25 Novel antibodies for identification, selection, and manipulation of T cells expressing Whitlow linker-containing CARs
Year: 2025
Type: conference-abstract
Abstract: Background The translational study of chimeric antigen receptor (CAR) T cell function, persistence, immunophenotype, and spatial localization after infusion is crucial for understanding factors that influence clinical outcomes. However, research has been limited by a lack of optimized tools to reliably detect CAR-engineered cells. To address this, we developed a novel platform to generate monoclonal antibodies (mAbs) targeting the Whitlow linker peptide, which is the most common linker incorporated in single-chain variable fragments (scFvs) of CAR constructs.Methods Using recombinant proteins and scFv linker peptides as immunogens, we generated murine mAbs against the Whitlow linker peptide, capable of binding cells expressing Whitlow linker-containing CARs in both fresh and formalin-fixed paraffin-embedded (FFPE) tissues. We evaluated these antibodies in multiple in vitro translational applications relevant to CAR T cell research and manufacturing.Results We identified five unique mAbs with specific reactivity against the Whitlow linker ( figure 1). Affinity testing with surface plasmon resonance and 3-D modeling were used to gain insight into binding properties of each mAb to the Whitlow peptide antigen. One mAb was characterized in depth (1B4A1), demonstrating comparable capacity to identify CAR T cells in peripheral blood compared to other methods using anti-idiotype antibodies or recombinant CAR-target proteins. In contrast, the anti-Whitlow mAb detects cells expressing Whitlow linker-containing CARs with different antigen specificities, including those harboring the widely employed anti-CD19 FMC63 scFv as well as others, such as those targeting BCMA or CD33. Importantly, the anti-Whitlow mAb identified CAR T cells in situ in archival FFPE tissues (figure 2). A DNA-barcoded format enabled spatial characterization and immunophenotyping of CAR T cells in situ in highly multiplexed immunohistochemistry (IHC). One mAb was found to significantly stimulate CAR T cells upon antibody binding in vitro. Using both FACS and magnetic bead protocols, we demonstrated the feasibility of anti-Whitlow mAb-mediated selective enrichment of CAR-expressing T cells and potential utility in manufacturing workflows.Conclusions Five anti-Whitlow mAb were discovered and exhibited distinct structural and functional properties that can be leveraged for multiple applications, providing versatile tools for detection and manipulation of CAR T cells in a broad range of clinical and preclinical CAR T cell products.Abstract 25 Figure 1Monoclonal anti-linker antibodies bind CAR-engineered T cells expressing the Whitlow peptide. Flow cytometry histogram of FMC63-Whitlow or FMC63-G4S CAR T cells (A) or 1H7-Whitlow or 1H7-G4S CAR T cells (B) with untransduced T cells (grey) stained with each of five anti-Whitlow mAbs (red). tEGFR and tCD19 transduction markers (green)Abstract 25 Figure 2Anti-Whitlow mAbs can detect CAR T cells in situ in archival tissue by mIHC in FFPE tissue. mIHC staining for detection of three different commercial CAR engineered T cells in archival FFPE patient biopsies from patients treated with. (A) liso-cel (B) axi-cel (C) ide-cel
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W4415899023