Fusion proteins between ligands for NKG2D and CD20-directed single-chain variable fragments sensitize lymphoma cells for natural killer cell-mediated lysis and enhance antibody-dependent cellular cytotoxicity

Kellner, Christian; Hallack, Daniela; Glorius, Pia; Staudinger, Matthias; Nodehi, Sahar Mohseni; Weers, Michel de; Winkel, Jan G. J. van de; Parren, Paul W.H.I.; Stauch, Martina; Valerius, Thomas; Repp, Roland; Humpe, Andreas; Gramatzki, Martin; Peipp, Matthias

doi:10.1038/leu.2011.288

Cited by 34 publications

(57 citation statements)

References 11 publications

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“…However, these strategies did not allow tumor-specific NK cell activation. In another more tumor-specific approach, tumor cells were opsonized with bifunctional Ab-based fusion proteins containing ligands addressing NKG2D (25)(26)(27). Fusion proteins containing scFvs against tumor-associated Ags such as CD20, CD33, or CD138 were able to mediate NK cell cytotoxicity.…”

Section: Discussionmentioning

confidence: 99%

“…To analyze the signaling properties of NKp30 and NKG2D, B7-H6:7D8 was compared with ULBP2:7D8, a bifunctional fusion protein containing the NKG2D-specific ligand ULBP2 and the CD20 scFv 7D8 (27). Similarly to B7-H6:7D8, ULBP2:7D8 improved recognition of Ramos cells (as evidenced by augmented numbers of activated NK cells with increased CD69 expression levels) and induced NK cell degranulation (as verified by CD107a exposure; Fig.…”

Section: B7-h6:7d8 Synergizes With the Nkg2d-directed Recombinant Immmentioning

confidence: 99%

“…Cytotoxicity was analyzed in standard 4-h 51 Cr-release assays performed in 96-well microtiter plates in a total volume of 200 ml as described (27,28). Human NK cells were used as effector cells at E:T ratios of 10:1 and 20:1 in experiments with cell lines and freshly isolated tumor cells, respectively.…”

Section: Cytotoxicity Assaysmentioning

confidence: 99%

“…The cDNA sequence encoding the natural secretion leader and the ECD of B7-H6 (amino acids 1-267) (15) was de novo synthesized (Entelechon, Regensburg, Germany) and ligated as NheI/NotI cassettes into vector pSecTag2/ULBP2:7D8-myc-his (27) generating vector pSecTag2/B7-H6:7D8-myc-his. The expression vector for B7-H6:4D5 was constructed by exchanging the sequences for scFv 7D8 against those encoding scFv 4D5 (28).…”

Section: Generation Of Recombinant Proteinsmentioning

confidence: 99%

“…Alternatively, tumor cells were exogenously coated with such danger signals by using recombinant immunoligands. These are bifunctional fusion proteins consisting of a stimulatory ligand and a tumor-directed Ab fragment that represent an attractive class of molecules with immunomodulatory functions (25)(26)(27). However, most attempts have focused on the NKG2D ligand system, whereas to date NKp30 and other NCRs have not been investigated as stimulatory molecules for potential therapeutic purposes.…”

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confidence: 99%

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Mimicking an Induced Self Phenotype by Coating Lymphomas with the NKp30 Ligand B7-H6 Promotes NK Cell Cytotoxicity

Kellner

Maurer

Hallack

et al. 2012

The Journal of Immunology

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Induced self expression of the NKp30 ligand B7-H6 facilitates NK cell-mediated elimination of stressed cells. A fusion protein consisting of the ectodomain of B7-H6 and the CD20 single-chain fragment variable 7D8 was generated to mimic an induced self phenotype required for NK cell-mediated target cell elimination. B7-H6:7D8 had bifunctional properties as reflected by its ability to simultaneously bind to the CD20 Ag and to the NKp30 receptor. B7-H6:7D8 bound by CD20+ lymphoma cells activated human NK cells and triggered degranulation. Consequently, the immunoligand B7-H6:7D8 induced killing of lymphoma-derived cell lines as well as fresh tumor cells from chronic lymphocytic leukemia or lymphoma patients. B7-H6:7D8 was active at nanomolar concentrations in a strictly Ag-specific manner and required interaction with both CD20 and NKp30. Remarkably, NK cell cytotoxicity was further augmented by concomitant activation of Fcγ receptor IIIa or NK group 2 member D. Thus, B7-H6:7D8 acted synergistically with the CD20 Ab rituximab and the immunoligand ULBP2:7D8, which was similarly designed as B7-H6:7D8 but engaging the NK group 2 member D receptor. In conclusion, to our knowledge, B7-H6:7D8 represents the first Ab-based molecule stimulating NKp30-mediated NK cell cytotoxicity for therapeutic purposes and provides proof of concept that Ag-specific NKp30 engagement may represent an innovative strategy to enhance antitumoral NK cell cytotoxicity.

