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Chemically defined, cell-free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination

Dr. Max L., Birnstiel et. al.

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Gene Ther Mol Biol Vol 1, 309-321. March, 1998.


Chemically defined, cell-free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination


Michael Buschle1, Walter Schmidt1, Manfred Berger1, Gotthold Schaffner1, Robert Kurzbauer1, Iris Killisch1, Julia-Kristina Tiedemann1, Barbara Trska1, Helen Kirlappos1, Karl Mechtler1, Franz Schilcher2, Cornelia Gabler3, and Max L. Birnstiel1

1Research Institute of Molecular Pathology, Vienna Biocenter, Dr. Bohrgasse 7, 1030 Vienna, Austria

2Institute of Pathology and Forensic Veterinary Medicine, University of Veterinary Medicine Vienna, Josef Baumanngasse 1, 1210 Vienna, Austria

3Institute of Nutrition, University of Veterinary Medicine Vienna, Josef Baumanngasse 1, 1210 Vienna, Austria

Correspondence: Max L. Birnstiel, Tel: (+43-1) 797 30-474, Fax: (+43-1) 797 30-490, E-mail: birnstiel@nt.imp.univie.ac.at



With the advent of gene therapy there has been a revival of immunotherapy of cancer. Preclinical studies with the so called tumor vaccines – syngeneic, irradiated tumor cells secreting cytokines – are at present entering clinical trials and hold much promise for efficacious treatment, maybe even cures, of cancer. However, autologous whole cell vaccines which are cytokine gene-modified are expensive and difficult to standardize. In addition, autologous tumor cell cultures, and hence tumor vaccines, cannot be prepared for all patients. For these reasons we have investigated alternative schemes for vaccination against cancer. We have developed vaccines on the basis of tumor antigen-derived peptides using a method, we called ”transloading”, to transfer peptides efficiently into cells. Such vaccines are chemically well defined and inexpensive to produce. We show in a preclinical mouse model system for mastocytoma, and to a somewhat lesser extent for melanoma, that peptide vaccines give excellent protection against tumor take, provided tumor antigen peptides are injected subcutaneously in conjunction with polycations like polyarginine. Immunohistological investigations on thin sections show that three days after injection, the vaccine bolus is heavily infiltrated by antigen presenting cells which take up peptides. We posit that such antigen presenting cells then migrate into draining lymph nodes where they activate naive T cells to become anti-tumor cytotoxic lymphocytes (CTL). The consequences of strong dependency of peptide sequence on the HLA-type of patients and possible remedies are discussed. One avenue to be tested in mouse models is the injection of mixtures of peptides covering a multiplicity of MHC-specific peptides. Another is the production of recombinant proteins as vaccines whose application is considerably less dependent on HLA-types of patients to be treated. In addition, such proteins would be expected to contain both class I and class II restricted peptides. Alternatively, artificial proteins incorporating many CTL epitopes of known tumor antigens, including members of the MAGE family, thus yielding a SUPERMAGE vaccine with utility for several human HLA-types could be designed. The prophylactic application of peptide/polyepitope vaccines can be envisaged.


I. Tumor-specific and tumor-associated antigens

After the great successes of vaccination against bacterial and viral infections, physicians and scientists have attempted to harness the exquisite specificity, sophistication and power of immunology to combat cancer, but the results hitherto have been rather disappointing and have led to a rather pessimistic assessment of immunotherapy of cancer. Indeed, at certain periods the postulate emerged that cancers were not immunogenic. Recent molecular and immunological investigations, however, have revealed the existence of tumor antigens in several tumor types (Boon et al., 1994; Boon and van der Bruggen, 1996; Rosenberg, 1997). This has led to the conclusion, that most, and perhaps all tumors are indeed immunogenic, but that the response of the immune system to tumor antigens is inadequate and in need of enhancement if immunotherapy of cancer is to be attempted.

Peptides derived from tumor antigens, mostly presented by cancer cells in the MHC class I context (Boon et al., 1994; Boon and van der Bruggen, 1996; Robbins and Kawakami, 1996; Rosenberg, 1997) can be classified as having been derived from tumor specific (TSAs) or tumor associated antigens (TAAs). They have been identified mostly by means of CTL clones obtained from cancer patients and by gene cloning or by peptide elution followed by tandem mass spectrometry analysis (Boon et al., 1994; Slingluff et al., 1994; Wang and Rosenberg, 1996). As shown recently by Kinzler and Vogelstein’s lab, cancerous transformation leads to extensive reprogramming of the cancer cell in that as many as 500 mRNA types are either up or down regulated (Zhang et al., 1997). As a consequence, cancer cells can express tumor associated antigens which are unconnected with the gene expression programme of the parental cell type from which the cancer cell arose.

Thus it is known for melanoma, that malignant cells express, amongst other TSA and TAA, the MAGE, BAGE and GAGE family of genes which are normally expressed in male germline cells and placenta (Boon and van der Bruggen, 1996) and thus are clearly self-proteins. Because germ line cells are immunologically privileged and do not express MHC surface receptors, clonal selection presumably cannot act to eliminate MAGE, BAGE and GAGE specific CTLs under normal circumstances. The frequent appearance of these related genes in tumors different from melanoma such as head and neck cancer, lung cancer or bladder carcinoma may be a consequence of random demethylation of genes in cancer cells and activation of their promoters by demethylation, as has been shown for the MAGE-1 promoter (De Smet et al., 1996). Another interesting example of TAAs in melanoma cells are the so called differentiation antigens tyrosinase, tyrosinase related proteins 1 and 2 (TRP-1, TRP-2) and other proteins normally encountered during melanocyte differentiation (see Table I and Boon et al., 1997; Rosenberg, 1997). Interestingly, the immune system treats these proteins, dysregulated and expressed in the ”wrong” context of tumor cells, as foreign antigens. Despite potential earlier clonal selection by the presumed interaction of the immune system with melanocytes, cytotoxic T cells which recognise peptides derived from these proteins are generated and hence are reactive against melanoma cells.

