6310 Vol. 9, 6310–6315, December 15, 2003 Clinical Cancer Research The Biology Behind Cancer Immunotherapy Targeting Survivin Commentary re: V. Pisarev et al., Full-Length Dominant-Negative Survivin for Cancer Immunotherapy. Clin. Cancer Res., 9: 6523–6533, 2003. John C. Reed1 and Darcy B. Wilson2 human tumor cell lines using antisense methods, or interfering 1The Burnham Institute and 2The Torrey Pines Institute for with the function of the endogenous Survivin protein by ectopic Molecular Studies, La Jolla, California expression of dominant-negative Survivin mutants, results in arrested cell division and apoptosis, implying that tumor cells cannot replicate and survive without this important and nonre- The Survivin protein regulates both cell division and cell dundant protein. Consequently, it stands to reason that tumors survival and is overexpressed in the vast majority of human could not easily escape therapeutic interventions targeting Sur- cancers (1). In fact, the gene encoding Survivin is notable for its vivin by simply shutting-off its expression. high degree of tumor-specific expression, ranking among the top With these issues in mind, the Gabrilovich group set out to five most tumor-specific genes in the human genome based on ask whether peptides-derived from the Survivin protein could comparisons of the number of times survivin transcripts appear stimulate cytolytic T-cell responses in vitro, using peripheral in tumors compared with normal cells and tissues (reviewed in blood lymphocytes (PBLs) derived from normal volunteers and Ref. 2). In normal cells, Survivin is produced only in small prostate cancer patients. The strategy they used involved infect- amounts and only briefly during mitosis. However, in tumors, the Survivin protein is continuously present at excess levels, ing autologous dendritic cells (DCs) with recombinant adeno- suppressing apoptosis and aiding in cell division (reviewed in virus expressing the full-length Survivin protein in the hope that Ref. 3). Consequently, Survivin has emerged as a hot target for at least some of the endogenously processed peptides from cancer therapy, with a variety of strategies already articulated Survivin could be displayed in immunogenic forms on HLA-A2, for nullifying Survivin in tumors, including (a) small-molecule the most common of the HLA class I antigens expressed in antagonists that block interactions of Survivin with critical Caucasian populations. To avoid any potential pro-oncogenic partner proteins (4–6); (b) antisense oligonucleotides that re- side effects, a dominant-negative mutant of Survivin [containing duce survivin expression; (c) ribozyme-mediated inhibition of a single amino acid substitution (T34A)] was used instead of the survivin expression (7); and (d) gene therapy using dominant- wild-type protein for in vitro immunizations (8). negative mutants of Survivin that induce cell cycle arrest and Elegant studies of the mechanisms of antigen processing apoptosis (8). In this issue of the Clinical Cancer Research, the and presentation have revealed that immunogenic peptides of Gabrilovich group [Pisarev et al. (9)] describe preclinical stud- 8–9 amino acids in length are displayed on cell surface HLA ies aimed at yet another strategy for exploiting Survivin as a class I molecules after degradation of intracellular proteins via cancer target, namely, using survivin to create a tumor vaccine. specialized proteasomes, and transport of the resultant peptides Inducing cell-specific immunity against tumors represents to the endoplasmic reticulum where they associate with class I a long-standing goal of cancer immunology (10). To be suc- antigens and then are transported to the plasma membrane (Ref. cessful, several conditions must be met, including identifying 12; Fig. 1). Thus, the 142-amino-acid human survivin protein tumor-associated antigens (TAAs) that are capable of being could theoretically generate 136 nonamer peptides. Rather than presented on human leukocyte class I or II antigens (HLA-I or testing all those possible peptide sequences to see whether they -II) and stimulating host T cells using the available repertoire of are reflected in the repertoire of T-cell responses made to T-cell antigen receptors (reviewed in Ref. 10). Ideally, such full-length Survivin, the Gabrilovich group took some educated TAAs should be displayed only on tumor cells and not normal guesses. Using data from the literature (13, 14) and predictive cells, or, at least, such antigens should be expressed only on computer algorithms, they surmised that three peptide sequences nonessential normal tissues if tumor specificity is impossible contained within Survivin ought to be generated in antigen- (e.g., breast, prostate, ovary, testes). Furthermore, the