CANCER RESEARCH | TUMOR BIOLOGYAND IMMUNOLOGY

Exploiting Preexisting Immunity to Enhance OncolyticCancer Immunotherapy A C

Siri T€ahtinen1, Sara Feola1, CristianCapasso1, Netta Laustio1, ChristianneGroeneveldt2, ErkkoO. Yl€osm€aki1,Leena Yl€osm€aki1, Beatriz Martins1, Manlio Fusciello1, Marta Medeot3, Maria Tagliamonte4, Jacopo Chiaro1,Firas Hamdan1, Karita Peltonen1, Tuuli Ranki5, Luigi Buonaguro4, and Vincenzo Cerullo1,6,7

ABSTRACT◥

Because of the high coverage of international vaccination pro-grams, most people worldwide have been vaccinated against com-mon pathogens, leading to acquired pathogen-specific immunitywith a robust memory T-cell repertoire. Although CD8þ antitumorcytotoxic T lymphocytes (CTL) are the preferred effectors of cancerimmunotherapy, CD4þ T-cell help is also required for an optimalantitumor immune response to occur. Hence, we investigatedwhether the pathogen-related CD4þ T-cell memory populationscould be reengaged to support the CTLs, converting a weak primaryantitumor immune response into a stronger secondary one. To thisend, we used our PeptiCRAd technology that consists of an onco-lytic adenovirus coated withMHC-I–restricted tumor-specific pep-tides and developed it further by introducing pathogen-specificMHC-II–restricted peptides. Mice preimmunized with tetanusvaccine were challenged with B16.OVA tumors and treated withthe newly developed hybrid TT-OVA-PeptiCRAd containing bothtetanus toxoid- and tumor-specific peptides. Treatment with the

hybrid PeptiCRAd significantly enhanced antitumor efficacy andinduced TT-specific, CD40 ligand-expressing CD4þ T helpercells and maturation of antigen-presenting cells. Importantly,this approach could be extended to naturally occurring tumorpeptides (both tumor-associated antigens and neoantigens), aswell as to other pathogens beyond tetanus, highlighting theusefulness of this technique to take full advantage of CD4þ

memory T-cell repertoires when designing immunotherapeutictreatment regimens. Finally, the antitumor effect was even moreprominent when combined with the immune checkpoint inhib-itor anti–PD-1, strengthening the rationale behind combinationtherapy with oncolytic viruses.

Significance: These findings establish a novel technology thatenhances oncolytic cancer immunotherapy by capitalizing onpre-acquired immunity to pathogens to convert a weak antitumorimmune response into a much stronger one.

IntroductionProphylactic vaccinations are among the most effective forms

of medical interventions with direct clinical and health economicbenefits, with the eradication of common deadly infectious diseasesbeing the most obvious example (1). Most vaccines rely on the useof attenuated pathogens or parts of them and evoke a robust T-cellmemory repertoire directed against the pathogen, with the CD4þ

T cells dominating the memory response (2, 3). Following immu-nization, na€�ve T cells differentiate into T effector memory (TEM)

cells that are rapidly re-called by encountering the antigen, and intoT central memory (TCM) cells that are found mainly in lymphoidorgans and are not immediately triggered in response to patho-gens (4). A strong repertoire of memory T cells against pathogensincluded in the national vaccine programs exists in the worldwidepopulation (5).

The efficacy of cancer immunotherapy relies on the generation ofspecific antitumor CD8þ T cells that recognize peptides presented onthe MHC-I (6). Effective antitumor activity requires fast T-cell medi-ated responses, which is highlighted for example by clinical successwith the chimeric antigen receptor (CAR) T cells targeting CD19 in B-cell malignancies (7). Importantly, it has been shown that the coop-eration of CD4þ and CD8þ T cells is required for efficient antitumorimmunity to occur (8). Indeed, CD4þ T cells provide signals thatimprove the functionality of CD8þ T cells within the tumor micro-environment (TME; ref. 9) and their depletion prior to tumor chal-lenge results in complete loss of tumor rejection in murine tumormodels (10).

