molecular insights into the development of t cell-based immunotherapy for prostate cancer
TRANSCRIPT
Molecular insights into thedevelopment of T cell-basedimmunotherapy for prostatecancerExpert Rev. Clin. Immunol. Early online, 1–11 (2014)
Baijun Dong1,Laurie J Minze2,Wei Xue*1 andWenhao Chen*2
1Department of Urology, Renji Hospital,
Shanghai Jiao Tong University School of
Medicine, Shanghai 200127, P.R.,
China2Department of Surgery,
Immunobiology Research Center,
Houston Methodist Research Institute,
Houston Methodist Hospital, Houston,
TX 77030, USA
*Authors for correspondence:
Tel.: +1 712 441 2173
Fax: +1 713 441 7439
Using a patient’s own immune system to fight cancer is a highly active area of cancerresearch. Four years ago, sipuleucel-T became the first approved cancer vaccine, which wasdeveloped to enhance T-cell immunity against metastatic castration-resistant prostate cancer.Other prostate cancer vaccines, including a viral-based vaccine PROSTVAC-VF and a cellularvaccine GVAX, are in development. Moreover, several clinical trials are investigating the roleof immune checkpoint blockade in the treatment of prostate cancer. Ipilimumab andnivolumab are potent T cell checkpoint inhibitors that reverse immunologic tolerance inmultiple types of cancers. Here we discuss the mechanisms underlying antitumor T cellresponses as well as the development of immunotherapies for prostate cancer.
KEYWORDS: cancer vaccine • immune checkpoint • immunotherapy • prostate cancer • sipuleucel-T • T cells
Prostate cancer is the second leading cause ofcancer death in US men and the sixth leadingcause of cancer death in males worldwide.Most prostate cancers are adenocarcinoma andare slow growing. If the cancer remains withinthe prostate, treatment options include watch-ful waiting, active surveillance, radiation ther-apy and surgery. Over time, some cancersadvance and metastasize to the bones, lymphnodes and other parts of the body. At thisstage, androgen deprivation (hormonal) ther-apy causes disease remission in 80–90% ofpatients, as the cancer cells depend on andro-gens for growth and survival. The first-lineandrogen deprivation therapy for metastaticprostate cancer includes luteinizing hormone-releasing hormone (LHRH) agonists and anti-androgens [1]. Nevertheless, most cancersbecome castration resistant after 1–3 years.There was no therapy that had been shownto extend survival for patients with castration-resistant prostate cancer (CRPC) until theyear 2004, when the chemotherapy drug doce-taxel was approved for the treatment of meta-static prostate cancer. Docetaxel provides anoverall survival benefit for patients with CRPCand prolongs median survival by 2–3months [2].
In April 2010, sipuleucel-T (Provenge; Den-dreon, Seattle, WA, USA) was approved bythe US FDA for treating men with metastaticCRPC with no or minimal symptoms. It pro-vides a 4-month median survival benefit forpatients with CRPC and has no clinically sig-nificant negative effect on quality of life [3].Sipuleucel-T is a cancer vaccine that stimulatesthe ability of the patient’s immune system toattack cancer. The production of sipuleucel-Tinvolves the use of patient’s peripheral bloodmononuclear blood cells (PBMCs), whichincludes antigen-presenting cells (APCs) thatdisplay antigens complexed with MHC pro-teins on their surfaces and present antigens toT cells. These APCs are enriched and stimu-lated ex vivo with a fusion protein-linkingprostatic acid phosphatase (PAP) and GM-CSF and are then transferred back to patientsto induce T-cell responses against prostate can-cer cells [4].
Since June 2010, four additional agents havebeen approved for treating CRPC, including achemotherapeutic cabazitaxel, a radiopharma-ceutical radium-223 and two hormonal thera-pies (abiraterone and enzalutamide). Inparticular, increased expression or activity ofandrogen receptor (AR) in CRPC is crucial for
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responding to residual androgens and tumor growth despite cas-tration [1]. Enzalutamide is an AR inhibitor with high-affinitybinding to AR, which affects AR nuclear translocation and func-tion. In August 2012, the FDA approved the use of enzalutamidein patients with CRPC who had previously received docetaxel,based on the results obtained from the Phase III AFFIRM trial [5].The other new hormonal therapy abiraterone is a potent andselective inhibitor for a microsomal enzyme cytochromeP450C17, which is crucial for the biosynthesis of androgen andadrenal hormones. Abiraterone has been approved for treatingCRPC in both pre-chemotherapy and post-docetaxel settings,based on the results of Phase III trials COU-301 andCOU-302 [5–7]. The sequential use of the available therapeuticsfor treating CRPC is currently under development. In this review,we focus on discussing the mechanisms underlying antitumorT-cell response, followed by the immunotherapeutic approachesthat have been or are being studied in clinical trials of CRPC.
T-cell immunity to prostate cancerCancers, as abnormal tissues, contain various tumor-specificantigens (TSAs) and tumor-associated antigens (TAAs) that canbe recognized by T cells. Here we discuss the factors that regu-late T-cell immunity to cancer in general and to prostate cancerin particular.
