Abstract
To harness the cytotoxic capacity of immune cells for the treatment of solid tumors, we developed tetravalent, bispecific tandem diabody (TandAb) antibodies that recognize EGFRvIII, the deletion variant III of the epidermal growth factor receptor (EGFR), and CD3 on T-cells, thereby directing immune cells to eliminate EGFRvIII-positive tumor cells. Using phage display, we identified scFv antibodies selectively binding to EGFRvIII. These highly EGFRvIII-specific, fully human scFv were substantially improved by affinity maturation, achieving KDs in the picomolar range, and were used to construct a set of bispecific EGFRvIII-targeting TandAbs with a broad range of binding and cytotoxic properties. These antibodies exhibited an exquisite specificity for a distinguished epitope in the N-terminal portion of EGFRvIII, as shown on recombinant antigen in Western Blot, SPR, and ELISA, as well as on antigen-expressing cells in FACS assays, and did not bind to the wild-type EGFR. High-affinity EGFRvIII/CD3 TandAbs were most potent in killing assays, displaying cytotoxicity toward EGFRvIII-expressing CHO, F98 glioma, or human DK-MG cells with EC50 values in the range of 1–10 pM in vitro. They also demonstrated dose-dependent growth control in vivo in an EGFRvIII-positive subcutaneous xenograft tumor model. Together with the tumor-exclusive expression of EGFRvIII, the EGFRvIII/CD3 TandAbs’ high specificity and strictly target-dependent activation with no off-target activity provide an opportunity to target tumor cells and spare normal tissues, thereby reducing the side effects associated with other anti-EGFR therapies. In summary, EGFRvIII/CD3 TandAbs are highly attractive therapeutic antibody candidates for selective immunotherapy of EGFRvIII-positive tumors.
Highlights
The human epidermal growth factor receptor (HER) family consists of four members: epidermal growth factor receptor (EGFR), HER2/neu, HER3, and HER4 [1, 2]
All EGFRvIII-specific scFv were isolated from the part of the library, that was cloned from donor-derived Variable heavy chain (VH) and Variable light chain (VL) domains, featuring IgM-specific priming for the amplification of VH encoding sequences as described previously [35, 36]
Two parallel selection strategies were applied with EGFRvIII-Fc being either immobilized on a plastic surface or captured by Protein G beads after incubation with the phage library in solution
Summary
The human epidermal growth factor receptor (HER) family consists of four members: epidermal growth factor receptor (EGFR), HER2/neu, HER3, and HER4 [1, 2] In normal tissue, they are activated when specific ligands bind the extracellular domains (ECD) of EGFR, HER3, or HER4 leading to hetero- or homo-dimerization and subsequent activation of their kinase domains through conformational changes and intracellular tyrosine phosphorylation [3]. They are activated when specific ligands bind the extracellular domains (ECD) of EGFR, HER3, or HER4 leading to hetero- or homo-dimerization and subsequent activation of their kinase domains through conformational changes and intracellular tyrosine phosphorylation [3] The function of these receptors is dysregulated under pathophysiological conditions such as in colorectal cancer, TandAbs Targeting EGFRvIII-Positive Tumors head and neck cancer, lung cancer, and glioblastoma, and it is well established that this contributes to malignant transformation. While its expression has originally been described in up to 60% of glioblastoma multiforme (GBM) patients and numerous groups have found this tumor-specific mutation in other cancers such as squamous cell carcinoma of the head and neck (HNSCC), prostate, breast, or lung (NSCLC), EGFRvIII is not expressed in normal tissue, making it an ideal target for cancer therapy [11, 12]
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