Perspectives on anti-CD47 antibody treatment for experimental cancer.

In PNAS, an article by Tseng et al. continues to examine an important and unique therapeutic manipulation that controls the growth of tumor cells in mice (1). This manipulation consists of using a monoclonal antibody specific to the membrane protein CD47 to block its biological activity: blocking CD47 reduces tumor growth by enabling macrophages to phagocytose the cancer cells (2–4). In this study, a previously undescribed effect of the anti-CD47 antibody treatment is reported, which is that it results in the activation of the CD8 set of T lymphocytes, a set important for the killing of tumor cells. What is CD47? It is a protein of about 50 kDa consisting of an extracellular Ig domain and five membrane-spanning segments with a small cytoplasmic tail (5). CD47 is expressed on the surface of all cells, but is particularly prominent in a variety of cancer cells (2, 5). Indeed, one of the first biochemical characterizations was made on ovarian carcinomas that express CD47 at high levels (6). Early studies called attention to CD47 as a protein promoting the interaction among integrins, adhesion molecules involved in cellular communications (7). Subsequent studies confirmed a number of interactions of CD47 with various adhesion molecules among leukocytes in diverse biological reactions (reviewed in ref. 5). An important breakthrough in identifying a key role of this molecule was a report by Oldenberg et al. when they examined cells from mutant mice that lacked the CD47 protein (8). Red blood cells lacking the CD47 proteins injected into normal mice were rapidly engulfed by the macrophages of the spleen, but those red cells expressing CD47 were not taken up. In other words, the presence of CD47 in a cell was a deterrent to phagocytosis. The study of Oldenberg et al. (8) was a breakthrough finding because it indicated that CD47 was acting as a molecule that regulated the recognition of “self.” Simply stated, the study indicates that expression of a level of CD47 by a cell will not favor its uptake by macrophages. Macrophages do not phagocytose their own cells: self-proteins avoid phagocytosis because they have CD47 as a “don’t eat me” signal, aside from lacking surface proteins that have complementary receptors on macrophages required for the uptake. CD47 is known to engage a receptor protein of the macrophage called signal regulatory protein-α (SIRP-α) (reviewed in ref. 5). Engagement of SIRP-α leads to inhibition of phagocytosis through an activation of a cellular phosphatase that inhibits cytoskeletal proteins. Phagocytosis of particle cells or pathogens is a complex process that involves membrane interactions and the rearrangement of intracellular organelles and cytoskeletal proteins to engulf the material (Fig. 1). CD47 is one of two molecular systems in which cells distinguish self from altered-self by the absence of a normally expressed protein. This capacity to distinguish self by the absence of a surface protein is the “missing self hypothesis” (9), first shown for natural killer cells, a cell lineage that patrols tissues and is important in early antiviral and antitumor resistance. In the case of natural killer cells, the cell-surface molecules are class I histocompatibility molecules interacting with receptors in the natural killer cells (10). Studies in the Weissman laboratory recognized the features of many cancer cells in displaying large amounts of CD47, and by doing this evading recognition by the macrophages (2). Indeed, in experimental systems in which tumor cells were transplanted into mice, the growth of the tumor was controlled by injection of antibodies that neutralized CD47 (2–4, 11). These experiments only examined the interactions of the innate cellular system with the cancer cells: the cancer cells had been injected into immunodeficient mice. Thus, blocking the antiphagocytic properties of CD47, the molecules that confer the “don’t eat me” signal enabled the innate cell system represented mainly by macrophages to attack the tumor. (Macrophages, natural killer cells together with dendritic cells, granulocytes, and mast cells constitute the innate system, which participates in diverse reactions early in the immune response to pathogens as well as during the inflammatory reactions that follow. This innate system interacts in a truly symbiotic fashion with the adaptive cellular system of T and B cells.) There is a second component of cancer cells that makes it possible for blockade of CD47 to be effective (12) (Fig. 1). As a result of stress or their intrinsic reactivity, many tumors abnormally express proteins that have complementary receptors on phagocytes, which favor their phagocytosis. One of these proteins is calreticulin, a protein normally expressed in the endoplasmic reticulum. Calreticulin acts as a chaperone of unfolded proteins in the endoplasmic reticulum, but it can be translocated to plasma membrane, where it is found in high levels in Fig. 1. (A) Macrophages ignore normal cells as a result of negative interactions in which the CD47–SIRP-α pair promote the “don’t eat me” signal. Cancer cells show high levels of CD47 and avoid phagocytosis despite having a higher level of calreticulin (12) (B), but blocking CD47 with antibody favors their uptake (C ).