Highly Specific and Effective Targeting of EGFRvIII-Positive Tumors with TandAb Antibodies

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.

A Rational Designed Novel Bispecific Antibody for the Treatment of GBM

“…In our in vivo animal experiments, we used immunodeficient mice to imitate the immune-cell-infiltrating tumor microenvironment by reestablishing their immune systems with mixed huPBMCs and tumor cells, based on previous studies [28,37,38]. The tumor inhibitory ability of EGFRvIII-BsAb was verified.…”

Lessons learned from rindopepimut treatment in patients with EGFRvIII-expressing glioblastoma

“…The negative findings align with other clinical pilot studies treating solid tumors with CAR-T cells (11), which is contrary to the success treating non-solid hematologic malignancies targeting a critical cell-surface ligand expressed by 100% of tumor cells (12). Future next-generation approaches targeting EGFRvIII, include the evaluation of bispecific antibodies specific to both EGFRvIII and CD3 (13). Overall, interest remains in the testing of next-generation mAb approaches for EGFRvIII and EGFRwt targeting, although a global target that would provide universal GBM targetability remains elusive.…”

Single-Chain Variable Fragment-Based Bispecific Antibodies: Hitting Two Targets with One Sophisticated Arrow

“…Due to this deletion, EGFRvIII is constitutively active in a ligand-independent fashion, and its aberrant signaling is associated with glioma growth and progression 53 . Two different groups individually developed TandAb and BiTE molecules targeting EGFRvIII and CD3 54, 55. The particular feature of both bsAbs is their EGFRvIII-specific scFvs.…”

“…The particular feature of both bsAbs is their EGFRvIII-specific scFvs. By the phage display technology and fully human antibody libraries, Ellwanger et al 54 . isolated a low-affinity scFv antibody, Li3G30, identifying EGFRvIII.…”

“…Furthermore, they demonstrated that EGFRvIII×CD3 TandAbs with high-affinity binding to CD3 did not activate T cells co-cultured with EGFRvIII-negative cells. In a dose-dependent manner, the TandAbs could inhibit the tumor growth in a tumor xenograft model derived from the EGFRvIII-positive cell line 54 …”