Overview of antitumor T-cell response
T cells are the master regulators that control the quality andquantity of adaptive immune responses. T cells encountertumor antigens at two major anatomic locations, secondarylymphoid organs and tumors. Mature naive T cells initially rec-ognize tumor antigens in lymphoid organs. In the presence ofsufficient ‘danger’ and inflammatory signals, antigen-respondingT cells are activated and differentiated into effector T cells.When effector T cells infiltrate into tumors, they re-encounterthe tumor antigens and kill the target cancer cells that expressthe antigens. On the other hand, the tumor microenvironmentcontains numerous inhibitory factors that impair T-cell func-tion and render immune tolerance, which is a state of unre-sponsiveness of the immune system to specific substances ortissues. Here, we will discuss antitumor T-cell response as wellas tumor-induced immune tolerance.
Tumor antigenicity
Cancer is induced by a series of genetic alterations and abnor-mal differentiation of cells, which lead to the expression of pep-tide TSAs and TAAs. For instance, during cancer development,unique peptide fragments (e.g., p53 mutations) are produced asTSAs and present only in tumors. By contrast, cancer-testis(CT) antigens are TAAs as they are also expressed in some nor-mal tissues, including immune-privileged testis, fetal ovary andplacental trophoblasts. Other groups of TAAs include the dif-ferentiation antigens that are expressed in tumors and the nor-mal tissue of origin as well as the overexpressed antigens thatare expressed in a wide variety of normal tissues and are overex-pressed in tumors [8].
Most antigens identified in prostate cancer are TAAs [9]. Forinstance, prostate-specific antigen (PSA), a member of thekallikrein-related peptidase family, is secreted almost exclusivelyby the epithelial cells of the prostate gland. PSA is present inthe blood at very low levels in healthy subjects, whereas bloodPSA level is elevated in most cases of prostate cancer. PAP,another major TAA of prostate cancer, is also secreted by bothnormal prostate epithelial cells and malignant prostate tissues.Some studies indicated that low PAP mRNA levels can also bedetected in non-prostatic tissues, such as placenta, kidney andtestis. Similarly, prostate-specific membrane antigen (PSMA), azinc metalloenzyme that resides in membranes, is expressed100- to1000-fold higher in prostate glands than in non-prostatic tissues. Other identified TAAs of prostate cancerinclude prostate stem cell antigen, T-cell receptor (TCR)gamma alternate reading frame protein, transient receptorpotential-p8 and six-transmembrane epithelial antigen of theprostate 1 [9].
Peripheral mature T cells express their own unique TCRconsisting of an a and a b chain, which recognize specific‘foreign’ peptide antigens (epitopes) presented by MHC mole-cules. T-cell clones recognizing self-antigens with high-affinityinteractions between the TCR and self-antigen/MHC ligandsare generally deleted in the thymus during their maturation,whereas clones with low-affinity interactions may escape thymicdeletion and enter into periphery. In other words, self-antigenshave weak immunogenicity and do not provoke strong immuneresponse due to the lack of high-affinity T-cell clones. In the-ory, most antigens identified from prostate cancers are TAAswith weak immunogenicity. Some TAAs may be considered as‘novel’ epitopes if they have not been previously exposed to theT cells. TSAs are truly novel epitopes to mature T cells due togenetic alterations in tumors. The advanced technology inexome sequencing has been applied to looking for potentialTSAs. Mass spectroscopy has been used to identify the epitopeseluted directly from MHC molecules [10]. These approachesmay help in identifying prostate cancer antigens that caninduce potent T-cell response.
Maturation of dendritic cells
T cells depend on APCs to pick up and present cognate anti-gens to them. In the context of prostate cancer, the APC vac-cine, sipuleucel-T, is a therapeutic option for CRPC. A betterunderstanding of APC function in cancer will further facilitatethe development of innovative APC vaccines.
Dendritic cells (DCs) are ‘professional’ APCs that are veryefficient at displaying antigen on their membrane MHC andpresenting antigen to T cells upon the interaction betweenTCR and the antigen/MHC complex. The lineage commit-ment of DCs is controlled by hematopoietic cytokines, such asFlt3L and GM-CSF [11]. Committed DCs stay in peripheraltissues and the blood in a relative immature state. The matura-tion and activation of these DCs require various ‘danger’ sig-nals from pathogens and dying tissue cells. In the presence ofpathogens, pathogen-associated molecular patterns (PAMPs;
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e.g., bacterial lipopolysaccharide) are recognized by Toll-likereceptors (TLRs) and other pattern recognition receptors,which initiate the maturation process of DCs. Similarly, celldeath through necrosis stimulates DCs by releasing damage-associated molecular pattern (DAMPs). Pro-inflammatory cyto-kines (e.g., IL-1b and TNF-a) further trigger DC maturationin peripheral tissues in an autocrine or paracrine fashion. Uponmaturation, antigen-carrying DCs leave the peripheral tissuesand enter draining lymph nodes to prime antigen-specificT cells. Activated CD4 T helper (Th) cells in turn induce fur-ther DC maturation via the CD40L–CD40 interaction [11].
