Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody... Show moreAntibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8+ T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo. Show less
Wulp, W. van der; Remst, D.F.G.; Kester, M.G.D.; Hagedoorn, R.S.; Parren, P.W.H.I.; Kasteren, S.I.; ... ; Heemskerk, M.H.M. 2023
Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody... Show moreAntibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8+ T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo. Show less
King, L.A.; Toffoli, E.C.; Veth, M.; Iglesias-Guimarais, V.; Slot, M.C.; Amsen, D.; ... ; Vliet, H.J. van der 2023
Vy9V82 T cells are effector cells with proven antitumor efficacy against a broad range of cancers. This study aimed to assess the antitumor activity and safety of a bispecific antibody directing... Show moreVy9V82 T cells are effector cells with proven antitumor efficacy against a broad range of cancers. This study aimed to assess the antitumor activity and safety of a bispecific antibody directing Vy9V82 T cells to EGFR-expressing tumors. An EGFR-V82 bispecific T-cell engager (bsTCE) was generated, and its capacity to activate Vy9V82 T cells and trigger antitumor activity was tested in multiple in vitro, in vivo, and ex vivo models. Studies to explore safety were conducted using cross-reactive surrogate engagers in nonhuman primates (NHP). We found that Vy9V82 T cells from peripheral blood and tumor specimens of patients with EGFR thorn cancers had a distinct immune checkpoint expres-sion profile characterized by low levels of PD-1, LAG-3, and TIM-3. Vy9V82 T cells could be activated by EGFR-V82 bsTCEs to mediate lysis of various EGFR thorn patient-derived tumor samples,and substantial tumor growth inhibition and improved survival were observed in in vivo xenograft mouse models using peripheral blood mononuclear cells (PBMC) as effector cells. EGFR-V82 bsTCEs exerted preferential activity toward EGFR thorn tumor cells and induced downstream activation of CD4 thorn and CD8 thorn T cells and natural killer (NK) cells without concomitant activation of suppressive regulatory T cells observed with EGFR-CD3 bsTCEs. Administration of fully cross-reactive and half-life extended surrogate engagers to NHPs did not trigger signals in the safety parameters that were assessed. Considering the effector and immune-activating properties of Vy9V82 T cells, the preclinical efficacy data and acceptable safety profile reported here provide a solid basis for testing EGFR-V82 bsTCEs in patients with EGFR thorn malignancies. Show less
Wulp, W. van der; Gram, A.M.; Bleijlevens, B.; Hagedoorn, R.S.; Araman, M.C.; Kim, R.Q.; ... ; Heemskerk, M.H.M. 2023
Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects... Show moreTherapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells. Show less
Lameris, R.; Ruben, J.M.; Iglesias-Guimarais, V.; Jong, M. de; Veth, M.; Bovenkamp, F.S. van de; ... ; Vliet, H.J. van der 2023
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... Show moreBispecific 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 immuno-suppressive regulatory T cells that limit efficacy. The development of Vy9V52-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 V52-TCR-specific VHH, we create a bsTCE with trispecific properties, which engages not only Vy9V52-T cells but also type 1 NKT cells to CD1d+ tumors and triggers robust proin-flammatory 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 Vy9V52-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-y5 bsTCE in NHPs shows Vy9V52-T cell engagement and excellent tolerability. Based on these results, CD1d-V52 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML. Show less
