Gene-based vaccines for immunotherapy of prostate cancer

Gene Ther Mol Biol Vol 8, 465-474, 2004

Gene-based vaccines for immunotherapy of prostate cancer - lessons from the past

Review Article

Milcho Mincheff* and Serguei Zoubak

The George Washington University Medical Center __________________________________________________________________________________

*Correspondence: Milcho Mincheff, Head, Tumor Immunology Laboratory, Department of Medicine, The George Washington Medical Center, 2300 Eye Street, N.W., Ross Hall 705, Washington, DC 20037; Tel: 202 994 7765; fax: 202 994 0465; e-mail: mcamsm@gwumc.edu

Key words: PSMA, Gene-based vaccine, immunodominance, CTLA-4

Abbreviations: activation-inducible TNF receptor, (AITR); antigen-presenting cells, (APCs); cytotoxic T lymphocyte antigen 4, (CTLA-4); delayed type hypersensitivity, (DTH); glucocorticoid-induced tumor necrosis factor receptor, (GITR); GITR ligand, (GITR-L); prostate acidic phosphatase, (PAP); prostate-specific membrane antigen, (PSMA); "secreted" prostate-specific membrane antigen, (sPMSA); T cell receptor, (TCR); truncated prostate-specific membrane antigen, (tPSMA); tumor-infiltrating lymphocytes, (TILs); tyrosinase-related protein-1, (TRP-1)

Supported in part by the American Foundation for Biolological Research and by the Bulgarian Foundation for Biomedical Research.

Supported in part by grant N00014-00-1-0787 from the Office of Naval Research.

Supported in part by award No DAMD17-02-1-0239. The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014 is the awarding and administering acquisition office.

The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. For purpose of this article, information includes news releases, articles, manuscripts, brochures, advertisements, still and motion pictures, speeches, trade association proceedings etc.

Received: 15 October 2004; Accepted: 15 November 2004; electronically published: November 2004

Summary

Gene-based vaccination in its current mode of application is effective in breaking tolerance to a self- or tumor-associated antigen, but the response is narrow and restricted to few of the potential epitopes due to immunodominance. In cancer, immunodominance carries the risk of inefficient immune surveillance due to loss of MHC alleles or point mutations in the recognized sequences. We have found that a T cell response to sub-dominant epitopes can be primed with transfected dendritic cells in which the newly expressed antigen is purposefully targeted for proteasomal degradation. Beginning in May 1998, we performed a phase I/II clinical trial for immunotherapy of prostate cancer that targeted the prostate-specific membrane antigen (PSMA). The primary objective of the study was to determine the safety of the described vaccines after repeated intradermal injections , since using PSMA as a target could be seriously offset by the development of autoimmunity . So far, six years since the study has begun, no patient has experienced any short- or long-term side effects, including anti-DNA antibody. Twenty-nine patients from this random population were treated solely by immunotherapy. Eighteen of them had biochemical recurrence following radical prostatectomy and eleven responded to the therapy with a PSA drop exceeding 50% of pre-therapy value. Patients with advanced disease and distant metastases were not influenced by the immunotherapy despite the fact that they all showed signs of T cell immunity towards PSMA. We found, however, that the post-vaccination T cell response was directed against only two of the potential 4 PSMA epitopes that had high affinity for binding. At least in vitro, priming with one of our vaccines led to a poly-epitope response. Unfortunately, even in such instances, consequent exposure to poly-epitope expressing dendritic cells during re-immunization led to selection of an immunodominant clone. To alleviate immunodominance and decrease tumor evasion due to loss of antigenic determinants, a poly-epitope T cell response would need to be maintained. Ensuring such a cytotoxic T cell response, therefore, would require either construction of separate epitope encoding vectors for boosting, an approach with limited therapeutic application, or identifying conditions during boosting that would restrict immunodominance. CD4 T cell depletion, GITR-L signaling or CTLA-4 all show promise in achieving this goal.

I. Introduction

A. Tumor antigen recognition

Evidence that the immune system recognizes tumor antigens is supported by the existence of tumor infiltrating lymphocytes but, since cancer cells fail to establish and support an effective immune milieu, tumors often prevail and survive. Worsening the problem is the fact that recognition of cancer antigens on tumor cells seems to evoke a tolerant state by induction of anergy in antigen-reactive T cells. In the past few years it has become increasingly evident that induction of tissue-specific autoimmunity can lead to tumor destruction. Initially Coulie and colleagues discovered that the target for a melanoma-specific CD8+ T cell clone grown from a melanoma patient was wild-type tyrosinase, a melanosomal enzyme selectively expressed in melanocytes. Subsequently, a number of investigators found that their melanoma-specific CD8+ T cells indeed recognized melanocyte-specific antigens rather than melanoma-specific antigens . Most of these antigens appear to be normal melanosomal proteins, and a number of them, including tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and glycoprotein 100 (gp100), are involved in melanin biosynthesis. Other melanosomal proteins such as MART1/Melan A have no known function but are nonetheless melanocyte-specific tissue differentiation antigens. As time progressed, evidence accumulated that the dominant targets of immune responses against tumors were tissue-specific or differentiation antigens. In contrast, recognition of peptides derived from unique tumor-specific mutations represented infrequent reactivities . Similar analysis of the specificities of tumor-infiltrating lymphocytes (TILs) in prostate cancer biopsies also revealed responses against tissue-specific antigens . Possible targets included the prostate-specific membrane antigen (PSMA) , the prostate-specific antigen (PSA) and prostate acidic phosphatase (PAP) .

