Combination strategies for enhancing the efficacy of immunotherapy in cancer patients

Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Thymosins in Health and Disease

Combination strategies for enhancing the efficacy ofimmunotherapy in cancer patients

Federica Moschella, Enrico Proietti, Imerio Capone, and Filippo BelardelliDepartment of Cell Biology and Neurosciences, Istituto Superiore di Sanita, Rome, Italy

Address for correspondence: Filippo Belardelli, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita,Viale Regina Elena 299, 00161 Rome, Italy. [email protected]

A major challenge in cancer immunotherapy is the identification of effective strategies for enhancing its clinicalefficacy. One approach is based on adjuvants capable of breaking tolerance against tumor-associated antigens.Interferon-α (IFN-α), an antiviral cytokine with a long record of clinical use, has recently been shown to act as aneffective adjuvant in cancer patients. Notably, a special interest is currently focused on the use of dendritic cells (DC)generated in the presence of IFN-α (IFN-DC) for the preparation of anticancer vaccines. An additional approachfor enhancing the response to immunotherapy relies on its combination with chemotherapy. In fact, an ensembleof results from both studies in animal models and pilot clinical trials suggest that certain chemotherapeutic agentscan act, under defined conditions, as strong adjuvants for enhancing the efficacy of immunotherapy. These resultsopen new opportunities for designing mechanism-based combination therapies involving both chemotherapy andnew-generation cancer vaccines, including IFN-DC-based vaccines.

Keywords: immunotherapy; cancer; chemoimmunotherapy; IFN-alpha; cyclophosphamide

Introduction

Immunotherapy represents a valuable approach,complementary to standard therapies, for the treat-ment of cancer, based on its specificity and lim-ited associated toxicity.1 Nevertheless, in spiteof the significant progress achieved in understand-ing the basic immunologic mechanisms responsiblefor the antitumor efficacy of immunotherapy in an-imal models, there has been a poor translation intothe clinical setting and currently no vaccination reg-imen is indicated as a standard anticancer therapy.2

In fact, although the immune response can play asignificant role in controlling tumor growth, as sug-gested by the correlation between clinical outcomeand tumor infiltration by T cells,3 tumor cells tend toevade the immune system through different mecha-nisms, including: (a) the recruitment of suppressivecells of the adaptive and innate immune system, suchas regulatory T cells (Tregs)4 and myeloid-derivedsuppressor cells,5 and (b) the selection of tumor es-cape variants, characterized by loss of expression oftumor associated antigens (TAAs), downregulation

of MHC/costimulatory molecules and secretion ofimmunosuppressive factors.

Over the last decade, many studies, focused onunderstanding the mechanisms of immunoregula-tion and the dynamics of host–tumor interactions,have led to the design of novel strategies capa-ble of breaking tumor-induced tolerance, thus im-proving the immunological and clinical response tocancer immunotherapy. These approaches, whichincluded depletion of Tregs and blockade of thenegative signaling receptor CTLA4, while keepingthe promise of overcoming tumor-induced toler-ance, have shown considerable toxicity in terms ofautoimmune or autoinflammatory side effects.6,7

In this paper, the most recent therapeutic strate-gies devised to overcome tumor resistance toimmunotherapy and characterized by a tolera-ble associated toxicity are reviewed, with specialemphasis on the combination of immunotherapywith chemotherapy and on the use of interferon-�(IFN-�) both as a vaccine adjuvant and as an in-ducer of dendritic cells (DCs) to be used in cancervaccination.

doi: 10.1111/j.1749-6632.2010.05464.xAnn. N.Y. Acad. Sci. 1194 (2010) 169–178 c© 2010 New York Academy of Sciences. 169

IFN-α or chemotherapy to boost cancer immunotherapy Moschella et al.