Although the central role of CD4þ T cells in T-cell mediatedimmunity is well recognized, it is still unclear how to optimallyutilize the interplay between CD4þ and CD8þ T-cell populations incancer treatment strategies (8). To this end, our aim was toinvestigate how to exploit the pathogen-specific T-cell memoryreservoir, mainly CD4þ T cells, to strengthen the antitumor CD8þ

CTL response.We utilized our PeptiCRAd platform that is based on oncolytic

adenovirus coated with MHC-specific peptides (11) to evaluate theeffect of reengagement of pathogen-specific CD4þ memory T cells onantitumor CD8þ T-cell responses in mice preimmunized with vac-cines specific for human pathogens. Our hypothesis was that antigen-

1Drug Research Program ImmunoViroTherapy Lab (IVT), Faculty of PharmacyiCAN Digital Precision Cancer Medicine Flagship, Helsinki University, Viikinkaari5E, Finland. 2Faculty of Science, Leiden University, Leiden, the Netherlands.3Department of Pharmaceutical and Pharmacological Sciences, University ofPadova, Padova, Italy. 4Cancer Immunoregulatory, Istituto Nazionale Tumori(IRCCS) G. Pascale, Naples, Italy. 5ValoTherapeutics, Helsinki, Finland. 6HelsinkiInstitute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland.7Department of Molecular Medicine and Medical Biotechnology, Naples Univer-sity “Federico II”, Naples, Italy.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

S. T€ahtinen and S. Feola contributed equally to this article.

Corresponding Author: Vincenzo Cerullo, University of Helsinki, P.O. Box 56,Helsinki 00790, Finland. Phone: 3585-0318-5754; Fax: 3589-1912-5610; E-mail:vincenzo.cerullo@helsinki.fi

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doi: 10.1158/0008-5472.CAN-19-2062

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presenting cells (APC) would process the virus and tumor- andpathogen-specific peptides linked to its surface and present thetumor-specific epitopes to CD8þ T cells and the pathogen-specificepitopes to memory CD4þ T cells that would then sustain the CD8þ

T cell–mediated immune response as a bystander effect (12).We investigated the feasibility of our approach in na€�ve or tetanus-

preimmunized immunocompetent mice engrafted with B16.OVAtumors. Mice were treated with intratumoral injections of PeptiCRAdcoated with SIINFEKL (CD8þ T cell epitope of chicken ovalbumin)and tetanus toxoid (CD4þ T-cell epitope) peptides. As hypothesized,we observed a superior antitumor response in mice preimmunizedwith the tetanus vaccine and treated with TT-OVA-PeptiCRAd.Interestingly, in na€�ve mice, the superiority of TT-OVA-PeptiCRAdover control treatments was lost, highlighting the prerequisite of thepreexisting immunity to exploit the CD4þ T memory. We validatedthis strategy by targeting different pathogens (Diphtheria and Pertus-sis), by targeting different tumor antigens (both tumor-associatedantigens and tumor neoantigens) and by combining them with acheckpoint inhibitor treatment (anti–PD-1 antibody). Consistent withour previous results, engagement of CD4þ T cells by Diphtheria-Pertussis–specific MHC-II–restricted peptides resulted in a slowertumor growth in preimmunized mice. In addition, a more robusteffector memory CD4þ T-cell infiltration was observed in the TME oftreated animals when compared with control animals. This responsecorrelated with the level of CD8þ antigen-specific tumor-infiltratinglymphocytes (TIL) and the level of tumor growth control. These resultsindicated that the proposed mechanism of action is not restricted totetanus, but the principle could be applied to other vaccineformulations.

Thus, our data suggest that the preexisting CD4þ memory T-cellrepertoire can be exploited to support the antitumor CTL response;moreover, our findings contribute to the knowledge on how togenerate an optimal T-cell response against tumors, which is the keyfor the next major improvement in cancer immunotherapy.

Materials and MethodsStudy design

The main goal of this study is revoking the CD4þ T-cell anti-pathogen memory repertoire to boost the antitumor response. First,our hypothesis was verified in tetanus immunized B16.OVA bearingmice compared with the na€�ve. To demonstrate that the use of thememory repertoire gave an advantage over the na€�ve, the miceimmunologic background was examined. Subsequently, we validat-ed our hypothesis using a clinically relevant tumor peptide incombination with immune checkpoint inhibitor. Finally, the exper-iment was repeated with a different type of vaccine, offering aconceptual framework. The control and treatments groups arespecified in the figure legends. Animal number for each study typewas determined by the investigators (each treatment group had notless of n ¼ 8 mice). Animals were randomly allocated to the controland the treatment groups.

