Protein tumor antigens

Protein tumor antigens Pramod

Sinai School of Medicine, New York, USA

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1991, 3:654-658

N Tsuboi, T Takashima, T Torigac, K Kikuchi, abstract 1437, Proc Am Assoc Cancer Research 1991, Vo132).

Introduction The focus of this article is on tumor antigens that elicit tu-

mor resistance or T-cell or antibody response in the host of tumor origin, or in a syngeneic host. Most tumor antigens or tumor-associated antigens are so defined because of their preferential distribution or enhanced expression in tumors or by anti-tumor antibodies generated in xenogeneic systems. Although useful in diagnosis or prognosis, such antigens do not elicit immunity against cancer and are outside the scope of this article. Antigens of transforming viruses are also not included because in contrast to autologous and syngeneic tumor antigens, they are foreign antigens. Immunogenic tumor antigens are defined by tumor transplantation (individually distinct antigens of chemicallyinduced tumors), antibody response (~53 antigen, gp95/p97 antigen of human melanoma) and T-cell response (e.g. antigens of P815 mastocytoma, mucins).

Antigens detected by tumor transplantation Immunogenic antigens of chemically induced tumors were demonstrated by tumor transplantation almost half a century ago and remain to date the most convincing examples of tumor-specific antigens [ 11. They have been sought most insistently in methylcholanthrene (MCA)-induced sarcomas of inbred mice and were identified independently as cell-surface glycoproteins of %kD size (gp%) [2] and as intracellular antigens of 84-86kD size (p84/86) [3]. Gp96 and p84/86 antigens share considerable homology with each other [4*] and belong to the family of stress-induced or heat shock proteins (hsp) [ 3,501. Involvement of hsp70 and gp%-like hsps in tumor immunity has also been observed in tumors obtained by transfection of Iibroblasts with oncogenes [6] (N Sato,

A striking feature of tumor rejection antigens of inbred mice is their individual distinctness: a tumor elicits immunity against itself but not against another tumor, even if both tumors are induced by the same carcinogen and in the same animal [I]. The gp% and p84/86 antigens also display this specificity and elicit immunity only against the tumors from which they are derived [2,3]. In contrast to this functional specificity, gp% and p84/86 genes do not show any tumor-specific DNA sequence polymorphism [ 4*,7,8*]. Because hsps are known for their ability to bind to a diverse array of molecules [ 91, it has been suggested that gp% and p84/86 are not antigenic per se, but are carriers of immunogenic peptides and the specificity of immunogenicity resides in the peptides rather than in the carrier [e]. Preliminary screening of a peptide epitope library [lo] with gp% supports this possibility (our unpublished data). A large proportion of gp% molecules resides in the endoplasmic reticulum - the presumed site of peptide charging of major histocompatibility complex (MHC) class I antigens [ 111 - and a role for gp% in presentation of peptides to MHC class I antigens has been proposed [4*,12*]. If this turns out to be correct, a long standing problem in cancer immunology will be on its way to resolution.

Serologically detected immunogenic tumor antigens Anti-tumor antibodies are often detected in tumor-bearing or tumor-immunized hosts. In most instances, these antibodies are not tumor specific and recognize proteins in normal tissues as well; it is diflkult to determine if such an antibody response is truly a reflection of tumor immunogenic&y or if it is a result of the breaking of toler-

Abbreviations CT-cytotoxic

654

T lymphocyte; MCA-methylcholanthrene;

hspheat

shock protein; MHC-major

@ Current Biology Ltd ISSN0952-7915

histocompatibility complex.

Protein tumor antinens

ante to normal antigens. Tumor-specific antibodies have been occasionally detected in tumor-immunized animals 113,141 but the structural basis of immunogenic&y is unresolved in these instances. The gp95/p97 antigen of human melanoma provides the clearest example of a tumor-specific antigen detected by autologous antibody. A 95 kD molecule was detected in melanoma patient’s serum on autologous melanoma, but not on other melanomas [ 151. However, monoclonal antibodies against purified gp95/p97 detect a 95 kD molecule on other melanomas and on some normal tissues. The patient antibody apparently detects a new epitope on a common gp95/p97 molecule [ 161. Autologous typing of sera of other melanoma patients has uncovered a number of such tumor-specific antibody reactivities [ 171. Molecules recognized by these antibodies have not been identified but it appears likely that the observations with the gp95/p97 are not an exception. An entire class of tumor-specific mutations that elicit specific antibody responses therefore awaits characterization. The p53 tumor suppressor molecule is another exam ple of a serologically detected tumor antigen. Antibod ies to p53 were found in sera of mice immunized with MCA-induced sarcomas and p53 molecules were precipitated from a number of transformed but not normal cells [18]. p53 molecules from a panel of tumors were compared by two-dimensional tryptic peptide mapping [ 191: the peptide maps of p53 derived from virally transformed cells appeared very similar. In contrast, p53 molecules obtained from four antigenically distinct MCA-induced sarcomas showed that although their lingerprints were generally similar, p53 antigens from each of the tumors seem to include peptides that are unique to each tumor. Jay et al [19] suggested for the first time that “muta tional events involving different regions of the p53 stuctural gene might also generate extensive polymorphism of the p53 product” and raised the possibility that vanation in p53 structure might be the basis for the indi vidually distinct immunogenicity of MCA-induced tumors. These studies anticipated by over a decade the recent discovery that the p53 gene is a hot spot for mutations in a broad spectrum of human cancers [ 20**]. Humoral and cellular immunogenic&y of p53 molecules of human carcers is clearly a propitious area of investigation (see Perspective).