In the context of prostate cancer, the human prostate cancercell line LNCaP has been shown to inhibit the in vitro genera-tion and maturation of DCs [12]. Indeed, a significant reductionin DC count was observed in the peripheral blood of patientswith metastatic prostate cancer when compared to that ofhealthy controls [13]. Tumor-associated DCs may even suppressantitumor immunity. Watkins et al. suggested that a transcrip-tion factor Forkhead box O3 mediated the immune suppressivefunction of tumor-associated DCs [14]. Hence, DC vaccinesaim to generate optimally activated and antigen-loaded APCsby manipulating cancer patient’s DCs ex vivo.
T-cell activation & effector function
Peripheral mature T cells mainly include CD4+ Th and CD8+
cytotoxic T (CTL) cells. CD4+ T cells are designated to assistother immune cells through direct cell–cell interaction andthrough the release of cytokines. CD8+ T cells are equipped todestroy virally infected cells and tumor cells. Naive T cells areprimed in the secondary lymphoid organs, where they receiveactivation signals upon engagement of the TCR by the cognatepeptide–MHC complex on APCs and interaction of CD28-B7co-stimulatory molecules. Intriguingly, T-cell activation leads toincreased expression of cytotoxic T-lymphocyte antigen4 (CTLA4), which is similar to CD28 and also binds toB7 molecules on APCs. By contrast, CTLA4 transmits aninhibitory signal to T cells. Modulation of co-stimulatory andinhibitory signals of T-cell activation has been extensively inves-tigated as an attractive approach for developing novel immuneinterventions [15].
Tumor infiltration by T cells is a good prognostic marker incertain types of cancers [16,17], but not in others (e.g., renal cellcarcinoma [18]). In the context of prostate cancer, a high ratioof CD8+ CTLs to Treg cells in the tumor is associated withfavorable prognosis [19]. Moreover, adoptive transfer of CD8+-enriched tumor infiltrating lymphocytes can mediate regressionof metastatic cancer [20]. A recent paper indicated thatIL-9-skewed CD8+ cytotoxic T (Tc9) cells elicited greater anti-tumor responses against advanced melanoma than that of type-I CD8+ Tc1 cells, though adoptive transferred Tc9 cells stilldifferentiated into IFNg- and granzyme B-producing Tc1-likeeffector cells [21]. Hence, a better understanding of the mecha-nism underlying differentiation of CD8+ CTLs may improvethe efficacy of adoptive T-cell therapy for cancer. CD4+ T cellsare mainly studied for their role as helpers for CD8+ CTLs.
High expression levels of Th1-related factors (e.g., IFNg ,IL-12, Tbet, IRF1 and Stat1) in cancer are considered asimmune signatures correlating with good prognosis [22]. CD4+
T cells are capable of mediating tumor rejection [23,24]. In addi-tion, natural killer (NK) cells also have powerful cytotoxicactivity. NK cell function and effect of tumor environment onNK cells have recently been reviewed by Vitale et al. [25]. Giventhe evidence that CD4+ T, CD8+ T and NK cells are criticalfor mediating antitumor immunity, attention should be paid tothese cell types when developing immunotherapies for cancer.
Tumor-induced immune tolerance
Tumor immunogenicity varies highly, not only between differ-ent types of cancers, but also between cancers of the same typein different individuals [26]. The presence of tumor antigensdoes not always provoke antitumor immune response. Evenwhen T cells recognize tumor antigens and infiltrate intotumors, many of them do not proliferate, secrete cytokines andterminally differentiate into CTLs [27,28]. In addition to theweak immunogenic nature of TAAs, tumors utilize multiplefactors to resist immune attack. For instance, the ability oftumors to escape from antitumor immune response is oftenassociated with the impaired expression of MHC I moleculeson surface of cancer cells, preventing the MHC-restricted kill-ing of cancer cells by CTLs [29].
Spranger et al. have recently shown that certain tumor typesexhibited high expression of indoleamine-2,3-dioxygenase(IDO) and PD-L1/B7-H1 and recruited high frequency ofCD4+Foxp3+ Treg cells that suppress immune response. Thepresence of these inhibitory pathways followed, rather than pre-ceded, CD8+ T-cell infiltration. The authors suggested thatCD8+ T-cell infiltration led to production of CCR4-bindingchemokines in tumors, which in turn recruit Treg cells. More-over, IFN-g produced by infiltrating CD8+ T cells up-regulatedthe expression of IDO and PD-L1 in tumors. Hence, thedevelopment of certain immunosuppressive pathways in tumorsis a consequence of the initial antitumor immune responses [30].By contrast, in a PTEN-null murine prostate cancer model,Garcia et al. showed an expansion of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSCs) in the prostate immediatelyfollowing epithelium-specific PTEN deletion. They found thatloss of PTEN in the epithelium leads to a significant up-regulation of Csf-1 and IL-1b, two molecules known to induceMDSC expansion. Those prostatic MDSCs potently suppressT-cell proliferation and express high levels of arginase 1 andinducible nitric oxide synthase. Therefore, intrinsic characteris-tics of cancer cells (e.g., PTEN deletion) are also involved in thedevelopment of immunosuppressive environment in tumors [31].
