMed  CAS  Google Scholar 

  47. Baum C, Dullmann J, Li Z, Fehse B, Meyer J, Williams DA, von Kalle C (2003) Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 101:2099–2114

    Article  PubMed  CAS  Google Scholar 

  48. Bonini C, Ferrari G, Verzeletti S, Servida P, Zappone E, Ruggieri L, Ponzoni M, Rossini S, Mavilio F, Traversari C, Bordignon C (1997) HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 276:1719–1724

    Article  PubMed  CAS  Google Scholar 

  49. Junker K, Koehl U, Zimmerman S, Stein S, Schwabe D, Klingebiel T, Grez M (2003) Kinetics of cell death in T lymphocytes genetically modified with two novel suicide fusion genes. Gene Ther 10:1189–1197

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Barbara Schnierle for providing pEFIRES-P vector, Dr. Annette Romanski for BV173 and NALM-6 cells, Dr. Byoung S. Kwon for anti-4-1BB antibody BBK-1, Daniela Bott for isolation of primary B and NK cells, Dr. Brigitte Rüster for help with microscopical analysis, Dipl. Ing. Nicola Krzossok for help with NK-92 cytotoxicity assays, Dr. Boris Brill, Sabrina Lehmen and Christiane Peter for help with animal experiments, and Dr. Markus Biburger for helpful suggestions. This work was supported in part by research grant 102386/10-2244 from Deutsche Krebshilfe. G. Maki was supported by grant CLL-63119, Section of Hematology, Rush University Medical Center.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Winfried S. Wels.

Electronic supplementary material

Below is the link to the electronic supplementary material.

262_2007_383_MOESM1_ESM.pdf

Supplementary Fig. S1 Recombinant scFv(Leu-16) binds to CD20 expressing lymphoma cells. a For periplasmic expression of CD20-specific scFv(Leu-16) under control of the IPTGinducible tac promoter, cDNA fragments encoding heavy (VH) and light chain variable domains (VL) of monoclonal antibody Leu-16 were connected by a flexible linker sequence and fused to the ompA signal peptide (SP) in the bacterial expression vector pSW50. Synthetic FLAG- and Myc-tags are included at N- and C-termini of the gene product. b The presence of ErbB2-specific control protein scFv(FRP5) (lane 1) and CD20-specific scFv(Leu-16) (lane 2) in periplasmic extracts was confirmed by SDS-PAGE and immunoblot analysis with FLAG-tag specific Mab M2. c Binding of recombinant scFv molecules to CD20 expressing but ErbB2-negative Raji lymphoma cells (upper panel), and ErbB2 expressing but CD20-negative SKBR3 breast carcinoma cells was analyzed by flow cytometry with Myc-tag specific Mab 9E10 and FITC-conjugated secondary antibody. CD20-specific Mab L27 served as a control. (PDF 59 kb)

262_2007_383_MOESM2_ESM.pdf

Supplementary Fig. S2 Granzyme B activity is required for target cell killing by NK-92-scFv(Leu-16)-ζ cells. NK-92-scFv(Leu-16)-ζ cells were incubated with 100 μM of the serine protease inhibitor DCI (3,4-dichloroisocoumarin) (Roche, Mannheim, Germany) in X-VIVO 10 medium for 1 h at 37°C, before their cytotoxic activity towards CD20 expressing NIH3T3-CD20 cells was analyzed in a 3 h MTT cytotoxicity assay as described in the methods section (E/T ratio of 10:1). Untreated NK-92 and NK-92-scFv(Leu-16)-ζ cells, and NK-92-scFv(Leu-16)-ζ cells treated with DMSO served as controls. The relative number of viable target cells is expressed in % of NIH3T3-CD20 grown in the absence of NK cells (set to 100 %). Mean values of triplicate samples are shown. The standard deviation is indicated by error bars. At the concentration applied DCI was not toxic to NK-92 cells as evaluated by propidium iodide staining (data not shown). (PDF 8.85 kb)

Supplementary movie Selectivity and kinetics of target cell killing. NIH3T3-CD20 cells transduced with a retroviral vector encoding enhanced green fluorescent protein (eGFP) were mixed at a 1:1 ratio with parental NIH3T3 cells and grown overnight. Then NK-92-scFv(Leu-16)-ζ cells were added at an effector to target ratio of 1:1, microscopic images of a single field were taken at 1.5 min intervals for 6.4 h, and assembled into a QuickTime movie at 10 frames per second. At the beginning of the movie, a fluorescence microscopic image of eGFP- and CD20-positive NIH3T3-CD20(eGFP), and eGFP- and CD20-negative NIH3T3 cells before addition of NK cells is shown. Exemplary NIH3T3-CD20(eGFP) cells are indicated by white circles, exemplary parental NIH3T3 cells by black arrows. Selected images from this experiment are also shown in Fig. 5. (MOV 2.33 mb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Müller, T., Uherek, C., Maki, G. et al. Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells. Cancer Immunol Immunother 57, 411–423 (2008). https://doi.org/10.1007/s00262-007-0383-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00262-007-0383-3

Keywords

Navigation