Immunogenic tumor antigens recognized by T lymphocytes

T-cell response plays a significant, if not the dominant role in tumor immunity. However, there are a number of serious hurdles in structural identification of detemrinants recognized by T cells. Methods that have been eminently successful in identification of serologically-defined antigens - immunoprecipitation, western blotting and others - are not helpful in identification of T-cell-defined antigens largely because of the cell-bound nature of the

Srivastava

T-cell antigen receptor and the MHC-restriction of specific T-cell recognition. Cloned tumor-specific T-cell reactivities were first identified as early as 1983 121,221but the identity of T-cell-defined antigens has, for the most part, remained in the dark. A small number of T-cell defmed tumor antigens have now begun to be characterized. Turn- antigens of the P815 mastocytoma were the first T-cell recognized antigens to be delined structurally. When P815 cells are mutagenized in vitro and mutant clones isolated, the clones are found to be so immunogenic that they do not form tumors in immunocompetent animals. Each clone (Turn- variant) elicits transplantation immunity specific to itself and the parent tumor but not to others. Cytotoxic T lymphocytes (CTLs) generated against the variants show the same specificity 1231. CTLdefined antigen on the variant P9lA was characterized by transfection of P9lA DNA into a syngeneic recipient and screening the transfected clones for their ability to be recognized by the CTLs against P9lA [24]. This antigen turned out to be a 60 kD protein that differs from its counterpart in normal cells by a single amino acid. This initial success was repeated with Turn - variants, P198 and P35B and antigens of the two variants were found to diller from normal counterparts by a single amino acid. The P198, P35B and P9lA genes do not share any homology [ 25*,26*]. One significant point of difference was observed among the three antigenic systems. The mutated peptide from P9lA could render non-target cells sensitive to lysis, whereas the corresponding normal peptide could not. Similar results were seen in P198 except that both peptides could compete for binding to a class I molecule. Presumably, the Turn- mutation in P9lA enabled the peptide to bind to the presenting molecule, whereas in P198 the normal and mutant peptides bind to the presenting molecule, but there is tolerance to the normal peptide. In P35B, the mutated and the normal peptide could sensitize the Pl.HTR cells to lysis. As the P35B mutation removes a serine, it might inhibit Oglycosylation, which may render the native peptide unpresentable. These results demonstrate interesting nuances of antigen presentation by MHC class I molecules. It was observed during the characterization of Turn- mutations, that the Turn- phenotype could be transfected not only by the whole gene but also by subgenic fragments of the turn- gene cloned in non-expression vectors [24,25*,26*,27]. Chomez et al. [28*] examined this phenomenon and showed that expression of subgenic fragments is not the result of homologous recombination of the fragment into the turn+ gene and it does not require any promoters on the 5’end of the subgenic fragments as transfection of the fragments without any vectors at all also confers the appropriate phenotype. There is evidence that an ATG codon located upstream of the turn- mutation may act as one of the translation initiation sites and may contribute to the production of antigenic peptides. The authors conclude that it is very difficult to decide whether this phenomenon “bears any revelance to the production of antigenic peptides by intact genes or whether the relevant processes operate only with transfected DNA”.

