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

Abstract

Bispecific antibodies (bsab) offer a promising approach for optimizing antibody-based therapies. In the present study, [(CD20)(2)xCD16], a recombinant CD20- and CD16-directed bsab in the tribody format, was designed to optimize recruitment of FcγRIII (CD16)-positive effector cells. [(CD20)(2)xCD16] 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)(2)xCD16] 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)(2)xCD16], recruiting CD16-positive effector cells, may represent promising candidates for clinical development.

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

“…Although the observed cytotoxic effects were lower in comparison with experiments performed in allogeneic settings, measurable cytotoxicity was induced. However, reduced efficiencies in such 3-to 4-h 51 Cr-release experiments in autologous settings are common and have been reported for rituximab and other CD20-specific Ab derivatives (27,36,46).…”

Novel Bispecific Antibodies Increase γδ T-Cell Cytotoxicity against Pancreatic Cancer Cells

“…Cloning cassettes for the integration in expression vectors allowing generation of bispecific antibodies in the tribody format were de novo synthesized (Entelechon GmbH). Secretion leaders and appropriate restriction sites were introduced to allow directional cloning into expression vectors coding for a [(Her2) 2 xCD16] tribody described earlier (29). The CD16-binding variable regions were replaced by the 7A5 variable regions, resulting in two expression vectors: pSEC-Tag2-Her2-scFv-HC-Vg9-VH and pSECTag2-Her2-scFv-LC-Vg9-VL coding the heavy chain and light chain derivative, respectively.…”

“…The tribody was purified from supernatant as described (29,30). 51 Cr-release assay Cytotoxicity against PDAC cell lines was analyzed in a standard 4 hours 51 Cr-release assay as described (31).…”

“…Lenti-X 293T cells were transfected with the respective expression vectors coding for the tribody [(Her2) 2 (29). The tribody was purified from supernatant as described (29,30).…”

Single-Domain Antibody–Based and Linker-Free Bispecific Antibodies Targeting FcγRIII Induce Potent Antitumor Activity without Recruiting Regulatory T Cells

“…Although it is difficult to assess the contribution of each of these mechanisms in their in vivo efficacy, clinical trial results support an important role of antibody-dependent cell-mediated cytotoxicity (ADCC) for both lymphomas and solid tumors (3). The affinity between the Fc portion of human IgG1 and FcgRIIIa (CD16a), an activating receptor mostly expressed by natural killer (NK) cells, monocytes, and macrophages, has a profound impact on ADCC exerted by antibodies (4,5). Correlation of clinical responses to mAb therapy with FcgRIIIa polymorphism has been observed with more favorable response in patients homozygous for the high-affinity FcgRIIIa (V158; refs.…”