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COMMENTARY

Perspectives on anti-CD47 antibodytreatment for experimental cancerEmil R. Unanue

1

Department of Pathology and Immunology, Washington University School of Medicine,

St. Louis, MO 63110

In PNAS, an article by Tseng et al. continuesto examine an important and unique thera-peutic manipulation that controls the growthof tumor cells in mice (1). This manipulationconsists of using a monoclonal antibody spe-cic to the membrane protein CD47 to blockits biological activity: blocking CD47 reducestumor growth by enabling macrophages to

phagocytose the cancer cells (2

4). In thisstudy, a previously undescribed effect of theanti-CD47 antibody treatment is reported,which is that it results in the activation ofthe CD8 set of T lymphocytes, a set impor-tant for the killing of tumor cells.

What is CD47? It is a protein of about 50kDa consisting of an extracellular Ig do-main and ve membrane-spanning segmentswith a small cytoplasmic tail (5). CD47 is ex-pressed on the surface of all cells, but is par-ticularly prominent in a variety of cancer cells(2, 5). Indeed, one of the rst biochemical

characterizations was made on ovarian carci-nomas that express CD47 at high levels (6).Early studies called attention to CD47 as aprotein promoting the interaction amongintegrins, adhesion molecules involved incellular communications (7). Subsequentstudies conrmed a number of interactionsof CD47 with various adhesion moleculesamong leukocytes in diverse biological reac-tions (reviewed in ref. 5). An importantbreakthrough in identifying a key role of thismolecule was a report by Oldenberg et al.when they examined cells from mutant micethat lacked the CD47 protein (8). Red bloodcells lacking the CD47 proteins injected intonormal mice were rapidly engulfed by themacrophages of the spleen, but those red cellsexpressing CD47 were not taken up. In otherwords, the presence of CD47 in a cell was adeterrent to phagocytosis.

The study of Oldenberg et al. (8) was abreakthrough nding because it indicatedthat CD47 was acting as a molecule thatregulated the recognition ofself.Simplystated, the study indicates that expressionof a level of CD47 by a cell will not favor itsuptake by macrophages. Macrophages do

not phagocytose their own cells: self-proteinsavoid phagocytosis because they have CD47as a dont eat mesignal, aside from lack-ing surface proteins that have complemen-tary receptors on macrophages required forthe uptake. CD47 is known to engage areceptor protein of the macrophage calledsignal regulatory protein- (SIRP-) (re-

viewed in ref. 5). Engagement of SIRP-

leads to inhibition of phagocytosis throughan activation of a cellular phosphatase thatinhibits cytoskeletal proteins. Phagocyto-sis of particle cells or pathogens is a complexprocess that involves membrane interactionsand the rearrangement of intracellular organ-elles and cytoskeletal proteins to engulf thematerial (Fig. 1).

CD47 is one of two molecular systems inwhich cells distinguish self from altered-selfby the absence of a normally expressed pro-tein. This capacity to distinguish self by the

absence of a surface protein is the

missingself hypothesis(9), rst shown for naturalkiller cells, a cell lineage that patrols tissuesand is important in early antiviral and anti-tumor resistance. In the case of natural killercells, the cell-surface molecules are class Ihistocompatibility molecules interacting withreceptors in the natural killer cells (10).

Studies in the Weissman laboratory rec-ognized the features of many cancer cells indisplaying large amounts of CD47, and bydoing this evading recognition by the macro-phages (2). Indeed, in experimental systemsin which tumor cells were transplanted intomice, the growth of the tumor was controlledby injection of antibodies that neutralizedCD47 (24, 11). These experiments only ex-amined the interactions of the innate cellularsystem with the cancer cells: the cancer cellshad been injected into immunodecientmice. Thus, blocking the antiphagocyticproperties of CD47, the molecules that conferthe dont eat mesignal enabled the innatecell system represented mainly by macro-phages to attack the tumor. (Macrophages,natural killer cells together with dendriticcells, granulocytes, and mast cells constitute

the innate system, which participates in di-verse reactions early in the immune responseto pathogens as well as during the inamma-tory reactions that follow. This innate systeminteracts in a truly symbiotic fashion withthe adaptive cellular system of T and B cells.)