The findings that the existing anti-tumor immune responses are predominantly targeting tissue-specific antigens open a new venue for cancer immunotherapy. In practical terms, however, harnessing autoimmunity for cancer therapy presents several problems:

i. Identification of a target antigen or a combination thereof that will confer protection. In a recent study performed in mice, anti-TRP-1 but not anti-TRP-2 or anti-gp-100 specific T cells induced vitiligo and anti-tumor immunity . This may have been true for the particular mouse strain in that study but it does show that targeting a single antigen based on analysis of T cell responses from tumor bearing patients or animals may be misleading. It also shows the shortcomings of using a single peptide derived from a tissue specific antigen for raising sustained autoimmunity sufficient to eradicate tumor. A cancer vaccine against a multitude of peptides against a tissue-specific antigen will definitely offer some advantages. This approach is strengthened by the discovery that, as is the case with different animal strains, particular autoantigen in different people may manifest different ability to break tolerance and induce autoimmunity .

ii. The prostate-specific membrane antigen (PSMA) is a type II integral membrane glycoprotein with a molecular weight of ~100 kDa . It has a folate hydrolase, as well as neuropeptidase activity. PSMA is highly expressed in benign prostate secretory-acinar epithelium, prostatic intraepithelial neoplasia and prostate adenocarcinoma . There is good evidence that PSMA expression is increased in high Gleason score tumors and in hormone-refractory tumor cells , which makes it an excellent target for immunotherapy. More recently, weak expression has been described in several normal tissues such as a subset of proximal renal tubules, duodenal and colonic mucosa. A shorter, alternatively spliced cytosolic form of PSMA, named PSM', is the predominant form expressed in benign prostate epithelium . Recently PSMA expression has been detected in tumor neovasculature , as well as in other healthy tissues both in human and in mice .

II. Clinical trial

Breaking of tolerance to tissue-specific antigens requires presentation of antigen to T cells by specialized, antigen presenting cells: the dendritic cells. This can be performed by a procedure known as naked DNA immunization. We have already performed a clinical trial on immunotherapy of prostate cancer using this approach and we have demonstrated its safety. Beginning in May 1998, we performed in Sofia, Bulgaria, a phase I/II clinical trial for immunotherapy of prostate cancer that targeted the prostate-specific membrane antigen (PSMA). The primary objective of the study was to determine the safety of the described vaccines after repeated intradermal injections , since using PSMA as a target could be seriously offset by the development of autoimmunity .

Sixty-five patients were accessed into the study and were repeatedly immunized. Fifty-nine of them were in the study for a period between 2.5 and 3 years. No patient experienced short or long-term side effects including the development of anti-DNA antibody . We also found that repeated local intradermal injection of rHuGM-CSF (Sargramostim) was a safe procedure and was well tolerated. Heterologous immunization regimen that consisted of two initial intradermal immunizations at 3-week intervals with a cocktail consisting of 200 m g plasmid DNA and 9 IU/m² b.s.a., followed by a recombinant adenoviral boost (5x108 PFUs of Ad5PSMA) led to uniform immunization as judged by the development of delayed type hypersensitivity reaction (DTH) to PSMA. DTH was measured 24 and 48 hours following intradermal injection of the plasmid immunization cocktail and was compared to reactions developing after intradermal injection at two separate sites of plasmid cocktail that contained the empty plasmid backbone instead, or of GM-CSF only. The patients were heterogeneous with regard to local advancement of disease, presence of distant metastases, or hormone treatment and refractoriness, which does not permit unequivocal interpretation of the results. Nevertheless, several responders to the immunotherapy could be identified. Twenty-nine patients from this random population were treated solely by immunotherapy (Table 1). Eighteen of them had biochemical recurrence following radical prostatectomy and eleven responded to the therapy with a PSA drop exceeding 50% of pre-therapy value (Table 1). In contrast, only one of the 11 patients with advanced metastatic disease was influenced by IT with the PSA remaining flat at 10-13 ng/ml and a decrease in bone pains. The remaining 10 patients experienced disease progression despite immunizations.

The PSA curve of a typical responder to immunotherapy is shown on Figure 3. The patient was prostatectomized in January, 1996, Gleason score 5, negative margins. Biochemical recurrence was first detected in February, 1999. Immunotherapy, consisting of two plasmid immunizations followed by a recombinant adenoviral boost was initiated in March, 1999. Regular boosts were performed at 3-4 month intervals alternating between the plasmid DNA and the adenoviral vector.

Patients with advanced disease and distant metastases were not influenced by the immunotherapy despite the fact that they all showed signs of T cell immunity towards PSMA. Anti-PSMA immunity was assayed by the presence of PSMA-reactive, g IFN-producing T cells in their peripheral blood (Figure 2).

