26 p53-Based Immunotherapy of Cancer

From: Cancer Drug Discovery and Development: Cancer Drug Resistance Edited by: B. Teicher © Humana Press Inc., Totowa, NJ

491

p53-Based Immunotherapy of Cancer

Albert B. DeLeo, ^{P h D}

C

ONTENTS

I

NTRODUCTION

C

ANCER

V

ACCINES

P

^RECLINICAL

M

^URINE

S

TUDIES OF P

53-B

^ASED

I

MMUNOTHERAPY

P

^RECLINICAL

D

EVELOPMENT OF P

53-B

^ASED

I

MMUNOTHERAPY

C

^RITICAL

I

^{SSUES AND}

C

^ONCERNS

T

^HAT

C

ONFRONT THE

C

^LINICAL

I

NTRODUCTION OF P

53-B

^ASED

C

^ANCER

V

^ACCINES

C

^LINICAL

T

^{RIALS OF P}

53-B

^ASED

I

MMUNOTHERAPY OF

P

ATIENTS

W

ITH

C

ANCER

S

UMMARY

A

CKNOWLEDGMENTS

R

^EFERENCES

S

UMMARY

In recent years, there has been an increasing awareness that the immune system, in particular the T-cell component, plays a significant role in tumor eradication. Advances in molecular immunology and identification of T-cell-defined human tumor antigens have accelerated the development of vaccines to promote T-cell-mediated antitumor immune responses. In general, many shared human tumor antigens are derived from proteins overexpressed or derepressed in tumors relative to normal cells. Alteration in the tumor suppressor gene product, p53, is one of the most common events in human cancers, but mutant p53-based immunotherapy would require “custom-made” vaccines for use in relatively few patients. Because most mutations in p53 are associated with accumulation or “overexpression” of mutant p53 in the cytosol, the protein is more readily available for antigenic processing and presentation than are the low levels of p53 molecules expressed in normal cells. A vaccine targeting wild-type sequence (wt) or nonmutant sequence peptides derived from altered p53 molecules, therefore, is a more attractive approach for developing broadly applicable cancer vaccines.

Extensive preclinical murine tumor model studies using peptide-based and DNA vaccines have demonstrated that wt p53-based vaccines can induce tumor eradication in the absence of deleterious antitumor autoimmune side effects. Like any T-cell-based immunotherapy, effective p53-based immunotherapy will be dependent on patients’

responsiveness to wt p53 peptides and the ability of their tumors to present these pep- tides for T-cell recognition. These and other issues and concerns related to p53-based

26

vaccines are discussed together with a brief summary of the initial clinical trials of p53- based immunotherapy.

Key Words: p53; immunotherapy; vaccines; dendritic cells; CTL; Th; peptides;

immunoselection; immunotherapy.

1. INTRODUCTION

The p53 tumor suppressor gene product was identified 25 yr ago as a transformation- related antigen, using antibodies present in the sera of mice immunized against chemi- cally induced sarcomas (1). It was characterized as being expressed at elevated levels in murine tumors induced with irradiation, RNA and DNA viruses, and chemical carcino- gens. Of the nontransformed cells/tissues tested for expression, only thymocytes and mitogen-activated lymphocytes showed low but detected levels of the antigen (2). Since then, genetic events leading to loss of function of p53 have been identified as the most fre- quently occurring event associated with oncogenesis (3–5). Whereas genetic alterations of the p53 gene, namely missense mutations, are considered the primary cause leading to loss of its function, alterations in gene products of several pathways that are critical for regulation of p53 can also result in loss of its function. Several approaches for restoring the normal p53 function of regulating the cell cycle and reversing the transformation phenotype of cancer cells are being actively pursued. They involve focusing on p53 gene replacement, identifying pharmacological agents capable of restoring mutated p53 to its normal conformation and functional activities, and viruses that are lytic to cells harboring mutant p53 (6–8). Another approach can be traced to the origins of the identification of p53, namely that as a tumor antigen, p53 could be targeted with cancer vaccines.

2. CANCER VACCINES

Preclinical murine and clinical evidence indicates that the immune system plays a major role in host defense against progressive tumor growth and drives the concept of developing immunotherapy to augment the antitumor immune responsiveness of patients with cancer in order to eradicate their tumors (9). Although all elements of the immune system are involved in tumor eradication, it has been shown to be primarily dependent on T-cell-mediated antitumor responses. Whereas CD8

⁺

cytotoxic T-lymphocytes (CTLs) are considered the critical effectors for tumor eradication, CD4

⁺

T-lymphocytes or T- helper (Th) cells have been shown to be required for expansion and maintenance of these effectors (10–13). Both T-cell subsets recognize short peptides or “epitopes” derived from proteins that are presented on the cell surface in association with class I or II human leukocyte antigen (HLA) molecules (14,15). A major focus of cancer immunotherapy, therefore, has been to develop vaccines that would induce and/or expand CTL-mediated antitumor immune responses.

2.1. T-Cell-Defined Tumor-Associated Antigens

Nearly all the human tumor antigens being used in developing cancer vaccines are

“shared” tumor-associated determinants. They represent nonmutated peptides derived

from three distinct groups of proteins (16). One group is derived from proteins that are

expressed in the testes, but not normal cells. Epigenetic and/or genetic events result in

activation of genes encoding these “cancer–testes” or “cancer germline” proteins. Their

lack of expression on normal cells and inappropriate expression in wide range of tumors

makes them immunologically “nonself” in nature and enhances their potential for use in cancer vaccines. The other two groups of tumor-associated antigen (TAAs) are “self- antigens,” and can be distinguished by their patterns of expression in tumors and normal adult cells. One group consists of tissue-specific or differentiation antigens that are overexpressed in tumors relative to normal cells, whereas the other represents antigens derived from a variety of gene products involved in cell cycle regulation. Loss of their functional activities is a critical event in transformation. Many of these proteins are products of oncogenes or tumor suppressor genes; p53 is a prime example of the latter group of gene products. The identification of melanoma-associated antigens and the development and clinical introduction of melanoma vaccines has accelerated the effort to develop vaccines for more widely occurring types of cancer, namely carcinomas of the breast, colon, and lung. An obvious candidate for such vaccines is p53.

2.2. p53: A TAA

Following the serological identification of p53 as a transformation-related murine tumor antigen (1), a subsequent study by Crawford and colleagues identified anti-p53 immunoglobulin (Ig)G antibodies in the sera of some patients with cancer (17). Because an IgG response against a protein like p53 requires the participation of CD4

⁺

Th cells as well as B-cells, that study and subsequent others have established the immunogenicity of p53 in humans and the presence, in some patients, of T-cell-mediated anti-p53 responses (18–21).