The encoded proteins of oncoviral genes, mutated oncogenes and tumor suppressor genes or mutated cellular genes, are tumor specific (Table I). Examples for the first are the oncoproteins of the Epstein-Barr virus (Rickinson and Moss, 1997) or E6 and E7 of HPV16 (Tindle, 1996), for the second and third the mutated Ras or p53 proteins (Disis and Cheever, 1996) and for the last are connexin (Mandelboim et al., 1994; Mandelboim et al., 1995), Her 2/neu (Disis and Cheever, 1997), MUC 1 (Finn et al., 1995), CDK4 (Wolfel et al., 1995) and ß-catenin (Robbins et al., 1996). Being tumor specific, any of these have the potential of eliciting anti-tumor immunity when used as a vaccine.


II. TSA and TAA peptide vaccines

Previous attempts at creating tumor vaccines have concentrated on the stimulation of the immune response by cytokines. The great advantage of using cytokine-secreting autologous tumor cells as vaccines is that in this strategy, unidentified and unknown TSAs and TAAs are presented to the immune system in a milieu of high local cytokine concentration (Pardoll, 1995; Zatloukal et al., 1995). The most widely used cytokine is IL-2, the level of which is highly critical for the outcome of the vaccination as indicated in preclinical studies using the M3 melanoma model (Figure 1a and Schmidt et al., 1995). We further determined cytokine optima in the B16/F10 model where the number of metastases were scored. The IL-2 optimum for B16/F10 appears to be higher (3,300 units IL-2/vaccine; Figure 1b) as compared to 1,500 units IL-2/vaccine for the M3 model (Figure 1a and Schmidt et al., 1995). As shown previously for the M3 system, GM-CSF shows no dosis maximum, but both in the M3 and B16/F10 model, protection increases with cytokine dosage (Figure 1c). In the case of IFN-g it appears that a dose of 165 ng/vaccine yields best protection (Figure 1c).

It has been the experience of different groups that despite the great potential that autologous cancer vaccines have, the procedure is cumbersome and expensive, and of limited utility since tissue cultures can only be prepared in roughly 2/3 of the cases for malignant melanoma (E. Wagner, Vienna, unpubl. results). These circumstances call for alternative strategies.

Syngeneic dendritic cells pulsed with tumor antigen peptides matched to the MHC type of the mice strains have repeatedly been shown to protect animals against tumor take (Celluzzi et al., 1996; Mayordomo et al., 1996; Mayordomo et al., 1995; Paglia et al., 1996; Porgador et al., 1996). This approach has also been used in a clinical setting: lymphoma patients were treated with dendritic cells pulsed with autologous idiotype protein (Hsu et al., 1996; Hsu et al., 1997). The results of these studies are promising.

In a seminal investigation, Boon and coworkers vaccinated fifteen terminally-ill melanoma patients with a HLA-A1 matched immunogenic MAGE-3 peptide and detected clinical benefits in five of them in that some tumors regressed (Marchand et al., 1995). This result is even more remarkable, because no adjuvants of any kind were used. Curiously, despite these clinical effects, no

Table I: (previous page) Human tumor specific and tumor associated antigens recognized by T cells known to date. After Boon et al., 1997; Robbins and Kawakami, 1996; Rosenberg, 1997; Wang et al., 1995 with adaptions.

Figure 1: Cytokine dosage effects in experimental murine melanoma systems.

Figure 1a:  IL-2 dosage curve as determined in DBA/2 mice (8-10 animals/group) with prophylactic treatment against M-3 melanoma tumor challenge (Schmidt et al., 1995). Mice were vaccinated twice at weekly intervals (105 irradiated cells/vaccination/animal) and challenged one week after the second vaccination (3 x 105 viable M-3 melanoma cells). Reproduction of a previously published Figure (Schmidt et al., 1995).

IL-2 (Figure 1b), GM-CSF and IFN-g (Figure 1c) dosage effects in the treatment of experimental B16-F10 lung metastases in C57BL/6J mice (eight animals/group). Animals were vaccinated three times (105 irradiated cells/vaccination/animal). Lung metastases were established by intraveneous injection of 104 B16-F10 cells three days before the first vaccination. After 28 days lung metastases were scored and the number obtained for all eight animals of the control group receiving mock-transfected B16-F10 was set to 100 % (n=215).



peptide specific CTLs could be detected in vaccinated patients. However, as reported at this conference, no clinical effects were detected in a repeat experiment by the Parmiani group (Milano).

Following in the footsteps of Boon et al. (Marchand et al., 1995) we have carried out a preclinical study in mice for the efficacious vaccination against tumor take in both the murine M3 melanoma and the P815 mastocytoma model system using small TAA-peptides matched to the MHC-type of the experimental animals (Schmidt et al., 1997a). Since no known tumor antigens are available for the M3 system, it was ascertained by means of RT-PCR that M3 cells expressed tyrosinase and tyrosinase related protein-1 (TRP-1) which are known as TAAs in human melanomas. Much progress has been made in establishing the binding rules for small peptides to MHC class I molecules (Rammensee et al., 1995), therefore,  potential tumor antigen peptides can be predicted up to a certain extent by computer analysis. Six potential H-2Kd binding peptides are found in tyrosinase (Table II, see also (Schmidt et al., 1997a) and four in TRP-1 (Table II). Two each of these two classes of peptides were arbitrarily selected (Table II) and combined for subcutaneous injection into mice. In the case of the P815 system it was known that a ”self”-peptide SYFPEITHI derived from the JAK1 tyrosine kinase protein occupied an unusually high proportion of 5% of all MHC class I molecules on P815 cancer cells (Harpur et al., 1993; Rammensee et al., 1995) and this peptide was therefore judged to represent a useful target for immunological rejection.