The use of IFN-α as a vaccine adjuvant and inthe generation of novel DC-based cancervaccinesScreening of tumor gene expression libraries us-ing patients’ sera and cancer-specific T cells, alongwith genomic and proteomic analysis of tumor cells,has brought to the discovery of a large number ofTAAs, providing the opportunity to evaluate nu-merous anticancer vaccines in different malignantdiseases. Nevertheless, peptide vaccines adminis-tered with incomplete Freund’s adjuvant usually arenot sufficiently immunogenic to elicit a robust and,more importantly, a clinically efficacious antitumorimmune response in the absence of exogenous cy-tokines. Furthermore, antitumor vaccines requirethe combination with potent adjuvants able to acti-vate the innate immune system.

Today, new attention is given to IFN-� as an im-portant factor modulating both the innate and theadaptive immunity.8 IFN-� are multifunctional cy-tokines belonging to type I IFNs and exerting a vari-ety of biological effects, ranging from the inhibitionof virus replication to the reduction of tumor cellgrowth. All this justified its intense use for the treat-ment of viral diseases, such as hepatitis C, as wellas of different types of hematologic malignanciesand solid tumors, including chronic myelogenousleukemia, Kaposi’s sarcoma, renal carcinoma, andmelanoma.

Early studies in mouse tumor models showed thatmice transplanted with syngeneic highly metastatictumors had significantly increased survival timeswhen treated with IFN-�/�.9,10 Similar results wereobtained when IFN was administered to mice trans-planted with either IFN-sensitive or IFN-resistantsyngeneic tumor cells, showing that a major rolefor the generation of a long-lasting antitumor re-sponse was played by the effects of type I IFNson host immune cells.9,10 New knowledge on thesemechanisms came from studies carried out withgenetically modified tumor cells releasing type IIFNs. By using different experimental tumor mod-els, it was shown that IFN-�-secreting tumor cellswere efficiently rejected by an IFN-induced host-mediated immune response and elicited an anti-tumor immunity against the subsequent challengewith metastatic parental cells (reviewed in Ref. 11).Noteworthy, tumor rejection required an intact im-mune system, as it did not occur in immunode-ficient mice.11 Moreover, tumor rejection strictly

depended on the induction of CD8+ memory Tcells, as also suggested by the expansion of memory-phenotype CD44hi CD8+ T cells observed in bothspleen and lymph nodes following the injection ofIFN-�-producing tumor cells12 as well as by a recentstudy on the IFN adjuvant activity in a melanomamodel of peptide vaccination.13 IFN-� was alsoshown to play a role in the polarization of T-helpercells toward a Th-1 subtype, as well as in the induc-tion of in vivo proliferation and long-term survivalof CD8+ T cells in response to specific antigens (re-viewed in Ref. 10).

A different set of studies carried out in our lab-oratory led to the comprehension of the effects ex-erted by IFN-� on the differentiation/activation ofDCs, suggesting that such effects may play a ma-jor role in the induction of IFN-induced antitu-mor immunity. In particular, IFN-� was shown topromote the rapid differentiation of granulocyte-macrophage colony-stimulating factor (GM-CSF)-treated human monocytes into DCs endowed withpotent functional activities (IFN-DC).14 IFN-DCsshowed the phenotype of partially mature DCswith regard to the expression of surface mark-ers and migratory response to chemokines.14 Awealth of studies, elucidating the distinct featuresof IFN-DCs as compared to conventional imma-ture DCs (differentiated with GM-CSF and IL-4),suggested that these cells can represent a valuabletool to be used in cancer immunotherapy.15 In fact,IFN-DCs exhibit a strong migratory behavior andinduce a potent primary antibody response andIFN-� production after injection into severe com-bined immunodeficient (SCID) mice reconstitutedwith human peripheral blood leukocytes (PBL).16

Remarkably, IFN-DCs also express TRAIL (TNF-related apoptosis-inducing ligand),14 and specif-ically kill and internalize TRAIL-sensitive tumorcell lines.17 Moreover, IFN-DCs were shown to bemore potent than conventional IL-4-DCs in stim-ulating a protective antiviral CTL response, whenpulsed with inactivated HIV-1 and used to immu-nize hu-PBL-SCID mice,18 and in inducing cross-priming of CD8+ T cells against exogenous viralantigens.19

