Marcil, A. | Search bioRxiv

bioRxiv2024

Broadly Reactive Anti-VHH Antibodies for Characterizing, Blocking, or Activating Nanobody-Based CAR-T Cells

Production of chimeric antigen receptor T cell (CAR-T) therapies is dependent on the use of antibody reagents to label, isolate, and/or expand T cell products. We sought to create antibody-based tools that directly target the variable domain of heavy-chain only antibodies (VHH or nanobody) used in some CAR molecules. Two murine antibodies were identified which bind to distinct epitopes in the conserved framework regions of llama-derived VHHs, and not to human VH domains. We produced a high-quality dual-clonal anti-VHH antibody product which reacts with over 98% of VHH proteins, regardless of their antigenic specificity. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus could be used for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies potently inhibited VHH-CAR function, blocking CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH antibodies could also be applied for activation and expansion of VHH CAR-T cells, inducing 730-fold mean expansion, >94% CAR purity, with retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen specific activity without functional exhaustion. Overall, these data identify a useful new tool for understanding and manipulating VHH-based CAR-T cells. Funding SourceThis work was funded by the National Research Council Canada Disruptive Technology Solutions Cell and Gene Therapy challenge program, and BioCanRx Declaration of interestsThe anti-VHH antibodies reported here are the subject of a provisional patent application by the National Research Council of Canada

immunology↗

bioRxiv2022

A Simplified Function-First Method for the Discovery and Optimization of Bispecific Immune Engaging Antibodies

Bi-specific T-cell engager antibodies (BITEs) are synthetic soluble molecules derived from antibodies that induce active contact between T-cells and other target cells in the body. BITE therapeutics have shown great promise for the treatment of various forms of cancer; however, the current development process for BITEs is time consuming and costly. BITE development requires empirical testing and characterization of the individual antigen binding domains, followed by extensive engineering and optimization in bi-specific molecular format to generate a molecule with strong biological activity and appropriate characteristics for clinical development. Here, we sought to create a cost efficient high-throughput method for creating and evaluating BITEs using a simplified function first approach to identify bioactive molecules without purification. Using a plasmid with a modular structure to allow high efficiency exchange of either binder arm, we established a simple method to combine many novel tumour-targeting single chain variable (scFv) domains with the well-characterized OKT3 scFv CD3-targeting domain. After generating these novel plasmids, we demonstrate two systems for high throughput functional screening of BITE molecules based on Jurkat T cells (referred to as BITE-J). Using BITE-J we evaluate four EGFRvIII BITEs, identifying two constructs with superior activity. We then confirmed this activity in primary T cells, where novel EGFRvIII-BITEs induced T cell activation and antigen selective tumor killing. We also demonstrate that we can similarly exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BITEs, we were able to identify highly active BITE molecules with desirable properties for downstream development. In summary, BITE-J presents a low cost, high-throughput method for the rapid assessment of novel BITE molecules without the need for purification and quantification.

immunology↗