The escape of tumor cells from immune surveillance despite presence of anti-PSMA T cell immunity in those patients could be mediated through a number of mechanisms:

III. Tumor evasion

A. Tumor evasion

Especially in advanced disease with a big tumor load, can be mediated through multiple pathways . Tumor cells secrete lymphokines such as TGF-b and VEGF which suppress dendritic cell and T cell function . Fas-L and other apoptosis inducing agents are expressed on tumor cells and induce programmed cell death in infiltrating lymphocytes .

Figure 1. Serum PSA of a patient following radical prostatectomy (1996), biochemical recurrence (January, 1999) and immunotherapy (March 1999 — August 2000). SDs represent three separate determination of PSA in serum derived from three venipunctures on three consecutive days.

(P-thPSMA plasmid; Ad5 — Ad5PSMA)

Figure 2. . g -interferon-positive CD8+T cells following 6-hour stimulation of peripheral blood from HLA-A2+ cancer patients with HLA-A2-specific, PSMA-derived peptide (MMNDQLMFL). Cells were stained using the FastImmune CD8 intracellular cytokine detection kit. Diamonds — prior to immunization (control), squares — post immunization. Data are from five different experiments involving five patients.

B. Immunodominance

The response of the host immune system to onlya few of the many possible epitopes in an antigen, additionally exacerbates the problem . We find gene-based vaccination in its current mode of application effective in breaking tolerance to a self-antigen, but the boosting narrows and restricts the response to few of the potential epitopes . For example, the post-vaccination T cell response of some of the HLA A2 patients from the clinical trial performed by us was directed against only two of the potential 4 PSMA peptide motifs that had high affinity for binding (Figure 3).

Table 1. Results from a clinical trial on DNA immunization for immunotherapy of prostate cancer

Outcome	Immunotherapy only
Outcome	Post-Prostatectomy	Distant metastases
Disease Progression	7	10
*Improvement (Responders to Therapy)**	11	1
Total Number of Patients	18	11

Responders* — Decrease of PSA exceeding 50% of initial value, decrease in bone pains (where applicable).

Figure 3. g -IFN-positive CD3+ T cells following 6-hour stimulation of peripheral blood of HLA-A2+ prostate cancer patients. The following PSMA peptides were identified by BIMAS to bind with high affinity to HLA A2, synthesized and tested in an in vitro assay: MMNDQLMFL (PSMA₆₆₃), ALFDIESKV (PSMA₇₁₁), LMFLERAFI (PSMA₆₆₈) and GIUDALFDI (PSMA₇₀₇). Legend: Stimulation was performed by a) squares — PSMA₆₆₃, b) diamonds — PSMA₇₁₁, c) triangles — PSMA₆₆₈. Results with PSMA₇₀₇ are not shown but are comparable to pre-immunization values (see Figure 1). Data are from three separate experiments with blood from one patient. Cells were stained using the FastImmune CD8 intracellular cytokine detection kit.

Immunodominance ensures the tight specificity of the immune reaction and prevents untoward autoimmunity . However, it carries the risk of inefficient immune surveillance in cases such as cancer in which mutations of the epitope or downregulation of MHC alleles occur . Malignant transformation and tumor progression are frequently associated with loss of HLA class I antigens.For example, a recent review of the literature reported that ~15%and ~55% of surgically removed primary and metastatic melanomalesions, respectively, were not stained in immunohistochemicalreactions by monoclonal antibodies to monomorphic determinants of HLA class I antigens.Loss or reduced HLA class I antigen expression enables tumorcells to evade the host's immune response and downregulationof HLA class I antigens in metastases from patients with malignant melanoma is associated with poorer prognosis .

Numerous factors combine to establish an immunodominance hierarchy . They include among others:

Lack of T cells that are responsive to a sub-dominant epitope

Low affinity of the epitope for binding to MHC

Ineffective generation and transport of sub-dominant epitopes by APCs

Intrinsic control of CD8 T cells to respond to sub-dominant epitopes

Extrinsic regulatory networks (T regulatory cells) .

We concentrated our efforts on studying the effects of the extrinsic regulatory networks, particularly CTLA-4 and GITR-L signaling and T regulatory cell influence on the establishment of immunodominance during priming and boosting with a gene-based vaccine.

1. Immunodominance and CTLA-4 inhibition

A homologue of CD28, CTLA-4 also binds to the B-7 family members but inhibits T cell activation . Mice lacking CTLA-4 reveal a striking phenotype of polyclonal T cell activation and tissue infiltration which results in death by 3-4 weeks of age, indicating a powerful regulatory role for CTLA-4 . Weak signals through the T cell receptor (TCR) are prompt to inhibition and, at least in vitro, no CTL stimulation to subdominant epitopes occurs if CTLA-4 is not inhibited . Alternatively, CTLA-4 may act as a non-signaling "decoy" receptor reducing the available ligand for CD28 costimulation . No matter what the mechanism is, inhibition of CTLA-4 may alleviate immunodominance and thus improve the efficacy of anti-tumor vaccines.