The endothelial and stromal cells in a tumor microenviron-ment play an important barrier role in preventing T-cell infil-tration. A recent report indicated that overexpression of theendothelin B receptor is associated with the absence of infiltrat-ing lymphocytes in advanced-stage epithelial ovarian cancer.In mice, endothelin B receptor blockade augments T-cell hom-ing to tumors [32]. Another paper showed that the regulator of
Molecular insights into the development of T cell-based immunotherapy for prostate cancer Review
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G-protein signaling 5 is responsible for the abnormal tumorvascular morphology. Deletion of Rgs5 results in vascular nor-malization and enhances influx of immune cells into tumorparenchyma [33]. Moreover, Kraman et al. have described that astromal cell type in human cancers expresses fibroblast activa-tion protein-a (FAP). Depletion of FAP-expressing cells per-mits immunological control of tumor growth by a processinvolving IFN-g and TNF-a [34]. The stromal extracellularmatrix also affects migration of T cells and antitumor immu-nity. A real-time imaging study provided evidence that alignedfibers in perivascular regions and around tumor epithelial cellregions limit T cells from entering tumors [35].
Taking all of this into account, T-cell recognition of tumorantigens is not sufficient to induce destruction of tumor [36].The tumor environment renders tumor-reactive T cells nonre-sponsive [37]. Tumors represent the complexity of biologicalnetworks involving tissue injury/repair, angiogenesis, extracel-lular remodeling, tumor growth and immune cell infiltration.Understanding the cross-talk between tumor and immunecells will reveal the mechanisms underlying tumor immunesuppression.
T cell response to prostate cancer
TAAs from prostate cancers can be recog-nized by human T cells. In vitro studieshave identified numerous HLA-restrictedhuman CTLs recognizing peptides derivedfrom the prostate TAAs [9]. To define theT-cell response to prostate cancer,Drake et al. created Pro-HA mice thatstrictly express the model antigen influenzahemagglutinin (HA) in the prostate. Theprostate glands of these mice are ignoredby the adoptively transferred HA-specificCD4+ T cells. When Pro-HA mice arecrossed with TRAMP mice that developspontaneous prostate cancer, tumorigene-sis induces a tolerogenic T-cell responsewith abortive proliferation. The authorsfurther indicated that androgen ablationmitigates the tolerance of transferredHA-specific T cells, which expand anddevelop effector function [38]. Similarly,Arredouani et al. developed HLA-A*0201/human PSA-double transgenic mice andshowed that androgen ablation augmentsanti-PSA T-cell response [39]. However, T-cell function enhanced by androgen abla-tion is transient. Tang et al. used anothertumor antigen model. They suggested thatTreg cells expand following androgen abla-tion, preventing a strong and durableT-cell response [40]. Together, a betterunderstanding of T-cell response to pros-tate cancer will further facilitate the devel-opment of effective immunotherapies.
Immunotherapies for prostate cancerThe following sections focus on the discussion of the immuno-therapeutic approaches that aim to enhance antitumor T-cellresponses.
Cancer vaccines
The goal of cancer vaccines is to enhance the ability of T cellsto respond to tumor antigens and eliminate malignant cells.Because APCs present antigens to initiate T-cell immunity,they are frequently exploited for inventing novel cancer vac-cines. Sipuleucel-T was developed under this strategy and dem-onstrated a 4-month median survival benefit for patients withCRPC. During the ex vivo sipuleucel-T processing, autologousAPCs enriched from patient’s PBMCs are activated with afusion protein linking PAP and GM-CSF. PAP is a prostatecancer TAA, whereas GM-CSF promotes the mature process ofmonocytes and DCs. After 2 days of culture, cells are infusedback into patients to promote antitumor T-cell response [4].Thus, sipuleucel-T is an autologous cellular therapy that com-poses of antigen-loaded and activated APCs (FIGURE 1). Treatment
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Figure 1. Potential mechanisms of action for prostate cancer vaccines andimmune checkpoint blockades. DCs are professional APCs that present antigens toT cells. Naive T cells are primed in the secondary lymphoid organs, where they receiveactivation signals upon engagement of the TCR by the tumor antigen–MHC complex onAPCs and interaction of CD28-B7 co-stimulatory molecules. Activated T effector cellsinfiltrate tumors and kill tumor cells in a MHC-restricted manner. Immunotherapeuticapproaches aim to enhance antitumor T cell responses. (A) Sipuleucel-T therapy involvesan ex vivo processing, which stimulates the maturation of autologous APCs with a fusionprotein linking PAP and GM-CSF. Upon infusion PAP–GM-CSF loaded APCs activatePAP-reactive T cells in patients; (B) PROSTVAC-VF utilizes a vaccinia virus encoding amodified PSA and three T-cell stimulatory molecules, B7.1, ICAM-1 and LFA-3. Uponinjection, the vaccinia virus infects DCs and somatic cells, both of which may in turn acti-vate PSA-reactive T cells; (C) CTLA4 expressed on the surface of activated T cells bindsB7 molecules on APCs to restrain T-cell activation. Blockade of CTLA4 using mAbs aug-ments the amplitude of T-cell responses and (D) PD1 expression is up-regulated on acti-vated T cells and maintained on tissue infiltrating T cells. Binding of PD1 with PDL1 ontumor cells down-regulate the activity of T cells. Blockade of PD1 using mAbs promotesthe effector T cells to attack tumor cells.APCs: Antigen-presenting cells; DCs: Dendritic cells; PAP: Prostatic acid phosphatase;PSA: Prostate-specific antigen; TCR: T-cell receptor.
