3.1. Anti-CD16 Bispecific Antibodies
NK cells are innate effector immune cells playing a role in eliminating malignant cells and pathogens. They play a role in early cancer killing and immunosurveillance [49
]. NK cell infiltration within tumor is a good prognosis in cancer patients [50
]. They mediate direct killing of tumor cells independently of antigen presentation. NK cells are also important mediators of the adaptive immune response through cytokines and chemokine release, dendritic cells recruitment and T cell activation [51
CD16 (FcγRIII) is a receptor for IgG1 and IgG3 Fc fragment expressed on NK cells, macrophages and γδT cells and mediates ADCC and phagocytosis of antibody-opsonized cells. NK cell-mediated ADCC is an important factor in the success of anti-tumor antigens antibody therapies and CD16 polymorphisms with higher affinity for Fc fragment are correlated with improved response to these treatments [54
]. Fc engineering has become a major approach to improve efficiency of therapeutic antibodies [58
]. As stated previously, the presence or absence of an Fc fragment can impact the action of nanobody-based constructs [44
]. The engagement of CD16 also activates NK cell proliferation and functions independently of the ADCC effect through PI3K/MAPK pathways, without the need of a costimulatory signal [59
]. The use of CD16 × TAA bispecific antibodies rather than Fc fragments was proposed to avoid binding to other Fc receptors, and sensitivity to CD16 polymorphisms. C21, A nanobody with high affinity for CD16 (10 nM, as determined by Biacore) was able to induce IL-2 and IFNγ secretion by NK cells in vitro after multimerization by sdAb biotinylation and incubation with streptavidin [61
]. Different bispecific formats using the C21 nanobody are being developed to achieve this dual effect to improve tumor targeted therapy and allow a better recruitment of NK cells.
: The smallest bispecific nanobody format consists in a tandem of two nanobodies linked head to tail by a short peptide linker. Wang et al. constructed two tandems based on the anti-CD16 nanobody C21 and an anti-MUC-1 nanobody with a 2 amino acid linker (GS) [62
] or an anti-CEA nanobody with a larger linker ((G4
]. The major interests of this format are an effective and low-cost production in bacterial systems and a high stability compared to classical scFvs. In both studies, the authors observed a high in vitro cytotoxicity and in vivo tumor growth inhibition in NOD/SCID mice xenografted with MUC-1+
colon carcinoma line LS174T, humanized by PBMC injection and treated daily with bispecific antibody injections. Therapeutic molecules based on this format would thus require PEGylation or the addition of an anti-albumin binding domain to increase their serum half-life, or the use of infusion pumps to deliver a constant flow rate, such as those used for blinatumomab in the clinic.
: The so-called bsFab format, combining the C21 nanobody with an anti-CEA nanobody through CH1-Cκ heterodimerization motif was developed [64
]. The team showed that albeit the bsFab alone could not activate CD16-transfected Jurkat cells, it could induce IL-2 and IFN-γ secretion through CD16 clustering in the presence of CEA+
colon carcinoma cells (LS174T). The bsFab showed potent in vitro NK cytotoxicity against CEA+
cancer cells independently of CD16 polymorphisms, leading to tumor growth inhibition in vivo. Such bispecific formats might constitute a promising approach to specifically activate NK cells within tumor tissues. A similar construct using C21 nanobody with an anti-Her2 nanobody was compared to Trastuzumab [65
]. The anti-Her2 bsFab showed increased cytokine release in vitro and similar tumor growth inhibition in vivo on Her2+
breast cancer cell lines SK-BR-3 and BT 474. Interestingly the bsFab also led to NK cell activating and tumor growth inhibition using the trastuzumab-refractive Her2low
cell line MCF-7 model.
: A bispecific CD16 × CEA was generated using anti-CD16 and anti-CEA nanobodies linked to two different mutants of an IgG1 to produce a bispecific Fc via the knob-into-hole technology [66
]. This construct resulted in tumor growth inhibition in mice models. Unfortunately, the half-life of this molecule was not assessed in this study but can be expected to be higher than bsFab or tandem formats.
: Wang et al. used the S-Fab format by combining an anti-CD16 Nanobody with the Fab of the anti-Her2 mAb Trastuzumab [67
] or Pertuzumab [68
]. The authors compared the efficiency of both constructs and showed that the pertuzumab-based construct was efficient at lower doses, but both induced potent tumor cell killing in vitro and reduced tumor growth in vivo. Interestingly the 2 antibodies used together seemed to synergize on Her2med
cell line LS174T. An anti-Glypican 3 (GPC3) × anti-CD16 antibody was developed using the same S-Fab format. GPC3 is a tumor antigen overexpressed in hepatocellular carcinomas (HCC) with low normal tissue expression. In this video article, the authors described the process of production of this antibody and showed an effective in vitro cytotoxicity effect on HCC cell lines [69