For the effective formation of immune synapses resulting in T-cell-mediated tumor cell lysis, the scDb/scFv-Fc format with small interdomain spacing in the (1-2)+1?and (2-1)+1 configuration showed efficient killing of medium HER3-expressing cancer cells while sparing target cells with low HER3 expression with an additional preferable safety profile due to low cytokine secretion

For the effective formation of immune synapses resulting in T-cell-mediated tumor cell lysis, the scDb/scFv-Fc format with small interdomain spacing in the (1-2)+1?and (2-1)+1 configuration showed efficient killing of medium HER3-expressing cancer cells while sparing target cells with low HER3 expression with an additional preferable safety profile due to low cytokine secretion. Acknowledgments We would like to thank Nadine Heidel and Sabine Mnkel for excellent technical assistance. Footnotes Contributors: NA, LK and OS performed cloning, protein expression and purification, biochemical analysis, binding studies and bioactivity assays. cells to determine T-cell activation, T-cell proliferation and PBMC-mediated cancer cell lysis of HER3-positive cell lines by the trivalent, bispecific antibodies. Results In this study, we developed trivalent, bispecific antibodies comprising a silenced Fc region for T-cell retargeting to HER3-expressing tumor cells, combining a bivalent single-chain diabody (scDb) fused to a first heterodimerizing Fc chain with either an Fab or scFv fused to a second heterodimerizing Fc chain. All these HER3-targeting T-cell engagers Rabbit Polyclonal to ROR2 comprising two binding sites for HER3 and one binding site for CD3 mediated target cell killing. However, format and orientation of binding sites influenced efficacy of target cell binding, target cell-dependent T-cell activation and T-cell-mediated target cell killing. Beneficial effects were seen when the CD3 binding site was located in the scDb moiety. These molecules showed efficient killing of medium HER3-expressing cancer cells with very low induction of cytokine release, while sparing target cells with low or undetectable HER3 expression. Conclusion Our study demonstrates that these trivalent, bispecific antibodies represent formats with superior interdomain spacing resulting in efficient target cell killing Indaconitin and a potential advantageous safety profile due to very low cytokine release. Keywords: immunotherapy, immunotherapy, active Background The ErbB family member HER3 has been reported to play an essential part in cancer progression, and elevated manifestation has been shown to correlate with worse overall survival.1 2 Furthermore, it has been demonstrated that upregulation of HER3 is an important resistance mechanism on epidermal growth element receptor (EGFR) and HER2-targeted therapy.3C5 More than two dozen antibodies targeting HER3 are currently investigated in preclinical trials, 6 7 mostly interfering with ligand binding and/or receptor dimerization.8 However, there is still no authorized treatment focusing on HER3. Since monoclonal antibodies9 10 as well as therapeutic methods including bispecific antibodies for dual focusing on of HER3 and another member of the EGFR family11 have not shown improved restorative activity in medical trials, restorative strategies such as HER3-directed antibody-drug conjugates12C14 have been developed, uncoupling the restorative activity from receptor signaling. Major histocompatibility complex (MHC)-self-employed crosslinking of tumor cells and T-cells by bispecific antibodies represents a rapidly expanding treatment strategy in malignancy therapies.15C17 Bispecific T-cell engagers are characterized by simultaneous binding of a tumor-associated antigen (TAA) and, in most cases, the CD3 chain of the T-cell receptor (TCR)/CD3 complex, leading to the close apposition of target and effector cell resulting in activation of the T-cell. Secretion of cytokines and cytotoxic effector proteins from the T-cell eventually results in killing of the targeted tumor cell. Bispecific T-cell engagers are an established therapeutic strategy for the treatment of hematologic malignancies, for example, blinatumomab, a small bispecific T-cell engager (BiTE) focusing on CD19 and CD3, authorized for the treatment of acute lymphoblastic leukemia.18 However, Indaconitin bispecific T-cell engagers face several challenges when it comes to their application for the treatment of solid tumors, including the attack of non-tumor cells with low expression level of Indaconitin the TAA and/or systemic cytokine-associated adverse events.16 Recent studies have shown that an avidity-mediated specificity gain through bivalent binding to the TAA can be achieved using novel formats having a 2+1 stoichiometry.19C23 Additionally, formats in the 2+1 stoichiometry circumvent unspecific or non-targeted CD3-crosslinking and T-cell activation by monovalent binding to the result in molecule CD3 on T-cells.17 Indaconitin 24C26 We have recently demonstrated that a small trivalent, bispecific single-chain diabody (scDb)-scFv showed first-class binding to target expressing tumor cells translating into more efficient target cell killing by peripheral blood mononuclear cells (PBMCs).27 Small bispecific formats such as BiTEs,28 dual-affinity re-targeting antibodies (DARTs)29 and scDb30 have been reported to mediate limited contacts between target cell and T-cells because of the small size and the short distance between the two binding sites, resulting in efficient T-cell activation. However, their pharmacokinetic properties are characterized by a very short serum half-life and continuous infusion is necessary.31 32 In the present study, trivalent, bispecific Fc-comprising anti-HER3anti-CD3 antibodies were generated by combining an scDb molecule and an scFv or Fab fragment having a silenced heterodimerizing Fc.