The ensemble of these studies, corroborated bythe formal demonstration that type I IFNs pos-sess strong in vivo adjuvant activity, markedly en-hancing the antibody response and immunologi-cal memory against a poorly immunogenic soluble

170 Ann. N.Y. Acad. Sci. 1194 (2010) 169–178 c© 2010 New York Academy of Sciences.

Moschella et al. IFN-α or chemotherapy to boost cancer immunotherapy

protein,20 paved the way for the clinical evaluation ofthe potentialities of IFN-� as a vaccine adjuvant.21

Although a clinical trial employing this rationale,conducted in healthy individuals vaccinated againsthepatitis B virus (HBV), did not show a vaccineadjuvant activity of IFN-�,22 the results of a morerecent study demonstrated that this cytokine canpotentiate the antibody response to HBV vaccine inhemodialysis patients.23 Thus, it is possible to spec-ulate that IFN-� can act as an effective immunopo-tentiator especially in immunocompromised sub-jects, as cancer patients can be.

A recent pilot phase I-II clinical trial promoted byour group, aimed at assessing the immune adjuvanteffects of IFN-� administered in combination withpeptide-based vaccination in stage IV melanomapatients, showed a consistent enhancement of cir-culating CD8+ T cells able to recognize the vaccinepeptides and MART-1+gp100+ melanoma cells.24

This vaccination regimen induced an increasedfrequency of CD45RA+CCR7− (terminally differ-entiated effectors) and CD45RA−CCR7− (effectormemory) cells and significantly augmented the per-centage of CD14+ monocytes, and particularly ofthe CD14+CD16+ cell fraction, expressing high lev-els of CD40 and CD86.24 The results of this pi-lot trial, along with the recent observation that inmelanoma patients treated with high-dose IFN-�2bthere was a statistically significant correlation be-tween the development of autoimmune manifesta-tions and the improvement of relapse-free time andoverall survival,25 represent an important startingpoint for considering a new clinical use of this cy-tokine aimed at potentiating the effectiveness of can-cer vaccines. In this regard, it should be mentioned,however, that a multicenter phase II trial, assessingthe immunological and clinical efficacy of multiepi-tope melanoma vaccines alone or in combinationwith high-dose IFN-� (or GM-CSF), showed that,although there was a trend toward an augmentationof the immunologic response to the vaccine by IFN-� (the immune response rate was 43.2% with IFNtreatment in comparison with 25% without IFN),this difference was not statistically significant.26 Theapparently different results obtained in these twostudies might be attributed to the differences in IFNregimens, pointing out to the critical importanceexerted by dosage and timing of administration ofIFN-�, with respect to the vaccine, in influencingboth clinical and immunological outcome. Thus, it

is possible to speculate that although in our pilotstudy24 3 MU of IFN-�, administered in close spa-tial and temporal proximity to the peptide vaccine,efficiently activated DCs and T cells, the systemicadministration of high-dose IFN-�2b (10 MU/m2

three times a week) used in the study performedby Kirkwood’s group26 could have induced somecounterregulatory mechanisms limiting the overallimmune response. Moreover, in the first study pa-tients received natural leukocyte IFN (Alfaferone),which is a mixture of different subtypes of type I IFNand is therefore more similar to the IFN producedby the immune system, while the second study wasconducted with a recombinant cytokine (IFN-�2b).Another important factor to be considered is thatnot all patients may be equally prone to respondto IFN-�, highlighting the need for the identifica-tion of predictive markers that permit selection ofpatients who are most likely to benefit from IFN-�administration.27