A key function of p53 is to prevent DNA replication following DNA damage because of a genotoxic event, such as irradiation (3). It does so by blocking replication until DNA repair has occurred. In normal cells, therefore, wild-type (wt) p53 molecules are seques- tered in the nucleus and have a relatively short half-life. Genetic alterations in p53, which result in loss of p53 function, have been shown to be the most frequently occurring genetic event associated with human cancer (5). At least 50% of all human tumors analyzed contain genetic alterations in p53. Most are missense mutations, in exons 5–8, which encode the DNA binding region of the molecule. In studies in which all the p53 exons (exons 2–11) as well as intron/exon junctions have been analyzed, as was recently done for a group of squamous cell carcinoma of the head and neck (SCCHN) tumors (22,23), the incidence of genetic defects can approach 80%. Missense mutation of p53, however, is frequently associated with stabilization (increased half-life) of mutated p53 molecules, resulting in accumulation or “overexpression” in the cytosol of tumors (24).

As the accumulation of mutated p53 in tumors resembles the overexpression or derepres- sion phenotype associated with many shared tumor-associated tissue/differentiation melanoma antigens targeted with vaccines (10,17), it was hypothesized that the accumu- lation of mutant p53 molecules in the tumor cytosol would enhance processing and presentation of p53-derived peptides for CTL recognition and eradication (24).

2.3. Two Classes of T-Cell-Defined p53 Peptides

In contrast to most other tumor associated antigens (TAAs), however, two classes of

epitopes can de derived from a mutant p53 molecule can be presented, an epitope con-

taining the missense mutation, which would be nonself and a unique tumor-specific

antigen and an array of epitopes composed of nonmutated, wt peptide sequences derived

from the rest of the mutant molecule (Fig. 1). The latter would be “self-TAAs.” Although

mutant peptides should be highly immunogenic and induce robust antitumor responses,

the constraints of antigen processing and presentation limit their presentation to tumors

of only a few individuals that express the appropriate class I HLA molecules capable of presenting the mutation. Consequently, vaccines targeting a mutant peptide would essen- tially need to be “custom-made” for an individual patient and of limited applicability. In contrast, due the polymorphisms of HLA molecules, there is a much greater probability that one or more wt p53 peptides can be presented for T-cell recognition by tumors expressing any given class I HLA allele. These wt p53 peptides represent shared TAAs, and vaccines targeting them would be broadly applicable (24,25). Although they would be targeting self-epitopes and presumably would induce less robust antitumor responses than the mutant p53 epitopes, wt p53-based vaccines represent a practical approach to developing a broadly applicable cancer vaccine.

3. PRECLINICAL MURINE STUDIES OF

P

53-BASED IMMUNOTHERAPY The demonstration that a wt p53 peptide-based dendritic cell vaccine induced rejection of a transplanted chemically induced tumor established the potential efficacy of p53- based vaccines as broadly applicable for use in immunotherapy of cancer. Since then, studies utilizing mice and murine tumor models have been continually used to evaluate the efficacies of various types of p53-based vaccines, including DNA vaccines, as well as the roles that tolerance to “self-p53” peptides and the potential of inducing autoim- mune might have in vaccine-induced, anti-p53 immune responses.

3.1. p53-Based Immunotherapy of Mice Bearing Transplanted Tumors The study by Majordomo et al. (25), which used the same tumor model systems that were used to serologically identify p53, was the first to demonstrate the ability of a wt p53 peptide to induce tumor rejection of a chemically induced tumor in mice. The study was also one of the first studies that established dendritic cells, considered the professional antigen-presenting cell, as the vehicle of choice for tumor peptide based vaccines. Fur- thermore, no “antiself” autoimmune side effects were detected in wt p53-immunized mice. As in any vaccine development program, optimization of the immunogen and vaccine vehicle are critical. A wide range of p53-based vaccines and immunization protocols has been evaluated in the past decade. Murine studies have shown that effective anti-wt p53 T-cell-mediated antitumor responses could be induced by: (1) wt p53 pep-

Fig. 1. Two classes of cytotoxic T-lymphocyte (CTL)/defined p53 tumor peptides.

tides or recombinant p53 protein admixed with chemical adjuvants or pulsed onto bone marrow-derived dendritic cells (DCs) (25,26), as well as (2) DCs transfected with nonviral plasmids or viruses encoding intact p53 or fragments (27,28). In addition, p53 nonviral plasmids DNA vaccines biolistically (gene gun) delivered as well as recombinant viral vectors expressing p53 have shown to be effective in inducing antitumor immunity (29,30).

Comparisons of the anti-wt p53 CTL responses of wt mice and p53 null mice to mouse and human p53 have been critical in demonstrating the extent of tolerance that exists to self-wt p53 peptides, and reducing fears that anti-wt p53 antitumor immune responses might be associated with deleterious autoimmune responses as well (31,32). The finding that anti-wt p53 CTL responses in p53

^+/+

mice display a low-to-intermediate affinity, whereas those generated in p53 null mice are of high affinity was a clear indication of the extent to which tolerance to self-p53 epitopes exists in mice. The fact that antihuman p53 CTLs generated in normal and HLA-A2-transgenic mice display higher affinities for their ligands than do antimouse p53 CTLs induced in the same mice further demonstrated the level of tolerance to wt p53 epitopes in mice. Subsequently, adoptive transfer of high- affinity anti-wt p53 CTLs derived from p53 null mice to tumor-bearing p53

^+/+

mice showed that these effectors were very effective in inducing tumor eradication (33–35).

Although the high-affinity anti-p53 CTLs were capable of recognizing mitogen-acti- vated T-cells, there was no evidence of autoimmunity in the treated mice. More recently, several reports employing administration of anti-CD40 antibody and/or cytosine-phos- phate-guanine oligonucleotides in combination with wt p53-based vaccines have dem- onstrated that these agents can enhance the induction/expansion of anti-wt p53 CTLs, but not their avidity (36–38).

