Matthias Staudinger

Combined Fc-protein- and Fc-glyco-engineering of scFv-Fc fusion proteins synergistically enhances CD16a binding but does not further enhance NK-cell mediated ADCC.

Protein- or glyco-engineering of antibody molecules can be used to enhance Fc-mediated effector functions. ScFv-Fc fusion proteins (scFv-Fc) represent interesting antibody derivatives due to their relatively simple design and increased tissue penetration. Here, the impact of protein- and glyco-engineering on ADCC potency of a panel of human IgG1-based scFv-Fc was tested. Three matched sets of scFv-Fc variants targeting CD7, CD20 or HLA class II and optimized for CD16a binding by mutagenesis, lack of core-fucose, or their combination, were generated and functionally tested in comparison to the corresponding wild type scFv-Fc. Antigen binding activity was not compromised by altered glycosylation or Fc mutagenesis, whereas Fc binding to CD16a was significantly enhanced in the order: non-core fucosylated/Fc-mutated double-engineered≫Fc-mutated≥non-core-fucosylated>wild-type IgG1-Fc. All engineered variants triggered potent ADCC with up to 100-fold reduced EC50 values compared to non-engineered variants. Interestingly, double-engineered variants were similarly effective in triggering ADCC compared to single-engineered variants irrespective of their 1 log greater CD16a binding affinity. Thus, these data demonstrate that protein- and glyco-engineering enhances NK-cell mediated ADCC of scFv-Fc similarly and show that enhancing CD16a affinity beyond a certain threshold does not result in a further increase of NK-cell mediated ADCC.

Mitochondrial electroporation and in organello RNA editing of chimeric atp6 transcripts.

The Sorghum bicolor atp6-1 gene and chimeric atp6 genes with additional maize sequences were introduced into isolated maize mitochondria via electroporation. Transcripts isolated after in vitro incubation of the transformed organelles were then analysed for RNA editing. Transcripts of the S. bicolor atp6-1 gene, and the RNAs obtained from most of chimeric sorghum-maize atp6 gene constructs tested, were not edited. However, the transcript of one engineered chimeric gene comprising the 5′untranslated sequence and a segment of the N-terminal ORF of the maize atp6 combined with the sorghum atp6 core ORF and 3′untranslated sequence was found to be partially edited. We were able to exclude low RNA stability or insufficient editing capacity as the reason for failure to edit in the other instances. Instead, the data indicate that the maize sequence in the edited fusion transcript provides a structural motif or binding site for a transcript-specific editing factor.

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

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

The novel tribody [(CD20) 2 xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells

Bispecific antibodies (bsab) offer a promising approach for optimizing antibody-based therapies. In the present study, [(CD20)2xCD16], a recombinant CD20- and CD16-directed bsab in the tribody format, was designed to optimize recruitment of FcγRIII (CD16)-positive effector cells. [(CD20)2xCD16] retained the antigen specificities of the parental monoclonal antibodies and binding to FcγRIIIa was not compromised by the F/V polymorphism at amino-acid position 158. [(CD20)2xCD16] mediated potent lysis of lymphoma cell lines and freshly isolated tumor cells from patients, even at low picomolar concentrations (∼10 pM). Irrespective of the CD16a allotype, potency as well as efficacy of lysis obtained with the tribody was significantly higher than lysis triggered by rituximab. Tumor cell killing also occurred when autologous NK cells were used as effector cells. Compared with rituximab, the tribody demonstrated depletion of autologous B cells in ex vivo whole blood assays at 100-fold lower antibody concentration. In mice with a reconstituted humanized hematopoietic system, established by transplantation of human CD34-positive cord blood cells, this novel tribody significantly depleted autologous human B cells. Thus, tribodies such as [(CD20)2xCD16], recruiting CD16-positive effector cells, may represent promising candidates for clinical development.

Enhanced ADCC activity of affinity maturated and Fc-engineered mini-antibodies directed against the AML stem cell antigen CD96.

CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential.

HER2-specific immunoligands engaging NKp30 or NKp80 trigger NK-cell-mediated lysis of tumor cells and enhance antibody-dependent cell-mediated cytotoxicity

NK cells detect tumors through activating surface receptors, which bind self-antigens that are frequently expressed upon malignant transformation. To increase the recognition of tumor cells, the extracellular domains of ligands of the activating NK cell receptors NKp30, NKp80 and DNAM-1 (i.e. B7-H6, AICL and PVR, respectively) were fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2), which is displayed by various solid tumors. The resulting immunoligands, designated B7-H6:HER2-scFv, AICL:HER2-scFv, and PVR:HER2-scFv, respectively, bound HER2 and the addressed NK cell receptor. However, whereas B7-H6:HER2-scFv and AICL:HER2-scFv triggered NK cells to kill HER2-positive breast cancer cells at nanomolar concentrations, PVR:HER2-scFv was not efficacious. Moreover, NK cell cytotoxicity was enhanced synergistically when B7-H6:HER2-scFv or AICL:HER2-scFv were applied in combination with another HER2-specific immunoligand engaging the stimulatory receptor NKG2D. In contrast, no improvements were achieved by combining B7-H6:HER2-scFv with AICL:HER2-scFv. Additionally, B7-H6:HER2-scFv and AICL:HER2-scFv enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by the therapeutic antibodies trastuzumab and cetuximab synergistically, with B7-H6:HER2-scFv exhibiting a higher efficacy. In summary, antibody-derived proteins engaging NKp30 or NKp80 may represent attractive biologics to further enhance anti-tumor NK cell responses and may provide an innovative approach to sensitize tumor cells for antibody-based immunotherapy.

Strategies for purging CD96(+) stem cells in vitro and in vivo: New avenues for autologous stem cell transplantation in acute myeloid leukemia.

The persistence of leukemic stem cells (LSCs) in acute myeloid leukemia (AML) patients receiving chemotherapy may be responsible for the high frequency of relapse. The selective elimination of CD96+ AML-LSCs by means of CD96-specific monoclonal antibodies may be a promising therapeutic approach and revitalize autologous hematopoietic progenitor cell transplantation.

In vitro activation of hTERT-specific T cell responses in lung cancer patients following chemotherapy

OBJECTIVE The aim of this study was to examine chemotherapy concomitant in vitro activation of human telomerase reverse transcriptase (hTERT)-specific T cell responses in peripheral blood mononuclear cell (PBMC) samples of patients with advanced non-small cell lung cancer (NSCLC). METHODS PBMCs depleted of regulatory T cells were stimulated by peptide loaded dendritic cells (DC) matured either by application of cytokines (cDC) or a Toll-like receptor 7/8-agonist combined with a soluble CD40-ligand (ligDC). The hTERT peptide-specific T cell responses were assessed using flow cytometry for intracellular interferon-γ (IFN-γ). RESULTS After cDC activation, T cells producing IFN-γ in response to hTERT were found in PBMC samples of 4 patients. In 2 of these patients the hTERT-specific T cell responses were further increased after ligDC application. However, PBMC of 3 other patients showed little or no induction of hTERT-specific T cell responses as a result of the methods applied during this study. CONCLUSIONS These results indicate, that concomitant to chemotherapy hTERT-specific T cell responses can be activated in PBMC of NSCLC patients in vitro. This activation can be further increased by ligDC though the number of responding patients is still limited.