cancer immunology: the search for specificityâ€”g. h. a. clowes … · cancer immunology is...

[CANCER RESEARCH 41, 361-375, February 1981]0008-5472/81 /0041-OOOOS02.00

Cancer Immunology: The Search for Specificityâ€”G. H. A. Clowes MemorialLecture1

Lloyd J. Old

Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Fig. 1.

Abstract

The major focus of cancer immunology has shifted awayfrom arguments about the validity of the immunosurveillancetheory of cancer to the more basic question of tumor-specific

antigens. Despite vast effort aimed at demonstrating suchantigens, their existence in the generality of cancer remainsunproven. Serological analysis of three tumor types, mouseleukemia, mouse sarcoma, and human malignant melanoma,has received most attention, and a rudimentary classificationof the surface antigens expressed by these tumors has begunto emerge. The prime candidates for antigens that can beconsidered tumor specific are the few instances of Class 1antigens that have now been serologically defined on mouse

' Presented May 30 at the 1980 meeting of the American Association for

Cancer Research, in San Diego, Calif.

and human tumors. These antigens show an absolute restriction to individual tumors, not being demonstrable on any othernormal or malignant cell type. Biochemical and genetic characterization of Class 1 antigens represents an essential nextstep in evaluating the significance of these antigens. Thesurprising features of the Thymus Leukemia (TL) antigens ofthe mouse provide insight into the genetic origin of another keyclass of tumor antigens, in this case antigens with characteristicproperties of both differentiation antigens and tumor-specific

antigens. In normal mice, TL antigens are restricted to cells inthe thymus, and strains differ with regard to expression versusnonexpression of TL antigens. Genetic information for TL isuniversal in the mouse, however, as leukemias developing inmice that normally lack TL are found to express TL.

What is clear from the past two decades of research incancer immunology is that a far more detailed knowledge ofsurface antigens of tumor cells will be necessary before wecan begin to assess the possibility of Â¡mmunological control ofcancer.

I am, of course, deeply pleased to have been asked to deliverthe 20th Annual Clowes Lecture honoring the memory of agreat pioneer in cancer research. Dr. Clowes clearly saw thepotential of immunological approaches to cancer and wouldhave been delighted with the surge of interest in cancer immunology in the recent past. Two of my distinguished predecessors in this series of lectures, Dr. Lloyd Law (58) and Dr.George Klein (52), dealt with aspects of cancer immunology intheir Clowes Lectures and reviewed the evidence for immunereactions to cancer coming from the study of transplantationantigens and the effect of immunosuppression on cancer susceptibility. My intention will be to stress findings coming fromthe use of serological techniques in the analysis of surfaceantigens of cancer cells and to see what this can tell us aboutimmune reactions to cancer in mice and humans.

In choosing the title for this lecture, my intention was to focuson the issue of specificity in relation to key questions thatconcern cancer immunologists. I use the term specificity in twoways, one in a more general sense that immunologists, particularly serologists, employ the term and the other in referenceto cancer antigens and the current status of efforts to definecancer-specific antigens. To the serologist, defining the specificity of a serological reaction is of overriding concern, the aimof such analysis being to identify the antigenic determinantsrecognized by antibodies. The extraordinary power of antibodies to make fine distinctions between molecules and to do sorapidly and with great sensitivity quickly expanded the horizonsof serology from its origins in the study of microbes andimmunity to infectious diseases to the use of antibodies asgenetic and biochemical probes. Karl Landsteiner, in a careerthat spanned over 50 years, was the central figure in this

FEBRUARY 1981 361

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L. J. Old

development. Although a chemist by training, his massive contributions, ranging from his discovery of blood group antigensto his classic analysis of the specificity of antibodies to artificialhaptens, resulted from a clear understanding and appreciationof the power of serological methods. Given techniques of thisresolving power, it is no surprise that cancer has been theobject of so much scrutiny by serologists. The fascinationstemmed, of course, from the expectation that antibodies mightreveal something specific, something unique about the cancercell, and the search that began in the last century for thephilosopher's stone of cancer immunology, the cancer-specific

antigen, continues unabated to this day in what surely is one ofthe longest uninterrupted lines of inquiry in cancer research.Although much of interest and value has been uncovered, anddespite innumerable claims to the contrary, the elusive cancer-

specific antigen has yet to be identified. The recurring storyover the decades has been that claims for cancer specificityare quickly found to be based on an incomplete or inadequateanalysis of serological specificity. Where serological specificityis first established, cancer specificity disappears.

In tracing the origins of current interest in cancer immunology, these early serological studies of cancer by heteroimmunesera played little role. Rather, it was the development of theinbred mouse and the resulting analysis and understanding ofthe genetics of transplantation immunity that provided thesound foundations on which the field was to grow. The discovery by Ludwik Gross (35), Edward Foley (26), and RichmondPrehn (80) that inbred mice could be immunized against transplants of chemically induced sarcomas arising in animals of thesame strain initiated the modern era of cancer immunologyand, as Peter Gorer put it, lifted the "unrelieved gloom" that

had come to be associated with the study of cancer immunity.And it is Peter Gorer, the brilliant serologist and mouse geneticist whose analysis of the H-2 complex of the mouse led to the

rapid growth of knowledge about histocompatibility antigens inanimals and humans, who has had an equally dominant influence on the course that cancer immunology, particularly cancer serology, was to take over the past 25 years.

Major Topics of Interest in Cancer Immunology

The rapid growth of interest in tumor immunology has led tothe field's becoming widely diversified, the only apparent link

among the different approaches being the application of im-munological techniques to the problems of cancer. Nevertheless, a number of clear themes have emerged over the yearsand, although there is extensive overlap, these can be groupedin the folllowing way (Table 1).

In the area of cancer immunogenetics, the coupling of serological probes and genetic analysis has proved to be a powerfulway to answer basic questions about cancer, such as theidentification of genes involved in cancer susceptibility and thedefinition of genes coding for surface antigens of cancer cells,a subject we will discuss in detail later. The early discovery byLilly ef al. (59) that genes in the MHC2 exert a profound

Table 1

Major topics of interest in cancer immunology

2 The abbreviations used are: MHC. major histocompatibility complex; MuLV,murine leukemia virus; FeLV, feline leukemia virus; EBV, Epstein-Barr virus;BCG, Bacillus Calmette-GuÃ©rÃ¬n;C. parvum, Corynebacterium parvum; TL, Thymus Leukemia antigen; gp70, MulV glycoprotein with a molecular weight of70,000; TNF. tumor necrosis factor; Clg, cold-insoluble globulin.

Immunogenetics Genetic determinants of cancer susceptibility, immunity to cancer, and cancer antigens.

Immunovirology Serological analysis of structural and nonstructuralcomponents of oncogenic viruses.

Immune reactions to oncogenic viruses and virus-transformed cells.

Seroepidemiology of oncogenic viruses in animalsand humans.

Immunochemistry Identification, isolation, and characterization ofcancer antigens.

Immunobiology Definition and regulation of cellular and humoralimmune reactions to cancer.

Immunodeficiency and cancer susceptibility.Immunological escape mechanisms.

Immunodiagnosis, Use of cancer antigens in cancer detection, ther-Immunotherapy, apy, and prevention.Immunoprevention Immunoregulators (microbial, synthetic, lympho-

kines, and monokines), monoclonal antibodies,and cloned T-cells in cancer therapy.

influence on susceptibility to leukemia in the mouse has led toa massive and continuing effort to find a comparable association between MHC antigens and cancer susceptibility in humans. Insight into the possible mechanism involved in MHCcontrol of leukemias came from the subsequent realization thatMHC genes also control immune recognition and response toa variety of antigens, both natural and synthetic (4), and thereis now active interest in defining the role of MHC-linked genes

in immune reactions to cancer antigens.In the study of viruses, there has always been a strong

dependence on immunological techniques and probes, andthis is certainly also true in the study of oncogenic viruses. Therapid advances in viral oncology have been dependent in largemeasure on the serological reagents that have been developedto structural and nonstructural components of RNA and DMAtumor viruses, starting from the work of Huebner and colleagues in defining the T-antigens of papovaviruses (7) andadenoviruses (42) and the group-specific antigens of avianretroviruses (41 ), and the work of our group in defining group-

specific antigens (29), interspecies antigens (28), and cellsurface antigens (77, 93) coded for by MuLV. Serologicalanalysis of FeLV and EBV resulted in reagents that permittedwide-scale seroepidemiological studies to be performed, and

these have led to unexpected findings of singular importance.As Hardy ef al. (36) discovered, much to the surprise of thoseworking with mouse leukemia viruses, FeLV is transmitted in ahorizontal fashion, from cat to cat, infection being followed bya greatly increased risk of leukemia development. In the caseof EBV, seroepidemiology not only proved the associationbetween EBV and Burkitt's lymphoma (25) but also revealed

the link between EBV and carcinoma of the postnasal space(75), and infectious mononucleosis (38), associations that weretotally unexpected.