3.2. p53-Based Immunotherapy of Mice Bearing Primary Chemically Induced Tumors

As insightful as the murine tumor model studies using transplantable tumor were, they did not mimic the relatively long-term tumor-immune system interactions that occur in hosts bearing primary tumors. Methylcholanthrene (MCA) is one of several polycyclic hydrocarbons that have been used routinely to induce tumors in experimental laboratory animals, especially inbred strains of mice and rats. It is also a major environmental pollutant and has been implicated as a causative agent in human cancers. MCA induces murine tumors within 6 mo of exposure. These tumors have a high incidence of genetic alterations in p53, and many are sensitive to wt p53-specific CTLs (25). In addition to being a carcinogen, MCA is also as an immunosuppressive effect on the mice, which is prominent within weeks of its administration and persists for approx 3 mo (39–41).

We recently reported the results of p53-based immunization of MCA-treated mice

employing peptide-pulsed DC and DNA vaccines administered in protection, therapy,

and combination protection/therapy protocols (42). The results indicate that the efficacy

of p53-based immunization relative to reducing tumor incidence was severely compro-

mised by vaccine-induced “tumor escape.” As compared to tumors induced in

nonimmunized mice, a higher incidence of “epitope-loss” tumors was detected in tumors

from the immunized mice. The increase in tumor escape arose as a consequence of either

increased frequencies of mutations within/flanking p53 epitope-coding regions and/or

downregulation of expression of H2 molecules, the class I major histocompatiblity com-

plex molecules that present these epitopes for CTL recognition. One must note that the

conditions of immunizing and inducing anti-p53 immune response in the presence of a

potent carcinogen are ideal for promoting immunoselection of epitope-loss tumors. These

findings are consistent with current views of immunoselection occurring in patients receiving tumor peptide-based immunotherapy and warrant further evaluation of p53- based immunization in the MCA and other primary murine tumor model systems.

4. PRECLINICAL DEVELOPMENT OF

P

53-BASED IMMUNOTHERAPY Development of p53-based immunotherapy has greatly benefited from the knowledge and insights gained from many of the preclinical studies in murine tumor model systems.

In the course of a decade, it has progressed from identification of the first CTL-defined wt p53 peptide to clinical introduction of several types of wt p53-based vaccines. The use of high-affinity anti-wt p53 T-cells derived from p53 null mice and HLA-A2.1-transgenic p53 null mice has been shown to be very effective in inducing tumor eradication. Although of high-affinity, these cells do not react with normal cells and did not induce any detect- able evidence of autoimmunity. Consequently, the concept of genetically engineering high-affinity anti-p53 human T-cell effectors by transfecting peripheral blood mono- nuclear cells (PBMCs), with cDNA encoding the T-cell receptor (TCR) derived from antihuman p53 murine CTLs is being actively pursued (43).

4.1. CTL-Defined Human wt p53 Peptides

Unlike many of the presently identified CTL-defined human tumor peptides, nearly all of the wt p53 peptides have been identified by “reverse immunology,” namely using algorithm-predictions of putative class I HLA-binding peptides (44) and immunizing HLA transgenic mice and/or in vitro stimulation (IVS) of PBMCs obtained from normal donors (45–53). The majority of those identify are HLA-A*0201 (HLA-A2)-restricted epitopes, although an HLA-A24-restricted wt p53 has also been identified. In this man- ner, wt p53

65–73

, wt p53

149–157

, wt p53

189–196

, wt p53

217–225

, and wt p53

264–173

epitopes were identified. Several of these peptides are being use in p53-based vaccine trials for cancer patients expressing the HLA-A2.1 allele.

4.2. Th Cell-Defined wt p53 Peptides

The identification of anti-p53 IgG antibodies in the sera of some patients with cancer is indicative of anti-p53 CD4

⁺

Th cell responses having been induced in these individuals.

Unfortunately, it is also associated with a poor prognosis, which might be attributable to a predominating Th2 antitumor immune response in these patients rather than the Th1- biased response that is generally associated with tumor eradication. Preclinical studies have demonstrated that vaccines employing Th- as well as CTL-defined epitopes derived from the same tumor antigen show enhanced efficacy because of the established role of the antigen-specific CD4

⁺

T-cells in the induction and maintenance of effective antitumor immunity (54,55). Consequently, the identification of Th cell-defined p53 peptides would be useful not only for enhancing the efficacy of p53-based immunization, but also to possibly “reverse” the Th2-biased responses of p53 sero-positive patients.

Several in vitro-based studies have focused on proliferative T-cell-mediated responses to intact p53 protein or p53 peptides relative to anti-p53 antibody production in patients with cancer (19–22,56), but none identified the T-cell-defined epitopes. The study of Fujita et al. (57) identified several immunogenic HLA class II-restricted wt p53 peptides.

The abilities of these peptides to be naturally presented, however, were not established

in their study. In our recent study, which utilized recombinant wt p53 protein-pulsed DCs

as the antigen presenting cell and algorithm-predicted HLA-DRB1*0401-binding 15- mer peptides (58), we identified wt p53

110–124

peptide as a naturally presented HLA- DRB1*0401-restricted epitope (59). In in vitro-based experiments using the autologous PCI-13 SCCHN system available in our laboratory, the addition of anti-wt p53

_110–124

CD4

⁺

T-cells to PBMCs was shown to increase the total number CD8+ T-cells in the IVS cultures and, more relevantly, enhance the induction of anti-PCI 13 effectors. This effect was dependent on the ratio PBMC/CD4 cells in the cultures. These results are consistent with the concept of developing a multiepitope p53 vaccine that would employ Th-defined as well as CTL-defined p53 peptides to maximize its efficacy.

5. CRITICAL ISSUES AND CONCERNS THAT CONFRONT THE CLINICAL INTRODUCTION OF

P

53-BASED CANCER VACCINES

Successful development of p53-based immunotherapy needs not only to overcome the general issues and concerns that conform any immunization targeting a self-TAA, but also several which are unique because of the complexity of this molecule and its role as a tumor suppressor. The two critical concerns of any tumor antigen-specific immuniza- tion are (1) the responsiveness of the patient to the immunization and (2) the ability of the patient’s tumor to present the targeted antigen (10). In addition to a general impairment of immunocompetency, which characterizes many patients with cancer, the issue of tolerance/anergy to wt p53 epitopes needs to be taken into account. Second, there is concern of knowing whether (a) tumor(s) being targeted with a p53-based vaccine can present the targeted wt p53 epitopes. In addition to direct mutation/deletion in p53 exons, which can influence processing and presentation of p53-derived epitopes (60), defects in any of the pathways involved in posttranslational modification and degradation of p53 promote “loss of p53 function” as well as processing and presentation of p53 epitopes (6,7). Finally, loss of function of p53 is considered an early event in an oncogenic process that can occur over decades. The induction of an anti-p53 antitumor immune response, during the early stages of oncogenesis even if it is not robust, could readily promote over a long period of time the immunoselection of p53 epitope-loss tumors (61–63). This would mimic the outgrowth of epitope loss: tumors that were enhanced in p53-immunized mice bearing primary chemically induced tumors (43), and might be particularly relevant to the concept of p53-based immunization of “high risk” individuals to prevent cancer.