Rationale for combining chemotherapy andimmunotherapy to enhance antitumorimmunityA different strategy to augment the antitumor effi-cacy of immunotherapy is represented by its com-bination with chemotherapy. Although these twotreatments have been generally regarded as antag-onistic options, because of the putative immuno-suppressive properties of chemotherapy, which isindeed cytotoxic for all dividing cells (includinglymphocytes), evidence of the immunopotentiat-ing ability of chemotherapy stemmed from severalearly studies on cyclophosphamide (CTX) (Fig. 1).CTX is a DNA alkylating agent, which has beenextensively used to treat hematological as well assolid malignancies. When given at an appropri-ate time, this antineoplastic agent was shown toaugment immunity rather than suppressing it.28

A pivotal study showed that the combination ofCTX with the adoptive transfer of tumor infiltrat-ing lymphocytes (TIL) mediated the elimination ofliver and lung metastases in tumor-bearing mice,while single treatments had a little impact on tu-mor growth.29 Further studies demonstrated thatCTX could enhance the effectiveness of adoptiveimmunotherapy also in CTX-resistant tumor mod-els and that the potent antitumor efficacy dependedon the removal of suppressor T cells.30 Notably,the combination of CTX with thymosin alpha 1

Ann. N.Y. Acad. Sci. 1194 (2010) 169–178 c© 2010 New York Academy of Sciences. 171


Figure 1. Selected reports highlighting the mechanisms whereby cyclophosphamide (CTX) enhances the antitumorefficacy of immunotherapy in animal models.

(T�1) and IFN-�/� demonstrated a synergistic an-titumor efficacy in mice bearing Lewis lung car-cinoma,31 Friend leukemia cells (FLC)32 and B16melanoma tumors,33 through an immune-mediatedmechanism involving NK, CD4+, and CD8+

T cells.In the following years several reports have shown

that some chemotherapeutic treatments (reviewedin Ref. 34), as well as irradiation,35 dependingon the dosage and timing of administration, candisplay either immunosuppressive or immunopo-tentiating effects and can augment the antitumorefficacy of both adoptive36,37 and active im-munotherapy.38 It is generally believed that thepotent therapeutic efficacy induced by the com-bination of chemotherapy with immunotherapydepends on homeostatic mechanisms, acting dur-ing the recovery phase that follows chemotherapy-mediated myelo-lymphodepletion that, as a by-stander effect, promote antitumor immunity.36,39

An early study from our group demonstrated thata single injection of CTX, followed by the adop-tive transfer of antitumor immune cells, could cureand prevent metastases of mice bearing four dif-

ferent highly metastatic tumors and that this effectdepended on the production of hitherto uncharac-terized soluble factors, which may sustain the prolif-eration, survival, and activity of transferred cells.36

Of note, the antitumor efficacy was abolished whenmice were treated with antibodies against IFN-�/�.A further study clarified that CTX treatment in-duces the expression of type I IFN in vivo, lead-ing to the expansion of CD4+ and CD8+ T cellsexhibiting a memory (CD44hi) phenotype.40 Therole played by CTX in favoring memory T cellswas later confirmed by the finding that prolongedlow-dose (metronomic) chemotherapy, despite di-minishing the number of proliferating tumor-specific CTLs, preserved CD43lo memory CD8+

T cells.41

Of interest, CTX (along with doxorubicin andpaclitaxel) was shown to enhance the antitumorimmune response of GM-CSF-secreting whole-cellvaccines in HER-2/neu tolerized mice, through amechanism involving the amplification of a Th-1neu-specific T-cell response.38 A Th-2 to Th-1 shiftin cytokine production in tumor-bearing animalstreated with low-dose chemotherapy has been also



described in a rat metastatic lymphoma model.42

Of note, two reports, showing that the administra-tion of CTX reduces the number43 and inhibits thefunctions44 of Tregs in tumor-bearing animals, rep-resented the formal proof that this treatment is ableto circumvent immune tolerance induced by tumorsand to tip the balance toward an effective antitumorimmunity.