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with sipuleucel-T leads to longer survival in patients withCRPC, but lacks in tumor regression or a delay in progres-sion [4,41,42]. This was also seen in prostate cancer patientstreated with PROSTVAC and melanoma patients treated withipilimumab [43,44]. Madan et al. have suggested that these treat-ments do not cause tumor size to diminish, but rather slowtheir growth rate over time [41]. Stein et al. further proposedthat novel paradigms are needed to assess therapeutic efficacyin solid tumors. They found that the ‘tumor growth rate con-stant’ correlates with patient survival and may be used as a newefficacy endpoint for clinical trials [43].
Another cellular vaccine approach is to inject patients withtumor-related cells. GVAX (BioSante Pharmaceuticals, Lincoln-shire, IL, USA) is composed of hormone-resistant PC-3 andhormone-sensitive LNCaP prostate adenocarcinoma cell lines.These allogeneic tumor cells are transduced to express GM-CSF and then irradiated. Results from Phase II clinical trialsindicated that GVAX was well tolerated in patients withCRPC [45], but two Phase III trials of GVAX were closed early.In one of the Phase III trials, more deaths occurred in theGVAX-treated group [46]. Currently, CTLA4 blockade in com-bination with GVAX is undergoing clinical trials and has beenshown to up-regulate CD40 expression on conventional DCsand induce the development of a PSMA-specific antibodyresponse [47]. LNCaP (expressing HLA-A*0201) transducedwith IL-2 and IFN-g has also been used to treat HLA-A*0201-matched patients with progressive CRPC. This vaccinestrategy was found to be safe and well tolerated and stimulatedT-cell response in the majority of patients [48]. The therapeuticeffect of whole cancer-cell vaccines remains to be determined.
Gene therapy approaches have been applied to induce theexpression of TAAs in vivo as vaccines. PROSTVAC-VF (BNImmuno Therapeutics, Mountain View, CA, USA) is a viral-based vaccine for prostate cancer. PROSTVAC-VF comprises aprimary vaccination using a recombinant vaccinia vector andmultiple booster vaccinations using a recombinant fowlpox vec-tor. Both vectors contain a modified PSA and three T-cellstimulatory molecules, including B7.1, ICAM-1 andLFA-3 (FIGURE 1). Upon PROSTVAC-VF treatment, patientswith a greater T-cell response to PSA showed survival advan-tage when compared to those with a lower T-cell response [49].In a Phase II trial that enrolled 125 patients with minimallysymptomatic CRPC, PROSTVAC-VF plus GM-CSF immuno-therapy was associated with an 8.5-month improvement inmedian overall survival in patients when compared to the con-trol empty vectors plus saline injections [50]. A Phase III trial ofPROSTVAC-VF in patients with metastatic CRPC is currentlyongoing. Recently, a plasmid DNA vaccine encoding PAP(without viral vectors) has been investigated in patients withrecurrent prostate cancer. When combined with GM-CSF, thisDNA vaccine has been shown to induce long-term PAP-spe-cific T-cell immunity in patients [51,52]. Moreover, direct vacci-nation with CureVac’s two-component mRNA (free andprotamine-complexed mRNA; CureVac GmbH, Tubingen,Germany) potently inhibits tumor growth in preclinical
models [53,54]. Phase I/II trials conducted in patients with pros-tate cancer have shown that the two-component mRNA vac-cines are safe and well tolerated in humans [54].
TAA-related peptides have also been utilized to induce anti-tumor T-cell response in CRPC patients. One intriguingapproach is to generate personalized peptide vaccination (PPV)by selecting two to four peptides for each individual based onthe preexisting host immunity to these antigens. In a recentPhase II trial enrolled with 100 CRPC patients, PPV was safeand well tolerated [55]. Another interesting approach is to use aDNA fusion-gene vaccination, which encodes an HLA-A2-binding PSMA27-35 epitope linked to a domain (DOM)from fragment C of tetanus toxin. The inclusion of the DOMepitope may allow for the sufficient generation of T-cell help.HLA-A2+ patients who received this vaccine exhibited DOM-specific CD4+ and PSMA27-specific CD8+ T-cell responses [56].