Cell lines and reagentsThe cell line B16.OVA, a mouse melanoma cell line expressing

chicken ovalbumin (OVA), was kindly provided by Professor RichardVile (Mayo Clinic, Rochester, MN) and cultured according toATCC recommendations. The cells were cultured in RPMI1640with low glucose and supplemented with 10% FBS, 1% antibiotics,1% L-glutamine, and 10%geneticin (G418). The cells were cultivated in37�C, 5% CO2 in a humidified atmosphere. The cell line JAWS II were

purchased from ATCC and cultured in a-MEM supplemented with20% FBS, 1% antibiotics, 1% L-glutamine in presence of 5 ng/mL ofGM-CSF.

All cell lines were cultured under appropriate conditions and wereroutinely tested for Mycoplasma contamination.

The following peptides purchased from Ontores BiotechnologiesCo. Ltd. were used throughout the study:

KKKKKKSIINFEKL (OVA),KKKKKKSVYDFFVWL (TRP2), KK-KKKQYIKANSKFIGITEL (Tetanus toxin), KKKKARYVSQQTR-ANPNPY (Pertussis), KKKKIQSKRFAPLYAVEAK (PolioMahoney),KKKKKKSPVYVGNGVHANLHV (Diphtheria), KKKKKKPVFA-GANYAAWAVNVAQVI (Diphtheria), ARYVSQQTRANPNPY(Pertussis), IQSKRFAPLYAVEAK (Polio Mahoney), SPVYVGNGV-HANLHV (Diphtheria), KKKKKKLCPGNKYEM (B16M27.2).

The peptide B16M27.2 was generated from the MHC-I–restrictedtumor neoantigen B16M27 (REGVELCPGNKYEMRRHGTTHSL-VIHD; ref. 13); the IEDB algorithm tool was used to predict the exactepitope that binds H2Db and contains the single point mutation.

Preimmunization of miceFor tetanus and diphtheria-tetanus-polio-pertussis vaccination,

4- to 6-week-old female C57BL/6 mice received a primary intra-muscular vaccination of Anatetall (GlaxoSmithKline: 8 IU in 100mL) or PolioBoostrix (GlaxoSmithKline: Diphtheria Toxoid 0.4 IUin 100 mL, Tetanus Toxoid 4 IU in 100 mL, Bordetella pertussisantigens: Pertussis Toxoid 1.6 mg in 100 mL, Hemagglutinin 1.6 mgin 100 mL and Pertactin 0.5 mg in 100 mL), respectively, admin-istered bilaterally into the quadricep muscle (50 mL per leg). Anintramuscular booster vaccination (50 mL) was administered twiceat an interval of two weeks. Mouse IgG antibody responses totetanus toxoid and diphtheria were measured by ELISA (XpressBio). Serum from immunized mice was harvested 5 days after thelast immunization and prior to the animal experiment.

PeptiCRAd complex formationOncolytic adenovirus and each epitope with a polyK tail (Ontores)

were mixed to prepare the PeptiCRAd complex. We mixed polyK-extended epitopes with Ad-5-D24-CpG for 15 minutes at roomtemperature prior to treatments with the PeptiCRAd complexes. Moredetails about the stability and formation of the complex can be found inour previous study (11).

Animal experiments and ethical permitsAll animal experiments were reviewed and approved by the Exper-

imental Animal Committee of the University of Helsinki and theProvincial Government of Southern Finland (license number ESAVI/9817/04.10.07/2016).

Four- to 6-week-old female C57BL/6JOlaHsd mice were obtainedfrom Envigo Laboratory. A total of 3 � 105 B16.OVA cells wereinjected subcutaneously into the right flank. Details about the scheduleof the treatment can be found in the figure legends. Viral dose was 1�109 vp/tumor complexed with 20 mg of a single peptide or with 10 mgþ10 mgmixture of two peptides. Intratumorally administrated Anatetallvaccine was dosed at 2 IU permouse. Checkpoint inhibitors were givenintraperitoneally at a dose of 100 mg/mouse.

Splenocyte restimulation assaySplenocytes from tumor-bearing mice treated with different treat-

ments regiments were harvested at the end of the experiment andprocessed into single-cell suspensions, after which, the splenocyteswere cocultured with TT peptide (QYIKANSKFIGITEL)-pulsed

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JAWSII cells for 6 hours in the presence of Brefeldin A (eBioscience).After restimulation, the cells were washed, fixed, and stained and thedata were acquired using BDLSRFORTESSA flow cytometer.