5.1. Immunological Tolerance and Autoimmunity

Effective immunity against self-tumor antigens, such as wt p53 peptides, must breach

the fine line that separates an antitumor immune response from a potentially deleterious

autoimmune response. This subtle distinction is particularly important in the case of wt

p53 epitopes. Most self-TAAs, such as those derived from tissue-specific or differentia-

tion antigens, have limited tissue distribution, and immune responses to them are gov-

erned by peripheral tolerance. In contrast, p53 is expressed by all nucleated cells and

readily available in the thymus for induction of tolerance to wt p53 epitopes (2). Based

on in vitro-based immunological studies involving PBMCs, it is apparent that only a

subset of normal donors and patients with cancer are responsive IVS with autologous

DCs pulsed with wt p53 peptides or transfected with adeno/wt p53 construct (48,64). The

DCs were chosen for this assay as they are considered the only antigen-presenting cell

capable of inducing antigen-specific responses from naive T-cells. The induced/expanded

anti-wt p53 CTLs display a low-to-intermediate affinity for their ligands and a limited

repertoire of TCR usage (48,65,66). The latter is quite evident following an analysis of TCRV β usage of anti-wt p53

264–272

CTLs. Despite the vast TCR repertoires theoretically available for any immunogen, a TCRV β immunoscope analysis of anti-wt p53

264–272

CTLs generated from PBMCs from HLA-A2

⁺

individuals showed restrictions in TCRV β family usage (66). Whether the weak immunogenicity of wt p53 peptides reflects deletion or anergy of anti-wt p53 T-cells is an open question, but preclinical murine studies have clearly demonstrated that the anti-wt p53 CTLs induced in p53 null mice are high affinity relative to those induced in normal p53

^+/+

mice (32,33). Despite their increased affinity, their adoptive transfer into normal mice did not result in deleterious autoimmune side effects, indicating that even the biochemical detectable levels of p53 expressed in some normal cells in the mice (2) are not sufficient to sensitize them to anti-p53 CTLs (25,67).

5.2. “Optimized” p53 Peptides

In many instances, the immunogenicity of a weakly immunogenic peptide can be enhanced by an amino acid exchange in the peptide sequence, which both increases its binding to HLA molecules and/or interaction with the TCR and results in an increased stabilization of the HLA/peptide/TCR complex. Ultimately, this results in an increase in the expansion of T-cells capable of recognizing the parental peptide (68–71). This ap- proach was successful in optimizing the immunogenicity of the wt p53

_264–272

and p53

_149–

157

peptides. In the case of the wt p53

_264–272

peptide (LLGRNSFEV), which contains a favorable amino acid (leucine) at anchor positions 2 and 9, the exchange of tryptophan for phenylalanine at position 7 of the peptide, F270W, did not increase its binding to HLA-A2.1 molecules, but did increase its immunogenicity. Presumably, this was be- cause of enhanced stability of the HLA/peptide/TCR complex, and was evidenced by an increased affinity for the parental peptide of anti-wt p53

264–272

CTLs induced using the optimized peptide. The amino acid exchange of a favorable anchor amino acid (leucine) for an unfavorable anchor amino acid (threonine) in position 2 of the wt p53

149–157

peptide (STPPPGTRV) increased its binding affinity to HLA-A2.1 molecules and its immuno- genicity (72).

5.3. Parameters Influencing Tumor Presentation of wt p53 Peptides for T-Cell Recognition

In addition to defects in antigen processing and presentation (73), which confront any

T-cell-based immunotherapy, the ability of tumors to present CTL-defined wt p53 pep-

tides appears to be more complicated than of any other shared TAA (Fig. 2). It first

arose with the differences in the sensitivities of three SCCHN cell lines to HLA-A2-

restricted, anti-wt p53

264–272

CTLs (45,46,48). The PCI-13 SCCHN cell line, which

displays the “classic” phenotype of accumulation of mutant p53 (E286K) associated with

presentation of wt p53

_264–272

peptide, was sensitive to anti-wt p53

_264–272

CTLs. In con-

trast, SCC-9, which expresses a deletion in p53 and does not accumulate p53, was sen-

sitive to lysis by the CTLs, whereas SCC-4, which displays the classic phenotype of

accumulating mutant p53 (P151M), was not. Subsequently, Theobald et al. established

that the commonly occurring p53 R273H mutation blocked processing of the wt

p53

264–272

peptide, and that MCF7 cells, which accumulate wt p53, presumably because

of defects in pathways regulating p53, did present the peptide (46,60). Further confound-

ing the situation is the demonstration by Vierboom et al. (67) that cells expressing the

oncogenic human papillomavirus (HPV) E6 protein, which functions by enhancing the degradation of p53 leading to “loss of function,” have a wt p53

⁺

/accumulation

^–

pheno- type, yet are sensitive to anti-wt p53 CTLs. The interaction of p53 with heat shock protein, which is apparently dependent on the conformation of the p53 molecules, influences proteasomal degradation of mutant p53 in tumors cells, and represents another set of parameters that can impact on wt p53 CTL-recognition of tumors. Clearly, a major focus of future research is to better assess the ability of tumors to present wt p53 peptides for T-cell recognition.

5.4. Implication of Immunoselection of p53 Epitope-Loss Tumors Nearly 18,000 human tumors have been analyzed for genetic alterations in p53, most of which are missense mutations (22). The class I HLA haplotypes of the patients from which these tumors were obtained from, however, are essentially unknown. One, therefore, is unable to readily assess whether a relationship exists between sites/nature of p53 mutations and the host’s class I HLA haplotype. Several years ago, the results of an analysis by Wiedenfeld et al. indicated the possible increase in the incidence of p53 epitope loss in lung tumors of HLA-A2

⁺

individuals (62). An analysis of the sites of p53 missense mutations expressed in 27 SSCHN obtained from HLA-A2

⁺

patients, 8/13 occurred within or immediately flanking one of three known CTL-defined wt p53 epitopes (74). Six of the missense mutations within CTL-defined epitopes, p53

_149–157

, p53

_217–225

, and p53

_264–273

, and one was a mutation at codon 273, which is known to block processing of the p53

_264–272

peptide (60). The eighth missense mutation was in codon 226, the codon immediately flanking the wt p53

217–225

epitope, which may also function like the R273H mutation in blocking epitope processing. Mutations in the p53

217–225

epitope at codon 220 were detected in 2/

27 tumors. Codons 273 and 220 are considered p53 mutational “hot spots.” Mutation at

p53 codon 273 is the most frequently detected p53 mutation in human cancers (~12%),

whereas mutation at codon 220 ranks sixth, with a frequency of approx 1% (5). These

values are independent of tumor type and do not take into account the HLA haplotypes

of the tumors. The frequency of mutations at p53 codon 220 in the HLA-A2

⁺

SCCHN we

Fig. 2. Presentation of wild-type (wt) p53264–272 peptide for cytotoxic T-lymphocyte recognition by tumor cells is associated with several distinct p53 genotypes and properties.

analyzed is well above its frequency in all the human tumors that have been analyzed.