Immunochemistry, a discipline that began with quantitativeprecipitation analysis and ultracentrifugai analysis of antibody,grew by quantum leaps as the newer techniques of carbohydrate and protein chemistry have been incorporated. Cell surface antigens identified by well-defined serological reagents

have been particularly amenable to study with these techniques, as the rapid accumulation of detailed information aboutthe structure of products of the major histocompatibility complex in mouse and humans has shown (66, 94).

As knowledge of the intricacies of the immune response has

362 CANCER RESEARCH VOL. 41



grown, with the findings of helper cells, suppressor cells,antigen presentation by macrophages, soluble regulatory andgrowth factors, associative recognition, and idiotype-antiidi-

otype networks, it can be expected that similar principles andsimilar regulatory influences will be found to apply to theimmune response to tumors. Understanding these principlesand how to manipulate the immune system in the direction offavoring tumor rejection is a major focus of interest to cancerimmunobiologists. Tumor cells are in no way unique in beingable to escape destruction as a consequence of their antige-

nicity; fetuses do so with regularity, and grafts of kidney and awhole host of parasites survive quite successfully in the face ofdemonstrable immune recognition. As the various strategiestumors use to escape immune destruction are understood,more and more rational approaches can be developed to studythe effect of natural and synthetic immunoregulatory agentsand active immunization with tumor cells and tumor antigens.

An underlying assumption in much of the work going on incancer immunology is that antigenic changes recognizable bythe host's immune system are an invariable accompaniment of

malignant transformation. Although this is frequently true oftumors induced by viruses and certain chemical carcinogens,the assumption is without foundation with regard to most spontaneous tumors of animals and the generality of human cancer.In my opinion, it is simply too soon to say whether tumor-

specific antigens, i.e., antigens with an absolute restriction tocancer, exist. Recent advances in cellular immunology, especially the ability to propagate and clone T-cells (30, 61 ) and in

serology with the development of hybridoma technology (56),represent powerful new ways to address this long-standing

issue. In the past few years, it has become popular in certainquarters to question the whole basis of immunological approaches to cancer. This negative view comes from the failureof transplantation tests to reveal any immunogenicity in a seriesof spontaneous mouse tumors (39) and from the finding thatimmunosuppressed animals, particularly athymic nude mice,do not have a high incidence of spontaneous or chemicallyinduced tumors (95). This, our critics say, shows that theÂ¡mmunosurveillance theory of cancer is incorrect, that paststudies demonstrating immunogenicity of certain tumors areartifactual, and that little can ever be expected of Â¡mmunother-apy, throwing in as an aside to bolster the argument commentsabout the general ineffectiveness of BCG and C. parvum inclinical trials. I find several things with which to disagree inthese conclusions. For one thing, the value of immunologicalapproaches to cancer has never, at least in my view, dependedon the validity of the immunosurveillance theory of cancer. Ifthere is a basic tenet in the field, it is that cancer cells areantigenically distinguishable from their normal progenitors,whether this leads to immune recognition or not, and, as I saidbefore, it is too soon to say anything definite one way oranother about that. Secondly, there is no reason to believe thatall cancer antigens will be recognized as transplantation antigens capable of eliciting cell rejection. In fact, with the manyescape routes available to tumor cells, it is surprising thatcancer antigens were ever demonstrable at all by tests fortransplantation resistance. And finally, the general ineffectiveness of BCG and other immunopotentiators gives no indicationwhatsoever as to the ultimate value of immunological approaches to cancer. All of us involved in early work with BCGknew that the antitumor effects in animal systems were modest

Cancer Immunology: Search for Specif/city

at best. To expect results on this first try in humans wasunrealistic.

Cell Surface Antigens of Mouse Leukemias

With these general comments about the origins and majorissues facing investigators in the field, I now want to turn to atopic that has occupied my colleagues and me for the past 20years, the serological analysis of the surface antigens of cancercells. Although cancer-specific antigens were what we set out

to find, the paths we take in research rarely go in direct orpredictable ways, and so it was with ours. What experiencehas taught us over and over is that the study of cancer cellstells us important and surprising things about normal cells andthat exploration of these unexpected findings become mightypreoccupations along the way. The antigens that we discoveredhave led us to inquire into aspects of gene regulation anddifferentiation, cellular and transplantation immunology, andviral biochemistry and genetics. In addition, as the rich diversityof antigens expressed on the surface of cancer cells becameevident, some of our preconceived ideas about what characteristics cancer-specific antigens would or should have, havehad to be modified.

The work I will draw on first to illustrate these points wasdone with mouse leukemia. There were several reasons for ouroriginal choice of this tumor type as the object of study. Forone thing, leukemia is a common neoplasm of the mouse; somestrains such as AKR have a predictably high incidence ofspontaneous leukemia, whereas other strains with a low incidence of spontaneous leukemia develop leukemias when exposed to X ray or to certain chemicals or hormones. Mostmouse leukemias are of T-cell origin and arise in the thymus,and this gives the serologist studying such leukemias theenviable advantage of being able to examine the neoplastic cellside by side with what is generally thought to be its normalcounterpart, the thymocyte. Thymocytes and leukemia cellsare easy to obtain in large number and in free-cell suspension,and this greatly facilitates serological analysis. Finally, lym-

phoid cells, both normal and neoplastic, are highly susceptibleto the lytic action of antibody and complement, and this makesthem sensitive targets in cytotoxic tests, the serological methodwe chose to use in our studies (Chart 1). The cytotoxic testwas developed by Gorer and O'Gorman (32) in 1956 to detect

H-2 antigens on nucleated cells. Although there are a numberof ways in which cell lysis by antibody and complement couldbe measured, Gorer used trypan blue uptake as the end point,and we continue to find this the best method to assess cell

Target cells Antibody Complement s-Cell Lysis

(Normal or leukemiacells)

(Conventional ormonoclonal antibody)

(Guinea pig orrabbit serum)

(Trypan blue)

Adaptation:; of the cytotoxic test

1) Detection and quant.tation of cytotoxic antibody

2) Detection and quantitation of cell surface antigens(quantitative and qualitative absorptions withviable cells, tissues, viruses or soluble antigens)

3) Mapping cell surface antigens (blocking test)

Chart 1. Description of the cytotoxic technique.

FEBRUARY 1981 363



L. J. Old

Immunizing cell Recipient Antiserum Target cell Cytotoxic test

H-2a

Chart 2. Immunization strategy for detection of TL antigens.

TLdetected

injury and death. With the various modifications and adaptations that have been made in the test over the years, it hasbecome an extremely sensitive, reliable, and versatile way todetect and analyze cytotoxic antibody. In fact, virtually everyantigen that has been defined serologically on nucleated cellsof the mouse was initially discovered through the application ofthis remarkably simple test.