2. CD4+CD25+ T cell depletion and cancer immunodominance

Enhanced priming to sub-dominant epitopes by CTLA-4 inhibition is at least partially mediated through the inhibition of CD4+CD25+ T cell function . These CD4+ T cells are a minor subpopulation (10%) that co-expresses the IL-2 receptor a -chain (CD25) and they can prevent both the induction and effector function of autoreactive T cells . Additionally, they suppress polyclonal T cell activation in vitro by inhibiting IL-2 production . Based on these data, we speculate that immunodominance that develops after re-immunization may be reduced by CD4+CD25+ T cell depletion prior to boosting.

3. CD4+CD25+ T cell regulation

Very little is known of the physiologic regulation of CD4+CD25+ T cells in vivo . Recent reports suggest that glucocorticoid-induced tumor necrosis factor receptor (GITR), also known as TNFRSF18 — a member of the TNF-nerve growth factor receptor gene superfamily — is predominantly expressed on CD4+CD25+ T cells and stimulation of GITR abrogates CD4+CD25+ T cell-mediated suppression . The gene encoding the natural ligand of murine GITR has been cloned and characterized. The putative GITR ligand (GITR-L) is composed of 173 amino acids with features resembling those of type II membrane proteins and is 51% identical to the human activation-inducible TNF receptor (AITR) ligand, TL6. Expression of the GITR-L is restricted to immature and mature splenic dendritic cells. GITR-L binds GITR expressed on HEK 293 cells and triggers NF-k B activation. Functional studies reveal that soluble CD8-GITR-L prevents CD4+CD25+ regulatory T-cell-mediated suppressive activities . Stimulation through this receptor has been shown to break immunologic tolerance , i.e. it acts similarly to CD4+CD25+ T cell depletion .

IV. Immunodominance during priming and boosting

A. "Truncated" vs. secreted vaccines (tVacs vs. sVacs). Dendritic cells transfected with truncated vaccines primes to both dominant and subdominant epitopes of the target antigen

To enhance priming to sub-dominant epitopes, we designed a vaccine (hPSMAT; truncated (tPSMA); tVac) whose product encoded for only the extracellular domain of PSMA. The product, expressed following transfection with this vector, is retained in the cytosol and is degraded by the proteasomes. For the "secreted" (sPMSA) vaccine, a signal peptide sequence was added to the expression cassette. The expressed protein following transfection with such vaccines is glycosylated and directed to the secretory pathway. Dendritic cells transfected in vitro with tVacs primed T cells to both dominant and subdominant epitopes . Subsequent boosting with antigen-presenting cells (APCs) that expressed both dominant and sub-dominant epitopes, however, narrowed the immune response to the dominant ones . Research from other groups has gained similar results . In all these instances, boosting with polyepitope encoding constructs resulted in failure to expand polyepitope CTLs. A likely explanation is that competition between T cells for antigen on individual APC leads to obscuring of responses to sub-dominant epitopes when both the dominant and subdominant epitopes are present on the same APC .

New vaccines (separate DNA vaccines encoding isolated dominant and subdominant epitopes might maximize epitope dispersal among APCs thus inducing broad immunity against numerous epitopes, dominant and subdominant. Due to the HLA polymorphism of the human population, however, construction of such separate vaccines is mainly of academic interest and will have limited therapeutic application. Different approaches for the maintenance of a poly-epitope CTL response following repeated boosting, therefore, are necessary. Some of those are listed below:

B. CTLA-4 inhibition and immunodominance. Addition of anti-CTLA-4 antibodies during priming alleviates immunodominance

We find that in vitro priming to subdominant responses is enhanced by CTLA-4 inhibition . Will similar CTLA-4 inhibition during in vivo re-immunization (boosting) preserve a poly-epitope CTL response ? What will be the cytokine production profile of the sub-dominant T cell clones? T1/T2 polarization (g -IFN vs. IL-4 secretion) has been shown to depend on the amount of the antigen and on the affinity of the peptide for MHC , with weaker signals promoting IL-4 secretion. CTLA-4 inhibition may promote T cell activation at instances of weak T cell receptor engagement . Will there be a difference in the cytokine profile of the sub-dominant clones raised by either minigene re-immunization or CTLA-4 inhibition? Will sub-dominant clones be cytotoxic to tumor cells?

C. CD4+CD25+ T cell prior to priming reduces immunodominance

Results from our laboratory show that the enhanced priming to sub-dominant epitopes by CTLA-4 inhibition is at least partially mediated through the inhibition of CD4+CD25+ T cell function . For obvious reasons, CD4+CD25+ T cell depletion prior to in vivo boosting may lead to serious side effects . Could alleviation of immunodominance be achieved by means other than CD4+CD25+ T cell depletion?

D. In some cases, GITR-signaling during priming reduces immunodominance

We find that while CD4+CD25+ T cell depletion prior to in vitro priming with sVacDCs alleviates immunodominance, co-transfection of dendritic cells with GITR-L does so in some but not all cases. Could immunodominance in vivo be restricted by GITR signaling? Could this be achieved by the co-administration of anti-GITR antibodies or by enhanced GITR-L co-expression during re-immunization? Preliminary results from our laboratory suggest that in some cases in vitro, co-transfection of dendritic cells with GITR-L alleviate immunodominance.

