Targeted delivery of proapoptotic peptides to tumor-associated macrophages improves survival

Most current cancer therapies focus on killing malignant cells, but these cells are often genetically unstable and can become resistant to chemotherapy. Tumor-associated macrophages (TAMs) facilitate disease progression by promoting angiogenesis and tumor cell growth, as well as by suppressing the adaptive immune response. TAMs are therefore potential targets for adjuvant anticancer therapies. However, resident macrophages are critical to host defense, and preferential ablation of TAMs remains challenging. Macrophage activation is broadly categorized as classically activated, or M1, and alternatively activated, or M2, and TAMs in the tumor microenvironment have been shown to adopt the anti-inflammatory, M2-like phenotype. To date, there are no methods for specific molecular targeting of TAMs. In this work, we report the discovery of a unique peptide sequence, M2pep, identified using a subtractive phage biopanning strategy against whole cells. The peptide preferentially binds to murine M2 cells, including TAMs, with low affinity for other leukocytes. Confocal imaging demonstrates the accumulation of M2pep in TAMs in vivo after tail vein injection. Finally, tail vein injection of an M2pep fusion peptide with a proapoptotic peptide delays mortality and selectively reduces the M2-like TAM population. This work therefore describes a molecularly targeted construct for murine TAMs and provides proof of concept of this approach as an anticancer treatment. In addition, M2pep is a useful tool for murine M2 macrophage identification and for modulating M2 macrophages in other murine models of disease involving M2 cells.Macrophages are phagocytic cells of our immune system that are found in almost all tissues. Originating from progenitor cells in bone marrow, circulating blood monocytes extravasate into tissues, where they differentiate into macrophages and can subsequently be shifted to diverse functional phenotypes by local environmental cues (1). These polarization states are broadly categorized as classically activated M1 macrophages or alternatively activated M2 macrophages. The M1 macrophage phenotype is induced by mediators, such as IFN-γ or LPS, resulting in a proinflammatory and microbicidal functional phenotype (2). In contrast, M2 macrophages are activated by IL-4 and IL-13, and they possess functional roles in resolution of inflammation and tissue remodeling (3).There are several chronic pathological findings associated with increased tissue levels of M2 macrophages, including cancer, late-stage atherosclerosis, fibrosis, and asthma (4). In cancer, populations of tumor-associated macrophages (TAMs) mediate immunosuppression and possess M2-like qualities, such as poor antigen presentation, promotion of angiogenesis, and tissue remodeling and repair, although there is heterogeneity in pathways and phenotypes in different tumors (5). TAMs also secrete the factors milk fat globule-EGF factor 8 and IL-6 that lead to tumorigenicity and drug resistance of cancer stem/initiating cells (6). The extent of TAM accumulation within tumors generally correlates with poor disease prognosis (7, 8). Indeed, the role of M2 macrophages in chronic inflammation and disease is increasingly appreciated, and the ability to modulate specific subsets of macrophage populations is a major focus of macrophage-targeted therapy (4). However, there is a dearth of available targeting entities that are specific for M2 macrophages.Several ligands have been used to target macrophage populations. The small molecules mannose and folate, which are ligands for the mannose receptor and folate receptor β, respectively, have been conjugated to drugs or drug carriers for macrophage delivery (9–11). However, receptors for both molecules are highly expressed in other cell types. Mannose receptor is a pathogen recognition receptor that is also used by dendritic cells (12). In addition to activated macrophages, folate binds to receptors on normal epithelial cells and tumor cells (13). Segers et al. (14) reported a peptide that binds the scavenger receptor-A on macrophages found within atherosclerotic plaques, but scavenger receptor-A is also expressed on all dendritic cells.Our goal is to develop an M2-targeted construct that can be used to ablate TAMs selectively but not other leukocytes. To achieve this goal, an M2-selective ligand was first identified by a library selection approach. Peptide phage library screening is a common method of identifying novel targeting ligands, and it has been used to select peptides for targeting tumor vasculature (15) and colon cancer (16) among several others (17). We therefore designed a subtractive phage panning strategy that uses bone marrow-derived, in vitro-activated macrophage subpopulations to increase binding specificity of a selected phage to the M2 macrophage subpopulation. We report here the discovery and evaluation of a peptide, called M2pep, which shows selective binding and internalization in M2 macrophages but with minimal M1 macrophage interaction. We demonstrate this peptide’s ability to identify murine M2-like TAMs within mixed cell populations isolated from mouse colon carcinoma tumors and to accumulate in TAMs after i.v. injection into tumor-bearing mice. We show that i.v. administration of M2pep fusion peptides with KLAKLAKKLAKLAK (KLA), a proapoptotic peptide, to tumor-bearing mice selectively reduces TAM populations and prolongs survival.