As a consequence of this focus on lymphoid cells, more isknown about the cell surface antigens of these cells than ofany other nucleated cell type in the mouse. At the onset of ourstudies, H-2, the major histocompatibility antigen of the mouse,

was the only antigen that had been serologically defined onmouse cells. One of the problems confronting us in the searchfor other antigens on leukemia cells was that antibodies to H-2

were the dominant ones formed after immunization, and theirpresence obscures the detection of other antibodies. To overcome this, a number of serological strategies have been developed (78); the most recent, elegant, and powerful is thehybridoma technique, which permits capture and expressionof plasma cells producing antibodies to a single specificity. Thefirst strategy we used to get around the concomitant presenceof H-2 antibodies led to the discovery of TL antigens (76). The

idea behind this method of immunization and testing is thatthere are shared leukemia-specific antigens occurring on leu-

kemias arising in different strains of mice (Chart 2). Leukemiacells from mice of strain A are used to immunize C57BL/6 (B6)mice, and this leads to the production of antibodies to H-2

antigens, to other alloantigens that distinguish A and B6 mice,and to the theoretical shared antigens of A and B6 leukemias.By testing these immune sera on B6 leukemia cells, the presence of antibodies to normal A strain H-2 alloantigens is irrel

evant because these are not present on B6 cells. Cytotoxicreactions with antisera produced by alloimmunization andtested on syngeneic leukemia cells indicate detection of sharedantigens, in this case TL. Table 2 shows some of the otherways in which antibodies to non-H-2 antigens were raised andinitially defined. These include immunization of H-2-compatibleor syngeneic partners and the removal of H-2 antibodies by in

vivo absorption, a technique developed by Gorer and Amos(31 ) in which H-2 antibodies are removed by a brief sojourn innormal mice expressing the relevant H-2 antigens. Naturallyoccurring antibodies to MuLV-related antigens are also valuable reagents that lack contaminating H-2 antibodies.

Let me choose three systems of antigens on the surface ofleukemia cells to illustrate how the serological definition ofthese antigens provided tools to investigate questions aboutnormal T-cell development and function and about leukemo-

genesis in the mouse.The first will be antigens belonging to the Lyt family (9) (Table

3). These antigens mark cells undergoing T-cell differentiation

and maturation; no other cell type is known to express Lyt

Table 2

General strategies for detection of mouse cell surface antigens

Method Antigen detected

Alloimmunization/syngeneic target cell

H-2-compatible immunizationSyngeneic immunization

In vivo absorptionNatural antibody

Hybridoma/monoclonal antibody

TLGCSAThy-1FMRG,xAbelsonLytG(AKSL2)

G(ERLD)

G(RADAl)

Previously defined systemsNew systems

Table 3

Lyt antigens of normal and leucemie mouse cellsRestricted to cells belonging to the T-cell lineage (differentiation antigens)

2 separate alloantigenic systems

Cell surface glycoproteins

Define T-cell subsets

Lyt-1 (chromosome 19)Lyt-2,3 (chromosome 6)

Lyt-1 (m.w. 67,000)Lyt-2,3 (m.w. 35,000)

Helper T-cells Lyt-1 *2~3~

Cytotoxic T-cells Lyt-1*2*3*

antigens. It is for this reason that Boyse and I designated theseantigens, and others that distinguish cells undergoing distinctpathways of differentiation, "differentiation antigens" (10).

Two systems of Lyt antigens have been characterized: Lyt-1

antigens, coded for by a locus with two alÃeleson chromosome19; and Lyt-2 and -3 antigens (each also with two alÃeles)

coded for by a single locus or closely linked loci on chromosome 6 (43). The presence of Lyt antigens on mouse leukemiasdefines these cells as having arisen from progenitors belongingto the T-cell lineage. Biochemical studies have indicated thatLyt-1 determinants reside on a glycoprotein with a molecularweight of 67,000 (23). Molecules with Lyt-2 and -3 determi

nants are also glycoproteins, but with a molecular weight of35,000 (22). It is not yet established whether Lyt-2 and Lyt-3

reside on the same molecule or on two distinct molecules.Enormous interest in Lyt antigens was generated by our findingthat T-cells with different functional activities could be distinguished on the basis of their Lyt phenotypes. Cytotoxic T-cellswere found to express Lyt-2 and -3 antigens (86), whereashelper T-cells did not (50). Although there is still some debateabout the presence of Lyt-1 antigens on killer cells, it appearsthat all T-cells express Lyt-1. Our current view of the differentpathways of T-cells is given in Chart 3. Precursor cells in thebone marrow programmed for T-cell development express H-2antigens but none of the T-cell differentiation antigens. After

migration to the thymus and presumably under the influence ofthymic hormones, these cells begin to manufacture T-cell




Bone Marrow Precursor Thymus

Cancer Immunology: Search for Specificity

Periphery Functional Activities

H-2

H-2

Helper T cellProduce TCGFRespond to Con ARecognize Mir and j_ region

products

Lyt-2,3

Thy-1 Thy-1

Cytotoxic T cellSuppressor T cellRespond to PHARecognize H-2D and H-2K

products

Chart 3. Modification in the surface phenotype of T-cells during T-cell differentiation. TCGF, T-cell growth factor; Con A, concanavalin A; PHA, phytohemagglutinin

A.

markers, namely, TL, Thy-1, and Lyt antigens. Although notfinally established, it appears that Lyt-1+ cells and Lyt-1 *2*3+

cells represent two separate T-cell lineages and are not derivedfrom a common Lyt-1 *2*3+ precursor. Peripheralization of

these T-cells results in loss of TL and acquisition of functionalproperties, i.e., alloreactivity, helper and killer activity, andresponse to certain mitogenic lectins. An intriguing new findingmade by Nakayama et al. (64, 65) is the ability of Lyt-2,3antibody to block alloantigen-induced T-cell proliferation andT-cell killing. The specificity of this blocking and the fact thatthe locus for Lyt-2 and -3 is tightly linked to genes determiningK chains of Â¡mmunoglobulins (33) raise the possibility that Lyt-

2,3 molecules may be involved in the construction of at leastone class of T-cell receptor.

Like Lyt antigens, TL antigens are T-cell differentiation anti

gens (78) (Table 4). Unlike Lyt antigens, their expression innormal mice is restricted to T-cells in the thymus. Not all mouse

strains express TL antigens, and this permitted genetic analysisshowing that the TL trait is determined by a locus on chromosome 17, less than two crossover units from H-2. This closegenetic association with H-2 is also mirrored in related bio

chemical properties, both having a molecular weight of 45,000and an association with ^-microglobulin. What makes the TLlocus of such interest in the study of leukemia is the findingthat TL may appear in the leukemias of strains that normallynever express TL during fetal or adult life. This anomalousappearance of TL antigens in leukemias of mice with a TL~

thymocyte phenotype has been taken as evidence for theuniversal presence of TL structural information in the mouse.Thus, the TL* and TL" phenotype of normal mice is not due to

presence versus absence of TL structural genes but rather toregulatory genes that control expression versus nonexpressionof TL. Leukemogenesis disrupts this regulatory mechanism andpermits the expression of normally silent TL genetic information. Although a great number of changes associated withcancer have been ascribed to abnormalities in gene control,such as appearance of embryonic or fetal characteristics,ectopie hormone production, and changes in enzyme levels,most if not all of these are quantitative rather than qualitativefeatures, reflecting possibly the phenotype of the normal cellpopulation from which the malignant cell arose. The appear-

Table 4

TL antigens of normal and leukemic mouse cells

Expression in normal mice restricted to thymocytes.TL* and TL mouse strains.

Determined by the Tla locus on chromosome 17.Cell surface glycoprotein, rn.w. 45,000 (associated with /32-microglobulin).TL * leukemias occur in TL ~ strains.

ance of TL antigens in the leukemias of TL mice is the clearestexample of a qualitative change in gene expression associatedwith cancer, and the frequency with which it occurs suggeststhat it is causally related to leukemogenesis. In fact, where ithas been possible to test because of a genetic marker identifying the TL lineage of normal cells, anomalous TL inductionhas been found to be an invariable accompaniment of leukemictransformation. The mechanism underlying this activation ofnormally silent genetic information is obscure, but mutationsinvolving TL-regulatory sequences, alteration in regulatory control of TL due to chromosomal insertion of leukemia virus genesor other types of insertional elements, and leukemia viruscapture and independent replication of TL structural genes arethree obvious possibilities (Chart 4). It should not be too longbefore the sort of molecular analysis that is giving insight intoimmunoglobulin and hemoglobin genes can be applied successfully to the analysis of TL regulation.