V. Conclusion

Immunotherapy is a safe, non-invasive, relatively inexpensive procedure that can avoid side effects that often result from surgical, cryosurgical or radiation therapy. Gene based vaccination is effective in breaking tolerance to tumor-associated antigens, but the response is directed towards few of the potential epitopes due to immunodominance. Tumor cells that have lost the immunodominant epitope due to mutations are no-longer recognized and evade immune surveillance. Designing a protocol for immunotherapy, therefore, necessitates stimulation of an immune response directed against a multitude of epitopes. Increasing the number of epitopes available for presentation to T cells is the initial step. It mandates increased degradation of the antigen following DNA immunization and we have already initiated experimentation directed at this . A logical continuation to the current work involves manipulation of the intimate mechanisms controlling the processes of stimulation and/or suppression of T cells recognizing the "sub-dominant" epitopes.

References

Bacich DJ, Pinto JT, Tong WP and Heston WD (2001) Cloning, expression, genomic localization, and enzymatic activities of the mouse homolog of prostate-specific membrane antigen/NAALADase/folate hydrolase. Mamm Genome 12, 117-123.

Bakker AB, Schreurs MW, de Boer AJ, Kawakami Y, Rosenberg SA, Adema GJ and Figdor CG (1994) Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J Exp Med 179, 1005-1009.

Baldwin KK, Trenchak BP, Altman JD and Davis MM (1999) Negative selection of T cells occurs throughout thymic development. J Immunol 163, 689-698.

Barouch DH, Craiu A, Santra S, Egan MA, Schmitz JE, Kuroda MJ, Fu TM, Nam JH, Wyatt LS, Lifton MA, Krivulka GR, Nickerson CE, Lord CI, Moss B, Lewis MG, Hirsch VM, Shiver JW and Letvin NL (2001) Elicitation of high-frequency cytotoxic T-lymphocyte responses against both dominant and subdominant simian-human immunodeficiency virus epitopes by DNA vaccination of rhesus monkeys. J Virol 75, 2462-2467.

Beck C, Schreiber H and Rowley D (2001) Role of TGF-b in immune-evasion of cancer. Microsc Res Tech 52, 387-395.

Belz GT, Stevenson PG and Doherty PC (2000) Contemporary analysis of MHC-related immunodominance hierarchies in the CD8+ T cell response to influenza A viruses. J Immunol 165, 2404-2409.

Boise LH and Thompson CB (1996) Hierarchical control of lymphocyte survival. Science 274, 67-68.

Carbone JE and Ohm DP (2002) Immune dysfunction in cancer patients. Oncology (Huntingt) 16, 11-18.

Cefai D, Favre L, Wattendorf E, Marti A, Jaggi R and Gimmi CD (2001) Role of Fas ligand expression in promoting escape from immune rejection in a spontaneous tumor model. Int J Cancer 91, 529-537.

Chang SS, Reuter VE, Heston WD, Bander NH, Grauer LS and Gaudin PB (1999) Five different anti-prostate-specific membrane antigen (PSMA) antibodies confirm PSMA expression in tumor-associated neovasculature. Cancer Res 59, 3192-3198.

Chen W, Anton LC, Bennink JR and Yewdell JW (2000) Dissecting the multifactorial causes of immunodominance in class I-restricted T cell responses to viruses. Immunity 12, 83-93.

Cordon-Cardo C, Fuks Z, Drobnjak M, Moreno C, Eisenbach L and Feldman M (1991) Expression of HLA-A,B,C antigens on primary and metastatic tumor cell populations of human carcinomas. Cancer Res 51, 6372-6380.

Coulie PG, Brichard V, Van Pel A, Wolfel T, Schneider J, Traversari C, Mattei S, De Plaen E, Lurquin C and Szikora JP (1994) A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 180, 35-42.

Coulie PG, 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 AL, Skipper J, Chen Y, Henderson RA, Darrow TL, Shabanowitz J, Engelhard VH, Hunt DF and Slingluff CL, Jr (1994) Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines. Science 264, 716-719.

de la Salle H, Zimmer J, Fricker D, Angenieux C, Cazenave JP, Okubo M, Maeda H, Plebani A, Tongio MM, Dormoy A and Hanau D (1999) HLA class I deficiencies due to mutations in subunit 1 of the peptide transporter TAP1. J Clin Invest 103, R9-R13.

Doyle AM, Mullen AC, Villarino AV, Hutchins AS, High FA, Lee HW, Thompson CB and Reiner SL (2001) Induction of cytotoxic T lymphocyte antigen 4 (CTLA-4) restricts clonal expansion of helper T cells. J Exp Med 194, 893-902.

Dunn GP, Bruce AT, Ikeda H, Old LJ and Schreiber RD (2002) Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol 3, 991-998.

Eder JP, Kantoff PW, Roper K, Xu GX, Bubley GJ, Boyden J, Gritz L, Mazzara G, Oh WK, Arlen P, Tsang KY, Panicali D, Schlom J and Kufe DW (2000) A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer. Clin Cancer Res 6, 1632-1638.

Ferrone S and Marincola FM (1995) Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance. Immunol Today 16, 487-494.

Firat H, Garcia-Pons F, Tourdot S, Pascolo S, Scardino A, Garcia Z, Michel ML, Jack RW, Jung G, Kosmatopoulos K, Mateo L, Suhrbier A, Lemonnier FA and Langlade-Demoyen P (1999) H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies. Eur J Immunol 29, 3112-3121.

