Abstract
Project I
Macrophage antibody-dependent cellular phagocytosis (ADCP) is a principal cytotoxic mechanism of CD20-targeting therapeutic monoclonal antibodies (mAbs). Although these antibodies induce rapid B cell clearance, durable efficacy is limited by finite macrophage effector capacity that promotes therapeutic resistance. While Fcγ receptor–mediated ADCP (fADCP) is well characterized, the contribution and constraints of complement-dependent ADCP (cADCP) remain poorly defined. Here, we used quantitative live-cell imaging of primary mouse macrophages, genetic disruption of Fcγ receptor signaling, and controlled modulation of complement activity to compare the kinetics, capacity, and exhaustion behavior of fADCP and cADCP. Relative to fADCP, cADCP displays delayed initiation but substantially greater cumulative target clearance. When engaged concurrently, fADCP and cADCP act additively, indicating functional non-redundancy. Notably, macrophages rendered refractory following fADCP retain full cADCP capacity, demonstrating that complement receptor–mediated phagocytosis bypasses Fcγ receptor–associated hypophagia. However, cADCP is also finite, as increasing target burden induces a distinct, dose-dependent state of complement-associated phagocytic exhaustion that is largely reversible within 24 hours and occurs independently of reduced CR3 or CR4 surface expression. Together, these findings establish cADCP as a quantitatively potent yet intrinsically constrained effector pathway and define pathway-specific limits on antibody driven phagocytosis. Effector exhaustion thus emerges as a fundamental bottleneck to sustained antibody-mediated cell clearance and a key consideration for improving the durability of ADCP-based therapies.
Project II
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BrCa) owing to its lack of targetable receptors and resistance to chemical and molecularly targeted therapeutic approaches. While chemotherapy and surgical resection remain the standard of care, these agents have significant side effects and varying patient outcomes. Thermally-ablative focused ultrasound (T-FUS) - a non-invasive and non-ionizing therapy that utilizes targeted acoustic energy to debulk tumors – has displayed immunomodulatory effects in BrCa. However, T-FUS as a monotherapy has had limited clinical efficacy in TNBC due to the presence of anti-inflammatory immunosuppressive myeloid cells (isMC). We hypothesized that elimination of isMC or initiating tumoricidal activity from them would lead to augmented activity of T-FUS. Thus, we interrogated the ability of myeloablative chemotherapies and antibodies; myeloid recruiting chemokine receptor blockade; and TLR agonists to remodel the tumor myeloid populations. Consistent with our previous studies, we found that while myeloablative chemotherapies decreased circulating isMC they had little impact on intratumoral isMC. In contrast, antibodies targeting Ly6C and Ly6G ablated intratumoral and systemic isMC, yet their effect was transient and was accompanied by a surprising depletion of T cells. While targeting CCR2, the dominant chemokine receptor for intratumoral isMC diminished a large subset of immunosuppressive cells within the TME, it also depleted T cells and dendritic cells. Contrary to previous studies, TLR stimulation failed to repolarize myeloid cells into a proinflammatory, tumoricidal phenotype but did lead to their depletion from the tumor microenvironment (TME) and mobilization of conventional dendritic cells to the draining lymph nodes. We therefore hypothesized that combining isMC depletion and TLR driven immune activation would enhance FUS efficacy; however, this combinatorial regimen did not enhance overall survival or control tumor volume after T-FUS treatment. Thus, the BrCa TME is highly resistant to approaches intended to remodel the myeloid cell
component which fail to synergize with T-FUS-mediated tumor ablation.