Flow cytometry analysisThe antibodies used are the following: TruStain Fcblock and anti-

CD8-FITC (eBioscience), Affymetrix (Thermo Fisher Scientific),Foxp3-PE (eBioscience), CD4-PeCy7 (eBioscience), CD3-PerCPCy5.5(eBioscience), IFNg-APC (eBioscience), CD40L-BV650 (BD Bios-ciences Bel Art Scienceware (Thermo Fisher Scientific), IFNg-FITC(BD), IL17A-PE (BD), CD4-PerCPCy5.5 (BD), IL4-APC (BD), CD44-V450 (BD), CD44-PE (eBioscience), CD4-PeCy7 (eBioscience), CD3-PerCPCy5.5 (eBioscience), CD62L-APC (eBioscience), CCR7-V450(BD), CD11c-FITC (BD), B220-PE (eBioscience), MHC-II(A-I/E-I)-PeCy7 (eBioscience), CD86-V450 (BD), CD40-APC (eBioscience),CD11b-PerCP-Cy5.5F4/80BV650 (BD), H-2Kb SVYDFFVWL-APC(ProImmune), CD8a-FITC (ProImmune). The data were acquiredusing BDLSRFORTESSA flow cytometer.

The data were analyzed calculating the ratio between the percentageof cells and the tumor volume.

Data were analyzed using FlowJo software v9.

IFNg ELISPOTIFNg ELISPOT assays were performed using a commercially avail-

able mouse ELISPOT reagent set (ImmunoSpot) and 20 ng/mL of eachpeptide was tested in in vitro stimulations of splenocytes at 37�C for72 hours. Spots were counted using an ELISPOT reader system(ImmunoSpot).

Data and materials availabilityAll data associated with this study are present in the article or in

Supplementary Materials. These data are available by request fromDrug Research Program ImmunoViroTherapy lab, University ofHelsinki (Helsinki, Finland).

Statistical analysisStatistical analysis was performed using Graphpad Prism 6.0

software (Graphpad Software Inc.). For animal experiment, two-way ANOVA with Tukey multiple comparisons test was used andP < 0.05 was considered statistically significant. All results areexpressed as the mean � SEM. Details about the statistical testsfor each experiment can be found in the corresponding figurelegend.

ResultsPreimmunization with tetanus vaccine boosts the antitumorresponse of a double-coated PeptiCRAd

We assessed the potential of engaging the CD4þ T-cell memory tothe concept of the PeptiCRAd vaccine platform (11, 14, 15) where wecoated an oncolytic adenovirus with both MHC-I–restricted tumor-specific peptides and MHC-II–restricted pathogen-specific peptides,and studied the effect in tumor-bearing mice preimmunized for thepathogen (Fig. 1A). Our hypothesis was that by adding the MHC-II–restricted pathogen-specific peptides to the PeptiCRAd platform, wewould provide a swifter and stronger T helper response, enhancing thetumor-specific CTL response.

First, we investigated the antitumor effect of PeptiCRAd in micepreimmunized with tetanus vaccine intramuscularly and bearing B16.OVA tumors, a melanoma model expressing chicken OVA as a modelantigen (16). The OVA-epitope was selected because it has a high

immunogenicity and hence provides a suitable model to analyze thegeneration of T-cell response (17). C57BL/6 mice were immunizedwith tetanus vaccine three times at 2-week intervals (Fig. 1B). Fiveweeks after the priming, serum samples were collected from mice andanti-tetanus antibody titer was measured to confirm the success of thevaccination (Fig. 1B; Supplementary Fig. S1A).

After tumor engraftment, mice were randomized and treated withPeptiCRAd coated with tumor-specific peptides (OVA-PeptiCRAd),tetanus-specific peptides (TT-PeptiCRAd), or both tetanus and OVApeptides (TT-OVA-PeptiCRAd). In addition, tetanus vaccine alone orin combination with OVA-PeptiCRAd was used to assess whetherintratumorally administrated commercial vaccine can affect tumorgrowth. All treatments were all delivered by intratumoral adminis-tration according to the regimen depicted in the Fig. 1B.