Fong L, Brockstedt D, Benike C, Breen JK, Strang G, Ruegg CL and Engleman EG (2001) Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy. J Immunol 167, 7150-7156.

Garrido F and Algarra I (2001) MHC antigens and tumor escape from immune surveillance. Adv Cancer Res 83, 117-158.

Gilboa E (1999a) How tumors escape immune destruction and what we can do about it. Cancer Immunol Immunother 48, 382-385.

Gilboa E (1999b) The makings of a tumor rejection antigen. Immunity 11, 263-270.

Gilboa E (2001) The risk of autoimmunity associated with tumor immunotherapy. Nat Immunol 2, 789-792.

Grauer LS, Lawler KD, Marignac JL, Kumar A, Goel AS and Wolfert RL (1998) Identification, purification, and subcellular localization of prostate-specific membrane antigen PSM' protein in the LNCaP prostatic carcinoma cell line. Cancer Res 58, 4787-4789.

Greene JL, Leytze GM, Emswiler J, Peach R, Bajorath J, Cosand W and Linsley PS (1996) Covalent dimerization of CD28/CTLA-4 and oligomerization of CD80/CD86 regulate T cell costimulatory interactions. J Biol Chem 271, 26762-26771.

Guntermann C and Alexander DR (2002) CTLA-4 suppresses proximal TCR signaling in resting human CD4(+) T cells by inhibiting ZAP-70 Tyr(319) phosphorylation: a potential role for tyrosine phosphatases. J Immunol 168, 4420-4429.

Hammer J, Sturniolo T and Sinigaglia F (1997) HLA class II peptide binding specificity and autoimmunity. Adv Immunol 66, 67-100.

Hicklin DJ, Wang Z, Arienti F, Rivoltini L, Parmiani G and Ferrone S (1998) b 2-Microglobulin mutations, HLA class I antigen loss, and tumor progression in melanoma. J Clin Invest 101, 2720-2729.

Hiraki A, Kaneshige T, Kiura K, Ueoka H, Yamane H, Tanaka M and Harada M (1999) Loss of HLA haplotype in lung cancer cell lines: implications for immunosurveillance of altered HLA class I/II phenotypes in lung cancer. Clin Cancer Res 5, 933-936.

Hislop AD, Annels NE, Gudgeon NH, Leese AM and Rickinson AB (2002) Epitope-specific evolution of human CD8(+) T cell responses from primary to persistent phases of Epstein-Barr virus infection. J Exp Med 195, 893-905.

Israeli RS, Powell CT, Fair WR and Heston WD (1993) Molecular cloning of a complementary DNA encoding a prostate-specific membrane antigen. Cancer Res 53, 227-230.

Kawakami Y, Eliyahu S, Delgado CH, Robbins PF, Sakaguchi K, Appella E, Yannelli JR, Adema GJ, Miki T and Rosenberg SA (1994) Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc Natl Acad Sci U S A 91, 6458-6462.

Kedl RM, Kappler JW and Marrack P (2003) Epitope dominance, competition and T cell affinity maturation. Curr Opin Immunol 15, 120-127.

Kersh EN, Shaw AS and Allen PM (1998) Fidelity of T cell activation through multistep T cell receptor zeta phosphorylation. Science 281, 572-575.

Kim JD, Choi BK, Bae JS, Lee UH, Han IS, Lee HW, Youn BS, Vinay DS and Kwon BS (2003) Cloning and characterization of GITR ligand. Genes Immun 4, 564-569.

Kim JJ, Trivedi NN, Wilson DM, Mahalingam S, Morrison L, Tsai A, Chattergoon MA, Dang K, Patel M, Ahn L, Boyer JD, Chalian AA, Schoemaker H, Kieber-Emmons T, Agadjanyan MA, Weiner DB and Shoemaker H (1998) Molecular and immunological analysis of genetic prostate specific antigen (PSA) vaccine. Oncogene 17, 3125-3135.

Koyama S, Koike N and Adachi S (2002) Expression of TNF-related apoptosis-inducing ligand (TRAIL) and its receptors in gastric carcinoma and tumor-infiltrating lymphocytes: a possible mechanism of immune evasion of the tumor. J Cancer Res Clin Oncol 128, 73-79.

Kumar V, Bhardwaj V, Soares L, Alexander J, Sette A and Sercarz E (1995) Major histocompatibility complex binding affinity of an antigenic determinant is crucial for the differential secretion of interleukin 4/5 or interferon g by T cells. Proc Natl Acad Sci U S A 92, 9510-9514.

Kwon B, Kim BS, Cho HR, Park JE and Kwon BS (2003) Involvement of tumor necrosis factor receptor superfamily(TNFRSF) members in the pathogenesis of inflammatory diseases. Exp Mol Med 35, 8-16.

Levings MK, Sangregorio R and Roncarolo MG (2001) Human cd25(+)cd4(+) t regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J Exp Med 193, 1295-1302.

Loirat D, Lemonnier FA and Michel ML (2000) Multiepitopic HLA-A*0201-restricted immune response against hepatitis B surface antigen after DNA-based immunization. J Immunol 165, 4748-4755.