To date, most immunotherapeutic trials for prostate cancerapply the vaccination strategy. The vaccines mentioned above aredesigned to treat patients who have already developed metastaticprostate cancers. At this stage, the patient’s own immune systemoften does not mount a strong attack against the tumor. Mostantigens identified from prostate cancer are TAAs, which, in the-ory, exhibit weak immunogenicity. Even when the immune cellsrecognize prostate cancer antigens, the tumor may establish anenvironment to suppress the antitumor immune responses. Theaim of vaccines is to prime antigen-specific responses that canovercome tumor’s immune suppression. Vaccines can be manip-ulated ex vivo to present selected antigens, enhance APC matura-tion and activity or transduce genes critical for T-cellpriming [4,48,50]. Adjuvants, delivery vehicles and routes of admin-istration also affect immunity induced by vaccines [57]. Forinstance, pre-conditioning of the vaccine injection site withinflammatory cytokines improves the migration of ex vivo gener-ated DCs to the lymph nodes where they activate T and Bcells [58]. Overall, the success of cancer vaccines is based not onlyon the vaccine design technology and antigen selection but also adetailed understanding of how immune cells and tumors interact.
Immune checkpoint blockade
Various surface molecules deliver inhibitory signals to immunecells as immune checkpoints, which are required to maintainimmune tolerance to self-tissues. Treg and activated T cellsexpress CTLA4 to impair antigen presentation and transmit aninhibitory signal to T cells. CTLA4 knockout or a specific defi-ciency of CTLA4 in Tregs leads to T-cell-mediated fatal auto-immunity in mice [59]. In 2010, Hodi et al. reported that animprovement in overall survival among patients with metastaticmelanoma has been achieved by administration of an anti-CTLA4 mAb, ipilimumab [44]. Immune-related adverse eventsoccurred in 10 to 15% of patients treated with ipilimumab,but most are reversible with appropriate treatment [44]. Whencombined with dacarbazine, ipilimumab also improved overallsurvival in patients with previously untreated metastatic mela-noma [60]. Ipilimumab gained FDA approval to treat melanomain early 2011.
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A pilot trial in metastatic CRPC indicated that ipilimumab wassafe and did not induce significant autoimmunity [61]. A Phase I/II study in patients with metastatic CRPC further indicated thatipilimumab plus radiotherapy exhibited clinical antitumor activitywith disease-manageable adverse events [62]. When combined withGVAX or a poxviral-based PSA vaccine, ipilimumab was also tol-erable and safe for patients with metastatic CRPA and did notexacerbate the immune-related adverse events [47,63]. Phase I andII studies of sipuleucel-T and ipilimumab as a combination ther-apy for prostate cancer are also ongoing [64,65]. Moreover, com-bined immunotherapy with ipilimumab and GM-CSF has beenshown to activate T cells that are specific for known TAAs [66] andpromote Ab response [67]. Most recently, Kwon et al. reportedresults from a Phase III trial of ipilimumab after radiotherapy inpatients with metastatic CRPC who progressed after docetaxelchemotherapy. A total of 799 patients were randomly assigned(399 to ipilimumab and 400 to placebo) to receive bone-directedradiotherapy followed by either 3-weekly ipilimumab (10 mg/kg)or placebo. The authors indicated that ipilimumab did notsignificantly improve overall survival in the primary analysis.Median overall survival was 11.2 months with ipilimumaband 10.0 months with placebo. However, some evidence ofantitumor activity with ipilimumab was identified, includingreductions in serum PSA concentration and improvedprogression-free survival [68]. The authors hypothesized that ipili-mumab might be more effective in patients with favorable prog-nostic features [68]. Ipilimumab is currently being evaluated in theCA184–095 Phase III trial [69], comparing the efficacy of ipilimu-mab versus placebo in asymptomatic or minimally symptomaticmCRPC. The results of this ongoing trial may demonstratewhether ipilimumab is effective in patients with favorableprognostic features.
PD-1 is an inhibitory receptor expressed on activated T cells.Interaction of PD-1 with its ligand PD-L1 serves as anothermajor immune checkpoint. While CTLA-4 prevents APCsfrom priming T cells to recognize tumors, PD-1 appears toprevent T cells from attacking cancer cells in peripheraltissues (FIGURE 1) [70]. Moreover, blocking CTLA-4 with ipilimu-mab improved overall survival in patients with advanced mela-noma [44,60], whereas blocking PD-1 with an antibodynivolumab produced durable regression of melanoma [71,72].Because nivolumab and ipilimumab work in different ways toenhance antitumor T-cell response, combined therapy withboth checkpoint inhibitors have the potential to be morepotent to destroy tumors. Indeed, nivolumab combined withipilimumab induced >80% tumor reduction in 53% of patientswith melanoma. Immune-related adverse events were generallyreversible [73]. The number of studies of nivolumab treatmenton CRPC is limited. Taube et al. have recently shown thatPD-L1 expression was abundant in melanoma, non-small-celllung carcinoma and renal cell carcinoma, but not CRPC. Theauthors suggested that tumor PD-L1 expression was correlatedwith response to PD-1 blockade [74]. It is of great interest tofurther determine the therapeutic effects of nivolumab alone ortogether with ipilimumab on CRPC.