Following therapy, TT-OVA-PeptiCRAd was superior to either oneof the single coated viruses in controlling the tumor growth in micepreimmunized with tetanus toxoid vaccine (Fig. 1C), suggesting thatthe anti-tetanus memory response indeed enhances the primaryimmune response elicited against the OVA antigen. The ability ofTT-coated PeptiCRAd to elicit mainly Th1-polarized CD4þ T-cellresponses was further corroborated by intracellular staining (Supple-mentary Fig. S1B). Less surprisingly, the approach worked also whenthe tetanus vaccine was reintroduced as a combination with OVA-PeptiCRAd (vaccine þ OVA-PeptiCRAd), whereas tetanus vaccinealone had no therapeutic efficacy (Fig. 1D). Notably, when comparingvaccine þ OVA-PeptiCRAd to OVA-PeptiCRAd, the latter showed asignificantly higher antitumor efficacy (P ¼ 0.05). This suggests thatthe effect was not caused by the adjuvant contained in the vaccine itselfbut rather by the presentation of tetanus-specific peptides onMHC-II,engaging CD4þ T cells to help the cytotoxic CD8þ T-cell response.

Interestingly, when the same experiment was performed in na€�vemice (mice that had not been preimmunized with tetanus vaccine), nostatistically significant differences were observed between OVA-PeptiCRAd and TT-OVA-PeptiCRAd (Fig. 1E).

These results demonstrate that the antitumor efficacy of our virus-based PeptiCRAd cancer nanovaccine is significantly enhanced if it issimultaneously coated also with peptides that are specific for apathogen for which a preexisting immunity exists.

The tetanus-specific memory response favorably shapes theimmune environment at the tumor site

To gain a deeper understanding of themode of action of the double-coated PeptiCRAd, wewanted to investigate the quality of the immuneresponse elicited by the different treatments. To this end, we analyzedthe frequency of different cell populations in the tumor by flowcytometry, most importantly the activated dendritic cells (DC), CD4þ

and CD8þ T cells with effector and memory phenotype and experi-enced and exhausted CD8þ effector T cells.

Interestingly, we found increased frequency of both total andactivated intratumoral DCs in all of the groups that had been treatedwith PeptiCRAd in the context of tetanus antigens (either coatedwith the TT peptide or coinjected with the whole vaccine; Fig. 2A;Supplementary Fig. S2). In contrast to these combination treatments,the use of vaccine alone led to poor induction of DCmaturation in theTME, suggesting that inclusion of an adenoviral adjuvant may becritical for a proper DC activation in this setting. Moreover, we sawincreased levels of CD4þ and CD8þ T cells in the tumors in all groupsof mice treated with PeptiCRAd (Fig. 2B and C), which is well in linewith what has previously been observed following treatments withvirus-based drugs (11, 14, 15, 18). Finally, we wanted to analyze thephenotype of these T cells. Majority of the TILs showed a T effector

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memory cell phenotype, with an increase in the frequency ofCD8þ andCD4þ TEMs in groups treated with TT-PeptiCRAd and OVA-TT-PeptiCRAd (Fig. 2D and E). Moreover, the expression level of T-cellimmunoglobulin and mucin-domain containing-3 (TIM3) on PD-1þ

TILs were assessed to study T-cell exhaustion (19–21). Interestingly,we observed a significantly lower frequency of exhausted CD8þ T cellsin the group ofmice treatedwith TT-OVA-PeptiCRAd comparedwiththe other groups, indicating that CD4þ T-cell help is required foroptimal CD8þ T-cell activity (Fig. 2F). We concluded that the tetanuspreexisting immunity improved the overall efficacy of the treatmentsubstantially bymodifying the immune environment at the tumor site,especially when the treatment was virus based and contained thetetanus vaccine or the tetanus peptides. Of note, the serotype 5 humanadenovirus used in these experiments is non-oncolytic in murinetumors, and therefore the effect on tumor control is solely based onantitumor immune response. To better elucidate this phenomenon, wereanalyzed all the datasets by stratifying the mice between respondersand non-responders and assessed again their immunologic responses.As expected, we observed a significant difference between the twogroups. Irrespective of the type of therapy, all responders hadan ongoing measurable immune response, highlighting the impor-tance of the immune system in controlling the tumor growth, regard-

less of what kind of treatment they had received. Importantly, themajority of these responders were found in the group of mice treatedwith TT-OVA-PeptiCRAd (Figs. 2A–F and 3A–E).