Ma H and Kapp JA (2001) Peptide affinity for MHC influences the phenotype of CD8(+) T cells primed in vivo. Cell Immunol 214, 89-96.

Manzotti CN, Tipping H, Perry LC, Mead KI, Blair PJ, Zheng Y and Sansom DM (2002) Inhibition of human T cell proliferation by CTLA-4 utilizes CD80 and requires CD25+ regulatory T cells. Eur J Immunol 32, 2888-2896.

Masteller EL, Chuang E, Mullen AC, Reiner SL and Thompson CB (2000) Structural analysis of CTLA-4 function in vivo. J Immunol 164, 5319-5327.

Mateo L, Gardner J, Chen Q, Schmidt C, Down M, Elliott SL, Pye SJ, Firat H, Lemonnier FA, Cebon J and Suhrbier A (1999) An HLA-A2 polyepitope vaccine for melanoma immunotherapy. J Immunol 163, 4058-4063.

McHugh RS, Whitters MJ, Piccirillo CA, Young DA, Shevach EM, Collins M and Byrne MC (2002) CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16, 311-323.

McNeel DG and Disis ML (2000) Tumor vaccines for the management of prostate cancer. Arch Immunol Ther Exp (Warsz) 48, 85-93.

Mincheff M, Altankova I, Zoubak S, Tchakarov S, Botev C, Petrov S, Krusteva E, Kurteva G, Kurtev P, Dimitrov V, Ilieva M, Georgiev G, Lissitchkov T, Chernozemski I and Meryman HT (2001) In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer--changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-g lymphokine profile. Crit Rev Oncol Hematol 39, 125-132.

Mincheff M, Tchakarov S, Zoubak S, Loukinov D, Botev C, Altankova I, Georgiev G, Petrov S and Meryman HT (2000a) Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer: a phase I/II clinical trial. Eur Urol 38, 208-217.

Mincheff M, Zoubak S, Altankova I, Tchakarov S, Makogonenko Y, Botev C, Ignatova I, Dimitrov R, Madarzhieva K, Hammett M, Pomakov Y, Meryman H and Lissitchkov T (2003) Human dendritic cells genetically engineered to express cytosolically retained fragment of prostate-specific membrane antigen prime cytotoxic T-cell responses to multiple epitopes. Cancer Gene Ther 10, 907-917.

Mincheff M, Zoubak S, Altankova I, Tchakarov S, Pogribnyy P, Makogonenko Y, Botev C and Meryman HT (2004) Depletion of CD25+ cells from human T-cell enriched fraction eliminates immunodominance during priming and boosting with genetically modified dendritic cells. Cancer Gene Ther, (accepted).

Mincheff M, Zoubak S and Meryman HT (2000b) Use of in Vitro and in Vivo Genetically Manipulated Cells for Immunotherapy of Cancer. In: Th Smit Sibinga C (ed). Proceedings of the 25^th International Symposium of Blood Transfusion, Groningen, The Netherlands, 1999. Kluwer Academic Publishers: Dordrecht, Boston and London, pp. 11-19.

Mo AX, van Lelyveld SF, Craiu A and Rock KL (2000) Sequences that flank subdominant and cryptic epitopes influence the proteolytic generation of MHC class I-presented peptides. J Immunol 164, 4003-4010.

Murphy G, Tjoa B, Ragde H, Kenny G and Boynton A (1996) Phase I clinical trial: T-cell therapy for prostate cancer using autologous dendritic cells pulsed with HLA-A0201-specific peptides from prostate-specific membrane antigen. Prostate 29, 371-380.

Murphy GP, Elgamal AA, Su SL, Bostwick DG and Holmes EH (1998) Current evaluation of the tissue localization and diagnostic utility of prostate specific membrane antigen. Cancer 83, 2259-2269.

Ng CS, Novick AC, Tannenbaum CS, Bukowski RM and Finke JH (2002) Mechanisms of immune evasion by renal cell carcinoma: tumor-induced T-lymphocyte apoptosis and NFkB suppression. Urology 59, 9-14.

Noel PJ, Boise LH and Thompson CB (1996) Regulation of T cell activation by CD28 and CTLA4. Adv Exp Med Biol 406, 209-217.

Ohm JE, Shurin MR, Esche C, Lotze MT, Carbone DP and Gabrilovich DI (1999) Effect of vascular endothelial growth factor and FLT3 ligand on dendritic cell generation in vivo. J Immunol 163, 3260-3268.

Overwijk WW, Lee DS, Surman DR, Irvine KR, Touloukian CE, Chan CC, Carroll MW, Moss B, Rosenberg SA and Restifo NP (1999) Vaccination with a recombinant vaccinia virus encoding a "self" antigen induces autoimmune vitiligo and tumor cell destruction in mice: requirement for CD4(+) T lymphocytes. Proc Natl Acad Sci U S A 96, 2982-2987.

Overwijk WW and Restifo NP (2000) Autoimmunity and the immunotherapy of cancer: targeting the "self" to destroy the "other". Crit Rev Immunol 20, 433-450.