Immunohistological investigations of the vaccination


Table II: Putative peptide antigens used in the M3 melanoma model. The sequences of mouse tyrosinase and tyrosinase related potein 1 (TRP-1) were searched for peptides fulfilling criteria necessary for binding to H-2Kd molecules (?Y??????L/I) (Rammensee et al., 1995).

Figure 2: Peptide immunization prevents tumor growth.

Figure 2a: P815 mastocytoma model. DBA/2 mice were immunized with the JAK-1 kinase derived peptide SYFPEITHI. Mice (8 animals per group) were vaccinated three times at weekly intervals and challenged one week after the last immunisation as described in (Schmidt et al., 1997a). Tumor cells stably transfected with a GM-CSF gene (P815 GM-CSF) served as gold standard for vaccine efficiency. Peptides were injected subcutaneously in PBS, emulsified in incomplete Freund’s adjuvant (IFA) or as a mixture of peptide and polyarginine (pArg).

Figure 2b: Model as in Figure 2a using the novel sorbitol containing injection medium.

Figure 2c: M3 melanoma model. Experimental metastases (104 parental M3 cells) were set 5 days before the first vaccination. Animals were vaccinated subcutaneously 3 times at weekly intervals with a mixture of tyrosinase and TRP-1 derived peptides (Table II). A cellular vaccine consisting of IL-2 gene transfected, irradiated M3 was used to assess the relative efficiency of the peptide vaccine.



site (Bannerji et al., 1994; Maass et al., 1995; Schmidt et al., 1997b) as well as complementary studies of others (Huang et al., 1994) have identified antigen presenting cells (APCs), rather than T cells, to play a pivotal role in initiating the cascade leading to the generation of tumor specific T cells. Our choice of the subcutaneous route for injection of the peptides was the logical consequence of the above cytological investigations in that it was our intention to elicit a cellular response based on APCs rather than a humoral immune reaction. APCs are present in skin at high frequency, with Langerhans constituting as much as 3-4% of the cell mass (Stingl, 1990). We surmised that these APCs could be engaged and thus initiate the immunological events which would lead to the production of anti-peptide (hence anti-tumor) CTLs capable of attacking the tumor cell challenge or pre-existing experimental metastases placed counterlaterally on the back of the mouse.

A trivial finding is that in physiological saline solutions tumor antigen peptides are often quite insoluble and are possibly inactivated by precipitation in the injection medium. This can be circumvented by dissolving the peptide at low ionic strength and making the solution isotonic by addition of sorbitol (unpublished result). The JAK 1 peptide by itself as well as peptide solutions to which incomplete Feund’s adjuvant (IFA) had been added did not elicit any protection against tumor take in the P815 test system (Fig 2a, see also Schmidt et al., 1997a). When peptide was dissolved in the novel sorbitol medium tumors developed somewhat slower, however, only administration of peptide in conjunction with polyarginine did result in protection levels comparable to a cellular vaccine secreting GM-CSF (Figure 2b). We hypothesize that the failure of peptides to elicit protection against tumor take may be caused by a lack of peptide uptake into APCs. Such a hypothesis is supported by the finding that at least in vitro APC do not take up small peptides efficiently, but do so, if polycations such as polylysine and polyarginine are included in the peptide medium (Buschle et al., 1997). Such an enhancement of peptide uptake can also be demonstrated to occur in vivo: Sectioning and immuno-staining the injection site after application of a fluorescently labelled small peptide clearly reveals importation of the small peptides into APCs when co-applied with polycations, but not, when these compounds are eliminated (Figure 3 and unpubl. results).

As shown in Figure 2b, the JAK 1 peptide administered in the sorbitol medium together with polyarginine was as good as a GM-CSF secreting cellular vaccine and better than peptide injected in isotonic saline (Figure 2a). Hence, under appropriate conditions, the potency of peptide vaccines can equal that of a cytokine secreting cellular vaccine in the P 815 model system. The M3 melanoma yields similar answers, although the first generation results show the peptides from tyrosinase and tyrosinase related protein to be less potent (Figure 2c and Schmidt et al., 1997a). The less efficient protection against tumor take seen in the M3 system may be explained by a poor immunodominance of the peptides arbitrarily selected for this experiment. Experiments with the novel sorbitol medium are in progress.

When comparing the peptide vaccines with cytokine secreting cellular vaccines, which have become the gold standard of immunotherapy of cancer, it may be concluded from the P815 experiments that peptide vaccines can be equally efficacious. The situation with the M3 experiment can only be judged once we have identified potent, immunodominant tumor antigen peptides. Also, as demonstrated by Boon’s group with a tumor antigen peptide (Marchand et al., 1995), humans may be less refractory to peptide vaccination so that even the incomplete protection seen in the M3 system may already be relevant for the human situation. However, the success of peptide vaccines will always depend on a cogent selection of peptides.

From the above we wish to conclude:

l. Peptide vaccines can be made to work as efficiently as cytokine secreting cellular vaccines.

2. By contrast with cellular vaccines, peptide vaccines are chemically defined and can be easily standardised.

3. They are inexpensive and are available for vaccination without delay and have a long shelf-life.

4. Although today’s focus is on developing vaccines to cure cancer or at least minimal residual disease of cancer, the availability of peptide vaccines has the potential to vaccinate prophylacticly to prevent the occurrence of cancer.