TL antigens have another remarkable feature, and that is theease with which these antigens can be stripped from the cellsurface by exposure to antibody (Chart 5). When we firstdiscovered the TL system, we expected that immunization withTL antigens would generate strong transplantation resistanceagainst TL* leukemias in TL" strains, judging from the high

titers of TL antibody that could be elicited in these mice. Muchto our surprise, TL* leukemias grew equally well in immunized

or unimmunized mice. The basis for this remarkable escapefrom immune destruction is a rapid loss of sensitivity to thecytotoxicity of TL antibody and complement following exposureto TL antibody either in vivo, by passive or active immunization,or in vitro. Loss of TL is reversible, full sensitivity to TL antibodyreturning after 1 to 2 cell divisions in the absence of TLantibody. We refer to this phenomenon of antigenic loss andreturn as "antigenic modulation" and subsequently found that

FEBRUARY 1981 365



L. J. Old

TL StrainsTL" Strains

Thymocytes or Leukemia cells Thymocytes

Mutation

Leukemia cells

integration

MuLV captureof TL structural genes

TL product No TL product

Chart 4. Regulation of TL expression.

TL product

Procedure

TL* leukemia-

TL Phenotype

- Exposure-toTLantibodyin vivoorin vitro

-Loss ofsensitivityto TL antibody andcomplement

â€¢â€¢Sensitivityreturnsalter 1-2 celldivisions in absenceof TL antibody

TL* -Â«.TL*

Chart 5. Antigenic modulation of TL antigens.

a similar process could be induced with surface immunoglob-

ulins but not with several other cell surface antigens. Antigenicmodulation can now be viewed in the light of recent knowledgethat many cell surface molecules undergo conformational andpositional changes as a consequence of antibody attachmentleading to patching, capping, and in some cases temporarydisappearance of the component from the cell surface. WhyTL and surface immunoglobulin are so particularly susceptibleto these antibody-induced changes is unknown but may be

related to the functional significance of these molecules.Before leaving this discussion of TL antigens, I want to stress

two lessons that we have learned from analyzing TL antigensthat are particularly relevant to our discussion of tumor antigens. One is that tumor antigens, like TL, can be either normaldifferentiation antigens or tumor-specific antigens, depending

on the genetic background of the host in which the tumorarises. The other is that not all tumor antigens can be detectedas transplantation antigens. The existence of antigens such asTL would have been missed had transplantation tests been theonly method used for their detection.

The last category of leukemia antigens I want to discuss arethose related to MuLV. MuLV genetic information is ubiquitousin the mouse, but whether these endogenous viral genes areexpressed or not and if expressed whether leukemia results, isdetermined by a variety of poorly understood viral and hostgene functions (60, 82). Endogenous MuLV of inbred mice fallinto three general classes: ecotropic MuLV, favoring replicationin mouse cells; xenotropic MuLV, replicating preferentially incells of foreign species; and the recently recognized dualtropicMuLV which replicate well in both mouse and non-mouse cells.Although this classification is based on viral host range, it isstrongly supported by interference and neutralization patterns,radioimmunoassays with isolated viral components, and pep-

tide and nucleotide mapping.Study of the cell surface antigens coded for by these viruses

began many years ago with the serological analysis of spontaneous and MuLV-induced leukemias (78). A summary of the

antigens that have been defined is given in Table 5. Both viralcore and envelope gp70 products have been detected on thecell surface, and the use of monoclonal antibodies promises toextend this list considerably. The four gp70-related antigenicsystems provide markers for each of the MuLV classes, withGix and G(RADAI>distinguishing two types of ecotropic MuLV,G(ERLD>identifying all xenotropic MuLV,3 and G(AKSL2>being a

marker for dualtropic viruses in AKR mice. Aside from theirvalue in typing viral isolates, including viral recombinants,analysis of these antigens has revealed the multiplicity ofresident gp70 genes in the mouse and the striking influencethat the state of cellular differentiation has on viral gene expression. Mouse strains differ in the pattern of gp70 determinantsnormally expressed on the surface of thymocytes (Chart 6).Three thymocyte phenotypes are found among low-leukemiainbred strains: some strains express no gp70; others expressonly G(ERi_D>;and others express both Gix and G<ERLD>-Normalthymocytes of high-leukemia-incidence strains, such as AKR

and C58, express all four of these gp70 determinants. G(AKSL2>is unique in that it has never been found in strains other thanthose with high leukemia incidence. The influence of cellulardifferentiation on expression of these gp70 traits is best illustrated in low-incidence strains, where replicating MuLV does

not make a contribution to the gp70 phenotype of the cellsurface (Table 6). G,x has the characteristics of a restricted-differentiation antigen in strain 129 mice, and this is also truefor the tissue distribution of gp70 determinants in other low-incidence mouse strains. Had we not known their relationshipto MuLV, these gp70 determinants would have been considered conventional differentiation antigens coded for by hostgenes, like TL and Lyt antigens. A critical but probably unanswerable question with regard to polymorphic gp70 determi-

Table 5

Cell surface antigens coded by naturally occurring MuLV

DesignationGCSAOnQ0MOA1]G(ERt_D)G(AKSL2>Relation

toMuLVstruc

tural componentp15,

p30gp70gp70gp70gp70Relation

toMuLVclassAll

classesEcotropicEcotropicXenotropicDualtropic

(MCF)Refs77,8970,

93.71789196

3 Y. Obata. E. Stockert. P. V. O'Donnell, A. B. DeLeo. H. W. Snyder, Jr., and

L. J. Old. GHin r. : a new cell surface antigen of the mouse related to the xenotropicclass of MuLV detected by naturally occurring antibody, manuscript in preparation.

ODD CANCER RESEARCH VOL. 41




Thymocytes oflow leukemia strains

Thymocytes ofhigh leukemia strains

GIX0 G(ERLD)O

G (RADA 1) Â© G (AKSL2) 0

Chart 6. Expression of MuLV gp70-related determinants on thymocytes ofdifferent mouse strains.

Table 6

Tissue distribution of G,x cell surface antigen

129 129-G,x AKR

ThymusSpleenLymph nodesBone marrowKidneyBrain

Leukemia

nants like G|Xon mouse cells is whether ancestral genes codingfor gp70 molecules were of cellular or viral origin. Do theyrepresent viral genes captured by the host or host genescaptured by the virus? Mice of the 129 strain appear to lackgenetic sequences related to ecotropic MuLV, and this explainswhy frequent attempts to isolate infectious ecotropic MuLVfrom strain 129 mice have failed. If G,x is of viral origin, thenonly part of the viral genome appears to have been integratedin strain 129 mice. On the other hand, G|X in strain 129 micemay represent the ancestral mouse gene whose original evolutionary history was unrelated to MuLV. From the study ofleukemia cells, we know that genetic information for Gtx, likegenetic information for TL, is ubiquitous in the mouse. Leuke-mias expressing GiX can be induced by X-ray in mouse strainssuch as 129-G|X" that normally never express this antigen in

any of their tissues. Under these conditions, GIXis a leukemia-

specific antigen. In some cases, MuLV coding for G|X can beisolated from G|X+ leukemias occurring in G,x~ strains, but in

others no MuLV can be found, indicating that G,x activation isnot necessarily associated with MuLV activation. In the high-leukemia AKR strain, where G,x and the other gp70 determinants are detected throughout life in many tissues, thymocytesshow an amplified expression of these antigens during the latepreleukemic period (48, 49) (Chart 7). This is not accompaniedby any change in the levels of ecotropic MuLV but is associatedwith the emergence of MuLV with dualtropic properties (37,48). These dualtropic MuLV, which have the unique propertyof inducing characteristic foci in mink cells (and thus the nameMCF), appear to arise as a consequence of recombinationalevents between ecotropic MuLV and other sequences in thecells, either viral or cellular. Although it has long been consid-

Expression of:

GIX- G(RADA1).