Palmowski MJ, Choi EM, Hermans IF, Gilbert SC, Chen JL, Gileadi U, Salio M, Van Pel A, Man S, Bonin E, Liljestrom P, Dunbar PR and Cerundolo V (2002) Competition between CTL narrows the immune response induced by prime-boost vaccination protocols. J Immunol 168, 4391-4398.

Pasche B (2001) Role of transforming growth factor b in cancer. J Cell Physiol 186, 153-168.

Piccirillo CA and Shevach EM (2001) Cutting edge: control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells. J Immunol 167, 1137-1140.

Rabinowitz JD, Beeson C, Wulfing C, Tate K, Allen PM, Davis MM and McConnell HM (1996) Altered T cell receptor ligands trigger a subset of early T cell signals. Immunity 5, 125-135.

Renneberg H, Friedetzky A, Konrad L, Kurek R, Weingartner K, Wennemuth G, Tunn UW and Aumuller G (1999) Prostate specific membrane antigen (PSM) is expressed in various human tissues: implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread. Urol Res 27, 23-27.

Rivoltini L, Barracchini KC, Viggiano V, Kawakami Y, Smith A, Mixon A, Restifo NP, Topalian SL, Simonis TB, Rosenberg SA and et al (1995) Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes. Cancer Res 55, 3149-3157.

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

Rodriguez F, Harkins S, Slifka MK and Whitton JL (2002) Immunodominance in virus-induced CD8(+) T-cell responses is dramatically modified by DNA immunization and is regulated by g interferon. J Virol 76, 4251-4259.

Sakaguchi S (2003) Regulatory T cells: mediating compromises between host and parasite. Nat Immunol 4, 10-11.

Sakaguchi S, Sakaguchi N, Asano M, Itoh M and Toda M (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a -chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155, 1151-1164.

Sanchez-Fueyo A, Weber M, Domenig C, Strom TB and Zheng XX (2002) Tracking the immunoregulatory mechanisms active during allograft tolerance. J Immunol 168, 2274-2281.

Sanda MG, Smith DC, Charles LG, Hwang C, Pienta KJ, Schlom J, Milenic D, Panicali D and Montie JE (1999) Recombinant vaccinia-PSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer. Urology 53, 260-266.

Schreiber H, Wu TH, Nachman J and Kast WM (2002) Immunodominance and tumor escape. Semin Cancer Biol 12, 25-31.

Schwartz JC, Zhang X, Fedorov AA, Nathenson SG and Almo SC (2001) Structural basis for co-stimulation by the human CTLA-4/B7-2 complex. Nature 410, 604-608.

Shah AH and Lee C (2000) TGF-b -based immunotherapy for cancer: breaching the tumor firewall. Prostate 45, 167-172.

Shevach EM (2001) Certified professionals: CD4(+)CD25(+) suppressor T cells. J Exp Med 193, F41-46.

Shimizu J, Yamazaki S, Takahashi T, Ishida Y and Sakaguchi S (2002) Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol 3, 135-142.

Smith SG, Patel PM, Porte J, Selby PJ and Jackson AM (2001) Human dendritic cells genetically engineered to express a melanoma polyepitope DNA vaccine induce multiple cytotoxic T-cell responses. Clin Cancer Res 7, 4253-4261.

Smyth MJ, Godfrey DI and Trapani JA (2001) A fresh look at tumor immunosurveillance and immunotherapy. Nat Immunol 2, 293-299.

Suri-Payer E, Amar AZ, Thornton AM and Shevach EM (1998) CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J Immunol 160, 1212-1218.

Thompson CB and Allison JP (1997) The emerging role of CTLA-4 as an immune attenuator. Immunity 7, 445-450.

Thornton AM and Shevach EM (1998) CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 188, 287-296.

Thornton AM and Shevach EM (2000) Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J Immunol 164, 183-190.

Troyer JK, Beckett ML and Wright GL, Jr (1995) Detection and characterization of the prostate-specific membrane antigen (PSMA) in tissue extracts and body fluids. Int J Cancer 62, 552-558.

van der Merwe PA, Bodian DL, Daenke S, Linsley P and Davis SJ (1997) CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics. J Exp Med 185, 393-403.

van Duinen SG, Ruiter DJ, Broecker EB, van der Velde EA, Sorg C, Welvaart K and Ferrone S (1988) Level of HLA antigens in locoregional metastases and clinical course of the disease in patients with melanoma. Cancer Res 48, 1019-1025.

Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, Thompson CB, Griesser H and Mak TW (1995) Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270, 985-988.

Wherry EJ, Puorro KA, Porgador A and Eisenlohr LC (1999) The induction of virus-specific CTL as a function of increasing epitope expression: responses rise steadily until excessively high levels of epitope are attained. J Immunol 163, 3735-3745.

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

Yewdell JW and Bennink JR (1999) Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol 17, 51-88.

Yin L, Poirier G, Neth O, Hsuan JJ, Totty NF and Stauss HJ (1993) Few peptides dominate cytotoxic T lymphocyte responses to single and multiple minor histocompatibility antigens. Int Immunol 5, 1003-1009.

Zinkernagel RM and Doherty PC (1979) MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness. Adv Immunol 27, 51-177.

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