TAA presenting cells and should be capable of binding HLA-A2. The should be an essential gene, so that tumors cannot avoid immune strategy thus involved in vitro stimulation of autologous PBLs surveillance simply by refusing to express it. with Survivin-adenovirus-infected DCs, followed by testing of Gene ablation studies in mice have confirmed that survivin the resulting activated T cells for their ability to recognize is an essential gene (11). Knocking-down survivin expression in HLA-A2-expressing cells pulsed with one of the three synthetic Survivin 9-mer peptides. The question asked was whether T cells could be enticed, at least in vitro, to respond to a self- antigen (Survivin), thus breaking tolerance. Secondary ques- Received 10/31/03; accepted 11/4/03. tions involved addressing the issues of whether such Survivin- Grant support: NIH CA-78040 and AG15402. specific T cells could kill either peptide-pulsed target cells or a Requests for reprints: John C. Reed, The Burnham Institute, 10901 tumor cell line that endogenously expresses Survivin and HLA- North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 646-3132; A2, and whether normal hematopoietic stem cells would be Fax: (858) 646-3194; E-mail: [email protected] or Darcy B. Wilson, Torrey Pines Institute for Molecular Studies, 3550 General affected inasmuch as they are among the nontransformed types Atomics Court, La Jolla, CA 92121-1122. Phone: (858) 455-3840; of cells known to express at least low levels of Survivin in vivo Fax:(858) 455-3804; E-mail: [email protected]. (15). Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. Clinical Cancer Research 6311 Fig. 1 Antigen processing and presentation. Intracellular proteins are proteolysed by proteosomes in the cytosol, and the resulting peptides are transported into the lumen of the endoplasmic reticulum via the TAP transporter. Peptides associate with HLA (human) or MHC (mouse) class-I antigens, which are transported eventually to the cell surface. PBLs derived from all four normal subjects and the four T-cell lines were tested on peptide-pulsed DCs, using the fre- cancer patients resulted in T-cell populations capable of re- quency of lymphokine-producing cells as a measure of response sponding to at least one of the three synthetic Survivin peptides, (“ELISPOT assay”), five of six HLA-A2-positive healthy do- after three rounds of successive stimulation with Survivin-ade- nors and all four HLA-A2-positive cancer patients showed only novirus-infected DCs. Moreover, seven of the eight PBL spec- an approximate doubling in the number of Survivin-responsive imens responded to two of the three Survivin peptides. Thus, T cells, compared with background controls. Similarly, cytolytic these findings argue that, at least in vitro, it is possible to break killing of peptide-pulsed cells by the stimulated T cells was tolerance and encourage T cells within the available repertoire relatively feeble, with only a ϳ10% increase in specific lysis, to respond to Survivin peptides in the context of HLA-A2. sometimes requiring high E:T ratios of 100:1. Of course, one of Although an encouraging observation, these findings only begin the limitations of these experiments is that the peptides chosen to scratch the surface and many questions remain to be ad- to measure responsiveness to Survivin are only guesses of what dressed toward the goal of establishing whether Survivin-based the T-cell repertoire might produce. Of the 136 theoretically tumor vaccines are likely to be safe and effective in cancer possible 9Јmer peptides represented in the human Survivin patients. protein sequence, only 3 were tested. On the basis of the First, regarding efficacy, from the information provided, it SYFPEITHI algorithm, which assesses peptide sequences for is unclear how efficiently Survivin-transduced DCs activated T HLA binding,3 probably at least five peptides in the Survivin cells within the available repertoire of PBLs of normal subjects sequence are candidate HLA-A2-binders. Thus, the actual fre- and prostate cancer patients. One would like to know some of quency of Survivin-responsive T cells in the normally HLA the details about the frequency of responding T cells in periph- heterozygous human population might be significantly higher eral blood and whether that frequency changes in cancer pa- than estimated from best-guess approaches, speaking to the need tients, to make comparisons of the merits of Survivin relative to for a more systematic survey of the possible “peptide space” in other TAAs that might have been used as an alternative (e.g., prostate-specific antigen; prostate-specific membrane antigen for prostate cancer or telomerase for many types of cancer).