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Bispecific T cell engagers (bsTCEs) hold great promise for cancer treatment but face challenges due to the induction of cytokine release syndrome (CRS), on-target off-tumor toxicity, and the engagement of immunosuppressive regulatory T cells that limit efficacy. The development of Vγ9Vδ2-T cell engagers may overcome these challenges by combining high therapeutic efficacy with limited toxicity. By linking a CD1d-specific single-domain antibody (VHH) to a Vδ2-TCR-specific VHH, we create a bsTCE with trispecific properties, which engages not only Vγ9Vδ2-T cells but also type 1 NKT cells to CD1d+ tumors and triggers robust proinflammatory cytokine production, effector cell expansion, and target cell lysis in vitro. We show that CD1d is expressed by the majority of patient MM, (myelo)monocytic AML, and CLL cells and that the bsTCE triggers type 1 NKT and Vγ9Vδ2-T cell-mediated antitumor activity against these patient tumor cells and improves survival in in vivo AML, MM, and T-ALL mouse models. Evaluation of a surrogate CD1d-γδ bsTCE in NHPs shows Vγ9Vδ2-T cell engagement and excellent tolerability. Based on these results, CD1d-Vδ2 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML.

Despite recent advances in treatment of B-acute lymphoblastic leukemia (B-ALL) there is still a need for novel targeted therapies. The tetraspanin CD9 is expressed in 60-80% of B-ALL and correlates with adverse prognosis. Recently, the mouse CD9 antibody ALB6 was shown to induce leukemia rejection in NOD/SCID mice. However, clinical development of ALB6 and other CD9-targeting antibodies was hampered by their CD9 mediated induction of platelet aggregation. It is known that CD9 is still expressed on tumor cells after treatment with chemotherapy or blinatumomab (Leung, 2019; Linder, 2016). AT1412 is a fully human antibody isolated from B cells of a patient that was cured from stage IV metastatic melanoma (Verdegaal, 2011). AT1412 targets CD9, without inducing platelet aggregation in vitro or thrombosis in cynomolgus monkeys after intravenous administration at therapeutic dose levels. By crystallography AT1412 was shown to bind a unique epitope preventing homodimerization of CD9, distinctly different from other CD9 antibodies. AT1412 binds a majority of patient B-ALL samples, but not T-ALL and induces ADCC and ADCP of CD9 positive B-ALL primary cells and the level of cytotoxicity significantly correlated with that of AT1412 binding. In both NSG and immunodeficient mice harboring a human immune system (HIS mice) AT1412 demonstrated a strong, dose-dependent tumor rejection of B-ALL, most pronounced in the extramedullary sites. In HIS mice AT1412 treatment led to an accumulation of T cells and CD14+ myeloid cells at the tumor sites. To support clinical development, pre-clinical safety of AT1412 was evaluated in cynomolgus monkeys. AT1412 demonstrated a half-life of 8.5 days, supporting 2-3 weekly administration in humans. Besides transient thrombocytopenia no other pathological deviations were observed. No effect on coagulation parameters, bruising or bleeding were observed macro- or microscopically. The thrombocytopenia is reversible, and its recovery accelerated in those animals developing anti-drug antibodies. Taken together, we demonstrate that CD9 on B-ALL cells can be successfully targeted by AT1412. AT1412 targets a unique epitope and does not induce thrombosis. Pre-clinical safety assessment is supporting that AT1412 can be safely administered. A First in Human clinical study is scheduled to start early 2021 in human solid tumors to determine safety and efficacy. AT1412 efficacy will be evaluated in B-ALL in expansion cohorts. Leung, K.T., Zhang, C., Chan, K.Y.Y., Li, K., Cheung, J.T.K., Ng, M.H.L., Zhang, X.-B., Sit, T., Lee, W.Y.W., Kang, W., et al. (2019). CD9 blockade suppresses disease progression of high-risk pediatric B-cell precursor acute lymphoblastic leukemia and enhances chemosensitivity. Leukemia. Linder, K., Gandhiraj, D., Hanmantgad, M., Seiter, K., and Liu, D. (2016). Complete remission after single agent blinatumomab in a patient with pre-B acute lymphoid leukemia relapsed and refractory to three prior regimens: HyperCVAD, high dose cytarabine mitoxantrone and CLAG. Exp. Hematol. Oncol. 5, 5-8. Verdegaal, E.M.E., Visser, M., Ramwadhdoebé, T.H., van der Minne, C.E., van Steijn, J. a Q.M.J., Kapiteijn, E., Haanen, J.B. a G., van der Burg, S.H., Nortier, J.W.R., and Osanto, S. (2011). Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha. Cancer Immunol. Immunother. 60, 953-963. Disclosures Schotte: AIMM Therapeutics: Current Employment, Current equity holder in private company. Villaudy:AIMM Therapeutics: Current Employment, Current equity holder in private company. Levie:AIMM Therapeutics: Current Employment, Current equity holder in private company. Go:AIMM Therapeutics: Current Employment, Current equity holder in private company. Yasuda:AIMM Therapeutics: Current Employment, Current equity holder in private company. Frankin:AIMM Therapeutics: Current Employment, Current equity holder in private company. Cercel:AIMM Therapeutics: Current Employment, Current equity holder in private company. van Hal-van Veen:AIMM Therapeutics: Current Employment, Current equity holder in private company. van de Berg:AIMM Therapeutics: Current Employment, Current equity holder in private company. Fatmawati:AIMM Therapeutics: Current Employment, Current equity holder in private company. Kedde:AIMM Therapeutics: Current Employment, Current equity holder in private company. Claassen:AIMM Therapeutics: Current Employment, Current equity holder in private company. Rijneveld:Amgen: Research Funding; Servier: Research Funding. Spits:AIMM Therapeutics: Current equity holder in private company, Ended employment in the past 24 months, Membership on an entity's Board of Directors or advisory committees. van Eenennaam:AIMM Therapeutics: Current Employment, Current equity holder in private company.