Figure 3: Immunohistological analysis of the injection site after application of peptide vaccines in the presence (A,B,C) or absence (D, E) of polyarginine.

Fluorescence labeled peptides were injected subcutaneously alone (D,E) or as a mixture of peptide with polyarginine (A,B,C). A, D: Fluorescence photomicrographs of injection sites 3 days after vaccination. Cryosections were stained with an antibody recognizing MHC class II followed by detection with a Texas Red tagged secondary antibody. Nuclei were counterstained with 4,6-diamidino-phenylindole (DAPI). B, C, E: Confocal microscopy of injection sites 7 days after vaccination. Sections were processed as above except that DAPI was omitted.


III. Cancer peptide vaccines and HLA heterogeneity

We are encouraged by these many positive aspects of peptide vaccines, but a discussion of this field would be incomplete without considering the downside and possible remedies thereof. The single most important impediment for the wide application of such vaccines is the dependence of the TSA and TAA peptide sequence on the HLA-type of the patient. Another type of heterogeneity one has to content with is the apparently diverse appearance of TAAs even within one class of tumors such as melanoma. Another issue which impinges on practicality of these vaccines, is whether different types of cancer share common antigens. The situation here is in flux and no final assessment is as yet possible. Each cancer type may ultimately require its own vaccine composition.

A possible solution to some of these problems, for instance TSA and TAA heterogeneity within one tumor type, is to work with peptide mixtures where it is assumed that mismatched peptides are simply inert. The same approach may be useful when dealing with HLA-heterogeneity although here the complexities are immense and vexing. One positive aspect is that certain HLA-types occur more frequently than others, as is the case for HLA-A2 which in Caucasians is encountered in nearly half of the population. It can be calculated that by combining HLA-A2 with other relatively frequent HLA-types like A1, A3 and A24 approximately 80% of the Caucasian population will be covered on statistical grounds. This proportion can be increased by adding rarer types, but with

diminishing returns and, in practice, 100% will never be attained.

TSAs and TAAs, as is the case for cellular proteins, are degraded in the cytoplasm by the proteasome and the resulting peptides transported into the endoplasmatic internal space by the transporter associated with antigen processing (TAP) (York and Rock, 1996). Association of the peptides binding tightly to the groove of the MHC class I molecule occurs in small cellular vesicles which then fuse with the cell membrane, thus exposing the peptide-charged MHC molecule to the extracellular environment. The consequence of such a MHC dependent selection of peptides is that HLA-dependence in peptide vaccines can be largely circumvented by using the entire TSA or TAA for vaccination. The task then becomes that of developing efficient procedures to import the unprocessed (recombinant) TSAs and TAAs into APCs. We have ascertained that the polycations also enhance the entry of large proteins into APCs (unpublished results) and hence this approach should be applicable to protein vaccination. One major advantage of such protein vaccines is that class I as well as MHC class II restricted epitopes are contained within the sequence of a given protein and thus can be presented following entry into APCs (Germain and Margulies, 1993; Kovacsovics Bankowski and Rock, 1995; Watts, 1997; York and Rock, 1996).


IV. Polyepitope proteins as vaccines

Melief et al. (Melief et al., 1996) have recently suggested a general utility of polyepitope proteins to vaccinate against cancer. We wish to point out that when a TAA protein is degraded and some of its peptides ”presented”, much of the protein sequence is dead-weight because peptides derived from most of the TAA sequences will not fit a given MHC molecule. Now, it has been demonstrated (An and Whitton, 1997; Thomson et al., 1996; Thomson et al., 1995; Whitton et al., 1993) that polyepitope proteins consisting entirely of contiguous short CTL epitopes are correctly processed by the immune system to yield CTLs against each of the epitopes. Assuming that this is a general principle we have constructed a gene encoding an artificial protein, “SUPERMAGE”, which consists entirely of known short peptides derived from known melanoma TAA and TSA capable of generating CTLs in different HLA contexts (Figure 4). Thus, it would be expected on the basis of the artificial SUPERMAGE sequence that such a protein could be used as a melanoma vaccine, in an HLA setting of A1, A2, A24, A31, B44, and Cw6. Thus a majority of melanoma patients, could be vaccinated with this antigen. It is not surprising that at this stage HLA-A2 peptides are over-represented in the SUPERMAGE protein, reflecting the present day state of the art in CTL analysis. The corresponding gene sequence shown in Figure 4 contains restriction sites within the coding sequence for easy modification of the protein sequence, as new epitopes become known. Large quantities of the SUPERMAGE protein could rather easily be produced using standard recombinant technologies including expression of SUPERMAGE in insect cells.

Our investigations of the injection sites following peptide vaccination revealed that invasion of the bolus of injected peptides requires several days (Figure 3a, b and c). By contrast, peptides injected without adjuvants rapidly diffuse from the injection site and are undetectable at time points beyond 24 hours (Figure 3d, e and unpublished results). At day 7 numerous APCs are loaded with flourescence labelled peptides, but  only when polyarginine is coinjected (Figures 3 b, c and e). Possibly, application of a larger polyepitope protein rather than peptide mixtures may enhance the efficiency of the vaccine by displaying decreased diffusion rates at least when injected subcutaneously. These ideas are now being tested in murine model systems. However, before such strategies including the generation of SUPERMAGE and related proteins of different kinds can be routinely implemented in man it will be necessary to establish the potency of individual epitopes (e.g. in form of peptides) and to determine what minimal number of each epitope in particular is necessary to elicit clinical benefits in different HLA contexts.