Adoptive T-cell transfer for cancer
Adoptive T-cell therapy involves the ex vivo manipulation ofautologous T cells and subsequent infusion of cells back intothe same patient. Adoptive T-cell therapy directly modulatesT-cell function, which is different from sipuleucel-T vaccinethat promotes the maturation and function of APCs. Forinstance, Zhang et al. isolated CD8+ T cells from mice primedwith TRAMP-C2 prostate cells and rendered them TGF-binsensitive by transducing a dominant-negative TGF-b type IIreceptor. Adoptive transfer of tumor-reactive TGF-b-insensitiveCD8+ T cells induced destruction of prostate cancer [75].
Another approach for adoptive T-cell therapy is to engineerperipheral T cells with a TCR that recognizes TAAs or TSAs.TCRg alternate reading frame protein (TRAP) is a TAA that isexpressed in normal prostate as well as in malignant prostateand breast. Hillerdal et al. transduced human peripheral bloodT cells with a TCR that recognizes the HLA-A2-restrictedTARP4-13 epitope. These engineered T cells exerted specifickilling of HLA-A2+ and TARP-expressing prostate cancer cellsin an in vitro assay [76]. Moreover, in a murine prostate cancermodel, sustained tumor regression was successfully induced byadoptive transfer of T cells that express both an engineeredTCR and the dominant-negative TGF-b type II receptor [77].
A relatively novel approach is the adoptive transfer of T cellsengineered with chimeric antigen receptors (CARs), which arefusion proteins consisting of extracellular antibody-type recog-nition domains and intracellular T-cell signaling domains. Inother words, CARs are artificial TCRs that provide T cellswith antibody-type recognition of cancer antigens. For instance,a single-chain fragment (scFv) of an anti-PSCA mAb was fusedto the b2 constant region of a TCR and to the CD3z-signalingdomain. When transduced into mouse CTLs, this CAR deliv-ered activating signals to CTLs and directed the specific killingof PSCA-positive tumor cells [78]. Recently, Ma et al. generatedPSMA-specific T cells engineered with an immunoglobulin-CD28-TCR (IgCD28TCR), which was designed to deliverboth TCR and co-stimulatory signals. T cells expressingIgCD28TCR mediated PSMA-specific cytotoxicity in vitro andsuppressed tumor growth in animal models [79].
Adoptive T-cell transfer and cancer vaccine are two funda-mentally different strategies to augment antitumor immunity.Cancer vaccines are active immunization and aimed at boostingthe patient’s own immune system to attack tumors. Vaccinesare easy to use and can be manipulated to adjust antigen-presenting activity to lymphocytes. However, it remains diffi-cult to define the subsequent antitumor T-cell response invaccinated cancer patients. Adoptive T-cell transfer is a type ofpassive immunization. T cells used for transfer are directlymanipulated to select specific antitumor clones and regulatecell function. In particular, T cells can be easily expandedin vitro, permitting sophisticated genetic manipulation. T cellsengineered with high-affinity TCRs and CARs provideimproved specificity for tumor antigens. T cells can also betransduced to express molecules that support co-stimulation,differentiation and migration [80]. Nevertheless, the techniques
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used to genetically engineer T cells are still in the developingstages. The safety of various engineered T-cell types needs tobe investigated. With the advances in technology and knowl-edge, engineered T cells that more accurately and potently tar-get tumor antigens will be developed.
Expert commentaryCancer vaccines for prostate cancer, such as sipuleucel-T, haveshown a survival benefit and a safety profile. To develop vac-cines with higher efficacy in inducing antitumor immunity, fur-ther progresses should be made in: vaccine delivery systems,such as plasmid DNA, viral vectors and manipulated DCs;selection of antigens, which relies on the development of noveltools to define TAA immunogenicity as well as therapeuticresponses and designing optimal combination therapy usingcancer vaccines and immunomodulatory agents. The samestrategies hold true for the development of novel adoptiveT-cell therapy, in which T cells, but not DCs, will be directlymanipulated.
Advanced melanomas are often infiltrated by T cells andmay utilize the immune checkpoints to suppress the infiltratingT cells. Immune checkpoint blockade thus re-activates theselocal T cells and potently induces tumor destruction. By con-trast, the therapeutic effects of PD-1 and CTLA4 blockaderemain undefined in prostate cancer. It is possible that CRPCcontains less T-cell infiltrates and resists immune checkpointblockade. Chemotherapy and radiotherapy induce immuno-genic tumor cell death, which have an important role in har-monizing antitumor immunity [81]. Thus, it is important todetermine the mechanism of action of immune checkpointblockade on CRPC following chemotherapy and radiotherapy.
Both cancer vaccine and immune checkpoint blockade aimto improve patient’s own immune system to fight cancer. Nev-ertheless, most studies of immunotherapy for prostate cancerare conducted in CRPC patients whose immune response toprostate TAAs (prior to immunotherapy) may continuouslydecline or become almost completely diminished due totumor-induced immune tolerance. Even after a cancer vaccineis given, it is generally difficult to determine the CTL responsesas well as to identify the biomarkers of immune activation [82].Hence, it remains unknown how cancer vaccine exerts specificantitumor T-cell response to modestly improve the overall sur-vival in CRPC patients.
