CD1d-specific single-domain antibodies for unconventional immunomodulation

Bispecific T cell engagers (bsTCEs) in clinical development and/or (recently) approved as standard of care, face challenges related to the bsTCE format (e.g half-life, tumor penetration, immunogenicity, stability) and targeted effector cells population, as CD3-directed bsTCE recruit T cell populations that either disproportionally contribute to toxicity or actively suppress efficacy. Development of next-generation bsTCE with an improved therapeutic window is therefore needed. This thesis describes the generation of a panel of CD1d specific VHH that differentially impact the CD1d-NKT cell axis and demonstrate that linkage of one specific anti-CD1d VHH with an anti-Vδ2 VHH resulted in a bsTCE with trispecific properties that engages type 1 NKT and Vγ9Vδ2-T cells to CD1d+ tumors. In chapter 2 we give a detailed description of a an efficient method to culture and expand type 1 NKT cells. In chapter 3 we describe the generation of 21 CD1d-specific VHHs via immunization of Lama glama, phage library construction, and selection of CD1d-specific clones. Functional evaluation of these CD1d-specific VHHs resulted in the identification of two VHHs able to induce maturation of dendritic cells, one VHH (VHH1D17) that enhanced annexin V binding (suggestive of early apoptosis) upon binding to CD1d+ malignant hematologic cells, and yet another VHH (VHH1D22) that inhibited activation of type 1 NKT cells by glycolipid-loaded CD1d. In chapter 4 we demonstrate that VHH1D17 mediated induction of annexin V binding on CD1d+ malignant hematologic cells is not reflective of early cell death but results from enhanced presentation of phosphatidylserine (PS) in CD1d. We demonstrate that CD1d(PS) can interact with immune regulatory molecules and induce T-cell immunoglobulin domain and mucin domain (TIM)-3 signaling on effector cells suggesting the existence of a functional CD1d(PS)-TIM-3-axis. In chapter 5 the ability of anti-CD1d VHH1D12 to trigger type 1 NKT cell activation is described. By using a structural and functional approach we demonstrate that VHH1D12 specifically stabilizes the human CD1d-type 1 NKT-TCR interaction and blocks (pro-tumor) diverse NKT cells, which translates directly into effective anti-tumor activity. In chapter 6, this anti-CD1d VHH1D12 is linked to a Vδ2-TCR-specific VHH to generate the CD1d-Vδ2 bsTCE. We subsequently demonstrate robust proinflammatory cytokine production, type 1 NKT and Vγ9Vδ2-T cell expansion, and anti-tumor activity to patient-derived MM, AML and CLL cells and in in vivo mouse models. Exploratory toxicology studies with a fully cross-reactive (surrogate) CD1d-γδ bsTCE in non-human primates (NHP) demonstrated good tolerability, and only low levels of the CRS related cytokine IL-6. Based on these results the CD1d-Vδ2 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML (ClinicalTrials.gov Identifier: NCT04887259). In chapter 7 our findings are discussed in the context of the current state of the research field as well as prospects for future research.