Obviously, more-extensive analyses of the HLA/p53 phenotypes of SCCHN and other types of cancers need to be done to determine the true significance of this observation.

Three distinct missense mutations in the p53

_149–157

epitope were detected in the study.

Two of these were nonconserved amino acid exchanges at the anchor positions of the peptide. Whether these mutations yield T-cell-defined mutant p53 epitopes is under investigation.

Overall, the skewed pattern of p53 missense mutation in the tumors of HLA-A2

⁺

patients with SCCHN (74) is highly suggestive of possible immunoselection that pro- motes the outgrowth of p53 epitope-loss tumors. It also implies that wt p53 peptides, although “self-antigens,” are surprising immunogenic. Given that mutation of p53 is considered an early event in development of SCCHN, the immunological pressure exerted over long periods by anti-wt p53 CTLs combined with the inherent genetic instability and heterogeneity of tumors could readily promote the outgrowth of p53 epitope-loss tumors in some patients.

A further implication that p53-related immunoselection in patients with cancer occurs is the inverse correlation between the frequencies of anti-wt p53

_264–272

tetramer

⁺

CD8

⁺

T-cells present in PBMCs obtained from HLA-A2

⁺

patients with SCCHN and the muta- tional site/level of p53 expressed in their tumors (74). The results obtained from the 27 patients divide these individuals into two groups, low tetramer

⁺

T-cell frequency/IVS nonresponsive vs high tetramer

⁺

T-cell frequency/IVS responsive. When this distinction was correlated to p53 immunohistochemistry and genotyping, the tumors of nonrespon- sive patients had a p53 phenotype traditionally consistent with a tumor’s ability to present the wt p53

_264–272

peptide (accumulation of mutant p53), whereas the responders had tumors expressing normal levels of wt p53 and, presumably, a low potential to present the epitope (see Fig. 2). Whereas these results are strongly supportive of immunoselection of epitope-loss tumors, they also are consistent with the possibility that the SCCHN tumors expressing wt p53 and associated with high frequencies of anti-wt p53 CTLs might also be HPV

⁺

. The expression of HPV E6 enhances the degradation of wt and mutant p53 and presentation of peptides derived from these molecules (67,75). The ability of an HPV

⁺

tumor to present the p53

_264–272

peptide for CTL recognition, therefore, need not require accumulation of p53. Obviously, the role of HPV in presentation of p53- derived epitopes required further investigation.

6. CLINICAL TRIALS OF

P

53-BASED IMMUNOTHERAPY OF PATIENTS WITH CANCER

In vitro-based studies using PBMCs obtained from normal donors and patients have

shown the utility of peptide or protein-pulsed DCs (36,47,60) or DCs transfected with

recombinant adenoviral constructs expressing p53 for induction/expansion of anti-wt

p53 CTLs and Th cells (68). As a result of these experiments, the concept of genetically

engineering high-affinity anti-p53 human T-cell effectors by transfecting PBMCs with

cDNA encoding the TCR derived from antihuman p53 murine CTLs is also being ac-

tively pursued (70). A number of p53-based vaccine clinical trails have been introduced

in Europe and the United States for patients with breast, colon, or ovarian carcinoma. The

initial findings of four of these trials have been reported. Vaccines consisting of p53

peptides admixed with chemical adjuvants or DC, recombinant viral vectors expressing

wt p53 as well as DCs transfected with adenoviral construct-expressing wt p53 were employed in these trials. The concept of replacing mutant p53 in a patient’s tumor with functional wt p53 delivered using a recombinant adenoviral/wt p53 construct preceded its use in p53-based immunotherapy. An unreported aspect of replacement adeno/p53 gene therapy trials is whether “bystander” antitumor immune responses are induced in the patients receiving this gene therapy (76). Induction of bystander anti-p53 immune responses has been shown to occur as a result of other types of cancer therapies not directly targeting p53, which also warrants further study (77,78).

Two HLA-A2-restricted wt p53 peptide-based vaccine trials have been initiated using peptide-pulsed DCs and/or peptides admixed with chemical adjuvants. The NCI p53 vaccine trial is for HLA-A2

⁺

patients with low-burden ovarian cancer (79). In this trial, one group of five patients received multiple monthly immunizations of the peptide pulsed onto autologous DCs and administered intravenously, whereas the second group of six patients received the peptide admixed with ISA-51 and GM-CSF and given subcutane- ously. Both groups of patients also received low dose interleukin 2 for 10 d, beginning with cycle 3 of the vaccination protocol. Immunological monitoring, using enzyme- linked immunosorbent and tetramer assays, showed the induction in individuals of both groups of anti-wt p53 CTL responses. This was accompanied by increased progression- free survival times. The other p53 peptide-pulsed DC trial was for HLA-A2

⁺

patients with advanced breast cancer (80). It consisted of a multiple p53 peptide-pulsed DC vaccine, which included three wt p53 epitopes mixed with three modified or “optimized” peptides, in addition to a generic pan-DR-binding protein peptide. The vaccine was administered together with interleukin 2. The immunological monitoring of PBMCs obtained from six patients receiving up to 10 immunizations was reported. The results show sporadic anti- p53 CTL responses and 1/6 patient showed a clinical response.

An advantage of using recombinant protein or viral vectors encoding p53 as immuno- gen is that it permits multiple CTL and Th epitopes to be presented, and gives the inves- tigator the option of monitoring the trial for identified HLA class I-restricted p53 epitopes or not. The initial results of the immunological monitoring of phase I/II immunization trials of patients using either a recombinant adenovirus or canarypox virus encoding wt p53 have been reported. Theobald et al. chose to focus on the responses of HLA-A2

⁺

patients treated with a recombinant adeno/p53 vaccine to the wt p53

264–272

peptide (81).

