Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter to the Editor
  • Published:

Simultaneous engagement of the activatory receptors NKG2D and CD3 for retargeting of effector cells to CD33-positive malignant cells

Leukemia volume 25, pages 1053–1056 (2011)Cite this article

Subjects

Acute myeloid leukemia (AML) is a cancer affecting both elderly people and children. The overall 3-year survival rate is around 30%. To prolong remission and survival of patients, there is a strong need for the development of novel treatment options. AML blasts of 85–90% of adult and pediatric patients express CD33 on their surface. Therefore, CD33 appears as promising target for immunotherapy.1 As surface-bound immune complexes from previously described anti-CD33 antibodies are rapidly uptaken by AML blasts within the first hours, previous anti-CD33 immunotargeting strategies were mainly based on toxic drugs, which were covalently bound to the anti-CD33 antibodies (for example, gemtuzumab ozogamicin, Mylotarg) and, thereby, co-delivered to the AML cells.2 Unfortunately, the European approval of gemtuzumab failed and recently the drug was also withdrawn from the American market for reasons of side effects.

To prove the functionality of both novel bispecific antibody constructs they were used for retargeting of their respective effector cells to AML cells. As effector cells we used in our study either freshly prepared PBMCs (containing non-activated T- and NK cells) or isolated CD8+ T cells or isolated pre-activated NK cells. All effector cells were prepared from blood samples of healthy donors with their informed consent and approval by the local institutional review board. CD8+ T cells were freshly isolated from PBMC by negative selection using CD8+ T cell isolation kit (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). NK cells were isolated using the human NK cell isolation kit II (Miltenyi Biotech GmbH). The isolated NK cells were expanded and activated for 6 days with 500 U per ml IL-2 and 6 μl antibody coupled beads (NK cell activation/expansion kit (Miltenyi Biotech GmbH). According to fluorescence-activated cell sorting staining (data not shown) more than 99% of the isolated CD8+ T cells or CD8+ T cells present in PBMCs were stained with an anti-NKG2D antibody, whereas the majority of CD4+ T cells were negative for NKG2D. As expected, the single-chain bispecific scBsTaFv CD33-CD3 is able to retarget PBMCs containing non-activated T cells to AML cells resulting in an efficient lysis of the target cells (Figure 1B, b2). AML cells from five independent patients were analyzed either in the presence (Figure 1B, b2, scBsTaFv CD33-CD3) or absence of the single-chain bispecific antibody (Figure 1B, b2, no protein). As also expected, the immuno-ligand ULB-scFv CD33 was capable of retargeting isolated pre-activated NK cells to AML cells (Figure 1C, c1,c2). More of interest, the immuno-ligand ULB-scFv CD33 is also capable of retargeting freshly isolated PBMCs (Figure 1D). The efficient lysis of AML cells from two representative patients is shown in Figure 1D (d1,d2). Lysis was observed in the presence (Figure 1D, ULB-scFv CD33) but not in the absence (Figure 1D, no protein) of the immuno-ligand. As both NK- and NKG2D+ CD8+ T cells could have contributed to the killing of AML cells by PBMCs in the presence of the immuno-ligand ULB-scFv CD33 (Figures 1E, F, e1, f1), we wanted to learn whether NKG2D+ T cells that are engaged with CD33-positive tumor cells via the bispecific scBsTaFv CD33-CD3 can be costimulated by the NKG2D immuno-ligand ULB-scFv CD33 (Figure 1E, e1). Representative data are shown in Figure 1E (e2–e4). As shown in Figure 1E, in the absence of both the CD33-CD3 diabody and the ULB-scFv CD33 immuno-ligand we could hardly detect any interferon (IFN)-γ (e2) or tumor necrosis factor (TNF)-α (e3) in the supernatant of freshly isolated non-stimulated CD8+ T cells when cocultured with CD33-positive tumor cells. In the presence of the bispecific CD33-CD3 diabody, we measured about 1200 pg/ml IFN-γ. In the presence of the immuno-ligand ULB-scFv CD33, the IFN-γ production was less compared with the bispecific CD33-CD3 diabody, but still reached a level of about 750 pg/ml. The combination of the bispecific CD33-CD3 diabody with the ULB-scFv CD33 was superior to the single compounds and resulted in the production of around 2200 pg/ml of IFN-γ. The combination of the single-chain bispecific CD33-CD3 diabody with the immuno-ligand ULB-scFv CD33 also resulted in the secretion of the highest amount of TNF-α (Figure 1E, e3, about 120 pg/ml). CD8+ T cells that were engaged with the CD33-positive tumor cells by the bispecific CD33-CD3 diabody alone produced significantly less TNF-α (about 60 pg/ml). Interestingly, NKG2D+ CD8+ T cells did not respond with a secretion of TNF-α in the presence of the immuno-ligand ULB-scFv CD33. Moreover, there is also no killing of AML cells by T cells in the presence of just the immuno-ligand (Figure 1E, e4). Furthermore, the killing capability of T cells in the presence of both bispecific anti-CD33 compounds is not significantly enhanced (Figure 1E, e4). These results which are in good agreement with previously published data6, 7 suggest that the signaling mediated by NKG2D can corroborate the signaling through the CD3 complex of the T cell receptor, but is not sufficient for killing of the target cell.6 Consequently, the killing of AML cells in the presence of the immuno-ligand ULB-scFv CD33 observed by PBMCs was mainly mediated by NK cells.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1