CD40L expressing TT-specific, Th1-polarized CD4þ T cells aredetected in secondary lymphoid organs following TT-OVA-PeptiCRAd therapy

To dissect the possible mechanism of the observed therapeuticefficacy, we assessed levels and phenotype of immune cells in second-ary lymphoid organs of preimmunizedmice. As expected, PeptiCRAd-treated mice showed expansion of CD4þ T-cell compartment both inthe spleen and in the draining lymph nodes (Fig. 4A; SupplementaryFig. S3A). More importantly, a significant increase of TT-specificCD4þ T cells expressing CD40 ligand (CD40L) was observed inTT-OVA–treated mice (Fig. 4B; Supplementary Fig. S3B). Majorityof these CD40Lþ cells were polarized toward Th1 phenotype, albeitsome TT-specific Foxp3þ T regulatory cells (Treg) were also detected(Supplementary Fig. S3C and S3D). Analysis of dLNs revealed that theintratumoral vaccination with TT-OVA-PeptiCRAd induced mainlyIFNg producing Th1memory cells in the expense of IL4 secreting Th2cells, whereas no differences was observed in IL17A producing Th17cells (Supplementary Fig. S3E–S3H). Because CD4þ T cell–associated

Figure 1.

Effect of recalling memory repertoire on murine model of melanoma. A, A schematic representation of the new hybrid PeptiCRAd system. A single adenovirus isloadedwith pathogen-specific peptides to evoke the preexistingmemory T-cell repertoire andwith tumor-specific peptides to evoke the antitumor T-cell repertoire.B, Treatment scheme. A total of 3� 105 B16.OVA cellswere injected into the right flank of na€�ve and tetanus preimmunized C57BL/6mice (n¼ 7–8). Treatmentsweregiven intratumorally four times (on days 9, 11, 13, and 15) as indicated in the figure. C–E, The B16.OVA tumor growth was followed until the end of the experiment inna€�ve and preimmunizedmice. The tumor size is presented as themean for each treatment� SEM and the statistical difference is shown in figure (statistical analysistwo-way ANOVA; � , P < 0.05; ��� , P < 0.001; ���� , P < 0.0001; ns, nonsignificant).

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CD40L has been shown to be important in stimulating cytotoxic CD8þ

T-cell responses (22, 23), we wanted to study whether we can seeCD40þ antigen-presenting cells. Indeed, when preimmunized micewere intratumorally treated with TT-OVA-PeptiCRAd, a significantlyhigher frequency of CD40þ expressing APCs was detected (Fig. 4C),further suggesting that double-coated PeptiCRAd stimulates TT-specific CD4þ memory T cells, that in turn could license professionalAPCs via CD40–CD40L interaction.

Combination with immune checkpoint inhibitors increases thenumber of responders and leads to complete tumor rejection

We have previously shown that combination of tumor-targetedPeptiCRAd with immune checkpoint inhibitors is synergistic in termsof improved antitumor efficacy (8). Thus, we wanted to assess whetherthe vaccine-induced preexisting immunity would further enhance thissynergy, particularly by increasing the frequency ofmice responding tothe therapy.

To test this hypothesis, we coated the virus with TT and tyrosinaserelated protein 2 (TRP2) peptides (TRP2180-188; ref. 24), which isnaturally occurring melanoma-associated antigen and hence moreclinically relevant epitope than OVA. Tetanus toxoid preimmunizedmice were implanted with subcutaneous tumors and treated intratu-morally with a PeptiCRAd coated with TRP2 peptides only (TRP2-PeptiCRAd) or with a PeptiCRAd coated with both TRP2 and TTpeptides (TT-TRP2-PeptiCRAd; Fig. 5A). Similarly, as in Fig. 1,we observed a significant inhibition of tumor growth in mice treatedwith the double-coated virus compared either to mock or to TRP2-PeptiCRAd groups (Fig. 5B).

Interestingly, when we combined the PeptiCRAd treatments with aPD-1 blocking mAb, we observed a significant increase in efficacy ofboth TRP2-PeptiCRAd and TT-TRP2-PeptiCRAd treatments

(Fig. 5C and D). However, the double-coated PeptiCRAd was stillmore effective than the virus coated with a single peptide in terms oftumor growth control (Supplementary Fig. S4A).

More importantly, inclusion of TT-specific peptides in the cancernanovaccine resulted in 75% response rate to anti–PD-1, whereas only28% of mice treated with TRP2-PeptiCRAd and PD-1 blockadeexperienced a complete tumor eradication (Fig. 5E; SupplementaryFig. S4B).One of the biggest advantages of combining oncolytic viruseswith checkpoint inhibitors is that the viruses in the tumor facilitate andincrease the T lymphocyte recruitment, thereby unleashing an unprec-edented activity of the monoclonal antibodies (25). Along this line, weobserved a significant increase in total and TRP-2–specific CD8þ TILsin mice treated with TT-TRP2-PeptiCRAd, when compared with thecontrol treatments (Fig. 5F and G).

