Markus Eser

RECRUIT-TandAbs®: harnessing the immune system to kill cancer cells

Tandem diabodies (TandAbs) are tetravalent bispecific molecules comprised of antibody variable domains with two binding sites for each antigen. RECRUIT-TandAbs can simultaneously engage an immune system effector cell, such as a natural killer cell or a cytotoxic T cell, and an antigen expressed specifically on a cancer cell, thus leading to killing of the cancer cell. Recruitment of immune effector cells is highly specific and mediated via binding of the TandAb to molecules expressed on the surface of these cells. Furthermore, the absence of an Fc domain allows TandAbs to avoid certain IgG-mediated side effects. With a molecular weight of approximately 110 kDa, TandAbs are far above the first-pass renal clearance limit, offering a pharmacokinetic advantage compared with smaller bispecific antibody formats. This article reviews the RECRUIT-TandAb technology and the therapeutic potential of these molecules.

Trispecific antibodies for CD16A-directed NK cell engagement and dual-targeting of tumor cells

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.

Abstract 4624: High affinity CD3 RECRUIT-TandAbs for T cell-mediated lysis of malignant CD19+ B cells

The TandAb technology enables the development of innovative antibody therapeutics for improved treatment of various diseases. This platform has been primarily applied to oncology and comprises of CD3 RECRUIT- and CD16A RECRUIT-TandAbs for activation of T and NK effector cells, respectively, and lysis of target cells expressing specific surface antigens. The CD3 RECRUIT-TandAb AFM11 is a human bispecific tetravalent antibody with two binding sites for the α-chain of CD3 and two binding sites for CD19. CD19 is expressed at early stages of B cell development and persists until the final differentiation into plasma cells. Thus, CD19 represents an attractive target for the treatment of various B cell malignancies including leukemias and lymphomas that lack CD20 expression or are refractory to anti-CD20 antibody therapies. In vitro cytotoxicity assays employing tumor cell lines demonstrate high potency of AFM11 in mediating target cell lysis: EC50 values are in the low to sub-picomolar range depending on CD19+ target cell lines and primary B-CLL tumor cells. Cytotoxic activity of tetravalent AFM11 is superior to those of alternative bivalent antibody formats. High affinity binding of AFM11 to CD3 and CD19 (low to sub-nanomolar range) contributes to efficacious T cell recruitment. This binding was determined by flow cytometry and by a label-free biosensor that measures real-time binding kinetics on live cells. Therapeutic recruitment of T cells has a potential for a devastating cytokine release if it results not in a site-specific but rather systemic activation. Thus, functional in vitro assays were used to demonstrate that high affinity and bivalent binding of AFM11 to CD3 is not sufficient for triggering activation signals in T cells. AFM11 activates T cells only in the presence of CD19+ target cells, and specifically mediates lysis of these cells without affecting antigen-negative bystander cells. In the absence of target cells and at high concentrations AFM11 induces down-modulation of the CD3/TCR complex from the surface of T cells. The latter may contribute additional safety to therapeutic potential of CD3 RECRUIT-TandAbs without affecting the efficacy, since AFM11-treated T cells can be re-engaged for target cell lysis. In vivo AFM11 demonstrated a robust dose-dependent inhibition of tumor growth in a study with Raji xenografts in NOD/scid mice and reconstituted human PBMC. Altogether, these data suggest that AFM11 is a highly efficacious novel drug candidate for the treatment of hematological malignancies and displays an advantageous safety profile. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4624. doi:1538-7445.AM2012-4624