A. DNA sequence



B. Amino acid sequence


























Mart-1/Melan A



Mart-1/Melan A







































Figure 4: Artificial, polyepitopic SUPERMAGE antigen for the treatment of human melanoma of the HLA haplotypes A1, A2, A24, A31, B44 and Cw6.

A) DNA sequence of the SUPERMAGE polypeptide.  5’ Eco R I and 3’ Xba I sequences were added for subcloning (underlined). A Sal I site was included for later modification of the molecule (underlined). The ATG start codon (bold) is preceeded by the sequence gccgcc to allowing efficient translation (Kozak, 1984). B) Amino acid sequence of the SUPERMAGE antigen (single letter amino acid code). C) Peptides used in the SUPERMAGE antigen. For references see Table II.



V. A schedule for curing of cancers ?

Looking far ahead and assuming that both cellular and at least one form of peptide/protein vaccination can be shown to be efficacious in patients, the following scheme shown in Figure 5 may ultimately apply. For immunotherapy of cancer, tumors would be resected or, if indicated, patients would undergo chemotherapy or radiation therapy in order to remove the cancerous tissue. In this way cancer would be reduced to a minimal residual disease. Blood cells or excised tumor tissue could be used to determine the HLA-type of the patient and the tumor tissue would be screened for any known TSA and TAA by RT-PCR or immunological techniques. If the appropriate peptides or multiepitope proteins are available, patients would be vaccinated with these defined vaccines. Because of the HLA heterogeneity, in practice probably around 80% of the patients (see above) can be anticipated to qualify for such a protocol, if the vaccine is tailored to the four most frequent HLA-types. Alternatively, patients could be treated with recombinant TSA, TAA proteins. If neither of the above are available, either because of a rare HLA genotype or lack of known antigens, the patients would proceed to treatment with either autologous or allogeneic cellular tumor vaccines, if either of these can be shown to be effective in the ongoing clinical trials. If there is no reoccurrence of metastases after peptide/protein vaccination, no further steps would be required. If there is

Figure 5: Potential treatment schedule for human cancers



reocurrence of tumor metastases, either because progression of tumors renders the vaccine ineffectual or the peptide epitopes prove insufficiently immunogenic, the patients would undergo remission-inducing conventional therapy followed by treatment with cellular vaccines at a later stage. It is hoped that by applying the above scheme at least select patient populations will benefit from immunotherapy of cancer.



We would like to thank Drs. Cotten and Bello-Fernandez for critical reading of the manuscript and Ivan Botto for preparation of antibodies. This work was supported in part by the Vienna Business Promotion Fund (WWFF).



Alexander, M., Salgaller, M. L., Celis, E., Sette, A., Barnes, W. A., Rosenberg, S. A., and Steller, M. A. (1996). Generation of tumor-specific cytolytic T lymphocytes from peripheral blood of cervical cancer patients by in vitro stimulation with a synthetic human papillomavirus type 16 E7 epitope. Am J Obstet Gynecol 175, 1586-1593.

An, L. L., and Whitton, J. L. (1997). A multivalent minigene vaccine, containing B-cell, cytotoxic T- lymphocyte, and Th epitopes from several microbes, induces appropriate responses in vivo and confers protection against more than one pathogen. J Virol 71, 2292-2302.

Bannerji, R., Arroyo, C. D., Cordon-Cardo, C., and Gilboa, E. (1994). The role of IL-2 secreted from genetically modified tumor cells in the establishment of antitumor immunity. J Immunol 152, 2324-2332.

Boel, P., Wildmann, C., Sensi, M. L., Brasseur, R., Renauld, J. C., Coulie, P., Boon, T., and van der Bruggen, P. (1995). BAGE: a new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes. Immunity 2, 167-175.

Boon, T., Cerottini, J. C., Van den Eynde, B., van der Bruggen, P., and Van Pel, A. (1994). Tumor antigens recognized by T lymphocytes. Annu Rev Immunol 12, 337-365.

Boon, T., Coulie, P. G., and Van den Eynde, B. (1997). Tumor antigens recognized by T cells. Immunol Today 18, 267-268.

Boon, T., and van der Bruggen, P. (1996). Human tumor antigens recognized by T lymphocytes. J Exp Med 183, 725-729.

Brichard, V. G., Herman, J., Van Pel, A., Wildmann, C., Gaugler, B., Wolfel, T., Boon, T., and Lethe, B. (1996). A tyrosinase nonapeptide presented by HLA-B44 is recognized on a human melanoma by autologous cytolytic T lymphocytes. Eur J Immunol 26, 224-230.

Buschle, M., Schmidt, W., Zauner, W., Mechtler, K., Trska, B., Kirlappos, H., and Birnstiel, M. L. (1997). Transloading of tumor antigen-derived peptides into antigen presenting cells. Proc Natl Acad Sci USA 94, 3256-3261.

Castelli, C., Storkus, W. J., Maeurer, M. J., Martin, D. M., Huang, E. C., Pramanik, B. N., Nagabhushan, T. L., Parmiani, G., and Lotze, M. T. (1995). Mass spectrometric identification of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes. J Exp Med 181, 363-368.

Celluzzi, C. M., Mayordomo, J. I., Storkus, W. J., Lotze, M. T., and Falo, L. D., Jr. (1996). Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity. J Exp Med 183, 283-287.

Coulie, P. G., Lehmann, F., Lethe, B., Herman, J., Lurquin, C., Andrawiss, M., and Boon, T. (1995). A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci U S A 92, 7976-7980.

Cox, A. L., Skipper, J., Chen, Y., Henderson, R. A., Darrow, T. L., Shabanowitz, J., Engelhard, V. H., Hunt, D. F., and Slingluff, C. L. (1994). Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines. Science 264, 716-719.

