To harness the potent tumor-killing capacity of T cells for the treatment of CD19 C malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19 C cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly targetdependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19 C malignancies with an advantageous safety risk profile and anticipated dosing regimen.

Bispecific antibodies that redirect the lytic activity of cytotoxic immune effector cells, such as T- and NK cells, onto tumor cells have emerged as a highly attractive and clinically validated treatment modality for hematological malignancies. Advancement of this therapeutic concept into solid tumor indications, however, is hampered by the scarcity of targetable antigens that are surface-expressed on tumor cells but demonstrate only limited expression on healthy tissues. To overcome this limitation, the concept of dual-targeting, i.e. the simultaneous targeting of two tumor-expressed surface antigens with limited co-expression on non-malignant cells, with multispecific antibodies has been proposed to increase tumor selectivity of antibody-induced effector cell cytotoxicity. Here, a novel CD16A (FcγRIIIa)-directed trispecific, tetravalent antibody format, termed aTriFlex, is described, that is capable of redirecting NK cell cytotoxicity to two surface-expressed antigens. Using a BCMA/CD200-based in vitro model system, the potential use of aTriFlex antibodies for dual-targeting and selective induction of NK cell-mediated target cell lysis was investigated. Bivalent bispecific target cell binding was found to result in significant avidity gains and up to 17-fold increased in vitro potency. These data suggest trispecific aTriFlex antibodies may support dual-targeting strategies to redirect NK cell cytotoxicity with increased selectivity to enable targeting of solid tumor antigens.

To harness the cytotoxic capacity of immune effector cells for the treatment of several types of solid tumors, we developed tetravalent bifunctional antibodies that recognize EGFRvIII, the deletion variant III of EGFR. Their second functionality binds with high affinity to CD3, thereby directing T-cells to eliminate EGFRvIII+ cancer cells. The expression of EGFRvIII on various solid tumor types, and its absence from healthy tissues, provides an opportunity to develop cytotoxic antibodies that solely target cancer; these would spare normal tissues, and substantially reduce the side effects associated with EGFR therapy. Using phage display, we identified scFvs that selectively bind to EGFRvIII but not to EGFR. The binding affinities of these highly EGFRvIII-specific antibodies were substantially improved, employing affinity maturation techniques, and achieved KDs in the 100pM range and lower. We engineered a panel of bispecific EGFRvIII/CD3 TandAbs with a broad range of binding and cytotoxic properties. Mono- and bivalent binding, specificity for both EGFRvIII and CD3, T cell-mediated cytotoxic activity, and target-mediated T-cell activation were characterized in a panel of in vitro assays. EGFRvIII/CD3 TandAbs exhibit exquisite specificity towards the EGFRvIII antigen in Western Blot, SPR, ELISA, and FACS assays of EGFRvIII+ cells. No specific binding was observed to recombinant EGFR antigen or to EGFR-expressing cells. Apparent affinities of EGFRvIII/CD3 TandAbs to EGFRvIII were up to 25fold improved relative to the monovalently binding scFvs, and achieved a KD of 11pM for the best binding EGFRvIII/CD3 TandAb. Improvement of the binding affinities in the bivalent TandAb format was largely due to slower dissociation. TandAbs with high affinity for EGFRvIII were most potent in killing assays, displaying cytotoxicity towards EGFRvIII-expressing F98 glioma and CHO cells with EC50 in the range of 1pM-10pM. No cytotoxicity was observed on EGFR+ cells or EGFRvIII- cells up to the maximally-evaluated TandAb concentration of 0.5μM (which is 100000 fold higher than the EC50) demonstrating the high selectivity of EGFRvIII/CD3 TandAbs for the tumor-specific EGFRvIII. High affinity binding to CD3 was necessary for efficacious T cell recruitment as shown by the correlation of CD3-binding and cytotoxic potency of EGFRvIII/CD3 TandAbs. Importantly, in the absence of EGFRvIII+ target cells in vitro, TandAbs did not elicit T cell activation, as demonstrated by their lack of proliferation: this specificity contributed to a good preclinical safety profile. Biophysical and pharmacological characterization of several candidates is currently ongoing, whereby the first EGFRvIII/CD3 TandAbs demonstrated a robust dose-dependent growth retardation of EGFRvIII+ subcutaneous xenograft tumors. In summary, EGFRvIII/CD3 TandAbs are specific and highly potent drug candidates for the treatment of EGFRvIII+ malignancies. Citation Format: Kristina Ellwanger, Uwe Reusch, Ivica Fucek, Michael Weichel, Carmen Herbrecht, Stefan Knackmuss, Erich Rajkovic, Vera Molkenthin, Eugene A. Zhukovsky. EGFRvIII T-cell TandAbs are specific and highly potent drug candidates for the treatment of a variety of solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2015-2471