In a recent study,45 we have shown that the syner-gism of chemotherapy and adoptive immunother-apy was dependent on CD4+ T cells and on thecooperation of transferred cells with the host im-mune system. Furthermore, CTX promoted themigration of specific tumor-immune lymphocytesto the tumor bed and induced the homeostaticproliferation/activation of transferred B and Tlymphocytes.45 A first characterization of the molec-ular mechanisms underlying the immunomodu-latory effects of CTX came from the analysis ofcytokine gene expression in the bone marrow oftreated mice. Optimal therapeutic responses to theadoptive transfer of immune cells were found tobe associated with the chemotherapy-mediated in-duction of a “cytokine storm” (including GM-CSF,IL-1 �, IL-7, IL-15, IL-2, IL-21, and IFN-�) occur-ring during the rebound phase after drug-inducedmyelo-lymphodepletion.45 Notably, the importanceof the increased expression of homeostatic cytokines(IL-7 and IL-15) was also suggested by the find-ing that the antitumor efficacy of the combina-tion of a sublethal total body irradiation (TBI)with the adoptive transfer of CD8+ T cells was im-paired in mice deficient of both cytokines.46 Wehave recently started a more extensive character-ization of CTX-mediated immunomodulation bymicroarray analysis of the changes in gene expres-sion induced by this drug in the bone marrow,spleen and PBLs of tumor-bearing mice. This studydemonstrated that CTX induces the upregulationof several genes involved in the regulation of theimmune response, such as chemokines, factors in-volved in the inflammatory and innate immuneresponses and genes modulating lymphocyte pro-liferation and activation. Of note, the modulationof gene expression is an early and transient phe-nomenon following chemotherapy, being activatedalready 1 day after treatment and finished by day5 (Moschella et al., unpublished results). Theseresults point out that the immunotherapeutic in-tervention should be performed early after treat-

ment, to consent to the chemotherapy-induced “cy-tokine storm” to shape and enhance the antitumorimmunity.

Taken together these reports proved that CTXrepresents a strong modulator of the adaptive im-mune response. More recent studies underscoredthe ability of this agent to influence also the innateimmune system. CTX is, in fact, able to increasethe number and the activation status of myeloidDCs through the induction of IFN-� and other in-flammatory cytokines.47 In addition, Radojcic andcolleagues found that CTX, through its myelosup-pressive action, perturbs DC homeostasis, leadingto the emergence of tumor-infiltrating DCs secret-ing more IL-12 and less IL-10, compared to thosefrom untreated tumor-bearing animals, and there-fore able to prime T-cell responses but not prolifer-ation of Tregs.48

Although chemotherapy-mediated apoptosis hashistorically been considered nonimmunogenic,hence not able to elicit an immune response, re-cent advances suggest that certain cytotoxic drugscan induce an immunogenic cell death dictated byspecific signals of danger.49 In fact, treatment witha selected panel of chemotherapeutic agents caninduce the release or exposure of tumor antigensas well as of damage-associated molecular patterns(DAMPs) (such as heat shock proteins, calretic-ulin, and HMGB1), that in turn determine apop-totic cell uptake by DC, maturation of DCs, tu-mor Ag cross-presentation, and ultimately T-cellactivation.50 These mechanisms have been exten-sively elucidated for anthracyclins51 and more stud-ies are needed to assess whether they take place alsoin the case of alkylating agents. The ensemble ofthese studies suggests that the postchemotherapyphase of immune system reconstitution may pro-vide a unique opportunity for immunotherapeuticintervention, given that the chemotherapy-inducedimmunomodulation may redirect the immune re-sponse from tumor-induced tolerance toward effec-tive immunity.

Moving toward a mechanism-based design ofchemoimmunotherapy clinical trialsThe results obtained in animal models led to thedesign of new clinical trials assessing the safety andefficacy of the combination of chemotherapy andimmunotherapy in cancer patients. Nevertheless,clinical research in chemoimmunotherapy is still in



its infancy, and the best schedules and modalities ofcombination are still under investigation.