De Smet, C., De Backer, O., Faraoni, I., Lurquin, C., Brasseur, F., and Boon, T. (1996). The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation. Proc Natl Acad Sci U S A 93, 7149-7153.

Disis, M. L., and Cheever, M. A. (1997). HER-2/neu protein: a target for antigen-specific immunotherapy of human cancer. Adv Cancer Res 71, 343-371.

Disis, M. L., and Cheever, M. A. (1996). Oncogenic proteins as tumor antigens. Curr Opin Immunol 8, 637-642.

Finn, O. J., Jerome, K. R., Henderson, R. A., Pecher, G., Domenech, N., Magarian Blander, J., and Barratt Boyes, S. M. (1995). MUC-1 epithelial tumor mucin-based immunity and cancer vaccines. Immunol Rev 145, 61-89.

Fisk, B., Blevins, T. L., Wharton, J. T., and Ioannides, C. G. (1995). Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines. J Exp Med 181, 2109-2117.

Gaugler, B., Van den Eynde, B., van der Bruggen, P., Romero, P., Gaforio, J. J., De Plaen, E., Lethe, B., Brasseur, F., and Boon, T. (1994). Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. J Exp Med 179, 921-930.

Germain, R. N., and Margulies, D. H. (1993). The biochemistry and cell biology of antigen processing and presentation. Annu Rev Immunol 11, 403-450.

Guilloux, Y., Lucas, S., Brichard, V. G., Van Pel, A., Viret, C., De Plaen, E., Brasseur, F., Lethe, B., Jotereau, F., and Boon, T. (1996). A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is encoded by an intron sequence of the N-acetylglucosaminyltransferase V gene. J Exp Med 183, 1173-1183.

Harpur, A. G., Zimiecki, A., Wilks, A. F., Falk, K., Rotzschke, O., and Rammensee, H. G. (1993). A prominent natural H-2 Kd ligand is derived from protein tyrosine kinase JAK1. Immunol Lett 35, 235-237.

Hsu, F. J., Benike, C., Fagnoni, F., Liles, T. M., Czerwinski, D., Taidi, B., Engleman, E. G., and Levy, R. (1996). Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med 2, 52-58.

Hsu, F. J., Caspar, C. B., Czerwinski, D., Kwak, L. W., Liles, T. M., Syrengelas, A., Taidi-Laskowski, B., and Levy, R. (1997). Tumor-specific idiotype vaccines in the treatment of patients with B- cell lymphoma--long-term results of a clinical trial. Blood 89, 3129-3135.

Huang, A. Y., Golumbek, P., Ahmadzadeh, M., Jaffee, E., Pardoll, D., and Levitsky, H. (1994). Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens. Science 264, 961-965.

Ikeda, H., Lehte, B., Lehmann, F., Van Baren, N., Baurain, J.-F., De Smet, C., Chambost, H., Vitale, M., Moretta, A., Boon, T., and Coulie, P. G. (1997). Characterization of an antigen that is recognized on a melanoma showing partial loss by CTL expressing an NK inhibitory receptor. Immunity 6, 199-208.

Ioannides, C. G., Fisk, B., Pollack, M. S., Frazier, M. L., Taylor Wharton, J., and Freedman, R. S. (1993). Cytotoxic T-cell clones isolated from ovarian tumour infiltrating lymphocytes recognize common determinants on non-ovarian tumour clones. Scand J Immunol 37, 413-424.

Kang, X., Kawakami, Y., el Gamil, M., Wang, R., Sakaguchi, K., Yannelli, J. R., Appella, E., Rosenberg, S. A., and Robbins, P. F. (1995). Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes. J Immunol 155, 1343-1348.

Kawakami, Y., Eliyahu, S., Delgado, C. H., Robbins, P. F., Sakaguchi, K., Appella, E., Yannelli, J. R., Adema, G. J., Miki, T., and Rosenberg, S. A. (1994). Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc Natl Acad Sci USA 91, 6458-6462.

Kawakami, Y., Eliyahu, S., Jennings, C., Sakaguchi, K., Kang, X., Southwood, S., Robbins, P. F., Sette, A., Appella, E., and Rosenberg, S. A. (1995). Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression. J Immunol 154, 3961-3968.

Kawakami, Y., Eliyahu, S., Sakaguchi, K., Robbins, P. F., Rivoltini, L., Yannelli, J. R., Appella, E., and Rosenberg, S. A. (1994). Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes. J Exp Med 180, 347-352.

Kovacsovics Bankowski, M., and Rock, K. L. (1995). A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 267, 243-246.

Kozak, M. (1984). Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res 12, 857-872.

Linehan, D. C., Goedegebuure, P. S., Peoples, G. E., Rogers, S. O., and Eberlein, T. J. (1995). Tumor-specific and HLA-A2-restricted cytolysis by tumor-associated lymphocytes in human metastatic breast cancer. J Immunol 155, 4486-4491.

Maass, G., Schmidt, W., Berger, M., Schilcher, F., Koszik, F., Schneeberger, A., Stingl, G., Birnstiel, M. L., and Schweighoffer, T. (1995). Priming of tumor-specific T cells in the draining lymph nodes after immunization with interleukin 2-secreting tumor cells: three consecutive stages may be required for successful tumor vaccination. Proc Natl Acad Sci U S A 92, 5540-5544.

Mandelboim, O., Berke, G., Fridkin, M., Feldman, M., Eisenstein, M., and Eisenbach, L. (1994). CTL induction by a tumour-associated antigen octapeptide derived from a murine lung carcinoma. Nature 369, 67-71.

