Receptor engineering for developing cancer immunotherapies
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Immunotherapy can be traced back as far as third century BC during China’s Quin dynasty, with records noting purposeful inoculations with variola minor virus to prevent smallpox disease. Immunotherapy seeks to harness and manipulate the body’s immune system and redirect its curative abilities against disease, infection, and cancer. In the 1960’s through 1970’s, the major players of the immune system, T cells, dendritic cells, and natural killer cells, were discovered. Bone marrow and stem cell transplants began to offer patients reliable treatments. Additionally, the role of alloreactive T cells was discovered through observations of transplant rejection and graft versus host disease (GvHD), showing the importance of HLA matching. Hybridoma technology was established by Kohler and Milstein, a method to identify and produce antigen-specific monoclonal antibodies (mAbs), leading to a whole new therapeutic market. In the next two decades, significant changes began with FDA approval of the first mAbs, approval of high dose IL-2 therapy, discovery of immune checkpoint molecules, and the first genetic engineering of T cells. ☐ Simultaneously in the 1990’s, tumor-specific T cell receptors (TCRs) and chimeric antigen receptors (CARs) were designed and showed promising results as the future of cell-based immunotherapies. Recently, remarkable results have been observed in patients with hematological malignancies treated with CAR T cells, up to 80% remission, leading to the approval of six CAR T cell therapies starting in 2017. Two major areas of focus in immunotherapy research are (1) identifying tumor antigens (TAs) and developing specific binding domains against them, and (2) developing more effective therapies against solid tumors. Here, we use receptor engineering to address these areas of concern. ☐ In chapters 2 and 3, we propose a design and establish a method for generating a novel T cell-based immunotherapy that redirects T cell alloreactivity to tumor sites while limiting normal tissue damage. Tumor-Activated Alloreactive T cells (TAATs) use a TA-sensing receptor linked to an intracellular transcription activator to control T cell alloreactivity (TCR expression). We use HLA-mismatched class-I+ breast cancer cells to expand alloreactive T cells and we show knocking out TCR expression is an effective approach to inhibiting T cell alloreactivity. ☐ In chapter 4, TAATs show functional abilities through restoring TCR expression and perform degranulation-mediated killing process, when stimulated with TA+ tumor cells. We show TAATs have strong cytotoxic killing responses against TA+/HLA-I+ tumor cells (cis killing) and TA-/HLA-I+ tumor cells (trans killing). Trans killing properties of TAATs allow elimination of all HLA-I+ tumor and non- tumor cells within the tumor environment, permitting complete tumor clearance. ☐ In chapter 5, progress was made towards establishing a solid tumor/GvHD mouse model for conducting in vivo studies for HER2-specific TAATs. HER2+ tumors are established and grow in transgenic NSG mice expressing human HLA-A2 allele. We first test treatment of tumor bearing mice with alloreactive T cells (AlloT) and show AlloT cells localize to tumors and suppress tumor growth. We also show in one mouse co-injected with tumor cells and AlloT cells, that they prevent establishment of tumors. ☐ Identifying and generating mAbs against membrane receptors using conventional approaches, like hybridoma technology, has proven challenging. In chapter 6, the Bait Receptor Approach was investigated as a novel method for identifying and producing mAbs against difficult receptor targets. With this approach, B cells specific for G protein-coupled receptor, CXCR4, and epidermal growth factor receptor, HER2, were selectively expanded in vitro. We show support of specific B cell expansion and establish a preliminary protocol for single-cell sorting and cloning of variable regions from B cell immunoglobulins. Cloned antibodies from CXCR4 and HER2 cultures are tested show no specificity against targets. Alternative approaches and future directions are discussed to improve the Bait Receptor Approach and increase specific B cell expansion. ☐ Overall, our data support our rationale for using TAATs to direct T cell alloreactivity to tumor sites. Receptor engineering was instrumental in designing and evaluating TAATs and their toxicities, and for investigating a novel approach for immunoglobulin discovery.
Description
Keywords
Immunotherapy, Tumor antigens, T cells