eIg-based bispecific T-cell engagers targeting EGFR: Format matters.

Bispecific antibodies are molecules with versatile modes of action and applications for therapy. They are commonly developed as T-cell engagers (TCE), which simultaneously target an antigen expressed by tumor cells and CD3 expressed by T-cells, thereby inducing T-cell-mediated target cell killing. There is growing evidence that the molecular composition and valency for the target antigen influence the activity of TCEs. Here, the eIg platform technology was used to generate a set of bispecific TCEs targeting epidermal growth factor receptors (EGFR) and CD3. These molecules either included or lacked an Fc region and exhibited one binding site for CD3 and either one or two binding sites for EGFR (1 + 1 or 2 + 1 formats) utilizing different molecular arrangements of the binding sites. In total, 11 different TCE formats were analyzed for binding to target cells and T cells, T cell-mediated killing of tumor cells, and for the activation of T cells (release of cytokines and proliferation of T-cells). Bivalent binding to EGFR strongly increased binding and T cell-mediated killing. However, the molecular composition and position of the CD3-binding arm also affected target cell killing, cytokine release, and T-cell proliferation. Our findings support that screening of a panel of formats is beneficial to identify the most potent bispecific TCE, and that format matters.

The eIg technology to generate Ig-like bispecific antibodies.

Bispecific antibodies have emerged as therapeutic molecules with a multitude of modes of action and applications. Here, we present a novel approach to solve the light-chain problem for the generation of bispecific Ig-like antibodies using the second constant domain of IgE (EHD2) genetically modified to force heterodimerization. This was achieved by introducing a C14S mutation in one domain and a C102S mutation in the other domain, which removed of one of the crossover disulfide bonds. Substituting the CH1 and CL domains of an antigen binding fragment (Fab) with these heterodimerizing EHD2 (hetEHD2) domains resulted in Fab-like building blocks (eFab). These eFabs were used to generate different bispecific antibodies of varying valency and molecular composition employing variable domains with different specificities and from different origins. Formats included bivalent bispecific IgG-like molecules (eIgs) and Fc-less Fab-eFab fusion proteins, as well as tri- and tetravalent Fab-eIg fusion proteins. All proteins, including bispecific antibodies for dual receptor targeting and for retargeting of T cells, efficiently assembled into functional molecules. Furthermore, none of the hetEHD2-comprising molecules showed binding to the two Fcε receptors and are thus most likely do not induce receptor cross-linking and activation. In summary, we established the eIg technology as a versatile and robust platform for the generation of bispecific antibodies of varying valency, geometry, and composition, suitable for numerous applications.Abbreviations: antibody drug conjugate (ADC), acute lymphocytic leukemia (ALL), constant domain of IgE (Cε), receptor of Cε (CεRI or CεRII), cluster of differentiation (CD), constant domain of heavy chain (CH), constant domain of light chain (CL), (single-chain) diabody ((sc)Db), diabody-immunoglobulin (Db-Ig), dynamic light scattering (DLS), Fragment antigen-binding (Fab), Fab with hetEHD2 (eFab), Fab-EHD2 with T121G in chain 1 and S10I in chain 2 (EFab), bispecific Ig domain containing hetEHD2 (eIg), extracellular domain (ECD), epidermal growth factor receptor 1, 2, 3 (EGFR, HER2, HER3), heavy chain domain 2 of IgE (EHD2), EHD2 domain with C102S (EHD2-1), EHD2 domain with C14S and N39Q (EHD2-2), (human or mouse) fragment crystalline ((hu or mo)Fc), heavy chain (HC), heterodimerized second domain of IgE (hetEHD2), high molecular weight (HMW), immunoglobulin (Ig), light chain (LC), liquid chromatography-mass spectrometry (LC-MS), mesenchymal epithelial transition factor (MET), heavy chain domain 2 of IgM (MHD2), peripheral blood mononuclear cell (PBMC), prolactin receptor (PRLP), Stokes radius (RS), single-chain Fragment variable (scFv), tumor necrosis factor (TNF), TNF receptor 2 (TNFR2), single-chain TNF-related apoptosis-inducing ligand (scTRAIL), variable domain of heavy chain (VH), variable domain of light chain (VL).

Fc-based Duokines: dual-acting costimulatory molecules comprising TNFSF ligands in the single-chain format fused to a heterodimerizing Fc (scDk-Fc).

Targeting costimulatory receptors of the tumor necrosis factor superfamily (TNFSF) to activate T-cells and promote anti-tumor T-cell function have emerged as a promising strategy in cancer immunotherapy. Previous studies have shown that combining two different members of the TNFSF resulted in dual-acting costimulatory molecules with the ability to activate two different receptors either on the same cell or on different cell types. To achieve prolonged plasma half-life and extended drug disposition, we have developed novel dual-acting molecules by fusing single-chain ligands of the TNFSF to heterodimerizing Fc chains (scDuokine-Fc, scDk-Fc). Incorporating costimulatory ligands of the TNF superfamily into a scDk-Fc molecule resulted in enhanced T-cell proliferation translating in an increased anti-tumor activity in combination with a primary T-cell-activating bispecific antibody. Our data show that the scDk-Fc molecules are potent immune-stimulatory molecules that are able to enhance T-cell mediated anti-tumor responses.