Abstract 2643: A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnTo harness the cytotoxic capacity of T cells for the treatment of EGFRvIII+ cancers, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 (or CD16A) and EGFRvIII, the EGFRvIII/CD3 (and EGFRvIII/CD16A) RECRUIT-TandAb. EGFR dysregulation has been linked to numerous cancers, and both small molecules and EGFR targeting antibodies have successfully reached the clinic. The deletion variant III (EGFRvIII), the most common mutant form of EGFR is expressed exclusively in cancer tissues contributing to oncogenic transformation. The restricted EGFRvIII expression on various solid tumor types provides an opportunity to develop cytotoxic antibodies that solely target cancer, sparing normal tissues, and substantially reduce the side effects associated with EGFR therapy.nnUsing phage display libraries, we identified scFvs that selectively bound to the mutated and not the native form of EGFR. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies; hence we engineered a panel of bispecific EGFRvIII/CD3 TandAbs, capable of T cell-recruitment, with a broad range of binding and cytotoxic properties. The TandAbs binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. They exhibited exquisite specificity towards EGFRvIII in ELISA and FACS assays, with no wild-type EGFR binding observed, up to the maximally evaluated TandAb concentration of 1 μM. The most potent TandAbs displayed cytotoxicity towards EGFRvIII expressing F98 glioma and CHO cells with EC50 = 25 pM. We also assayed the cytotoxicity of these TandAbs towards EGFR+ cells as a more sensitive probe of residual binding to the native form. No cytotoxicity was observed up to the maximally evaluated TandAb concentration of 0.5 μM. The cytotoxic potency of the EGFRvIII/CD3 TandAb was higher than that of a comparator bivalent bispecific antibody, constructed from different anti-EGFRvIII/CD3 moieties or of EGFRvIII/CD16A TandAbs, which recruit NK cells instead of cytotoxic T cells. EGFRvIII/CD3 TandAbs with high affinity binding to CD3 were most efficacious for T cell recruitment and tumor cell killing and yet, in the absence of EGFRvIII+ target cells in vitro, TandAbs did not elicit T cell activation, as measured by their lack of proliferation, contributing to a good preclinical safety profile. In vivo EGFRvIII/CD3 TandAbs demonstrated a robust dose-dependent growth retardation of EGFRvIII+ subcutaneous Xenograft tumors in NOD/scid mice reconstituted with human PBMC; this tumor growth inhibition was more pronounced than that associated with cetuximab. In summary, our data demonstrate the strict specificity and high potency of the anti-tumor cytotoxicity mediated by the EGFRvIII/CD3 TandAb, a novel highly potent drug candidate for the treatment of EGFRvIII+ malignancies.nnCitation Format: Eugene A. Zhukovsky, Kristina Ellwanger, Uwe Reusch, Markus Eser, Fionnuala McAleese, Ivica Fucek, Carmen Burkhardt, Stefan Knackmuss. A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2643. doi:10.1158/1538-7445.AM2014-2643

Abstract C163: A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers.

To harness the cytotoxic capacity of T cells for the treatment of EGFRvIII+ cancers, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 and EGFRvIII, the EGFRvIII/CD3 RECRUIT-TandAb. EGFR dysregulation has been linked to numerous cancers, and both small molecules and EGFR targeting antibodies have successfully reached the clinic. However, the antibodies approved for clinical use, and those in development, all share a severe side effect profile due to the broad normal tissue-expression of EGFR. The deletion variant III of EGFR (EGFRvIII), with truncated extracellular domain and ensuing ligand-independent constitutive activity, is the most common mutant form associated with oncogenic transformation. EGFRvIII is expressed exclusively in cancer tissues and is associated with various solid tumor types. The restricted EGFRvIII expression on cancer cells provides an opportunity to develop cytotoxic antibodies that solely target cancer, sparing normal tissues, and substantially reduce the side effects associated with EGFR therapy. Using phage display libraries, we identified anti-EGFRvIII antibodies that selectively bound to the mutated and not the native form of EGFR. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies; hence we engineered a panel of bispecific EGFRvIII/CD3 TandAbs, capable of T cell-recruitment, with a broad range of binding and cytotoxic properties. The TandAbs’ binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. They exhibited exquisite specificity towards EGFRvIII in ELISA and FACS assays, with no wild-type EGFR binding observed, up to the maximally evaluated TandAb concentration of 1 μM. The most potent TandAbs displayed cytotoxicity towards EGFRvIII expressing F98 glioma and CHO cells with EC50 = 25 pM. We also assayed the cytotoxicity of these TandAbs towards EGFR+ cells as a more sensitive probe of residual binding to the native form. No cytotoxicity was observed up to the maximally evaluated TandAb concentration of 0.5 μM. The cytotoxic potency of the EGFRvIII/CD3 TandAb was higher than that of a comparator bivalent bispecific antibody, constructed from different anti-EGFRvIII/CD3 moieties; moreover, the comparator was not strictly specific for EGFRvIII and had residual cytotoxicity, EC50 = 1 nM, towards EGFR+ cell lines. High affinity binding to CD3 was essential for efficacious T cell recruitment and yet, in the absence of EGFRvIII+ target cells in vitro, TandAbs did not elicit T cell activation, as measured by their lack of proliferation, contributing to a good preclinical safety profile. In summary, our data demonstrate the strict specificity and high potency of the anti-tumor cytotoxicity mediated by the EGFRvIII/CD3 TandAb, a novel highly potent drug candidate for the treatment of EGFRvIII+ malignancies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C163. Citation Format: Eugene A. Zhukovsky, Kristina Ellwanger, Uwe Reusch, Markus Eser, Ivica Fucek, Carmen Burkhardt. A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C163.

