Novel Chemical Immunotherapeutic Strategies for Stabilized Immune Recruitment to Cancer Targets

Abstract

Cancer immunotherapy utilizes re-engineered or innate immune machinery to noninvasively treat solid tumors and free malignant cells. Monoclonal antibodies (mAbs) and derivatives thereof currently dominate the cancer immunotherapeutic landscape. MAbs enact therapeutic function largely through formation of highly valent mAb-target cell binding interactions (opsonization) and subsequent recruitment of endogenous immune components (e.g.complement, T-cells, Natural Killer Cells, Macrophages) for immune-mediated target cell killing.However, these protein-based therapeutics carry inherent challenges including costly manufacturing, relatively poor patient accessibility, large dosing requirements, and potential immunogenicity and in-vivo instability. As an alternative, small molecule immune recruiters termed antibody recruiting molecules (ARMs) are capable of conscripting endogenous serum antibodies for target opsonization and subsequent immune recruitment. ARMs function via an antibody binding domain (ABD) capable of reversibly binding specific endogenous serum antibodies, in combination with a target binding domain (TBD) for cancer antigen binding. While ARMs lack pitfalls of mAb-based technology, they introduce novel difficulties including rapid invivo clearance and dependence on in-system formation of a strong ternary-complex (target cell : ARM : antibody). As such, ARMs struggle to stabilize immune recruitment in a serum-relevant setting. Instead, ARM function is largely limited to high target expressing cancer cells in the context of induced or model high affinity anti-ARM antibodies. To build on ARM and ARM-like technologies, this thesis introduces two novel small molecule immune recruiter platforms. In Chapter 2, Covalent Antibody Recruiting Molecules (cARMs) are introduced. These initial cARMs present a Dinitrophenyl (DNP) ABD for the covalent tagging of model anti-DNP antibodies with a cancer homing TBD. The result is a serum stable endogenous antibody capable of forming a simple binary complex (target cell : cARMantibody) for target cell opsonization and immune-mediated clearance. Initial studies validate cARM capacity to site-selectively label model SPE7 anti-DNP antibody in a pre-determined location (Lysine 59) on the scale of hours (k2 = kinact ≈ 10-4s-1). Further, TBD labelled antibodies demonstrate capacity to opsonize and induce immune mediated clearance of model target cells. As a continuation, Chapter 3 compares the ability of cARMs and ARMs to induce immune recruitment and subsequent immune-mediated clearance in a variety of immune contexts. Through varying anti-DNP : ABD affinity (two antibody sources investigated with Kd determined to be approx. 10-8 M and 10-7 M) and varying Immune effector : antibody affinity (targeted immune activation induced through either high affinity CD64 or lower affinity CD16α), cARM and ARM tools are used to probe stability requirements for target clearance in the context of small molecule immune recruiters. The inability of ARMs, and ability of cARMs to induce immune mediated target clearance in a variety of contexts suggests functional immune recruitment depend both on quantity and kinetic stability of recruited immune components. Finally, Chapter 4 introduces Polymeric Antibody Recruiting Molecules (pARMs). Initial pARMs present a multivalent display of DNP ABDs for recruitment of model SPE7 anti-DNP antibodies together with a multivalent display of TBDs for cancer targeting. Optimized pARMs mediated enhanced anti-cancer immune function against lower target expressing cells compared to analogous ARMs. Further, the initial set of pARMs presented in Chapter 4, together with results, can be used to guide the design of future multivalent small molecule immune recruiters.