655

656

Cancer

Characterization of Turn- antigens shows a method that can lead to definition of CTL-defined antigens of experimental and human cancers. This expectation has begun to be fulfilled and a tumor rejection antigen (PlA) of the P815 mastocytoma has just been characterized by the transfection method [29**,30*]. The PlA antigen is predicted to be a 224 amino-acid protein that shows no homology with any of the Turn- antigens. Surprisingly, the sequence of the PlA gene in P815 and normal DBA/2 cells is identical. However, PlA transcripts are not detected in any normal tissues or in a number of mastceU lines. The transcript is detected in a single mast-cell line L138.8A this line can be lysed by CTIs against the PlA antigen [30*], but it is not clear if it can immunize against the P815 mastocytoma. Van den Eynde et al. 129-l suggest the PlA antigen is immunogenic because of its very specific distribution and speculate that it may be an oncofetal antigen or differentiation antigen. It is of interest to recall here that these were among the earliest speculations regarding the identity of tumor antigens over 25 years ago [ 31 I. Regarding the CTL-defined immunogenic antigens of human cancers, one of the antigens recognized by autologous CTLs against the human melanoma line MZ2MEL has recently been characterized by the transfection method; the genetic basis of its antigenicity is presently under investigation (C Traversari et al., unpublished data). Biochemical approaches for identification of antigens recognized by T lymphocytes are also being explored [32*,33*]. The general strategy is to present proteins separated on a gel or expressed on a plaque to a T ceU in the presence of antigen-presenting cells and to monitor Tcell recognition. The rationale for presenting exogenous antigens to CD4+ T lymphocytes is of course clear but it is not obvious how the authors [33-l hoped to present exogenous antigens to CD8+T lymphocytes. No antigens were convincingly identified in either study but these efforts represent interesting beginnings of an important methodology. The single most important component in this methodology is the efficient presentation of exogenous antigens into the MHC class I endogenous presenta tion pathway. Possibilities of such presentation have been demonstrated with mode1 antigens by coating cells with peptides [34], hypertonic loading of extracts [35] and under normal conditions [36]. However, these methods are relatively inefficient as they require large quantities of model proteins or peptides; smaller quantities of whole or partially puriIied cell extracts are unsuccessful in sensitizing T lymphocytes (M Heike and PK Srivastava, unpublished data). Further advances in antigen presentation wilI pave the way for identification of peptides recognized by T lymphocytes. Among the T-cell recognized human tumor antigens, mucins of pancreatic, breast and colon cancers are perhaps the best characterized [37]. Because of their unique polyvaIent structure, mucins are able to stimulate CTL recognition in an apparently MHC-unrestricted fashion, even though the cytotoxicity is mediated through an afl T-cell receptor [38]. The CTIs recognize the polypeptide core of the heavily glycosylated mucin molecules [39-l.

Mucins are expressed abundantly in normal epithelium and on cancers and there is no evidence of tumor-associated genetic changes in the mucin genes. Thus, the structural basis of specificity of immunological recognition of mucins has been a major puzzle. Jerome et al. [39**] have recently addressed this question and have identified a mucin epitope expressed preferentially on malignant cells. They demonstrate that normal breast lines that express mucin but not the SM3 epitope are not lysed by tumor-reactive CTIs nor do they act as cold target inhibitors of lysis of SM3bearing malIgnant cells. The SM3 epitope appears to be generated as a result of an altered Olinked glycosylation of the mucin polypeptide (OJ Finn, personal communication) and this may provide an interesting example of the intluence of a specific posttranslational modification of a peptide on its presentability by an MHC antigen. Anti-mucin CTIs can be obtained from cancer patients with a relatively high efficiency and it is not clear why these CTIs do not eliminate cancer cells in uivo. Preliminary studies suggest that a relative lack of functional helper cells may be responsible for this paradox [40]. Resolution of these questions at the structural and cellular levels will make it possible to develop strategies to augment the human anti-tumor immune response in mutinous cancers.

Perspective It was an early belief that tumor-specific antigens are a special set of proteins that are induced as a result of malignant transformation and elicit tumor immunity. About two decades and several hundred publications later, that belief turns out to be substantially incorrect. It now appears that there are no tumor-specific molecules, but only tumor-specific epitopes of common molecules. Furthermore, the old idea that tumor antigens may only be lineage-specific differentiation antigens may be gaining new credibility. Finally, tumor-specific antigens were earlier sought primarily among cell-surface proteins. Recent advances in our understanding of antigen presentation by MHC class I antigens have rendered that dogma untenable and have replaced it with the new dogma: any alterations in a coding gene are potentially recognizable by the cellular immune system and alI proteins are potential tumor antigens. Each of the immunogenic antigens identified by transplantation assays or by T-cell reactivity can be interpreted in terms of this new canon. The vast openness (‘any protein is a tumor antigen’) of this new canon will no doubt now begin to be delined and constricted by the answers to questions such as are there common motifs among peptides presented by tumor MIX antigens? are there any hot spots for mutations? are the hot spots tumor-type specific? do specific environmental carcinogens induce characteristic mutation profiles? The recent discovery that p53 genes appear to be hot spots for mutations in a wide spectrum of human can cers is a fruitful indicator of events to come. It may also mark a dramatic U-turn in cancer immunology in that our

Protein tumor antigens Srivastava

attention is once again likely to be focused on common genetic alterations that accompany malignant transformation and we may yet rediscover the old dogma with new wisdom. In any case, a return to original principles marks a milestone in evolution of any pursuit.