G(ERLD)' G(AKSL2)

Levels of MuLV

2 month 6 month

Ecotropic MuLV

2 month 6 month

Chart 7. Age-related changes in preleukemic AKR mice.

ered that AKR ecotropic MuLV is the leukemogenic agent inAKR mice, recent studies with cloned AKR isolates indicatethat ecotropic MuLV are not leukemogenic. In contrast, anumber of dualtropic isolates from AKR mice have been foundto be strongly leukemogenic (16, 69, 72)," leading to the belief

that dualtropic MuLV are the proximal vectors of leukemoge-

nesis in AKR mice. This idea receives further support fromrecent work in our laboratory (92). Injection of SMX-1, a

nonleukemogenic dualtropic virus, early in life prevents spontaneous leukemia development in AKR mice, most probably byinterfering with the emergence, spread, or integration of leukemogenic dualtropic MuLV (Chart 8).

Although much progress has been made toward definingproducts of the transforming sequences of certain retroviruses,transformation-specific products have not been found in as

sociation with avian leukosis viruses or murine leukemia virusescausing T-cell leukemias, and the mechanism whereby these

viruses induce leukemia is still a mystery. In the case of AKRmice, the formation of recombinant MuLV through reassortmentof viral genes or capture of host genes may induce the regulatory disorders that lead normal cells to become neoplastic,and the insertion of novel molecules into the surface of cellscould be one of the ways in which this is brought about.Whatever the initiating events in AKR leukemia turn out to be,however, stable genetic change appears to be one of the endresults, and in the case of T-cell leukemias this is associatedwith a highly consistent cytogenetic abnormality, trisomy 15(20, 90).

Table 7 summarizes the current information about the categories of serologically demonstrable surface antigens in mouseleukemias of thymic origin. In our analysis, we have not yetfound antigens that are leukemia specific in the strictest sense,i.e., expression restricted to leukemia cells and never found onany normal cell. What we have encountered instead are antigens such as TL and certain gp70-related determinants that

behave as normal differentiation antigens in some mousestrains and as tumor-specific antigens in strains that do not

express these antigens during normal life. This situation arisesbecause TL and gp70 coding genes are universal in the mouse,but in some strains of mice these genes are never activated orderepressed. Malignant transformation, either directly or indirectly, leads to expression of these normally silent genes and,because their appearance is restricted to tumor cells in thesestrains, these products have the characteristics of transfor-

FEBRUARY 1981 367



L. J. Old

Table 8

Demonstration of individually distinct transplantation antigens on differentmethylcholanthrene induced sarcomas derived from the same inbred strain

200 250

AGE (Days)

Chart 8. Protective effect of SMX-1 on the development of spontaneous AKR

leukemia.

Table 7

Categories of sero/ogical/y demonstrable surface antigens on mouse leukemiasof thymic origin

Conventional alloantigensDifferentiation alloantigensMuLV-related antigensDerepression antigens

Mammary tumor virus-related anti

gensSpecies antigensTransformation-specific MuLV anti

gensEmbryonic and fetal antigensIndividually distinct (unique) antigensIdiotypic immunoglobulin antigensAnomalous H-2 antigens

H-2D, H-2KLyt-1,2,3, Thy-1, TL.1,2,3

GCSA, Glx. GfRADAn, G(ERUD). G<AKSL2

TL. 1,2,4 (in TL strains)GCSA, GÃ¬Â«.GIRADAII(in strains not

expressing MuLV-related anti

gens in normal tissues)ML

MSTANone defined

None definedNone definedNone definedNone defined

mation-specific products. The idea that normally silent geneticinformation is activated in tumor cells has become a popularone, and the occurrence of tumor antigens shared with fetal orembryonic tissue or with histocompatibility antigens of alloge-

neic mice has been taken as evidence that this occurs quitefrequently. With rare exceptions, however, these antigens havenot been rigorously defined, and in many cases trivial explanations for these findings have not been excluded.

Cell Surface Antigens of Chemically Induced MouseSarcomas

Because of the ease of working with mouse leukemias, wehave far more knowledge about the surface antigens of leukemia cells than we do about other tumor types of the mouse. Amajor challenge to tumor Â¡mmunologists is the serologicaldefinition of the individually distinct transplantation antigensthat were first detected on chemically induced sarcomas (26,35, 53, 74, 80) but are now known to exist on other tumors aswell (15, 63, 98). The remarkable feature of these transplantation antigens is their extraordinary polymorphism, with eachtumor having a transplantation antigen unique to itself despitecommon derivation from mice of the same inbred strain (Table8). Although this class of tumor antigens was recognized morethan 25 years ago, our knowledge of these antigens hasadvanced very little over the years, and this slow progress isdue to the fact that no reliable in vitro methods for detectingthese antigens have been developed. This has not been due tolack of attention or effort, as attested by the large literature on

Immunizedwith

Challenged with

+ , resistance to tumor challenge: -. no resistance to tumor challenge.

the subject, but rather to difficulties involved in demonstratingthe specificity of cellular or humoral reactions elicited in miceimmunized with these tumors. Because MuLV antigens arefrequently associated with chemically induced sarcomas, wenow know that past studies that did not take these antigensinto consideration are impossible to interpret. With advancesin MuLV serology, this problem can now be controlled. By farthe major difficulty confronting the serologist wanting to studythese individually distinct antigens of chemically induced tumors is the general experience that these antigens do not elicitdemonstrable humoral immunity despite prolonged immunization and a resulting high degree of resistance to challenge withtumor grafts. In this respect, these tumor antigens resemblemutant H-2 products that also give rise to strong transplantationimmunity but do not result in an antibody response (54). Thatthis may not be an absolute rule is shown by the fact that twoantigens with an exceedingly restricted distribution have beendefined on BALB/c methylcholanthrene-induced sarcomas

with antibody derived from hyperimmunized mice (17, 18)(Table 9). The detection of the Meth A and CMS4 antigensgives us the first serological probes to investigate the nature ofthese highly restricted cellular antigens (Table 10). One of thefirst questions to be answered is whether the serologicallydefined antigens correspond to the transplantation antigens,and evidence from experiments carried out with Lloyd Law andEttore Appella on the purification of Meth A antigen indicatesthat they are related (21). Structural comparisons of Meth Aand CMS4 antigens should reveal whether they are relatedproducts, and this will give clues to their derivation from asingle genetic locus or distinct loci. Another way to approachthe question of genetic specification is through the analysis ofsomatic cell hybrids. Dimi Pravtcheva in Frank Ruddle's labo

ratory has tentatively assigned the gene for Meth A antigen tothe telomeric region of chromosome 12, and a comparablestudy of CMS4 antigen is now in progress. Ever since theindividually distinct transplantation antigens of chemically induced tumors were discovered, there have been argumentsabout the significance of these antigens, particularly whetherthey are causally related to cancer or reflect secondarychanges induced by chemical carcinogens. That these antigens occur in spontaneous tumors as well as in tumors inducedby chemical agents and that they cannot be eliminated bystrong immunoselection pressure favor the idea that they havea causal relationship to the malignant phenotype. A different

' P. V. O'Donnell, E. Stockert, Y. Obata. and L. J. Old. Leukemogenic

properties of AKR dualtropic (MCF) viruses: amplification of murine leukemiavirus-related antigens on thymocytes and acceleration of leukemia developmentin AKR mice, manuscript submitted for publication.





Table 9

Distribution of serologically defined Meth A and CMS4 antigens on BALB/cmethylcholanthrene-induced sarcomas

SarcomasMeth

ACMS1CMS2CMS3CMS4CMS5CMS7CMS8CMS9CMS10CMS11CMS12CMS13CMS14CMS15CMS16CMS17CMS18CMS19CMS20CMS21Meth

AantigenÅ’]â€”â€”_-â€”â€”â€”â€”â€”â€”â€”â€”â€”â€”â€”â€”_â€”â€”-CMS4antigen-â€”â€”_CEâ€”â€”â€”â€”i

+1-â€”â€”â€”â€”â€”â€”â€”â€”-

Table 10

Nature and significance of serologically defined Meth A and CMS4 antigens

Relationship to transplantation antigens?Products of a single locus or multiple loci?

Structural studiesSomatic cell genetic analysis

Causally related to transformation or secondary changes induced by carcinogen?

PrÃ©existence before transformation as a polymorphic family of molecules distinguishing normal cells?