Although antiviral responses were detected, no significant CTL-responses to the epitope were detected. In contrast, entry criteria for patients with advanced colon carcinoma recruited for a clinical vaccine trial of canary pox virus encoding wt p53 was independent of their class I HLA haplotype. No deleterious autoimmune side effects were noted for monkeys treated with this p53 viral construct. The regimen was found to induce or augment humoral anti-p53 IgG responses in 3/16 patients and anti-p53 T-cell prolifera- tive responses in 4/16 (83,84). A clear distinction between the two trials is that the anti- p53 T-cell responses in the latter trial were not restricted to a defined wt p53 peptide;

instead, they were detected using mixtures of overlapping wt p53 peptides to stimulate

the T-cells. The assays were independent of the patients’ HLA haplotypes and identity

of the p53 epitopes. According to the National Cancer Institute, a third viral-based vac-

cine trial for patients with lung cancer modeled on murine studies using adenoviral/p53-

transfected dendritic cell vaccine (28) is in progress.

7. SUMMARY

Advances in molecular immunology combined with improved and more detailed immunological monitoring of patients are enhancing the development of cancer vac- cines. Compared to many of the other tumor antigens being targeted, p53 is unique in many respects. It is truly a self-antigen. It is expressed in all nucleated cells. Despite this, no evidence of deleterious autoimmune reactions has been detected in preclinical experi- mental laboratory animal studies and, more importantly, in patients with cancer partici- pating in early phase p53-based immunotherapy trials. This is because of the high level of tolerance that exists to p53 and the low levels of expression and rapid turnover of p53 in normal cells. Whether an “autoimmune response,” such as those that signal the efficacy of some melanoma vaccines, will be eventually evident in patients receiving extensive and prolonged p53-based immunotherapeutic regimens is a critical unknown. Further- more, p53 seems to have the ability to readily “dodge the immunological bullet.” The very nature of the genetic instability that is initiated by loss of function of p53 coupled with the pressure of immunoselection/editing as a result of anti-p53 immune responses rep- resent a combination of influences that promotes tumor escape and is, at the very least, challenging. Whether vaccine-induced immunoselection will occur in patients, as it does in mice bearing primary tumors, needs to be monitored. Nonetheless, p53-based vaccines appear to have a high potential for developing into a broadly applicable immunotherapy for cancer. Whereas nothing is impossible, development of p53-based immunotherapy is certainly more difficult that initially envisioned.

ACKNOWLEDGMENTS Grant support: NIH Grant PO-1 DE-12321.

REFERENCES

1. DeLeo AB, Jay G, Appella E, Dubois GC, Law LW, Old LJ. Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse. Proc Natl Acad Sci U S A 1979; 76:2420–2424.

2. Jay G, DeLeo, AB, Appella E, DuBois GC, Law LW, Khoury G, Old, LJ. A common transformation- related protein in murine sarcomas and leukemias, Cold Spring Harbor Sym Quant Biol. 1980; 44:659–664.

3. Harris CC. Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies. J Natl Cancer Inst 1996; 88:1442–1455.

4. Hollstein M, Shomer B, Greenblatt M, et al. Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation. Nucleic Acids Res 1996; 24:141–146.

5. Oliver M, Eeles R, Hollstein, M, Khan MA, Harris CC, Hainaut P. TP53 Database: new online mutation analysis and recommendations to users. Hum Mutat 2002; 19:607–614.

6. Lane D. p53 from pathway to therapy. Carcinogenesis 2004; 25:1077–1081.

7. Li M, Brooks CL, Kon N, Gu W. A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol Cell 2004;

13:879–886.

8. McCormick F. Cancer-specific viruses and the development of ONYX-015. Cancer Biol Ther 2003;

2:S157–S160.

9. Sogn JA. Tumor immunology: the glass is half full. Immunity 1998; 9:757–763.

10. Rosenberg SA. Shedding light on immunotherapy for cancer. N Engl J Med 2004; 350:1461–1463.

11. Toes RE, Ossendorp F, Offringa Melief CJM. CD4 T cells and their role in antitumor immune responses.

J Exp Med 1999; 189:753–756.

12. Hung K. Hayashi R, Lafond-Walker A, Lowenstein C, Pardoll D, Levitsky H. The central role of CD4(+) T cells in the antitumor immune response. J Exp Med 1998; 188:2357–2368.

13. Ossendorp F, Mengede E, Camps M, Filius R, Melief CJM. Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II nega- tive tumors. J Exp Med 1998; 187:693–702.

14. Rotzschke O, Falk K, Deres K, et al. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells. Nature 1990; 348:252–254.

15. Falk K, Rotzschke O, Stevanovic S, Jung G, Rammensee HG. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 1991; 351:290–296.

16. Renkvist N, Castelli C, Robbins PF, Parmiani G. A listing of human tumor antigens recognized by T cells. Cancer Immunol Immunother 2001; 50:3–15.

17. Crawford LV, Pim DC, Bulbrook RD. Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer. Int J Cancer 1982; 30:403–408.

18. Bourhis J, Lubin R, Roche B, et al. Analysis of p53 serum antibodies in patients with head and neck squamous cell carcinoma. J Natl Cancer Inst 1996; 88:1228–1233.

19. Soussi T. p53 Antibodies in the sera of patients with various types of cancer: a review. Cancer Res 2000;

60:1777–1788.

20. Houbiers JG, van der Burg SH, van de Watering LM, et al. Antibodies against p53 are associated with poor prognosis of colorectal cancer. Br J Cancer 1995; 72:637–641.

21. van der Burg SH, de Cock K, Menon AG, et al. Long lasting p53-specific T cell memory responses in the absence of anti-p53 antibodies in patients with resected primary colorectal cancer. Eur J Immunol 2001; 31:146–155.

22. Hauser U, Balz V, Carey TE, et al. Reliable detection of p53 aberrations in squamous cell carcinomas of the head and neck requires transcript analysis of the entire coding region. Head Neck 2002;

24:868–873.

23. Balz V, Scheckenbach K, Gotte K, Bockmuhl U, Petersen I, Bier H. Is the p53 inactivation frequency in squamous cell carcinomas of the head and neck underestimated? Analysis of p53 exons 2-11 and human papillomavirus 16/18 E6 transcripts in 123 unselected tumor specimens. Cancer Res 2003;

63:1188–1191.