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and recommended

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2.

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HI T, JOHN M, OLD LJ: Cell SurDEIEO AB, SHIKU H, TAKAHAS

Immunogenicity of Stress-induced Proteina Convergence of Two Evolutionary Pathways of Antigen Presentation? Seminars in Immunolo~ 1991, 3:57-64. The proposed role of gp% in charging MHC class I molecules in the endoplasmic reticulum is discussed and expanded upon and it is suggested that T-cell receptor recognition of MHC-peptide represents a convergence of two independent evolutionaty pathways of antigen presentation - by hsps and MHC cla..s I.

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REAL FX, MATES MJ, HOUGHTON AN, OETTGEN HF, LLOVD KO, OLD LJ: Class I (Unique) Tumor Antigens of Human Melanoma: Identification of a 90,OOOD CeII Surface Glycoprotein by Autologous Antibody. J Eap Med 1984, 160:121?1227.

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SR~VASTAVA PK, MAKI RG: Stress-induced Proteins in Immune Response to Cancer. Curr Tgp Microbial Immunol 1991, 167:10’+123. This paper describes the total evidence to date for involvement of hsps in NmOr immunity and puts forward a hypothesis explaining this role. It is suggested that the gp96 molecules are responsible for charging the MHC class I antigens with peptides in the endoplasmic reticulum. The question of cell-surface localization of hsps is discussed. 5. .

MAKI RG, OID LJ, SRRTASTAVA PK: Human Homologue of MurIne Tumor Rejection Antigen gp96: 5’ ReguIatory and Coding Regions and Relationships to Stress-induced Proteins. Proc Nat1 Acad Sci US4 1990, 87:5658-5662. Human gp% genes are shown to be classic hsps with a heat shock element in the 5’ flanking region and another in the first intron. The relationship of gp96 with similar molecules such as grp94, Erp99, hsplOO and hspl08 is discussed. 6.

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KNIJTH A, DANO~~KI B, OET~GEN HF, OLD LJ: Cell Mediated Cytotoxicity Against Autologous Malignant Melanoma: Analysis with IL-2 Dependent T Cell Cultures. Proc Natl Acud Sci USA 1984, 81:3511-3514.

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the Gene Coding for Turn- Transplantation Antigen P91k A Peptide Encoded by the Mutated Fxon is Recognized with Ld Cytolytic T Cells. Cefi 1989, 58:29%303. SVKORA JP, VANPEL A, B~UCHARDV, ANDRE M, VANBARENN,

HENRY P, BOON T: Structure of the Gene of TurnTransplantation Antigen P35B: Presence of a Point Mutation in the Antigenic AIlele. EMBO J 1990, 9:104-1050. The Nmgene of P35B is characterized by the transfection method and is seen to differ from its normal counterpart by a single point mutation (serine to asparagine) in exon 5. Fragments containing only exons 4 and 5 cloned in non-expression vectors are able to transfect the Turn- phenotype. Mutant and normal peptide are able to render nontarget .syngeneic cells susceptible to lysis by anti-P35B m. It is sug-

657

658

Cancer gested that the normaI peptide may not be presented, residue may be a site for OIinked glycosylation.

as the serine

SBILLE C, CHOMF.Z P, WILDMANNC, VANPEL A, DEPLUN E, ~~ARYANSKIJ, DE BERGEYCK V, Boon T: Structure of the Gene of Turn- Transplantation Antigen P198: a Point Mutation Generates a New Antigenic Peptide. J Ejcp Med 1990, 172:35-45. A similar study to [25*] except that the single point mutation (alanine to threonine) lies in exon 7. The mutant peptide can sensitize nontarget syngeneic ceils to Iysis by appropriate CTIs, and normal peptide cannot; however, both peptides can bind to a class I MHC molecule. It is suggested that the cellular immune system is tolerant to the normal peptide. 26. .

27.

28.