Abstract 1243: Bispecific TandAbs: a safe and potent platform for T cell-mediated killing of CD19+ cells.

CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for therapeutic antibodies that treat B cell malignancies. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies. The TandAb technology enables the development of bispecific antibody therapeutics for oncology indications that harness the cytotoxic nature of T cells. The CD3 RECRUIT-TandAb AFM11, a human bispecific tetravalent antibody with two binding sites for both CD3 and CD19, exhibits highly potent cytotoxic activity and possesses pharmacokinetic properties that are considerably improved relative to alternative bispecific formats. In vitro assays demonstrate high affinity binding of AFM11 to CD3 + and CD19 + cells leading to higher potency and efficacy of T cell recruitment and target cell lysis relative to alternative bispecific antibody formats(tandem scFv, diabody). Cytotoxicity assays show that AFM11 mediates target cell lysis by CD4 + and CD8 + T cells with EC 50 values in the low to sub-picomolar range that is independent of the target cell CD19 density. AFM11 exhibits nearly identical cytotoxicity across a broad range of effector-to-target ratios, from 5:1 to 1:5, and facilitates serial target cell killing by T cells. Binding of antibodies to CD3 on T cells may elicit devastating cytokine release if it results in systemic rather than target-specific activation of T cells. Indeed, the anti-CD3 antibody OKT3 exhibits high toxicity in humans due to non-specific activation of T cells. Thus, in vitro assays were used to assess AFM11 off-target activity. AFM11 activates T cells inly in the presence of CD19 + cells, resulting in induction of CD69 and CD25 expression and the on-target release of IFN-γ, TNF-α, IL-2, and IL-6. However, depletion of CD19 + cells from PBMC cultures completely abrogates AFM11-mediated activation marker expression, T cell proliferation, and cytokine release, and supports strict CD19 + target-dependent T cell activation. Conversely, OKT3, produced cytokine release and T cell proliferation in all cultures except purified T cells. AFM11-mediated target cell lysis is specific for and restricted to CD19 + target cells since CD19 − bystanders were not lysed in assays with mixed target populations. Binding of AFM11 to CD3 also did not induce anergy since its incubation with T cells for several days did not inhibit their potent cytotoxicity. AFM11 demonstrated a robust dose-dependent inhibition of growth of Raji tumor xenografts in NOD/scid mice reconstituted with human PBMC. Moreover, a single dose of AFM11 resulted in tumor growth inhibition similar to that produced by 5 daily injections. Altogether, these data suggest that AFM11 is a highly efficacious novel drug candidate for the treatment of hematological malignancies with advantages in both safety profile and anticipated dosing regimen. Citation Format: Eugene A. Zhukovsky, Uwe Reusch, Carmen Burkhardt, Stefan Knackmuss, Ivica Fucek, Markus Eser, Fionnuala McAleese, Kristina Ellwanger. Bispecific TandAbs: a safe and potent platform for T cell-mediated killing of CD19 + cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1243. doi:10.1158/1538-7445.AM2013-1243