Mandelboim, O., Vadai, E., Fridkin, M., Katz-Hillel, A., Feldman, M., Berke, G., and Eisenbach, L. (1995). Regression of established murine carcinoma metastases following vaccination with tumor-associated antigen peptides. Nature Med 1, 1179-1182.

Mandruzzato, S., Brasseur, F., Andry, G., Boon, T., and Bruggen, P. (1997). A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma. J Exp Med 186, 785-793.

Marchand, M., Weynants, P., Rankin, E., Arienti, F., Belli, F., Parmiani, G., Cascinelli, N., Bourlond, A., Vanwijck, R., Humblet, Y., Canon, J.-L., Laurent, C., Naeyaert, J.-M., Plagne, R., Deraemaeker, R., Knuth, A., Jäger, E., Brasseur, F., Herman, J., Coulie, P. G., and Boon, T. (1995). Tumor regression responses in melanoma patients treated with a peptide encoded by gene MAGE-3. Int J Cancer 63, 883-885.

Mayordomo, J. I., Loftus, D. J., Sakamoto, H., De Cesare, C. M., Appasamy, P. M., Lotze, M. T., Storkus, W. J., Appella, E., and DeLeo, A. B. (1996). Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines. J Exp Med 183, 1357-1365.

Mayordomo, J. I., Zorina, T., Storkus, W. J., Zitvogel, L., Celluzzi, C., Falo, L. D., Melief, C. J., Ildstad, S. T., Kast, W. M., Deleo, A. B., and Lotze, M. T. (1995). Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat Med 1, 1297-1302.

Melief, C. J., Offringa, R., Toes, R. E., and Kast, W. M. (1996). Peptide-based cancer vaccines. Curr Opin Immunol 8, 651-657.

Paglia, P., Chiodoni, C., Rodolfo, M., and Colombo, M. P. (1996). Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo. J Exp Med 183, 317-322.

Pardoll, D. M. (1995). Paracrine cytokine adjuvants in cancer immunotherapy. Annu Rev Immunol 13, 399-415.

Peoples, G. E., Goedegebuure, P. S., Smith, R., Linehan, D. C., Yoshino, I., and Eberlein, T. J. (1995). Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same HER2/neu-derived peptide. Proc Natl Acad Sci U S A 92, 432-436.

Porgador, A., Snyder, D., and Gilboa, E. (1996). Induction of antitumor immunity using bone marrow-generated dendritic cells. J Immunol 156, 2918-2926.

Rammensee, H. G., Friede, T., and Stevanoviic, S. (1995). MHC ligands and peptide motifs: first listing. Immunogenetics 41, 178-228.

Ras, E., van der Burg, S. H., Zegveld, S. T., Brandt, R. M., Kuppen, P. J., Offringa, R., Warnarr, S. O., van de Velde, C. J., and Melief, C. J. (1997). Identification of potential HLA-A *0201 restricted CTL epitopes derived from the epithelial cell adhesion molecule (Ep-CAM) and the carcinoembryonic antigen (CEA). Hum Immunol 53, 81-89.

Ressing, M. E., van Driel, W. J., Celis, E., Sette, A., Brandt, M. P., Hartman, M., Anholts, J. D., Schreuder, G. M., ter Harmsel, W. B., Fleuren, G. J., Trimbos, B. J., Kast, W. M., and Melief, C. J. (1996). Occasional memory cytotoxic T-cell responses of patients with human papillomavirus type 16-positive cervical lesions against a human leukocyte antigen-A *0201-restricted E7-encoded epitope. Cancer Res 56, 582-588.

Rickinson, A. B., and Moss, D. J. (1997). Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu Rev Immunol 15, 405-431.

Robbins, P. F., El Gamil, M., Li, Y. F., Kawakami, Y., Loftus, D., Appella, E., and Rosenberg, S. A. (1996). A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes. J Exp Med 183, 1185-1192.

Robbins, P. F., el Gamil, M., Li, Y. F., Topalian, S. L., Rivoltini, L., Sakaguchi, K., Appella, E., Kawakami, Y., and Rosenberg, S. A. (1995). Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes. J Immunol 154, 5944-5950.

Robbins, P. F., El-Gamil, M., Li, Y. F., Fitzgerald, E. B., Kawakami, Y., and Rosenberg, S. A. (1997). The intronic region of an incompletely spliced gp100 gene transcript encodes an epitope recognized by melanoma-reactive tumor-infiltrating lymphocytes. J Immunol 159, 303-308.

Robbins, P. F., and Kawakami, Y. (1996). Human tumor antigens recognized by T cells. Curr Opin Immunol 8, 628-636.

Rosenberg, S. A. (1997). Cancer vaccines based on the identification of genes encoding cancer regression antigens. Immunol Today 18, 175-182.

Schmidt, W., Buschle, M., Zauner, W., Kirlappos, H., Mechtler, K., Trska, B., and Birnstiel, M. L. (1997a). Cell-free tumor antigen peptide-based cancer vaccines. Proc Natl Acad Sci USA 94, 3262-3267.

Schmidt, W., Maass, G., Buschle, M., Schweighoffer, T., Berger, M., Herbst, E., Schilcher, F., and Birnstiel, M. L. (1997b). Generation of effective cancer vaccines genetically engineered to secrete cytokines using adenovirus-enhanced transferrinfection (AVET). Gene 190, 211-216.

Schmidt, W., Schweighoffer, T., Herbst, E., Maass, G., Berger, M., Schilcher, F., Schaffner, G., and Birnstiel, M. L. (1995). Cancer vaccines: the interleukin 2 dosage effect. Proc Natl Acad Sci U S A 92, 4711-4714.