One intriguing treatment strategy is to conduct immuno-therapy earlier on patients with non-metastatic, recurrentprostate cancer [82]. Elevated PSA levels may indicate cancerrecurrence after local therapy. There are several potentialadvantages to treat prostate cancer patients with immunother-apy at an early recurrent stage. For instance, cancer vaccineand immune checkpoint inhibitor boost adaptive immuneresponse, which is generally long-lasting [52]. Moreover, earlytreatment may also provide sufficient time to allow theevolvement of antitumor T-cell response, such as recognizeand attack new tumor antigens that are not found in the vac-cines (known as antigen/epitope spreading or antigen
cascade) [82]. Preclinical and clinical examples of antigenspreading have been found following cancer vaccines, andantigen spreading post vaccination is presumably induced byproteins released from dead tumor cells and presented to newT-cell clones [83].
Five-year viewFour years ago, therapeutic vaccine sipuleucel-T was approvedby FDA for patients with CRPC. Since then, four other treat-ment options for CRPC have become available, which may ele-vate the expectations in therapeutic effects of new vaccines.PROSTVAC-VF vaccine has delivered clinical benefit in pre-liminary trials, especially for CRPC patients exhibiting an indo-lent disease course. In the coming years, further clinical trialswill determine the role of PROSTVAC-VF in the treatment ofprostate cancer patients. Moreover, combination therapies willdrive the full potential of cancer immunotherapy. In theory,ipilimumab and nivolumab should also block the inhibitoryimmune checkpoints during cancer vaccination. No doubt,new trials will be attempted to determine the therapeutic effectsof combining immune checkpoint blockade and prostatecancer vaccines.
Much has been discovered about immune cell activation, dif-ferentiation and function. By using TRAMP and other mousemodels, researchers have also investigated the critical molecularmechanisms of antitumor immunity [77,84], the treatment strate-gies [85] and the effects of combination therapies on prostatecancer [86,87]. It remains challenging to establish suitable mousemodels to entirely mimic human cancer progression. Forinstance, by compounding mutations observed in human dis-ease (e.g., loss of PTEN and overexpression of Myc), micedevelop prostate cancer but rarely achieve the invasive diseasestate. Nevertheless, experiments based on these models haveprovided essential information on prostate cancer progres-sion [88]. Researchers need to find the best way of utilizing pre-clinical models along with the advances in immunology toinvent innovative immunotherapy for clinical use.
New knowledge is emerging at accelerated rates in the tumorimmunology field. For instance, many therapeutic targets formodulating immune responses are initially identified in vitro.Zhou et al. recently suggested that targets for immune modula-tion can be systematically discovered in vivo [89]. MemoryT cells are considered as ‘good’ cells for clearing malignantcells. Gattinoni et al. indicated that Wnt signaling promotedthe generation of self-renewing multipotent CD8+ memorystem cells, which exhibited proliferative and antitumor capaci-ties exceeding those of memory T cells [90]. Immunotherapyhas already become a standard treatment for cancer. Undoubt-edly, the efficacy of cancer immunotherapy for prostate cancerwill be further improved.
Financial & competing interests disclosure
This work was supported by American Heart Association Grant
11SDG7690000 and Fondation de la Recherche en Transplantation
Grant IIG201101. The authors have no relevant affiliations or financial
Molecular insights into the development of T cell-based immunotherapy for prostate cancer Review
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involvement with any organization or entity with a financial interest in
or financial conflict with the subject matter or materials discussed in the
manuscript. This includes employment, consultancies, honoraria, stock
ownership or options, expert testimony, grants or patents received or pend-
ing, or royalties.
No writing assistance was utilized in the production of this manuscript.
Key issues
• Prostate cancers, as abnormal tissues, contain tumor-associated antigens that can be recognized by human T cells.
• T cells control the quality and quantity of adaptive immune responses and depend on antigen-presenting cells to pick up and present
cognate antigens to them.
• T-cell activation leads to increased expression of cytotoxic T-lymphocyte antigen 4, which is an immune checkpoint protein transmitting
inhibitory signal to T cells.
• The development of immune checkpoint in tumors may be a consequence of antitumor immunity. IFN-g produced by infiltrating CD8+
T cells induces PD-L1 expression in tumors.
• Prostate cancer vaccine sipuleucel-T contains autologous antigen-presenting cells that are activated ex vivo with a fusion protein linking
prostatic acid phosphatase and GM-CSF.
• PROSTVAC-VF is a viral-based vaccine for prostate cancer. PROSTVAC-VF comprises two vectors, both of which contain a modified PSA
and three T-cell stimulatory molecules.
• Adoptive T-cell therapy involves the infusion of ex vivo manipulated T cells back into the same patient. T cells are either naturally
occurring or engineered with TCR and chimeric antigen receptors.
• Nivolumab combined with ipilimumab induced >80% tumor reduction in 53% of patients with melanoma. Immune-related adverse
events were generally reversible.
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