24. Nijman,HW, Van der Burg SH. Vierboom MP, Houbiers JG, Kast WM, Melief CJ. p53, a potential target for tumor-directed T cells. Immunol Lett 1994; 40:171–178.

25. Mayordomo JI, Loftus DJ, Sakamoto H, et al. Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines. J Exp Med 1996; 183:1357–1365.

26. Hilburger Ryan M, Abrams SI. Characterization of CD8+ cytotoxic T lymphocyte/tumor cell interac- tions reflecting recognition of an endogenously expressed murine wild-type p53 determinant. Cancer Immunol Immunother 2001; 49:603–612.

27. Tuting T, DeLeo AB, Lotze MT, Storkus WJ. Genetically modified bone marrow-derived dendritic cells expressing tumor-associated viral or “self” antigens induce antitumor immunity in vivo. Eur J Immunol 1997; 27:2702–2707.

28. Nikitina EY, Chada S, Muro-Cacho C, et al. An effective immunization and cancer treatment with activated dendritic cells transduced with full-length wild-type p53. Gene Ther 2002; 9:345–352.

29. Tuting T, Gambotto A, Robbins PD, Storkus WJ, DeLeo AB. Co-delivery of T helper 1-biasing cytokine genes enhances the efficacy of gene gun immunization of mice: studies with the model tumor antigen beta-galactosidase and the BALB/c Meth A p53 tumor-specific antigen. Gene Ther 1999; 6:629–636.

30. Putzer BM, Bramson JL, Addison CL, et al. Combination therapy with interleukin-2 and wild-type p53 expressed by adenoviral vectors potentiates tumor regression in a murine model of breast cancer. Hum Gene Ther 1998; 9:707–718.

31. Theobald M, Biggs J, Hernandez J, Lustgarten J, Labadie C, Sherman LA. Tolerance to p53 by A2.1- restricted cytotoxic T lymphocytes. J Exp Med 1997; 185:833–841.

32. Hernandez J, Lee PP, Davis MM, Sherman LA. The use of HLA A2.1/p53 peptide tetramers to visualize the impact of self tolerance on the TCR repertoire. J Immunol 2000; 164:596–602.

33. Vierboom MP, Nijman HW, Offringa R, et al. Tumor eradication by wild-type p53-specific cytotoxic T lymphocytes. J Exp Med 1997; 186:695–704.

34. Zwaveling S, Vierboom MP, Ferreira Mota SC, et al. Antitumor efficacy of wild-type p53-specific CD4(+) T-helper cells. Cancer Res 2002; 62:6187–6193.

35. McCarty TM, Liu X, Sun JY, Peralta EA, Diamond DJ, Ellenhorn JD. Targeting p53 for adoptive T-cell immunotherapy. Cancer Res 1998; 58:2601–2605.

36. Hernandez J, Ko A, Sherman LA. CTLA-4 blockade enhances the CTL responses to the p53 self-tumor antigen. J Immunol 2001; 166:3908–3914.

37. Espenschied J, Lamont J, Longmate J, et al. CTLA-4 blockade enhances the therapeutic effect of an attenuated poxvirus vaccine targeting p53 in an established murine tumor model. J Immunol 2003;

170:3401–3407.

38. Daftarian P, Song GY, Ali S, et al. Two distinct pathways of immuno-modulation improve potency of p53 immunization in rejecting established tumors. Cancer Res 2004; 64:5407–5414.

39. Wojdani A, Alfred LJ. Alterations in cell-mediated immune functions induced in mouse splenic lym- phocytes by polycyclic aromatic hydrocarbons. Cancer Res 1984; 44:942–945.

40. Burchiel SW, Luster MI. Signaling by environmental polycyclic aromatic hydrocarbons in human lymphocytes. Clin Immunol 2001; 98:2–10.

41. Lutz CT, Browne G, Petzold CR. Methylcholanthrene causes increased thymocyte apoptosis. Toxicol- ogy 1998; 128:151–167.

42. Cicinnati VR Dworacki G, Albers A, et al. Impact of p53-based immunization on primary chemically induced tumors. Int J Cancer 2004; 113:961–970.

43. Liu, X, Peralta, EA, Ellenhorn JD, Diamond DJ. Targeting of human p53-overexpressing tumor cells by an HLA A*0201-restricted murine T-cell receptor expressed in Jurkat T cells. Cancer Res 2000;

60:693–701.

44. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 1991; 50:213–219.

45. Ropke M, Hald J, Guldberg P, et al. Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide. Proc Natl Acad Sci U S A 1996; 93:14,704–14,707.

46. Theobald M, Biggs J, Dittmer D, Levine AJ, Sherman LA. Targeting p53 as a general tumor antigen.

Proc Natl Acad Sci U S A 1995; 92:11,993–11,997.

47. Gnjatic S, Cai Z, Viguier M, Chouaib S, Guillet JG, Choppin J. Accumulation of the p53 protein allows recognition by human CTL of a wild-type p53 epitope presented by breast carcinomas and melanomas.

J Immunol 1998; 160:328–333.

48. Chikamatsu K, Nakano K, Storkus WJ, et al. Generation of anti-p53 cytotoxic T lymphocytes from human peripheral blood using autologous dendritic cells. Clin Cancer Res 1999; 5:1281–1288.

49. Eura M, Chikamatsu K, Katsura F, et al. A wild-type sequence p53 peptide presented by HLA-A24 induces cytotoxic T lymphocytes that recognize squamous cell carcinomas of the head and neck. Clin Cancer Res 2000; 6:979–986.

50. McArdle SE, Rees RC, Mulcahy KA, Saba J, McIntyre CA, Murray AK. Induction of human cytotoxic T lymphocytes that preferentially recognise tumour cells bearing a conformational p53 mutant. Cancer Immunol Immunother 2000; 49:417–425.

51. Barfoed AM, Petersen TR, Kirkin AF, Thor Straten P, Claesson MH, Zeuthen J. Cytotoxic T-lympho- cyte clones, established by stimulation with the HLA-A2 binding p5365-73 wild type peptide loaded on dendritic cells in vitro, specifically recognize and lyse HLA-A2 tumour cells overexpressing the p53 protein. Scand J Immunol 2000; 51:128–133.

52. Schirle M, Keilholz W, Weber B, et al. Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach. Eur J Immunol 2000; 30:2216–2225.

53. Wurtzen PA, Pedersen LO, Poulsen HS, Claesson MH. Specific killing of P53 mutated tumor cell lines by a cross-reactive human HLA-A2-restricted P53-specific CTL line. Int J Cancer 2001; 93:855–861.