There is a second component of cancercells that makes it possible for blockade ofCD47 to be effective (12) (Fig. 1). As a resultof stress or their intrinsic reactivity, manytumors abnormally express proteins thathave complementary receptors on phago-

cytes, which favor their phagocytosis. Oneof these proteins is calreticulin, a protein nor-mally expressed in the endoplasmic reticu-lum. Calreticulin acts as a chaperone ofunfolded proteins in the endoplasmic re-ticulum, but it can be translocated to plasmamembrane, where it is found in high levels in

Fig. 1. (A) Macrophages ignore normal cells as a result

of negative interactions in which the CD47SIRP- pair

promote the dont 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).

Author contributions: E.R.U. wrote the paper.

The author declares no conict of interest.

See companion article on page 11103.

1E-mail:unanue@wustl.edu.

1088610887 | PNAS | July 2, 2013 | vol. 110 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1308463110

mailto:unanue@wustl.eduhttp://www.pnas.org/cgi/doi/10.1073/pnas.1308463110http://www.pnas.org/cgi/doi/10.1073/pnas.1308463110mailto:unanue@wustl.edu

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conditions of endoplasmic reticulum stress,cell death, and in cancer cells (13). Calreticu-lin interacts with the receptor protein LDLreceptor-like protein on the macrophage: thisis the eat memolecular pair (14). The com-bination of calreticulin together with the in-hibition of the CD47-Sirp-, the dont eatmepair, promotes the effective phagocytosis

of cells (14), and most prominently cancercells (12) (Fig. 1).The potential use of anti-CD47 antibody

treatment as a therapeutic modality for hu-man cancer would be greatly increased iflymphocytes reactive to the tumor would berecruited into the tumor-rejection reaction.This is the case for the anti-CD47 therapy, asthe Tseng et al. (1) article now shows. Spe-cically, the new experiments indicated thatin the presence of antibodies to CD47, mac-rophages engulfed tumor cells that expressedthe protein ovalbumin (i.e., tumor cells weremade to express this protein), which thenserved as a surrogate tumor antigen. CD4and CD8 T cells were available that reactedwith specic peptide segments in the ovalbu-min protein so they could be tested and usedas probes of a specic antitumor reaction.The macrophage phagocytosed the tumorcells and degraded the protein-selecting pep-tides, which were bound to histocompatibilitymolecules. Macrophages that phagocytosedthe cancer cells then were able to stimulatethe specic antiovalbumin CD8 T cells. Thisnding indicated that the macrophage hadpresented to the T-cell peptides from the

phagocytosed cancer cells, in this case pepti-des bound to class I histocompatibility mol-ecules. Such stimulation took place both inculture experiments but also in vivo. Indeed,the CD8 T cells stimulated by the macro-phages that engulfed the tumors after theantibody treatment controlled the growth ofthe tumor by killing the cancer cell, reduc-ing the tumor load. Interestingly, in thestudy (1) there was no stimulation of CD4T cells, indicating that there was differ-ential processing of the epitopes that gave

rise to peptides, which stimulated one or theother T-cell.

However, the ndings of Tseng et al. (1)bring up other important issues. The macro-phage handled the protein ovalbumin andpresented its immunogenic peptides into theclass I MHC system, making the point thatthe cross presentation, the phenomenon in

which engulfed proteins are able to be pre-sented by class I molecules, pertains to morethan one set of presenting cells and is notnecessarily the exclusive property of one spe-cialized set, such as the CD8 -dendritic cellsubset (15). The ndings also make a pointrecently ignored: that macrophages, the rstcells shown to present antigen (16), are in factexcellent presenting cells, depending muchon the circumstances of the immunologicalreaction (17).

In sum, there is one major message thatcomes from the study by Tseng et al. (1):that the anti-CD47 interaction with cancercells, together with macrophages, brings theadaptive T cells into the antitumor response,the symbiosis that is required between the

innate and the adaptive system for the im-mune system to operate at its best.