An early clinical trial in melanoma patientsshowed that pretreatment with CTX could signifi-cantly augment the DTH response to an autologoustumor cell vaccine.52 Of note, two patients receiv-ing the combined therapy experienced prolongeddisease-free survival. In the 1990s there were severalclinical trials based on the combination of low-doseCTX and vaccination in melanoma patients.53–55

The vaccine formulations used in these studies com-prised autologous irradiated tumor cells, known tu-mor antigens, and polyvalent allogeneic vaccines. Inall cases, no toxicity was observed and the combinedtreatment was able to induce DTH as well as hu-moral and cellular immune response to the vaccine.Although a trend toward clinical efficacy was ob-served in these studies, the small number of patients,the lack of a control group, and of randomizationdid not allow to draw definite conclusions. In a morerecent phase II clinical trial, the combination of low-dose CTX with an allogeneic vaccine (Melacine) andIFN-� was analyzed in metastatic melanoma pa-tients. Despite a low objective response rate (10.2%),this combination therapy showed tolerability anddisease stabilization for at least 16 weeks in 64%of patients.56 Further studies in melanoma patientssuggested that also in humans one of the princi-pal mechanisms accounting for the improvement ofthe antitumor response is the decrease in the num-ber of Tregs57 and suppression of their inhibitoryfunctions.58 The most effective combination ther-apy trial conducted so far was performed by Rosen-berg’s group, that reported an impressive objectiveclinical responses (51%) in metastatic melanomapatients receiving the adoptive transfer of in vitroexpanded autologous TILs, following nonmyeloab-lative conditioning therapy with fludarabine andCTX.59 Lymphodepletion is supposed to representone of the hallmarks of this approach and seems toplay a crucial role for response to immunotherapy.In fact, when the intensity of lymphodepletion wasincreased with the addition of either 2 or 12 Gy TBIto chemotherapy, the response rates were 52% and72%, respectively.60

In addition to melanoma, the combination ofCTX and immunotherapy has been exploited in sev-eral other tumors. A pilot study in prostate cancerpatients showed that 6 out of 16 patients, subjectedto chemoimmunotherapy after prostatectomy, had a

greater than 50% prolongation of prostate-specificdoubling time, which is a marker of disease pro-gression.61 A Phase II study in breast cancer pa-tients, comparing intravenous versus oral CTX ad-ministration to augment the efficacy of TheratopeSTn-KLH vaccine, showed a survival benefit onlyfor patients receiving the intravenous treatment.62

In nonsmall-cell lung cancer, the combination ofEGF-based cancer vaccine with low-dose CTX re-sulted in a marginal increased antibody response ascompared to the vaccine alone.63 Although the ob-served differences were not statistically significant,this study led to a second trial in which chemoim-munotherapy was given before and after standardfirst line chemotherapy. Remarkably, 92% of pa-tients showed an immunodominant antibody re-sponse against EGF and, more importantly, the sur-vival of patients correlated positively with antibodytiters.64 CTX was also shown to improve the efficacyof an allogeneic DC-based vaccine in patients withmetastatic renal cell carcinoma.65

The ensemble of these studies, while demonstrat-ing the feasibility of chemoimmunotherapy for can-cer treatment and the improvement of antitumorimmunity, did not establish yet its clinical efficacy.This may depend on the fact that patients with ad-vanced diseases, who have received multiple regi-mens of different chemotherapeutic agents, presenta severe impairment of their immune system. Thus,it may be advantageous to combine vaccine withchemotherapy earlier in the disease process, whenthe immune system is still highly functional, or inthe adjuvant setting after tumor resection. In or-der to combine immunotherapy with the standardtreatment of melanoma following tumor resection,we have recently performed a pilot clinical studyaimed at evaluating the safety and the antitumor im-mune response of the combination of dacarbazine(DTIC) and peptide-based vaccination in disease-free patients.66 Preclinical experience had shownthat also DTIC, which is a less toxic alkylating agentthan CTX, is able to potentiate the response to im-munotherapy in mouse tumor models (our unpub-lished observations). In our pilot study,66 a strong exvivo expansion of peptide-specific CD8+ T cells dis-playing a long-lasting effector memory phenotypeand ability to specifically lyse HLA-A2+/Melan-A+

tumor cell lines was observed only in patients pre-treated with DTIC with respect to patients treatedwith the vaccine alone. Global transcriptional