&XN T, VAN PEL A: T CeII Recognized Antigenic Peptides Derived from the CeIluIar Genome are not Protein Degradation Product but can be Generated Directly by Transcrip tion and Translation of Short Sub-genie Regions. A Hypothesis. Immunogenetics 1988, 2975-79. CHOMFZ P, DE P~AEN E, VAN PEL A, DE SMEULET C, SZIKORAJ-P, L~RQUIN C, IEBACQ-VERHEYDENA-M, BOON T: Efficient Ex-

pression of Turn- Antigen P91A by Transfected Sub-genie Fragments. Immunogenetics 1991, in press. This paper explores the phenomenon of expression of Turn- antigens by transfection with subgenic fragments. A number of possibilities (homologous recombination, promoter effect of Aanking vector sequences) are ruled out and the pepton hypothesis (see [27] ) is examined critically. The data are found not to support the hypothesis. The basis of expression of the sub-genie fragments is not elucidated but some possibilities are suggested (e.g. promoter effect of Banking regions of sites of insertion of the fragments). 29. ..

VAN DEN EYNDE B, LETHE B, VANPEL A, DEPIAEN E, BOON T: The Gene Coding for a Major Tumor Rejection Antigen of

Tumor p815 is IdeiificaI to the Normal Gene of Syngenic DBA/2 Mice. J Exp Med 1991, 173:1373-1384. The transfection method is used to isolate a tumor antigen of P815 mastocytoma. No differences are seen between the tumor and normal genes, but the PlA transcripts are not detected in normal tissues or a number of mast-cell lines. One mast-cell Line expresses the PlA transcript but the authors do not state if this line can immunize mice against the P815 tumor. The authors suggest that this normal protein is immunogeneic because of its restricted distribution. BOON T: Towards a Genetic Analysis of Tumor Rejection Antigens. Advances in Cancer Research 1991, in press. kis article presents a comprehensive and integrated account of the genetic approach to the identification of tumor rejection antigens that has been used in the author’s laboratory. 30.

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HERS!&’P, MELDRUMC, DANIEIS V, WERKMAN H, SNEDCON D:

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ofCanceredited by Finn OJ, Lotze M [book] Wiley-Liss lm, pp 343-349. Melanoma extracts separated by sodium dodecyfsulphate polyacrylamide gel electrophoresis and expression plaques of a melanoma cDNA library are transfected to nItroceUulose and the strips are used to stimulate CIX+ -cells in the presence of antigen-presenting cells. No convincing antigens or their genes are identified, but the methodology is promising. NC~TER M, S~HIRRMACHER V: Tumor Specific T CeII Clones Recognize Different Protein Determinants of Autologous Human Melanoma CeIIs. Int J Cancer 1990, 45834-841. A similar study to that carried out in [31]. Two CD4+ and one CD8+ cells are stimulated with electrophoretically separated antigens. No antigens are convincingly detected but the methodology is promising. It is not clear how the authors hoped to present exogenous antigens to class I restricted CD8+ T cells. 33. .

34.

MARYANSKI JL, PALU P, CSROTIINI JC, CORRADING: Synthetic

Peptides as Antigens and Competitors in Recognition by H-2 Restricted Cytolytic T CeIIs Specific for HLA J EXp Med 1988, 167:1391-1405. of Soluble Processing

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GENDLERS, TAYLOR-PAPADIMITRIOU J, DUHUG T, ROTHBARDJ, BURCHEU.J: A Highly Immunogenic Region of a Human PoIymorphic EpitheIiaI Mucin Expressed by Carcinomas is Made up of Tandem Repeats. J Biol Cbem 1988, 263:1282@12823.

38.

BARND DL, L&N M, METZGAR R, FINN OJ: Specific, MHCUnrestricted Recognition of Tumor-associated Mucins by Human Cytotoxic T Cells. Proc Nat1 Acad Sci USA 1989, a6:7159-7163.

JEROME KR, BARND DL, BENDT KM, BOYER CM, TAYIQRP.w~ulmuou J, MCKENZIE IFC, BAIT RC JR, FINN OJ: Cytotoxic T Lymphocytes Derived from Patients with Breast Adenocarcinoma Recognize an Epitope Present on the Protein Core of a Mucin Molecule Preferentially Expressed by MaIignant CeIIs. Cancer Res 1991, 51:29(X%2916. This is the first demonstration of a mucin T-cell epitope (SM3) expressed preferentialfy on malignant cells. As there are no tumor-associated genetic changes in the mucin gene, Olinked giycosylation appears to be responsible for generation of this epitope. This may provide an interesting example of the effect of post-translational modiication on the presentability of a peptide.

39. ..

40.

FINN OJ: T ceII recognition of human epithelial tumor mucins Indications for active immunotherapy. Proc Amer ican Axux Cancer Research 1991, 32:4W91.

PK Srivastava, Box 1215, Department of Pharmacology, School of Medicine, New York, New York 10029, USA

Mount Sinai