54. Casares N, Lasarte JJ, de Cerio AL, et al. Immunization with a tumor-associated CTL epitope plus a tumor-related or unrelated Th1 helper peptide elicits protective CTL immunity. Eur J Immunol 2001;

31:1780–1789.

55. Wang L, Miyahara Y, Kato T, Aota T, Kuribayashi K, Shiku H. Essential roles of tumor-derived helper T cell epitopes for an effective peptide-based tumor vaccine. Cancer Immun 2003; 3:16.

56. Tilkin AF, Lubin R, Soussi T, et al. Primary proliferative T cell response to wild-type p53 protein in patients with breast cancer. Eur J Immunol 1995; 25:1765–1769.

57. Fujita H, Senju S, Yokomizo H, et al. Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms. Eur J Immunol 1998;

28:305–316.

58. Brusic V, Rudy G, Honeyman, G, Hammer J, Harrison L. Prediction of MHC class II-binding peptides using an evolutionary algorithm and artificial neural network. Bioinformatics 1998; 14: 121–130.

59. Chikamatsu K, Albers A, Stanson J, et al. P53(110-124)-specific human CD4+ T-helper cells enhance in vitro generation and antitumor function of tumor-reactive CD8+ T cells. Cancer Res 2003;

63:3675–3681.

60. Theobald M, Ruppert T, Kuckelkorn U, et al. The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope. J Exp Med 1998; 188:1017–1028.

61. Wiedenfeld E A, Fernandez-Vina M, Berzofsky JA, Carbone DP. Evidence for selection against human lung cancers bearing p53 missense mutations which occur within the HLA A*0201 peptide consensus motif. Cancer Res 1994; 54:1175–1177.

62. Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD. Cancer immunoediting: from immunosurveillance to tumor. Nat Immunol 2002; 3:991–998.

63. Khong HT, Restifo NP. Natural selection of tumor variants in the generation of “tumor escape” pheno- types. Nat Immunol 2002; 3:999–1005.

64. Nikitina EY, Clark JI, Van Beynen J, et al. Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients. Clin Cancer Res 2001; 7:127–135.

65. Hoffmann TK, Nakano K, Elder EM, et al. Generation of T cells specific for the wild-type sequence p53(264-272) peptide in cancer patients: implications for immunoselection of epitope loss variants. J Immunol 2000; 165:5938–5944.

66. Hoffmann TK, Loftus DJ, Nakano K, et al. The ability of variant peptides to reverse the nonresponsiveness of T lymphocytes to the wild-type sequence p53(264-272) epitope. J Immunol 2002; 168:1338–1347.

67. Vierboom MP, Zwaveling S, Bos GMJ, et al. High steady-state levels of p53 are not a prerequisite for tumor eradication by wild-type p53-specific cytotoxic T lymphocytes. Cancer Res 2000; 60:5508–5513.

68. Boehncke WH, Takeshita T, Pendleton CD, et al. The importance of dominant negative effects of amino acid side chain substitution in peptide-MHC molecule interactions and T cell recognition. J Immunol 1993; 150:331–341.

69. Zaremba S, Barzaga E, Zhu M, Soares N, Tsang KY, Schlom J. Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen. Cancer Res 1997; 57:4570–4577.

70. Rivoltini L, Squarcina P, Loftus DJ, et al. A superagonist variant of peptide MART1/Melan A27-35 elicits anti-melanoma CD8+ T cells with enhanced functional characteristics: implication for more effective immunotherapy. Cancer Res 1999; 59:301–306.

71. Slansky JE, Rattis, FM, Boyd LF, et al. Enhanced antigen-specific antitumor immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex. Immunity 2000; 13:529–538.

72. Petersen TR, Buus S, Brunak S, Nissen MH, Sherman LA, Claesson MH. Identification and design of p53-derived HLA-A2-binding peptides with increased CTL immunogenicity. Scand J Immunol 2001;

53:357–364.

73. Marincola FM, Jaffee EM, Hicklin DJ, Ferrone S. Escape of human solid tumors from T-cell recognition:

molecular mechanisms and functional significance. Adv Immunol 2000; 74:181–273.

74. Hoffmann TK, Donnenberg AD, Finkelstein SD, et al. Frequencies of tetramer+ T cells specific for the wild-type sequence p53(264-272) peptide in the circulation of patients with head and neck cancer.

Cancer Res 2002; 62:3521–3529.

75. McKaig RG, Baric RS, Olshan AF. Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head Neck 1998; 20:250–265.

76. Waku T, Fujiwara T, Shao J, et al. Contribution of CD95 ligand-induced neutrophil infiltration to the bystander effect in p53 gene therapy for human cancer. J Immunol 2000; 165:5884–5890.

77. Disis ML, Goodell V, Schiffman K, Knutson KL. Humoral epitope-spreading following immunization with a her-2/neu Peptide based vaccine in cancer patients. J Clin Immunol 2004; 24:571–578.

78. Chakraborty M, Abrams SI, Coleman CN, Camphausen K, Schlom J, Hodge JW. External beam radia- tion of tumors alters phenotype of tumor cells to render them susceptible to vaccine-mediated T-cell killing. Cancer Res 2004; 64:4328–4337.

79. Herrin V, Behrens RJ, Achtar M, et al. Wild type p53 peptide vaccine can generate a specific immune response in low burden ovarian adenocarcinoma. 2003; ASCO Chicago, IL, U S A Abstract 67846.

80. Svane IM, Pedersen AE, Johnsen HE, et al. Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from a phase I study. Cancer Immunol Immunother 2004; 53:633–641.

81. Kuball J, Schuler M, Antunes Ferreira E, et al. Generating p53-specific cytotoxic T lymphocytes by recombinant adenoviral vector-based vaccination in mice, but not man. Gene Ther 2002; 9:833–843.

82. Rosenwirth B, Kuhn EM, Heeney JL, et al. Safety and immunogenicity of ALVAC wild-type human p53 (vCP207) by the intravenous route in rhesus macaques. Vaccine 2001; 19:1661–1670.

83. van der Burg SH, Menon AG, Redeker A, et al. Induction of p53-specific immune responses in colorectal cancer patients receiving a recombinant ALVAC-p53 candidate vaccine. Clin Cancer Res 2002;

8:1019–1027.

84. Menon AG, Kuppen PJ, van der Burg SH, et al. Safety of intravenous administration of a canarypox virus encoding the human wild-type p53 gene in colorectal cancer patients. Cancer Gene Ther 2003;

10:509–517.