Coded for by transfecting DMA?

view is that they are not tumor-specific antigens at all butrepresent an extremely polymorphic family of normal cell surface markers that distinguish individual cells one from another.Malignancy causes the clonal expansion of cells bearing suchmarkers, and because of the restricted nature of these antigensduring normal life, the immune system does not develop tolerance to such antigens and can recognize them as foreign onthe resulting tumor. Although this possibility has been approached experimentally (3, 24), it has not been ruled out. Therecent finding that DNA from chemically transformed cells cantransfer the malignant phenotype to nontransformed cells (85)provides a way to examine this relationship between antigenand malignancy. If the coding sequences for antigen and thecoding sequences for transformation are identical or closelylinked, they should cotransfer in such transfection experiments.

Cell Surface Antigens of Human Cancers

We now turn to a discussion of the antigens of human cancer.Although it is widely assumed that tumor-specific antigens have

been demonstrated in human cancer, there is no basis whatsoever for this belief. Heteroimmune sera that were initiallyclaimed to identify tumor-specific antigens have, on further

analysis, been found to be detecting quantitative differencesbetween tumors and normal tissues or differentiation antigenscharacterizing a step in the corresponding normal cell lineage.The vast literature that has been built around the study ofcellular and humoral immune reactions of cancer patients alsofails to provide critical evidence for claims of tumor specificity.

Aside from the fact that some of the uncertainty surroundingwork in this area is due simply to incomplete or inadequateanalysis, there is no doubt that establishing the specificity ofan immune reaction to human cancer cells represents anextremely difficult task. The human cancer immunologist doesnot have the inbred mouse to fall back on, with its ready supplyof immunized donors and normal tissues for specificity testingor the wide variety of genetically defined strains. Nor can heresort to the family studies that are such an essential part ofthe analysis of blood group antigens and histocompatibilityantigens. Without these advantages, how does the humancancer immunologist propose to prove the cancer specificityof reactions he observes? What I want to illustrate is how ourgroup has approached this issue and where we are in ourattempts to answer the two key questions of the field. Dohuman cancer-specific antigens exist, and if so do they elicit

an immune response in humans? Our initial decision to stressserological approaches to these questions had nothing to dowith prejudices about the relative importance of humoral versuscellular immune reactions against cancer. It was simply basedon the fact that defining the specificity of a serological reactionis far easier than doing so for a reaction involving lymphoidcells. Indeed, if MHC restriction of T-cells turns out to be the

case with tumor antigens as it is with other classes of cellsurface antigens (102), this adds yet another dimension ofcomplexity to defining the specificity of cellular immune reactions to human cancer antigens. Nevertheless, the discoveryof growth factors that permit the propagation and cloning ofspecifically reactive T-cells has set off a new search for cancer-specific T-cells in cancer patients, and it is not possible to say

where this will lead. Of course, these difficulties in defining thespecificity of T-cell reactions apply with equal, if not more,

force to the analysis of natural killer cells and other types ofcell-mediated killing.

In the evolution of our serological study of human cancer,we have attempted to develop as rigorous and comprehensivean approach as possible to the analysis of cell surface antigensand the issue of cancer specificity. The initial serologicalmethod that we set upon is referred to as autologous typingand has the following features (Table 11). Analysis is restrictedto the study of autologous reactions (i.e., reactions betweensera and tumor cells from the same patient) to eliminate thecontribution of alloantibodies to blood group antigens andhistocompatibility antigens and to detect antigens not expressed by any cells other than autologous tumor cells. Cultured cells, rather than cells directly from the tumor, are superior targets for the detection of cell surface antigens, andthe establishment of autologous tumor cell lines permits repeated serological testing over an extended period. Several

Table 11

Cell surface antigens of human cancers defined by autologous typing

Direct tests restricted to sera and tumor cells from the same individual (autologous combination).

Tissue culture lines as source of target cells (rather than cells directly from thetumor).

Parallel tests with several serological techniques.Absorption analysis to determine occurrence and quantity of antigen on cells

from various sources.Panel of cultured autologous normal cells for direct tests and absorption analy-

FEBRUARY 1981 369



L. J. Old

serological techniques are used in parallel to detect antibodiesbelonging to different immunoglobulin classes and subclasses.Finally, absorption analysis is relied on as the chief way todefine specificity, just as it has in the analysis of tumor antigensin the mouse. However, unlike work with inbred animals, normalcells from individuals with genetic identity to the tumor donorare not available except in the rare instance of identical twins.To overcome this problem, we culture as many different normalcell types as possible from the tumor donor. At the beginningof our studies, these included only skin fibroblasts and B-cells,but now other cell types can be cultured, including T-cells and

skin epithelium. These autologous normal cells (including peripheral blood cells), as well as allogeneic and xenogeneicnormal and malignant cells, represent the panel of absorbingcells used in our analysis. Malignant melanoma (12, 87, 88),astrocytoma (79), and renal cancer (97) have been the majortargets for our initial serological analysis, primarily becausethese tumors can be serially cultured in a fair proportion ofcases. Autologous typing of patients with acute leukemia (27)has also been possible, even though leukemia cells cannot beeasily adapted to tissue culture. Large numbers of viable leukemia cells can, however, be obtained directly from patientsand stored by cryopreservation for subsequent use as targets.Over the past seven years, more than 200 patients with thesetumor types have been analyzed by autologous typing. Theoutcome of this study has been the recognition that threeclasses of surface antigens can be defined by autologousantibody (Table 12). Class 1 antigens are restricted to theautologous tumor and cannot be found on autologous normalcells or on any other normal or malignant cell type. The sero-

logically defined Meth A antigen that we discussed previouslywould be a Class 1 antigen of mouse sarcoma. Class 2 antigensare shared tumor antigens, found on autologous as well as onallogeneic tumors of similar and in some cases dissimilarorigins. They are not detected on a wide variety of other tumortypes or on normal B-cells, kidney cells, or fibroblasts. In

contrast, Class 3 antigens are widely distributed on normal andmalignant cells, autologous, allogeneic, and xenogeneic. (Fetalcalf components adsorbed on the cell surface from the culturemedium would be classified as Class 3 antigens. These caneasily be identified by absorbing reactive sera with fetal calfserum or, more critically, determining whether the reactionpersists when the cells are grown in human serum rather thanin fetal calf serum.) By far, the largest number of reactionsdetected by autologous typing are due to antibodies directedagainst Class 3 antigens, and such antibodies in addition toalloantibodies undoubtedly account for the great majority ofpositive reactions recorded in past serological studies of humancancer and for many of the mistaken claims for tumor specificity. Antibodies to Class 3 antigens can give the impression of

Table 12

Classes of cell surface antigens of human cancers defined by autologoustyping

Class 1

Class 2

Class 3CharacteristicsIndividually

distinct (unique)tumor antigens

Shared tumorantigensNormal

cell surface antigensHeterologous serum compo

nentsDistributionRestricted

to autologous tumor cells

Present on autologous andsome allogeneic tumor cells

Widely distributed on normaland tumor cells

tumor specificity in direct tests because tumor cells may express higher levels of these antigens than do normal cells. Forthis reason, absorption tests are essential to distinguish Class3 antigens from Class 1 and Class 2 antigens.

