Novel Approaches for Tumor Targeting via Artificial Antigen Delivery
Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics
First Advisor
Glazer, Peter
Abstract
In cancer therapy, there is growing interest in antigen-targeted therapeutics, such as antibody drug conjugates (ADCs), and cell-based therapies, such as chimeric antigen receptor (CAR) T cells, to elevate immune system engagement within the cancer microenvironment. However, the paucity of tumor specific antigens and the challenge of tumor heterogeneity limit the efficacy of these therapies. Consequently, there is an unmet clinical need to discover antigen targets that are restricted to and are expressed within cancer cells.To overcome these challenges, we have developed a tumor-targeting peptide conjugate that allows for selective delivery of an antigenic peptide to cancer cells. Our approach exploits the tumor targeting and intracellular insertion properties of the pH low insertion peptide (pHLIP), which selectively targets tumors and inserts directionally into the membrane of cancer cells based on the unique acidic tumor microenvironment, sparing healthy tissue. To study artificial antigen delivery to cancer cells, we employed two different approaches for antigen conjugation to the pHLIP peptide. Our first strategy focused on conjugating the antigenic peptide SIINFEKL to the c-terminal end of pHLIP, designated pHLIP SIINFEKL, for intracellular delivery of the antigen. This approach was used to study the effects of T cell trafficking of SIINFEKL-specific CD8+ T cells (OT1) to sites of tumors in order to provide a proof-of-concept model utilizing pHLIP to modulate a cell-based therapy. Our second approach conjugated the viral antigen of hemagglutinin influenza A (HA) (YPYDVPDYA) to the N-terminus of pHLIP, designated pHLIP-HA, to investigate anti-HA antibody recruitment to the extracellular membrane of pHLIP-HA tagged cancer cells. Both strategies serve to develop pHLIP therapeutically to homogenize antigen landscapes for more effective targeting of cancer. To study how pHLIP can be used as a marker for T cell detection and cytotoxic activity, we prepared a pHLIP-peptide conjugate designed to deliver the SIINFEKL peptide, an immunogenic fragment of ovalbumin, to tumor cells in vivo. Once inserted into cells, the SIINFEKL peptide is released from the C-terminus of pHLIP via a cleavable linker and is then processed and class restricted for presentation and immune recognition through the major histocompatibility complex (MHC) I pathway. We observed that pHLIP-SIINFEKL was selectively delivered to cancer cells in vitro and to tumors in vivo using fluorescently labeled constructs. In vitro, treatment of melanoma cells with pHLIP-SIINFEKL resulted in recognition by OT1 T cells expressing an engineered T cell receptor specific for the SIINFEKL antigen, leading to T cell activation. Mechanistically, it was shown that this recognition by OT1 cells was abrogated by siRNA knockdown of the β2 microglobulin (β2m) component of MHC I in the target tumor cells, indicating that an intact antigen processing pathway in the cancer cells is necessary to mediate the effect of pHLIP-directed SIINFEKL delivery. In vivo, pHLIP-SIINFEKL treatment of tumor-bearing mice resulted in recruitment of OT1 T cells and suppression of tumor growth in two syngeneic tumor models in immunocompetent mice. There was no effect when using mutated peptides in place of either the pHLIP or SIINFEKL components of the conjugate. Similarly, to determine if pHLIP-HA can be used as an antibody-recruiting molecule (ARM), we conjugated the HA antigen to the N-terminus of pHLIP so that HA would remain extracellular to the cell upon pHLIP insertion. This enabled HA to act as a marker for antibody recruitment of an anti-HA antibody and stimulation of downstream immune effects through FC-mediated binding. We observed that upon pHLIP-HA insertion, anti-HA antibodies were able to traffic to these sites in vitro and in vivo. For HA detection, fragment antigen-binding (FAB) regions were more effective in detecting pHLIP-HA-labeled tumors when compared to a full-length anti-HA antibody due to enhanced permeability and retention (EPR effect). However, full length antibodies, both commercially available and generated in mice through vaccination strategies, were able to show downstream activation of antibody dependent cellular cytotoxicity (ADCC) and lead to therapeutic effect in two mouse model backgrounds where pHLIP-HA and anti-HA antibodies are present. Our results suggest that pHLIP can act as a bridge between cancer cells and adaptive immune response. Overall, these results suggest that pHLIP-mediated peptide delivery can be used to decorate tumor cells with a novel artificial antigen that can be targeted by cell-based therapies. We believe that this work could provide an avenue for expanding the use of ADCs and CAR T approaches to malignancies for which specific antigenic targets are needed
Recommended Citation
Yurkevicz, Annali, "Novel Approaches for Tumor Targeting via Artificial Antigen Delivery" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1409.
https://elischolar.library.yale.edu/gsas_dissertations/1409
