Immunotherapies have provided significant benefit for cancer patients. However, the use of highly potent immunotherapeutic agents is limited by associated toxicities that result from systemic immune activation. We have developed a rational approach to design therapeutics that are selectively activated in the tumor micro-environment. This approach has the potential to improve the safety and efficacy of cytokine-based therapies and immune checkpoint inhibitors, and can be applied broadly to protein-based therapeutics.

We have developed an inflammation-responsive hydrogel platform from small-molecule amphiphilic gelators, which we have identified through our screening of the FDA’s generally recognized as safe (GRAS) list of compounds. This platform can titrate drug release to the level of inflammation, ensuring that the biotherapeutic is released only when needed at a therapeutically relevant concentration. Another interesting characteristic of this platform is its selective adhesion to inflamed tissue, which results in high, local drug concentrations at the disease site, maximizing the therapeutic effect. This talk will discuss our previously published and currently ongoing work to advance the self-assembled hydrogel platform. I will also discuss another platform, which we have developed for blood-brain barrier pathophysiology-independent delivery of nucleic acid-based therapeutics in traumatic brain injury.

Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Although the properties of the BBB are manifested in the endothelial cells, transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. We are interested in identifying how the BBB interacts with the neural circuitry to regulate brain function and behavior, addressing the following questions: How does neural activity dynamically regulate the BBB? How do changes to the BBB regulate brain function and behavior? Are there regional specializations of the BBB that are important for local circuit function? Here we use a genomic, genetic, and molecular approach to elucidate these questions. We have found that neural activity robustly alters the gene expression of CNS endothelial cells, regulating key BBB properties, including efflux transport. We have further identified roles for vascular metabolism in regulating behavior, and have identified significant regional heterogeneity of the BBB.

In this talk, I will present how to use high-throughput microscopy to explore temporal dynamics of signaling in single cells. I am presenting a subset of antibodies targeting opioid receptors to examine the effect of treatment with opiates, such as morphine and fentanyl, that have played central roles in the worsening of the ‘Opioid Epidemic.’ The combination of high-throughput approaches and rigorous validation can lead to the identification of novel antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.

Adoptive T cell therapies using T cells expressing synthetic chimeric antigen receptors (CARs), or engineered native T cell receptors (TCRs) have been successful in treating some cancers. This approach harnesses the inherent potency of T cells and redirects their recognition toward cancer antigens. While many challenges remain (e.g. cross-reactivity with self-antigens and antigen escape), protein engineering can offer solutions for such issues.

We have recently modulated the endosomal trafficking behavior of a HER2-specific antibody-drug conjugate (ADC) to improve the efficiency of cytotoxic payload delivery to lysosomes. This approach, called ALTA technology (for ADCs with increased lysosomal trafficking activity), is expected to enable therapeutic efficacy using lower doses, thereby leading to decreased off-tumor toxicities. Consequently, this strategy may allow tumors with a broad range of HER2 expression levels to be targeted.

Antibodies have become an integral tool in the life sciences and yet very little is done to ensure that we are working with good and reliable antibodies. Because antibodies are characterized by what they bind rather than their physical characteristics there is still much we do not understand about reagents we use every day. At best they do not bind reliably, and at worst they bind to entirely different targets. Antibody sequencing has advanced dramatically over the past five years to the point where antibodies can be sequenced reliably and affordably. Historically antibody mixtures and polyclonal antibody samples could not be characterized, however, by applying novel proteomics techniques sequencing on these complex samples is finally possible.

Membrane proteins (MPs) such as GPCR, transporters, channels etc.. represent more than 60% of therapeutic targets. In my presentation I will provide a comprehensive overview of MP isolation tools. I will be speaking about expression, solubilization, purification & stabilization of unmutated MPs. The use of detergents, amphiphilic copolymers, nanodisc, extracellular vesicles or liposomes to successfully enable antibody discovery, FBDD and SBDD (NMR and Cryo-EM) of MPs will be presented. Finally, novel trends dealing with Machine learning and AI-based approaches will be discussed.

Antibodies are typically administered systemically and act throughout the body, which can cause on-target toxicity away from the disease site (tumor). To address this, we developed a strategy whereby an antibody can be de-activated by mutation, then re-awakened upon addition of a complementary ligand. Because our design is built upon conserved residues in the antibody framework, the same mutation/ligand pair can be used to modulate antigen binding in many different clinically relevant antibodies.

To understand the advantages and feasibility of KRAS-targeted degradation, we developed KRAS bioPROTACs - chimeric proteins consisting of high-affinity RAS binders fused to an E3 ligase. These provided definitive evidence for RAS degradability and elucidated the consequences of RAS degradation. These also informed on the degradation kinetics of KRAS mutants and prevalence of nucleotide states in WT/mutant KRAS. If delivery challenges can be addressed, RAS bioPROTACs may be exciting clinical candidates.

T cell-engaging bispecific antibodies have demonstrated potent cytotoxicity against cancer cells. This potency can engender off-tumor, on-target toxicity when targeting solid tumors. Maverick Therapeutics has developed a bispecific platform called COBRA™, which includes two active tumor-targeting domains and inactive T cell-engaging domains, that become active within the tumor microenvironment.  This presentation will demonstrate the efficacy of these molecules against human tumor cells in vitro and in vivo.

Recent technological advancements in HDX-MS have enabled the rapid investigation of biotherapeutic antibodies and their interactions. We are developing a sophisticated HDX-MS platform to support antibody development against difficult-to-analyze protein targets. Our automated HDX-MS technology, coupled with novel electrochemical reduction technology, allows for reproducible and high-throughput screening of the dynamic behavior of antigen-antibody complexes. Described here is the dynamic epitope mapping and conformational characterization by HDX-MS of two distinct antigenic targets. First, the screening of four antibodies against carbonic anhydrase IX (CA-IX), a membrane-bound anticancer target with a role in homeostasis of tumor microenvironments, is highlighted. Our HDX profiles delineated three distinct binding modes, each with its own unique therapeutic outcome. Second, we screened the binding of nanobodies to the large, complex, and dynamic insulin-like growth factor receptor (IGF1R). This receptor has recently been identified as a target for receptor mediated-transcytosis across the blood brain barrier. Using HDX-MS, we sought to investigate the relationship between binding epitope and crossing efficacy. These projects outline the unique contribution of HDX-MS to the antibody development pipeline, and demonstrate its applicability to probe increasingly complex protein systems.

The vision of structure-based drug design has long been to predict how ligands will influence the function of their targets. This is finally becoming a reality, thanks to advances in computational methods and technologies. I will present recent studies in which we used molecular dynamics simulations and machine learning to guide the design of ligands that selectively bind desired targets and selectively stimulate desired functions.