References

  1. O’Reilly MK, Paulson JC . Siglecs as targets for therapy in immune-cell-mediated disease. Trends Pharmacol Sci 2009; 30: 240–248.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Bross PF, Beitz J, Chen G, Chen XH, Duffy E, Kieffer L et al. Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin Cancer Res 2001; 7: 1490–1496.

    CAS  PubMed  Google Scholar 

  3. Singer H, Kellner C, Lanig H, Aigner M, Stockmeyer B, Oduncu F et al. Effective elimination of acute myeloid leukemic cells by recombinant bispecific antibody derivatives directed against CD33 and CD16. J Immunother 2010; 33: 599–608.

    Article  CAS  PubMed  Google Scholar 

  4. Baeuerle PA, Kufer P, Bargou R . BiTE: Teaching antibodies to engage T-cells for cancer therapy. Curr Opin Mol Ther 2009; 11: 22–30.

    CAS  PubMed  Google Scholar 

  5. von Strandmann EP, Hansen HP, Reiners KS, Schnell R, Borchmann P, Merkert S 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. Blood 2006; 107: 1955–1962.

    Article  PubMed  Google Scholar 

  6. Groh V, Rhinehart R, Randolph-Habecker J, Topp MS, Riddell SR, Spies T . Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat Immunol 2001; 2: 255–260.

    Article  CAS  PubMed  Google Scholar 

  7. Maccalli C, Scaramuzza S, Parmiani G . TNK cells (NKG2D+ CD8+ or CD4+ T lymphocytes) in the control of human tumors. Cancer Immunol Immunother 2009; 58: 801–808.

    Article  CAS  PubMed  Google Scholar 

  8. Carmo-Fonseca M, Pfeifer K, Schroder HC, Vaz MF, Fonseca JE, Muller WE et al. Identification of La ribonucleoproteins as a component of interchromatin granules. Exp Cell Res 1989; 185: 73–85.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Barbara Uteß, Christine Gräfe, Heidrun Gebauer, Livia Schulze and Martina Franke for excellent technical assistance.

Author information

Authors and Affiliations

  1. Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Dresden, Germany

    S Stamova, M Cartellieri, A Feldmann, C C Bippes, H Bartsch, R Wehner, M Schmitz, E P Rieber & M Bachmann

  2. Medizinische Klinik und Poliklinik I Universitätsklinikum Dresden, Medical Faculty Carl Gustav Carus, Technical University Dresden, Dresden, Germany

    M von Bonin, M Bornhäuser & G Ehninger

  3. From the Center for Regenerative Therapies Dresden, Technical University Dresden, Dresden, Germany

    M Bachmann

Authors
  1. S Stamova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M Cartellieri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A Feldmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C C Bippes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. H Bartsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R Wehner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M Schmitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M von Bonin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. M Bornhäuser
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. G Ehninger
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. E P Rieber
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. M Bachmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M Bachmann.

Ethics declarations

Competing interests

Drs S Stamova and M Bachmann have applied for patents related to the novel anti-CD33 abs and the novel linker module.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stamova, S., Cartellieri, M., Feldmann, A. et al. Simultaneous engagement of the activatory receptors NKG2D and CD3 for retargeting of effector cells to CD33-positive malignant cells. Leukemia 25, 1053–1056 (2011). https://doi.org/10.1038/leu.2011.42

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2011.42

This article is cited by

Search

Advanced search

Quick links