To further validate the model in a more therapeutically relevantsetting, we used a previously published, MHC-I restricted neoanti-gen B16-M27 as the tumor target (13) and double-coating Pepti-CRAd with the predicted B16-M27 short peptide together with thetetanus toxoid peptide. Following intratumoral treatment, signifi-cant tumor growth suppression was observed in TT-B16M27PeptiCRAd treated mice compared with B16M27 monocoatedPeptiCRAd mice (Fig. 5H), demonstrating that the platform canalso be applied to potentiate therapies based on induction ofneoantigen-specific T cells.

The preexisting immunity is a general mechanism to enhancethe antitumor response and reshapes the immunologic balancein T-cell repertoire

Because we observed that preexisting immunity to tetanus toxoidpotentiates the antitumor response of a double-coated PeptiCRAdalone and in combination with PD-1 blockade, we sought to further

Figure 2.

Immune cell component withinthe TME in preimmunized miceafter treatment. Flow cytometryanalysis of the tumor samples col-lected from mice preimmunizedwith tetanus at the end of theexperiment. The data are plottedas bar graphs and single valuesfor responders (green) and notresponders (black) are shown.A–E, The frequency of activatedDCs (A), CD8þ (B) and CD4þ T cells(C), and CD8þ and CD4þ effectormemory (CD44þCD62L�; D and E)T cells within the TME is reported(statistical analysis Kruskal–Wallistest ANOVA). F, Flow cytometryanalysis of the activation/exhaus-tion profile of the CD8þ T cellsin the tumors. The bar graph depictsgMFI mean of CD8þ T cells thatare antigen experienced (PD1þ)and exhausted (TIM3þ). Signifi-cance was assessed by two-tailedunpaired Student t test; � , P < 0.05;ns, nonsignificant.

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Figure 3.

Single tumor growth and immune cell componentwithin the TME.A, Tumor growth curve for eachmouse and one graph for each group are reportedwith the specifictreatment indicated in each graph. Tumor volumes were normalized against the values on the day of the first treatment and are presented as mean of percentage�SEM. The percentage displayed next to each graph shows the responders (green), defined as mice with a tumor volume lower than 400% (dashed line). Flowcytometry analysis of DC activation (B) and total CD8þ T cells (C), CD4þ T cells (D) and effector memory (CD44þCD62L�) CD4þ T cells (E) within the TME arereported for individual mice in green (responders) and black (nonresponders) among each group. The frequency of all the analyzed cell types was significantlyhigher in the responders compared with nonresponders. Significance was assessed by two tailed unpaired t test with Welch correction for DC activation andfor CD8þ and CD4þ T-cell infiltration analysis, and by two tailed unpaired t test for the effector memory T-cell infiltration. � , P ≤ 0.05; �� , P ≤ 0.01.

Figure 4.

Phenotype of immune cells in splenocytes. Splenocytes collected from preimmunizedmice were analyzed by flow cytometry to assess the level of TT-specific CD4þ

(A), TT-specific CD4þ expressing CD40L (B), and APCs exhibiting CD40 receptor (C). Statistical analysis ordinary one-way ANOVA; �� , P < 0.005; ���� , P < 0.0001.

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investigate whether our approach is valid also in the context oftetravalent vaccine. Polioboostrix is a tetravalent vaccine with ahigh coverage of 85% of infants immunized, making it an attractivestudy model (26). C57BL/6 mice were preimmunized with Polioboos-trix vaccine with the same immunization regime as before (Fig. 6A).Serum samples and splenocytes were collected and analyzed in order toconfirm the effectiveness in the immunization protocol. Tetravalentvaccine was found to efficiently generate both antibodies and CD4þT-cell specific for pertussis and diphtheria (Supplementary Fig. S5A–S5C). For the tumor growth analysis, B16.OVA tumors in na€�ve orpreimmunized mice were treated with anti–PD-1 antibody and Pep-tiCRAd coatedwithMHC-II–restrictedDiphtheria–Pertussis peptidesand MHC-I–restricted TRP2 peptides (DP-TRP2-PeptiCRAd). Con-sistent with our previous results, a superior antitumor response wasdetected in preimmunized treated with DP-TRP2-peptiCRAd andanti-PD1, whereas treatment efficacy was lost in na€�ve mice (Fig. 6B).

