Co-stimulation through 4-1BB has shown promising anti-tumor activity in preclinical models, but the development of 4-1BB agonistic antibodies in the clinic has been hampered by Fc-mediated liver toxicity. Here, we describe a novel CD19-targeted 4-1BB ligand designed to deliver a safe but potent 4-1BB agonist to effector T and NK cells with the goal to improve treatment of B cell malignancies. The antibody fusion protein is composed of split trimeric 4-1BB ligands and a tumor antigen targeting moiety recognizing CD19 fused to a silent Fc part (CD19-4-1BBL). The construct is devoid of FcgR-mediated crosslinking responsible for Fc-mediated toxicity and reintroduces 4-1BB hyperclustering upon binding to CD19 on B cells exclusively. In mice and cynomolgus monkeys, CD19-4-1BBL shows IgG-like pharmacokinetic properties. When cross-linked via CD19 on Non-Hodgkin Lymphoma cell lines or normal B cells, CD19-4-1BBL is biologically active in co-stimulating T cells. As 4-1BB is an inducible protein on activated T cells and NK cells, we combined CD19-4-1BBL with a T cell bispecific Ab targeting CD20 (CD20-TCB) to provide initial T cell activation while engaging with CD19+CD20+ tumor cells. In vitro, pre-treatment with CD20-TCB mediates the clustering of CD19-4-1BBL molecules to the interaction synapse of T cells and tumor cells. Live imaging revealed that the addition of CD19-4-1BBL induces significantly prolonged T cell-tumor contact (>30 min), leading to fast and efficient killing of tumor cells. In vivo in WSU-DLCL2-bearing human stem cell engrafted NSG mice (HSC-NSG) mice, CD20-TCB treatment quickly up-regulates 4-1BB on activated T cells, resulting in tumor growth inhibition. The combination of CD19-4-1BBL and CD20-TCB (used at a suboptimal dose) synergizes to eradicate the tumor completely. The combination induces strong T cell infiltration into the tumor, accompanied by an elevated CD8/Treg ratio, as compared to the monotherapies. Similarly, the combination also induces complete remission in a “difficult-to-treat” Nalm6 tumor model associated with low and patchy CD20 expression, mimicking patterns seen in the ABC subtype of DLBCL. Finally, the combination of CD19-4-1BBL and the ADCC-enhanced Type II CD20 antibody obinutuzumab induces complete tumor remissions in WSU-DLCL2 tumor-bearing HSC-NSG mice, confirming effective 4-1BB co-stimulation on NK cells. Taken together, tumor-targeted cross-linking of 4-1BB mediated by CD19-4-1BBL provides safe and efficient co-stimulation of T cells that are pre-activated by a TCB, or of NK cells pre-activated by an ADCC mediating antibody. This novel and effective combination immunotherapy warrants clinical investigation and offers possible “chemo-free” treatment of B cell malignancies.
Citation Format: Wei Xu, Johannes Sam, Mario Perro, John Challier, Christina Claus, Stanford Chen, Claudia Ferrara Koller, Michael Mølhøj, Stella Tournaviti, Marina Bacac, Tom Moore, Christian Klein, Pablo Umana. Design of CD19-4-1BBL, a novel CD19-targeted 4-1BB ligand for combination therapy with CD20 T-cell bispecific antibodies and CD20 antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 957.