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Section: Discussionmentioning

confidence: 99%

Section: B7-h6:7d8 Synergizes With the Nkg2d-directed Recombinant Immmentioning

confidence: 99%

Section: Cytotoxicity Assaysmentioning

confidence: 99%

Section: Generation Of Recombinant Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Mimicking an Induced Self Phenotype by Coating Lymphomas with the NKp30 Ligand B7-H6 Promotes NK Cell Cytotoxicity

Kellner

Maurer

Hallack

et al. 2012

The Journal of Immunology

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The bispecific immunoligand ULBP2‐aCEA redirects natural killer cells to tumor cells and reveals potent anti‐tumor activity against colon carcinoma

Rothe

Jachimowicz

Borchmann

et al. 2013

Intl Journal of Cancer

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NKG2D, an activating receptor expressed on NK cells and T cells, is critically involved in tumor immunosurveillance. In this study, we explored the potential therapeutic utility of the NKG2D ligand ULBP2 for the treatment of colon carcinoma. To this end we designed a fusion protein consisting of human ULBP2 and an antibody-derived single chain targeting the tumor carcinoembryonic antigen (CEA). The bispecific recombinant fusion protein re-directed NK cells towards malignant cells by binding to both, tumor cells and NK cells, and triggered NK cell-mediated target cell killing in vitro. Moreover, tumor growth was significantly delayed in a syngeneic colon carcinoma mouse model in response to immunoligand treatment. The anti-tumor activity could be attributed to the stimulation of immune cells with an elevated expression of the activation marker CD69 on NK, T and NKT cells and the infiltration of CD451 immune cells into the solid tumor. In summary, it was demonstrated that immunoligands provide specific tumor targeting by NK cells and exert anti-tumor activity in vitro and in vivo. This technology represents a novel immunotherapeutic strategy for solid tumors with the potential to be further developed for clinical applications.Colorectal cancer is fatal in advanced stages of the disease. Curative treatment options are commonly reserved for tumors not yet metastasized or with resectable metastasis. Colorectal cancer is susceptible to chemotherapy from the onset, but due to therapeutic pressure cancer cells usually become resistant to the standard therapies. In addition, the standard therapies are frequently associated with severe side effects. Thus, there is a high demand for targeted therapies. Most recently developed antibody-based immunotherapies including cetuximab, bevacizumab and the fully human antibody panitumumab have not only added new impulses to colorectal cancer therapy, but essentially improved clinical outcome. 1 They have shown to overcome resistance to conventional chemotherapy and have clearly demonstrated that colorectal carcinoma is accessible to antibody therapy.The potential of immune modulation as well as the role of antibody therapy to enhance a cell-based immunotherapy is currently evaluated. 2-4 Increasing attention has been drawn onto NK cells, which are part of the innate immune system and can attack malignant cells without prior antigen stimulation. 5,6 Numerous ways of tumor cell recognition by NK cells have been described. NK cell cytotoxicity against tumors can be triggered by cells lacking expression of self-recognized MHC class I molecules. This mechanism has been referred to as the "missing-self"-hypothesis and is controlled through a group of inhibitory receptors on the NK cell surface. 7 In addition, an anti-tumor immune response can be induced by the upregulation of ligands for triggering NK cell receptors on the surface of tumor cells. 8 This mechanism has been referred to as "induced-self" and provokes an effective immune response even in the presence of inhibitory signalling....

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Engineered Antibody Derivatives in Preclinical and Clinical Development

Kellner

Peipp

2013

Molecular and Cellular Mechanisms of Antibody Activity

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Fusion proteins between ligands for NKG2D and CD20-directed single-chain variable fragments sensitize lymphoma cells for natural killer cell-mediated lysis and enhance antibody-dependent cellular cytotoxicity

Cited by 34 publications

References 11 publications

Mimicking an Induced Self Phenotype by Coating Lymphomas with the NKp30 Ligand B7-H6 Promotes NK Cell Cytotoxicity

Mimicking an Induced Self Phenotype by Coating Lymphomas with the NKp30 Ligand B7-H6 Promotes NK Cell Cytotoxicity

The bispecific immunoligand ULBP2‐aCEA redirects natural killer cells to tumor cells and reveals potent anti‐tumor activity against colon carcinoma

Engineered Antibody Derivatives in Preclinical and Clinical Development

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