Fc-comprising scDb-based trivalent, bispecific T-cell engagers for selective killing of HER3-expressing cancer cells independent of cytokine release.

BACKGROUND: Bispecific T-cell engagers are an established therapeutic strategy for the treatment of hematologic malignancies but face several challenges when it comes to their application for the treatment of solid tumors, including on-target off-tumor adverse events. Employing an avidity-mediated specificity gain by introducing an additional binding moiety for the tumor-associated antigen can be achieved using formats with a 2+1 stoichiometry. METHODS: Besides biochemical characterization and validation of target cell binding to cancer cells with different HER3 expression, we used in vitro co-culture assays with human peripheral blood mononuclear cells (PBMCs) and HER3-expressing target 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 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 and a potential advantageous safety profile due to very low cytokine release.

A scDb-based trivalent bispecific antibody for T-cell-mediated killing of HER3-expressing cancer cells.

HER3 is a member of the EGF receptor family and elevated expression is associated with cancer progression and therapy resistance. HER3-specific T-cell engagers might be a suitable treatment option to circumvent the limited efficacy observed for HER3-blocking antibodies in clinical trials. In this study, we developed bispecific antibodies for T-cell retargeting to HER3-expressing tumor cells, utilizing either a single-chain diabody format (scDb) with one binding site for HER3 and one for CD3 on T-cells or a trivalent bispecific scDb-scFv fusion protein exhibiting an additional binding site for HER3. The scDb-scFv showed increased binding to HER3-expressing cancer cell lines compared to the scDb and consequently more effective T-cell activation and T-cell proliferation. Furthermore, the bivalent binding mode of the scDb-scFv for HER3 translated into more potent T-cell mediated cancer cell killing, and allowed to discriminate between moderate and low HER3-expressing target cells. Thus, our study demonstrated the applicability of HER3 for T-cell retargeting with bispecific antibodies, even at moderate expression levels, and the increased potency of an avidity-mediated specificity gain, potentially resulting in a wider safety window of bispecific T-cell engaging antibodies targeting HER3.

A bivalent, bispecific Dab-Fc antibody molecule for dual targeting of HER2 and HER3.

Dual targeting of surface receptors with bispecific antibodies is attracting increasing interest in cancer therapy. Here, we present a novel bivalent and bispecific antagonistic molecule (Dab-Fc) targeting human epidermal growth factors 2 and 3 (HER2 and HER3) derived from the Db-Ig platform, which was developed for the generation of multivalent and multispecific antibody molecules. Dab-Fc comprises the variable domains of the anti-HER2 antibody trastuzumab and the anti-HER3 antibody 3-43 assembled into a diabody-like structure stabilized by CH1 and CL domains and further fused to a human γ1 Fc region. The resulting Dab-Fc 2 × 3 molecule retained unhindered binding to both antigens and was able to bind both antigens sequentially. In cellular experiments, the Dab-Fc 2 × 3 molecule strongly bound to different tumor cell lines expressing HER2 and HER3 and was efficiently internalized. This was associated with potent inhibition of the proliferation and migration of these tumor cell lines. Furthermore, IgG-like pharmacokinetics and anti-tumoral activity were demonstrated in a xenograft tumor model of the gastric cancer cell-line NCI-N87. These results illustrate the suitability of our versatile Db-Ig platform technology for the generation of bivalent bispecific molecules, which has been successfully used here for the dual targeting of HER2 and HER3.

Inhibition of Tumor Cell Growth and Cancer Stem Cell Expansion by a Bispecific Antibody Targeting EGFR and HER3.

The frequent activation of HER3 signaling as a resistance mechanism to EGFR-targeted therapy has motivated the development of combination therapies that block more than one receptor tyrosine kinase. Here, we have developed a novel tetravalent, bispecific single-chain diabody-Fc fusion protein targeting EGFR and HER3 (also known as ErbB3) that integrates the antigen-binding sites of a humanized version of cetuximab as well as a recently developed anti-HER3 antibody, IgG 3-43. This bispecific antibody combines the binding and neutralizing properties of the parental antibodies, as observed in biochemical and in vitro two-dimensional and three-dimensional cell culture assays, and gave rise to long-lasting growth suppression in a subcutaneous xenograft head and neck tumor model. In triple-negative breast cancer (TNBC) cell lines, treatment with the bispecific antibody inhibited the proliferation and oncosphere formation efficiency driven by HER3 signaling. In an orthotopic MDA-MB-468 tumor model, this translated into antitumor effects superior to those obtained by the parental antibodies alone or in combination and was associated with a reduced number of cells with stem-like properties. These findings demonstrate that the bispecific antibody efficiently blocks not only TNBC proliferation, but also the survival and expansion of the cancer stem cell population, holding promise for further preclinical development.