DuoBody®-CD3xCD20 (GEN3013) is a bispecific antibody (bsAb), recognizing the T-cell antigen CD3 and the B-cell antigen CD20, that triggers potent T-cell-mediated lysis of CD20-expressing cells. DuoBody-CD3xCD20 is a full-length bispecific IgG1 generated by controlled Fab-arm exchange (cFAE) [1, 2] and contains an effector function-silenced Fc region. In vitro, DuoBody-CD3xCD20 induced potent activation, proliferation and cytotoxic activity of both CD4+ and CD8+ T cells in the presence of CD20-expressing cells, as measured by flow cytometry and bromodeoxyuridine (BrdU) incorporation assays. DuoBody-CD3xCD20 induced T-cell-mediated cytotoxicity towards a diverse panel of cell lines derived from various B-cell malignancies and endogenous B cells, with EC50 values in the low picomolar range (EC50: 0.2-5.0 pM). The CD20-specific antibody 7D8 [3-5] forms the basis for the CD20-specific Fab arm of DuoBody-CD3xCD20. To study the contribution of this specific Fab arm to the observed potency of DuoBody-CD3xCD20, we compared the target binding characteristics and the capacity to induce T-cell-mediated cytotoxicity of a CD3 bsAb based on 7D8, with CD3 bsAbs using B-cell targeting arms derived from alternative CD20 antibodies or from antibodies against other well-known B-cell membrane molecules CD22, CD24, CD37, CD70, CD79b, CD138 and HLA-DR. In addition, target expression levels of the B-cell targets were assessed in a panel of B-cell lines. Using a classic chromium release assay, the 7D8-based CD3 bsAb displayed cytotoxic activity superior to all other B-cell-targeting CD3 bsAbs tested, including alternative CD20-targeting CD3 bsAbs. This unique cytotoxic activity could not be explained by expression levels of the target antigen, nor by the binding affinity or epitope of the B-cell specific Fab arm. This illustrates the complexity of factors that determine the potency of CD3 bsAbs. The anti-tumor activity of DuoBody-CD3xCD20 was confirmed in vivo in humanized mouse models using three different B-cell lymphoma xenograft models, in prophylactic and therapeutic settings. Non-clinical safety studies with DuoBody-CD3xCD20 in cynomolgus monkeys demonstrated profound and long-lasting B-cell depletion (at least 70 days, at dose levels > 0.1 mg/kg) from both peripheral blood and lymphoid organs. B-cell depletion was reversible, with time to B-cell recovery correlating with the treatment dose. Notably, at the same dose level, B-cell depletion was comparable between subcutaneous and intravenous administration. Pharmacokinetic (PK) analysis demonstrated comparable bioavailability for the two administration routes, although peak plasma levels were lower and delayed after subcutaneous administration. Moreover, lower plasma cytokine levels were observed after subcutaneous administration. Based on these data, Genmab has initiated a First-in-Human clinical trial to evaluate the safety and preliminary efficacy of DuoBody-CD3xCD20 by subcutaneous administration in patients with B-cell malignancies. The study is currently enrolling (EudraCT No: 2017-001748-36). References Labrijn, A.F., et al., Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proc Natl Acad Sci U S A, 2013. 110(13): p. 5145-50. Labrijn, A.F., et al., Controlled Fab-arm exchange for the generation of stable bispecific IgG1. Nat Protoc, 2014. 9(10): p. 2450-63. Teeling, J.L., et al., Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood, 2004. 104(6): p. 1793-800. Teeling, J.L., et al., The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique Epitopes on CD20. Journal of Immunology, 2006. 177(1): p. 362-71. van Meerten, T., et al., HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20 low expressing tumor cells that resist rituximab-mediated lysis. Haematologica, 2010. 95(12): p. 2063-71. Disclosures Hiemstra: Genmab: Employment, Other: Warrants. Engelberts:Genmab: Employment, Other: Warrants. de Jong:Genmab: Employment, Other: Warrants. Schuurhuis:Genmab: Employment, Other: Warrants. Salcedo:Genmab: Employment, Other: Warrants. Verploegen:Genmab: Employment, Equity Ownership. van der Zee:Genmab: Employment, Other: Warrants. Gerritsen:Genmab: Employment, Other: Warrants. Losic:Genmab: Employment, Other: Warrants. Horbach:Genmab: Employment, Other: Warrants. Oliveri:Genmab: Employment, Other: Warrants. Lammerts van Bueren:Genmab: Employment, Other: Warrants. Autzen Usher:Genmab: Employment, Other: Warrants. Schuurman:Genmab: Employment, Other: Warrants. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Breij:Genmab: Employment, Equity Ownership.