Figure 2. Schematic representation of the scenario of events that could be exploited in the mechanism-based designof a trial of combination of chemotherapy and intratumoral injection of IFN-DC. The scheme represents the rationaleof a phase I clinical trial in melanoma patients, recently designed by our group. Patients will be first treated withchemotherapy that, as described in the text, should exert both a systemic effect on the patient and a local effecton the tumor mass. Chemotherapy should, in fact, promote a “cytokine storm” responsible for the homeostaticproliferation and activation of memory T and B cells and simultaneously induce the selective reduction of regulatoryT cells (Treg). At the same time the direct cytotoxic activity of chemotherapy should induce apoptosis and releaseof tumor antigens, altogether creating a suitable environment for the subsequent immunotherapeutic intervention.Following chemotherapy preconditioning of the patient, autologous IFN-DC will be injected directly at the tumorsite, where, given their powerful ability to take up apoptotic bodies and cross-present tumor antigens to CD8+ Tcells, they should lead to the activation of an effective antitumor immune response.

analysis carried out on patient peripheral bloodmononuclear cells (PBMC) revealed that DTIC in-duced, already one day after chemotherapy, an in-creased expression of immunoregulatory factors,that can account for the enhancement of tumorantigen-specific CD8+ T-cell responses. Notably, themost impressive expansion of antitumor effectormemory CD8+ T cells was observed in patients whodid not show tumor recurrence.66

Conclusions and perspectives

The results reviewed herein show that combina-tion therapies based on the use of IFN-� and/orchemotherapy in association with cancer vaccineshave the potential to overcome tumor-induced im-

munosuppression and to significantly alter the out-come of immunotherapy, provided that the designof combination relies on the knowledge of the effectsof both treatments in cancer patients. The encour-aging results obtained in our pilot clinical trial66 hasnow led to the design of a phase II study, aimedat assessing the immunological correlates as well asthe clinical efficacy of the combination of DTIC andpeptide-based vaccination in stage III/IV disease-free melanoma patients. Following the rationale il-lustrated in this review, vaccine peptides will beadministered in conjunction with 6 MU IFN-� asimmune adjuvant and will be injected one day afterchemotherapy (i.e., at the time of “cytokine storm”).

In addition to the use of type I IFNs as an adjuvantto be injected together with a soluble vaccine, the



aforementioned knowledge on the effects of IFN-�on DC biology could be exploited for the develop-ment of novel strategies of active immunotherapy.Therefore, a phase I clinical study will shortly beactivated by our group to evaluate the potential an-titumor efficacy of the combination of chemother-apy and intratumoral injection of IFN-DC. Therationale besides this idea stemming from preclin-ical studies67 is based on the particular efficiencyof IFN-DC in taking up apoptotic bodies, that aresupposed to be produced locally as a consequenceof chemotherapy, and cross-presenting tumor anti-gens to CD8+ T cells, as illustrated in Figure 2. Theimmunological results and clinical outcome of thesetwo studies will hopefully contribute to a betterunderstanding of the mechanisms whereby IFN-�and/or chemotherapy may overcome tumor resis-tance to immunotherapy, thus guiding the furtherdevelopment of mechanism-based design of moreeffective combination strategies.

Acknowledgments

The work in the authors’ laboratory was partiallysupported by grants from the Italian Association forResearch against Cancer (AIRC) and from the ItalianMinistry of Health (Integrated Project on Oncologyand “ISS Project for Alliance against Cancer”). Weare grateful to Anna Ferrigno, Cinzia Gasparrini,and Angela Fresolone for secretarial assistance.

Conflicts of interest

The authors declare no conflicts of interest.

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Combination strategies for enhancing the efficacy of immunotherapy in cancer patients

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