Cancer cells create an environment thatpermits their growth and curbs the actionof the innate and adaptive immune systems.Understanding the basis of these negativeeffects will permit a more rational attackon cancer. Immunotherapy against cancer

is a burgeoning

eld in oncology involving,among several therapeutic maneuvers, theuse of antibodies (18). These antibodieshave diverse targets, such as blocking an-giogenesis, or growth receptors on the tu-mors, or inhibitory interactions amonglymphocytes. The end result is that theantibodies foster the activation of cancer-specic lymphocytes: cancer cells can dis-play mutant proteins to which the immunesystem can react (19). The experimentalstudies establish that anti-CD47 therapy iseffective in various experimental systems, set-ting the rationale for translating these nd-ings into the cancer patient. Anti-CD47therapy should be part of the immunother-apy armamentarium.

1 Tseng D, et al. (2013) Anti-CD47 antibody-mediated phagocytosis

of cancer by macrophages primes an effective antitumor T-cell

response.Proc Natl Acad Sci USA 110:1110311108.

2 Chao MP, Weissman IL, Majeti R (2012) The CD47-SIRPpathway

in cancer immune evasion and potential therapeutic implications.

Curr Opin Immunol24(2):225232.

3 Kim D, et al. (2012) Anti-CD47 antibodies promote phagocytosis

and inhibit the growth of human myeloma cells. Leukemia26(12):

25382545.

4 Majeti R, et al. (2009) CD47 is an adverse prognostic factor and

therapeutic antibody target on human acute myeloid leukemia

stem cells. Cell138(2):286

299.5 Oldenborg PA (2013) CD47: A cell surface glycoprotein which

regulates multiple functions of hematopoietic cells in health and

disease. ISRN Hematol2013:614619.

6 Campbell IG, Freemont PS, Foulkes W, Trowsdale J (1992) An

ovarian tumor marker with homology to vaccinia virus contains an

IgV-like region and multiple transmembrane domains. Cancer Res

52(19):54165420.

7 Brown E, Hooper L, Ho T, Gresham H (1990) Integrin-associated

protein: A 50-kD plasma membrane antigen physically and functionally

associated with integrins. J Cell Biol111(6 Pt 1):27852794.

8 Oldenborg PA, et al. (2000) Role of CD47 as a marker of self on

red blood cells. Science 288(5473):20512054.

9 Krre K, Ljunggren HG, Piontek G, Kiessling R (1986) Selective

rejection of H-2-decient lymphoma variants suggests alternative

immune defence strategy. Nature 319(6055):675678.

10 Jonsson AH, Yokoyama WM (2009) Natural killer cell tolerance

licensing and other mechanisms. Adv Immunol101:2779.

11 Willingham SB, et al. (2012) The CD47-signal regulatory protein

alpha (SIRPa) interaction is a therapeutic target for human solid

tumors.Proc Natl Acad Sci USA 109(17):66626667.

12 Chao MP, et al. (2010) Calreticulin is the dominant pro-

phagocytic signal on multiple human cancers and is counterbalanced

by CD47. Sci Transl Med2(63):63ra94.

13 Obeid M, et al. (2007) Calreticulin exposure dictates the

immunogenicity of cancer cell death. Nat Med13(1):5461.

14 Gardai SJ, et al. (2005) Cell-surface calreticulin initiates clearance

of viable or apoptotic cells through trans-activation of LRP on thephagocyte.Cell123(2):321334.

15 Kamphorst AO, Guermonprez P, Dudziak D, Nussenzweig MC

(2010) Route of antigen uptake differentially impacts presentation by

dendritic cells and activated monocytes. J Immunol185(6):

34263435.

16 Unanue ER (1981) The regulatory role of macrophages in

antigenic stimulation. Part Two: Symbiotic relationship between

lymphocytes and macrophages. Adv Immunol31:1136.

17 Gray EE, Cyster JG (2012) Lymph node macrophages. J Innate

Immun4(5-6):424436.

18 Mellman I, Coukos G, Dranoff G (2011) Cancer immunotherapy

comes of age. Nature 480(7378):480489.

19 Matsushita H, et al. (2012) Cancer exome analysis reveals a

T-cell-dependent mechanism of cancer immunoediting. Nature

482(7385):400404.

Unanue PNAS | July 2, 2013 | vol. 110 | no. 27 | 10887

COMMENTARY