Biomedical Sciences ETDs

Publication Date

Spring 4-7-2017

Abstract

This dissertation is built upon the fundamental idea that the pre-B cell receptor (pre-BCR) is important to leukemia cell survival and a logical therapeutic target in B cell precursor acute lymphoblastic leukemia (BCP-ALL). The pre-BCR is expressed early at a specific stage during B cell development where it plays a central role in survival of healthy B lymphocytes. This receptor is composed of the membrane heavy chain (mIgμ) associated with surrogate light chain components, 5 and VpreB. Through the use of advanced imaging modalities, in particular two-color single particle tracking (SPT), we showed that pre-BCRs formed transient, homotypic interactions. These receptors displayed correlated motion at short separation distances that is consistent with the formation of dimers. These encounters were sufficient to generate an autonomous signal that helped to maintain leukemia blast survival. Incubation of the BCP-ALL cells with galectin-1, which can bind both lambda5 and glycans, led to aggregate formation that drastically slowed down the diffusion of pre-BCRs. This correlated to a downregulation of pre-BCR signaling through proximal Syk activation. Blocking galectin-1’s lectin-binding capability with saturating concentrations of beta-lactose partially restored diffusion to “tonic” levels. The introduction of this sugar also impacted the downstream signaling profile. Due to these differential diffusion and signaling profiles observed in experimental procedures we turned computational biology to gain additional structural insight. Molecular dynamics (MD) simulations supported the idea that the pre-BCR can allosterically control galectin-1’s binding capacity at its distal carbohydrate-binding site (CBS). We generated a monovalent Fab against the dimer interface of the pre-BCR using phage display technologies and showed that it not only could disrupt dimerization but also disrupted downstream signaling. Due to recent clinical advances in chimeric antigen receptor (CAR) T cell therapy, we reformatted our Fab into a scFv, the binding region of the CAR, using genetic cloning techniques. Through the use of computer-aided design we successfully generated a scFv with high affinity to the VpreB target. This antibody binding fragment was cloned into a lentiviral vector containing the CD28 co-stimulatory domain and immunoreceptor tyrosine-based activation motif (ITAM)-containing TCR-CD3. After efficient transduction of primary CD8+ T cells, we have shown efficient in vitro efficacy against BCP-ALL cells.

Keywords

B Cell Precursor Acute Lymphoblastic Leukemia, Chimeric Antigen Receptor T cell Therapy, Single-Particle Tracking, Molecular Dynamics, pre-B cell receptor, Immunology

Document Type

Thesis

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Bridget Wilson

Second Committee Member

Diane Lidke

Third Committee Member

Stuart Winter

Fourth Committee Member

S. Gnana Gnanakaran