Let me briefly review our findings with malignant melanoma.One hundred twenty melanoma cell lines have been establishedin our laboratory over the past seven years, and this representsa success rate of 20 to 25% for establishing long-term lines ofmelanoma. Autologous typing of sera from 75 melanoma patients showed IgG or IgM antibody in three-fourths of the

patients (Chart 9). Of the patients with antibody, four patientshad antibody identifying Class 1 antigens, five patients Class2 antigens, and 21 patients Class 3 antigens. The Class 1antigen that has received the greatest attention is AU (12)(Table 13). The patient whose sera defined this antigen was a51-year-old male with recurrent melanoma who had an unu

sually prolonged clinical course. Presence of antibody appeared to relate to presence rather than to absence of tumor,with tumor recurrence being associated with rising antibodytiter and tumor resection being associated with a falling titer.Peak antibody titers ranged up to I /256, and all antibodyresided in the IgG fraction. Despite these relatively high titers,it has not been possible to characterize AU antigen by theconventional radioimmunoprecipitation techniques that haveproven so useful for other cell surface antigens. Instead, wehave used antibody inhibition assays to follow antigen solubi-lization and characterization (11). AU antigen is easily solubi-

lized by papain, and molecular sizing shows that it has amolecular weight in the range of 25,000 to 40,000. The antigenis a glycoprotein (as indicated by its affinity for Lens culinarialectin), with no serological relation to HLA, la, or /?2-microglob-

ulin. Somatic cell hybrids formed by fusing AU melanoma cellswith Chinese hamster cells express AU antigen, and in segregating progeny the AU trait segregates independently of HLAand /?2-microglobulin. In one series of AU hybrids that has beenextensively analyzed by Lois Resnick and Dimi Pravtcheva, AUantigen appears to be coded for by a locus on chromosome19. In dealing with Class 1 tumor antigens of mice or humans,the major question that needs to be resolved is whether theyrepresent a family of structurally related molecules with polymorphic epitopes coded for by a single locus or totally unrelated molecules coded for by many loci. Structural studies andsomatic cell analysis of a series of Class 1 antigens should tellus which of these is true.

AutologousTyping ol MalignantMelanoma75 Patients(Stage II-IV)

Chart 9. Serological analysis of cell surface antigens of malignant melanomadefined by autologous typing.




Table 13

AU cell surface antigen of malignant melanoma

PatientAntibodyTiter

Antigen occurrenceAntigen characterization

Somatic cell hybrids

51-year-old male with recurrent melanoma.IgG.1/128-1 /256 (highest titer at time of tumor

recurrence).Class 1 (restricted to autologous melanoma).Glycoprotein (m.w. 25,000-40,000).Unrelated to HLA, la or /^-microglobulin.Expressed by AU melanoma x Chinese ham

ster hybrid cells.Tentative assignment of AU locus: chromosome

19.

AH antigen, the Class 2 melanoma antigen most extensivelyanalyzed, is defined by IgM antibody in the sera of a melanomapatient who has remained alive for six years after resection ofrecurrent melanoma (87, 99) (Table 14). It has been found on70% of melanomas and all astrocytomas but not on epithelialcancers, B-cell lines, or fibroblasts. Physical characterization

of AH antigen by Cliff Pukel and Kenneth Lloyd indicates thatit resides on a glycolipid moiety, and structural studies areunder way.

One of the major questions that can be raised about antibodyto Class 2 antigens is whether antibody is a direct consequenceof cancer development or whether antibody and cancer arecausally unrelated. To approach this, we have begun to analyzethe melanoma reactivity of sera from normal individuals. Aninitial survey of over 100 normal nontransfused males, ages 14to 68 years, has now been completed (40). In this population,naturally occurring IgG antibody to melanoma surface antigenswas rare, whereas IgM antibody was more common. IgM antibody in five of these normal individuals identified an antigenrelated to AH melanoma antigen, indicating that overt melanoma is not necessary for the development of AH antibody. Anextremely high-titered IgG antibody was found in another nor

mal individual in this series that defines a melanocyte differentiation antigen we have called Mel 1, expressed by normalmelanocytes and approximately 50% of the melanoma celllines.

This serological dissection of sera from melanoma patientsand from normal individuals tells us which melanoma surfacemolecules can be recognized as immunogenic by humans. Thehybridoma methodology in its current state tells us what theheterologous host, mouse or rat, recognizes as immunogenicon melanoma cells. Chart 10 summarizes information from ourongoing analysis of mouse monoclonal antibodies to human


melanoma cells (19). Monoclonal antibodies have now definednine systems of melanoma surface antigens, with six beingglycoproteins and three being glycolipids. Each shows a characteristic cellular distribution. None, however, defines as yet amelanoma-specific antigen. As a consequence of this intense

serological scrutiny by our group and by others (13, 57, 100,101), the list of melanoma antigens is growing rapidly, and wecan look forward to a comprehensive picture of the surfaceantigenic structure of melanoma cells in the near future.

There are many ways to put this information from autologoustyping, natural antibodies, and monoclonal antibodies to use.Defining the structure and genetic determinants of moleculesinvolved in the construction of the melanoma cell surface willundoubtedly give us new insights into melanocyte differentiation, just as definition of leukemia cell surface antigens gaveus new markers to study lymphocyte differentiation. With currently available markers, such as AH, Mel 1, la, and othersurface antigens defined by monoclonal antibodies, melanomas can be classified into several distinct subsets, and thisprobably relates to the stage in the melanoblast â€”Â»melanocyte

lineage when the transformation event took place. Relatingthese markers, individually or as a set, to biological and clinicalaspects of melanoma is an important task that has alreadybegun. Perhaps the most critical challenge for the tumor im-

munologist involves determining the range of melanoma surface molecules that are or can be made to be immunogenic inhumans. We already know from autologous typing that determinants, such as AU and AH, that have a high degree ofspecificity for melanoma cells can be recognized by melanomapatients. What we must ascertain is what clinical significancesuch antibodies have and why antibodies to antigens such asAH and Mel 1 are so rare in melanoma patients, despite thepresence of these antigens in over 50% or more of melanomas.In view of the demonstrable autoimmunogenicity of certainmelanoma surface components and the development now of

Table 14

AH cell surface antigen of malignant melanoma

Patient

AntibodyTiterAntigen occurrence

Antigen characterization

56-year-old male with recurrent melanomaFree of disease for 6 yr.

IgM.1/80 (without change over 5 yr).Class 2

22/32 melanomas8/8 astrocytomas0/25 epithelial cancers

Glycolipid.

Glycoproteins

Cell Panel

Melanoma

Epithelial Cancer

T-Cells

B-Cells

Fibroblasts

Fetal Brain

Melanocytes

gp45 gp28/34(HLA) (la) 9P95 gp150 M19 R8

I I

GlycolipidÂ«

Â°5 K9.|24 Â«24

I I

Chart 10. Current list of cell surface antigens of malignant melanoma defined by monoclonal antibodies.

FEBRUARY 1981 371



L. J. Old

serological methods with requisite resolving power to monitorthe specificity of humoral immune responses to melanomasurface antigens, it seems both justifiable and timely to reex-

plore the effects of active, specific immunization in melanomapatients with recurrent disease or whose primary tumor placesthem at high risk for recurrence. The idea of tumor cell vaccinesis as old as immunological thought itself, and repeated attemptshave been made over the years to alter the course of cancerby immunization with tumor cells themselves or extracts oftumor cells. Little if anything can be learned from the majorityof these past studies, involving as they usually did patients withadvanced cancer and using tumor growth or survival time asthe sole means to measure response. Without meaningfulmeasurements of the immune response of vaccinated patients,there is no way to know whether these individuals receivedimmunogenic vaccines. The first goal that Herbert Oettgen,Philip Livingston, and I have set in our melanoma vaccineprogram is to develop maximally immunogenic forms of melanoma antigens that we know from our past studies can berecognized by humans, using serological typing of vaccinatedpatients as the way to monitor immunogenicity (Table 15). Ourinitial studies have involved autologous or allogeneic whole-

cell vaccines. These did not elicit formation of AH or Mel 1antibody or antibodies recognizing Class 1 antigens. We arenow using membranes of autologous or allogeneic melanomasinfected with vesicular stomatitis virus to examine the possibilitythat viral infection increases the Â¡mmunogenicity of tumor antigens, as it has been reported to do in animals (2) and in skintests in humans (8). In the future, we plan to use cellularfractions enriched for melanoma glycoproteins or glycolipidsand interspecies or intraspecies hybrids expressing melanomaantigens. In this step-by-step analysis of the immunogenicity of

different vaccine preparations in groups of 10 to 15 patients,we are looking for vaccines that will result in an optimal antibody response to Class 1 or Class 2 melanoma antigens. Ifsuch a vaccine can be found, it will then be tested for therapeutic effects in appropriately larger series of patients. Thereis a possibility that these immunological manipulations aimedat raising the level of humoral immunity to melanoma cells mayresult in augmented rather than restricted tumor growth, theprecedent being the well-known phenomenon of immunological

enhancement of allogeneic tumors caused by antibody directedagainst the histocompatibility antigens of these tumors (44).Antibody-mediated immunological enhancement has never

been convincingly demonstrated with strictly syngeneic or autologous tumors in animal systems, nor has it been observedin any of the past attempts at active immunization againstcancer in humans. Furthermore, the popular belief that hasarisen over the past decade or so that cellular immunity againstcancer is a good thing and humoral immunity is bad is, in myopinion, without foundation. Nevertheless, accelerated tumor

Table 15Melanoma vaccine program: Memorial Sloan-Kettering Cancer Center

1. Autologous melanoma cells.2. Allogeneic melanoma cells expressing defined antigens.3. Vesicular stomatitis virus-infected melanoma cell lysates.4. Glycolipid or glycoprotein extracts of melanoma cells5. Chemically modified melanoma cells.6. Intra- or interspecies hybrids expressing melanoma antigens.7. Purified Class 1/Class 2 melanoma antigens defined by human sera or

monoclonal antibodies.

growth must be watched for, and this is an additional reasonfor keeping the groups of patients receiving each vaccinesmall.

