IMAT-ITCR Collaboration - Identification and development of T cell receptor mimic antibodies for high value neoantigen targets in triple negative breast cancer

点击翻译按钮获取中文摘要

Abstract, as submitted in the parent (R21 CA240220) application Major histocompatibility complexes (MHC), also termed Human Leukocyte Antigens (HLA) in humans are glycoproteins expressed on the surface of nucleated cells that act as proteomic scanning chips by providing insight into the status of cellular health. The recognition of antigen-presenting receptors by recombinant T-cell receptor (TCR)-like antibodies that mediate specific cancer cell killing forms the basis for newly emerging and very promising approaches to fight cancer that include antibody therapies, vaccines and cell-based immunotherapy. The success of these interventions depends on their personalization to a patient's biomarkers such as peptides presented by MHC-I molecules (pMHCs). The higher affinity binding of TCR-like antibodies to multiple pMHCs (higher avidity) can augment antitumor response significantly, up to a limit set by autoimmunity. The cell copy number of pMHCs targeted by specific TCR-like antibodies, is an important determinant of avidity and therefore of antitumor response. There is no easy way to quantify with current analytical technologies the number of pMHCs per cancer cell targeted by a specific TCR-like antibody. Common current methods for identifying antibody-ligand targeting include liquid chromatography with tandem mass spectrometry, ELISA, flow cytometry, immunohistochemistry and complement assays. These assays are challenged by low pMHC copy numbers often found in heterogeneous tumors. Our goal here is to develop a high-sensitivity nanosensor to quantify the copy number of pMHCs targeted by candidate TCR-like antibodies, enriched from only a few thousand (~104) cancer cells per assay. Working with low cell numbers will be essential for testing tumor pMHC heterogeneity from limited biopsy samples. Microfluidic isotachophoresis (ITP) will be integrated to the nanosensor to enable bound complex enrichment before detection. The nanosensor enables simultaneous quantification of size (optical signal) and effective charge (electrical signal). These bimodal data will provide independent measurements to verify whether an antibody forms a complex with the target ligand. In this proof of concept work, our overall hypothesis is that microfluidic ITP enrichment integrated with our sensor can detect individual TCR-like antibody-pMHC complexes isolated from ~104 cells derived from heterogeneous human tumor xenograft (PDX) tissues and distinguish specific binding from unbound protein, non-specific binding and aggregates to estimate targeted pMHC copy number per cancer cell. Accordingly, our Specific Aims are to (1a) determine sensor sensitivity limits to detect targeted complexes in pure protein solution, (1b) Implement ITP- based concentration enhancement and separation of antibody-pMHC complexes, and (2) Quantify copy number of targeted pMHCs enriched from cancer cells and PDX tumor tissues. Nanosensor sensitivity will be compared to ELISA, and mass spectroscopy will be used for pMHC target validation. Successful implementation of our Specific Aims will demonstrate how any feasibility gaps will be bridged to develop a clinical laboratory device in subsequent work for quantifying tumor pMHC expression heterogeneity to guide personalized immunotherapy.