Slingluff, C. L., Hunt, D. F., and Engelhard, V. H. (1994). Direct analysis of tumor-associated peptide antigens. Curr Opin Immunol 6, 733-740.

Stingl, G. (1990). Dendritic cells of the skin. Dermatol Clin 8, 673-679.

Thomson, S. A., Elliott, S. L., Sherritt, M. A., Sproat, K. W., Coupar, B. E., Scalzo, A. A., Forbes, C. A., Ladhams, A. M., Mo, X. Y., Tripp, R. A., Doherty, P. C., Moss, D. J., and Suhrbier, A. (1996). Recombinant polyepitope vaccines for the delivery of multiple CD8 cytotoxic T cell epitopes. J Immunol 157, 822-826.

Thomson, S. A., Khanna, R., Gardner, J., Burrows, S. R., Coupar, B., Moss, D. J., and Suhrbier, A. (1995). Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design. Proc Natl Acad Sci U S A 92, 5845-5849.

Tindle, R. W. (1996). Human papillomavirus vaccines for cervical cancer. Curr Opin Immunol 8, 643-650.

Topalian, S. L., Gonzales, M. I., Parkhurst, M., Li, Y. F., Southwood, S., Sette, A., Rosenberg, S. A., and Robbins, P. F. (1996). Melanoma-specific CD4+T cells recognize nonmutated HLA-DR-restricted tyrosinase epitopes. J Exp Med 183, 1965-1971.

Topalian, S. L., Rivoltini, L., Mancini, M., Markus, N. R., Robbins, P. F., Kawakami, Y., and Rosenberg, S. A. (1994). Human CD4+ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene. Proc Natl Acad Sci U S A 91, 9461-9465.

Traversari, C., van der Bruggen, P., Luescher, I. F., Lurquin, C., Chomez, P., Van Pel, A., De Plaen, E., Amar Costesec, A., and Boon, T. (1992). A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E. J Exp Med 176, 1453-1457.

Tsang, K. Y., Zaremba, S., Nieroda, C. A., Zhu, M. Z., Hamilton, J. M., and Schlom, J. (1995). Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst 87, 982-990.

Van den Eynde, B., Peeters, O., De Backer, O., Gaugler, B., Lucas, S., and Boon, T. (1995). A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma. J Exp Med 182, 689-698.

van der Bruggen, P., Bastin, J., Gajewski, T., Coulie, P. G., Boel, P., De Smet, C., Traversari, C., Townsend, A., and Boon, T. (1994). A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3. Eur J Immunol 24, 3038-3043.

van der Bruggen, P., Szikora, J.-P., Boël, P., Wildmann, C., Somville, M., Sensi, M., and Boon, T. (1994). Autologous cytolytic T lymphocytes recognize a MAGE-1 nonapeptide on melanomas expressing HLA-Cw*1601*. Eur J Immunol 24, 2134-2140.

Wang, R.-F., and Rosenberg, S. A. (1996). Human tumor antigens recognized by T lymphocytes: implications for cancer therapy. J Leukoc Biol 60, 296-309.

Wang, R. F., Appella, E., Kawakami, Y., Kang, X., and Rosenberg, S. A. (1996). Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes. J Exp Med 184, 2207-2216.

Wang, R. F., Parkhurst, M. R., Kawakami, Y., Robbins, P. F., and Rosenberg, S. A. (1996). Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen. J Exp Med 183, 1131-1140.

Wang, R. F., Robbins, P. F., Kawakami, Y., Kang, X. Q., and Rosenberg, S. A. (1995). Identification of a gene encoding a melanoma tumor antigen recognized by HLA-A31-restricted tumor-infiltrating lymphocytes [published erratum appears in J Exp Med 1995 Mar 1;181(3):1261]. J Exp Med 181, 799-804.

Watts, C. (1997). Capture and processing of exogenous antigens for presentation on MHC molecules. Annu Rev Immunol 15, 821-850.

Whitton, J. L., Sheng, N., Oldstone, M. B., and McKee, T. A. (1993). A "string-of-beads" vaccine, comprising linked minigenes, confers protection from lethal-dose virus challenge. J Virol 67, 348-352.

Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C., Klehmann-Hieb, E., De Plaen, E., Hankeln, T., Meyer zum Buschenfelde, K.-H., and Beach, D. (1995). A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 1281-1284.

Wolfel, T., Schneider, J., Meyer zum Buschenfelde, K. H., Rammensee, H. G., Rotzschke, O., and Falk, K. (1994). Isolation of naturally processed peptides recognized by cytolytic T lymphocytes (CTL) on human melanoma cells in association with HLA-A2.1. Int J Cancer 57, 413-418.

Wolfel, T., Van Pel, A., Brichard, V., Schneider, J., Seliger, B., Meyer zum Buschenfelde, K. H., and Boon, T. (1994). Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes. Eur J Immunol 24, 759-764.

York, I. A., and Rock, K. L. (1996). Antigen processing and presentation by the class I major histocompatibility complex. Annu Rev Immunol 14, 369-396.

Zatloukal, K., Schneeberger, A., Berger, M., Schmidt, W., Koszik, F., Kutil, R., Cotten, M., Wagner, E., Buschle, M., Maass, G., Payer, E., Stingl, G., and Birnstiel, M. L. (1995). Elicitation of a systemic and protective anti-melanoma immune response by an IL-2-based vaccine. Assessment of critical cellular and molecular parameters. J Immunol 154, 3406-3419.

Zhang, L., Zhou, W., Velculescu, V. E., Kern, S. E., Hruban, R. H., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. (1997). Gene expression profiles in normal and cancer cells. Science 276, 1268-1272.