Immunopotentiators, TNF, and Antileukemia Activity ofNormal Plasma

I have placed great emphasis in this presentation on theissue of specificity in the search for cancer-restricted antigensand in the focus on specific vaccines, and it is this emphasison specificity that I see becoming more and more the majorconcern of cancer immunology in the coming years. For thepast 15 years, the field has been virtually dominated by whathas been termed nonspecific approaches to manipulating theimmune response to cancer. The premise underlying this approach was that agents such as BCG and C. parvum potentiateimmunological reactivity on a global basis and that this resultsin a more effective specific immune reaction directed againstthe cancer. As appealing a concept as this is, its generalvalidity remains to be proved, and the sum result of clinicaltrials with BCG, C. parvum, and other such agents has led tocurrent disenchantment with this approach to cancer therapy.Nevertheless, there remains a body of solid observations fromboth clinical and animals studies that requires continued exploration, ranging from the indisputable long-term regressions of

human cancers associated with concomitant bacterial infectionor injection of mixed bacterial vaccines (67, 68), to the tumorcell destruction induced by local delayed hypersensitivity reactions (51, 62), to the clearly therapeutic benefit of intra-

tumoral BCG in certain animal systems (55, 81). An understanding of how these agents bring about these effects andwhat role, if any, specific immune reactions have in the processhas turned out to be far more difficult than originally expected.Nonetheless, a valuable outcome of these studies is informationabout how these agents affect the various components of theimmune system and how their use can manipulate the immuneresponse in selected ways, matters of direct relevance to thedevelopment of specific cancer vaccines with maximal effectiveness. There is evidence that some of the antitumor effectsof these agents are not the result of augmented specific immunity but are due to mediator molecules released by the hostthat have direct antitumor activity. The main support for thisidea comes from an analysis of the striking effect that lipopoly-saccharides or endotoxins derived from gram-negative bacteriahave on certain transplanted rodent tumors (84). Within amatter of hours after systemic injection of endotoxin, sensitivetumors undergo progressive hemorrhage and necrosis, leadingin some cases to total tumor regression. Although tumor regression is clearly immunologically mediated (5), the immediateeffect of endotoxin is not. Because endotoxin has no directlytic effect on tumor cells in vitro, the prevalent idea for manyyears was that hemorrhage and necrosis resulted from a directeffect of endotoxin on the vascular system of the tumor, causingvascular collapse, tumor anoxia, and subsequent tumor celldeath (1 ). The possibility that a mediator molecule was involvedin this phenomenon came from our finding that tumor hemor-rhagic necrosis could be transferred by the serum of endotoxin-treated mice that had been presensitized with BCG or C.parvum (14, 34, 73, 83). In contrast to endotoxin itself, serafrom such mice have direct inhibitory activity for cultured tumorcells of both mouse and human origin. The factor, which we




Table 16

Tumor necrosis factor

Source Serum of endotoxin-injected mice presensitized withBCG or C. parvum.

Characterization Glycoprotein (m.w. 40,000).Specific activity of purified TNF, 10" units/mg protein.

In vivo activity Hemorrhagic necrosis of Meth A and other transplanted mouse tumors.

In vitro activity Cytotoxic or cytostatic for a range of mouse and human cancer cells.

Cellular source Macrophage.

Source

In vivo activity

In vitro activityCharacterization

Table 17

Antileukemia factor of normal plasmaNormal plasma from C5 ' mouse strains and

from a variety of other species.Leukemia cell destruction in AKR mice, cats,

and dogs.Not demonstrable.C5 involved in antileukemia activity in AKR

mice.Antileukemia factor and Clg have related

properties.

called TNF, has now been purified by Katsuyuki Haranaka,Elizabeth Carswell, and Barbara Williamson and has the following characteristics (Table 16). TNF is a glycoprotein with amolecular weight of 40,000 that has both tumor-necrotizingactivity in vivo and tumor-killing activity in vitro. The specificactivity of purified TNF is 108 units/mg protein, placing it in the

activity range of interferon and other lymphokines and mon-

okines. The initial suggestion that its cellular source was macrophages came from the fact that mice had to be primed withagents such as BCG or C. parvum to produce TNF, and theseagents are known to cause extensive macrophage proliferation.In support of this idea, we have recently found that two clonedlines of mouse histiocytomas produce a molecule with TNFcharacteristics. Now that the molecule has been purified, somekey questions about TNF can be answered, such as relationship to other lymphocyte- and macrophage-derived factors,

basis for tumor cell selectivity, and possible involvement in thecytotoxic effect of activated macrophages and other classes ofkiller cells.

I want to end by mentioning another potent antitumor factorthat we have been studying for some time, in this case one thatexists in normal plasma (Table 17). It was first recognizedduring experiments carried out on the antitumor effect of interferon in AKR mice (45). For control purposes, we used normalmouse serum and were surprised to find that it had the sameeffect as did the interferon preparations. The antitumor factorwas widespread in other mammalian sera and plasma, but inthe mouse it was restricted to those strains having the completeset of complement components. Striking and rapid regressionsof cat lymphoma and dog lymphoma can also be induced byrepeated infusions of normal plasma (47). This antitumor effectof normal plasma in mouse, cat, and dog may well be thecounterpart of the well-documented partial or complete remis

sions seen in leukemic patients following blood transfusions(6). Although there is no doubt about the involvement of complement in the antileukemic effect of plasma in AKR mice,purification of the factor from normal mouse plasma (46) showsthat it has several characteristics in common with Clg, alsoknown as fibronectin. Because of this association, MichaelMosesson suggested that the cryoprecipitated fraction of nor-


mal plasma might be active, because this plasma fraction, inaddition to concentrating the antihemophilia factor, is also richin Clg. Cat cryoprecipitate has now been tested in cats withlymphoma by Gregory MacEwen and was found to be as activeas whole plasma; tests are under way in the clinic with humancryoprecipitate. The mechanism underlying this antitumor effect in animals is obscure. One possibility to account for theeffect in CS-deficient mice involves the replacement of themissing complement component, allowing preexisting but ineffective immune responses to become evident. Another hasto do with more rapid clearance of immune complexes, whichin some way interfere with immune destruction of leukemiacells. As yet, it has not been possible to demonstrate a directeffect of plasma, C5, or Clg on isolated leukemia cells in vitro.

Concluding Comment

There has been a rather general feeling that the host hasonly a limited capacity at best to rid itself of naturally arisingcancer cells. Our experience with TNF and the antileukemiafactor in normal plasma has led us to realize that this maygreatly underestimate the body's potential for selective elimi

nation of cancer cells. However, until we know how to directthe full force of specific immunity against tumor cells, the truemagnitude of this potential will remain unknown. In my view,the systematic analysis of cancer cells by serological techniques is still the most promising way to guide us to that goal.With the advances that have been made and the powerful newtools that are available, the cancer immunologist's long search

for specificity may finally be rewarded.

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1981;41:361-375. Cancer Res Lloyd J. Old Clowes Memorial Lecture

G. H. A.−−Cancer Immunology: The Search for Specificity

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