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Though Vγ9Vδ2-T cells constitute only a small fraction of the total T cell population in human peripheral blood, they play a vital role in tumor defense and are therefore of major interest to explore for cancer immunotherapy. Vγ9Vδ2-T cell-based cancer immunotherapeutic approaches developed so far have been generally well tolerated and were able to induce significant clinical responses. However, overall results were inconsistent, possibly due to the fact that these strategies induced systemic activation of Vγ9Vδ2-T cells without preferential accumulation and targeted activation in the tumor. Here we show that a novel bispecific nanobody-based construct targeting both Vγ9Vδ2-T cells and EGFR induced potent Vγ9Vδ2-T cell activation and subsequent tumor cell lysis bothin vitroand in anin vivomouse xenograft model. Tumor cell lysis was independent ofKRASandBRAFtumor mutation status and common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific nanobody, this immunotherapeutic approach can be applied to a large group of cancer patients. [ABSTRACT FROM PUBLISHER]

Ligation of the CD1d antigen-presenting molecule by monoclonal antibodies (mAbs) can trigger important biological functions. For therapeutic purposes camelid-derived variable domain of heavy-chain-only antibodies (VHH) have multiple advantages over mAbs because they are small, stable and have low immunogenicity. Here, we generated 21 human CD1d-specific VHH by immunizing Lama glama and subsequent phage display. Two clones induced maturation of dendritic cells, one clone induced early apoptosis in CD1d-expressing B lymphoblasts and multiple myeloma cells, and another clone blocked recognition of glycolipid-loaded CD1d by CD1d-restricted invariant natural killer T (iNKT) cells. In contrast to reported CD1d-specific mAbs, these CD1d-specific VHH have the unique characteristic that they induce specific and well-defined biological effects. This feature, combined with the above-indicated general advantages of VHH, make the CD1d-specific VHH generated here unique and useful tools to exploit both CD1d ligation as well as disruption of CD1d-iNKT interactions in the treatment of cancer or inflammatory disorders., (© 2016 John Wiley & Sons Ltd.)

Vγ9Vδ2-T cells constitute the predominant subset of γδ-T cells in human peripheral blood and have been shown to play an important role in antimicrobial and antitumor immune responses. Several efforts have been initiated to exploit these cells for cancer immunotherapy, e.g. by using phosphoantigens, adoptive cell transfer, and by a bispecific monoclonal antibody based approach. Here, we report the generation of a novel set of Vγ9Vδ2-T cell specific VHH (or nanobody). VHH have several advantages compared to conventional antibodies related to their small size, stability, ease of generating multispecific molecules and low immunogenicity. With high specificity and affinity, the anti-Vγ9Vδ2-T cell receptor VHHs are shown to be useful for FACS, MACS and immunocytochemistry. In addition, some VHH were found to specifically activate Vγ9Vδ2-T cells. Besides being of possible immunotherapeutic value, these single domain antibodies will be of great value in the further study of this important immune effector cell subset., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Over the past decades advances in bioengineering and expanded insight in tumor immunology have resulted in the emergence of novel bispecific antibody (bsAb) constructs that are capable of redirecting immune effector cells to the tumor microenvironment. (Pre-) clinical studies of various bsAb constructs have shown impressive results in terms of immune effector cell retargeting, target dependent activation and the induction of anti-tumor responses. This review summarizes recent advances in the field of bsAb-therapy and limitations that were encountered. Furthermore, we will discuss potential future developments that can be expected to take the bsAb approach successfully forward., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Vγ9Vδ2-T cells constitute a proinflammatory lymphocyte subpopulation with established antitumor activity. Phosphoantigens activate Vγ9Vδ2-T cells in vivo and in vitro. We studied whether the antitumor activity of Vγ9Vδ2-T cells can be potentiated by invariant NKT cells (iNKT), an important immunoregulatory T cell subset. When activated by the glycolipid α-galactosylceramide (α-GalCer), iNKT produce large amounts of cytokines involved in antitumor immune responses. Monocyte-derived dendritic cells were loaded with both phosphoantigens (using aminobisphosphonates) and α-GalCer during maturation and subsequently co-cultured with Vγ9Vδ2-T and iNKT cells. Aminobisphosphonates dose-dependently enhanced Vγ9Vδ2-T cell activation, and this was potentiated by α-GalCer-induced iNKT co-activation. iNKT co-activation also enhanced the IFN-γ production and cytolytic potential of Vγ9Vδ2-T cells against tumor cells. Using transwell experiments and neutralizing antibodies cross-talk between iNKT and Vγ9Vδ2-T cells was found to be mediated by TNF-α. Our data provide a rationale for combining both activating ligands to improve Vγ9Vδ2-T cell based approaches in cancer-immunotherapy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies have been successfully used, such as cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In our study, we aimed to improve the efficacy of these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single biparatopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or cetuximab to EGFR and that did not compete for each others' binding. A combination of nanobodies from both epitope groups into the biparatopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this biparatopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, monospecific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of biparatopic nanobody-based anticancer therapeutics may yield potent lead molecules for further development., (Copyright © 2011 UICC.)