These results confirm that the pathogen-specific preexisting immu-nity can enhance the antitumor response and that the mechanism ofaction is dependent on the memory T cells. Moreover, this effect is not

restricted to tetanus but is adaptable to other pathogens as well. Tofurther verify that the mechanism of action behind the enhancedtreatment efficacy using diphtheria and pertussis as the preimmuniz-ing vaccine, we analyzed the T-cell repertoire of the tumor draininglymph nodes, TME, and spleen. The frequency of na€�ve CD8þ T andCD4þ T cells was lower in the draining lymph nodes of the pre-immunized, DP-TRP2 PeptiCRAd-treated mice compared with thecontrol groups (Fig. 6C). Concomitantly, increased levels of CD4þ

TEM cells were observed in the draining lymph nodes and in the TMEof preimmunized mice compared with the na€�ve and mock treatedmice (Fig. 6D). In addition, a significant higher infiltration of TRP2-specific CD8þ T cells was seen in the tumor tissue of the immunizedmice when compared with the na€�ve mice (Supplementary Fig. S5D),and the level of CD4þTEM cells in the tumor and draining lymphnodesstrongly correlated with the intensity of the TRP2-specific TILresponse. Taken together, the double-coated PeptiCRAd vaccineplatform can be used to stimulate preacquired, pathogen-specificCD4þ T-cell immunity to help the generation of effective antitumorCD8þ T-cell responses.

Figure 5.

Synergistic effect between hybrid PeptiCRAd and aPD1. A and C, Treatment scheme. A total of 3 � 105 B16.OVA cells were injected into the right flank ofC57BL/6 mice (n ¼ 7–8) preimmunized with tetanus and the treatments were initiated on established tumors with either the hybrid PeptiCRAd only (A) or acombination with anti–PD-1 antibody (C). B and D,The tumor growth curve for mice treated without (B) or with (D) anti–PD-1 is represented as mean� SEM. E,Complete responses (i.e., the disappearance of the total tumor mass upon treatment) for each group is depicted as the percentage of responders from alltreated mice in a single group as well as the ratio of responding individuals to nonresponding individuals in a single group. F and G, Flow cytometry analysis ofCD8þ T cells (F) and of TRP2-specific CD8þ T cells (G) in the tumor from mice treated with TRP2-PeptiCRAd and TT-TRP2-PeptiCRAd. The results aredisplayed as a single dot for each individual. The control groups that received no peptide vaccine (mock and anti–PD-1 only) were pooled and are indicated as“no peptide”. H, B16.OVA bearing mice (n ¼ 7–8) were treated intratumorally four times (on days 9, 11, 13, and 15) with PeptiCRAd coated with the neoantigenB16.M27 with (TT-B16M27 PeptiCRAd) and without (B16M27 PeptiCRAd) tetanus toxoid peptide. Statistical analysis was assessed by two-way ANOVA withuncorrected Fisher LSD (B), Tukey multiple comparison test (D) and ordinary one-way ANOVA (F and G), two-way ANOVA (H). � , P ≤ 0.05; �� , P ≤ 0.01;��� , P ≤ 0.01; ���� , P ≤ 0.0001.

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DiscussionVaccines lead to the formation of an immunologic memory that is

able to deploy a much faster and more effective immune responsewhen reencountering the pathogen; in fact, the primary immuneresponse is rather weak and slow while the secondary immuneresponse is faster and more effective (27). When cancer vaccines(e.g., peptides, nanoparticles, virus-like particles) are used as thera-peutic vaccines and not as prophylactic vaccines, the immune response

observed after treatment is usually more similar to a primary than asecondary one. Among cancer vaccine strategies, oncolytic viruses arestrongly reemerging as leading biological drugs used to synergize withcheckpoint inhibitors given their ability to trigger tumor-specificT-cellresponses (18, 28, 29). The idea of utilizing mainly MHC-I–restrictedpeptides as vaccine regimens to induce antitumor CD8þ T-cellresponse has dominated the field of cancer immunotherapy. Onlyuntil recently, more attention has been directed toward exploiting the

Figure 6.

Hybrid PeptiCRAd and aPD1 effects in the context of tetravalent vaccine.A,A total of 3� 105 B16.OVA cells were injected into the right flank of C57BL/6mice (n¼ 8)preimmunized with PolioBoostrix vaccine. Treatments were initiated on established tumors (9 days after tumor implantation) and the mice were treated four timeswith DP-TRP2-PeptiCRAd (on days 9, 11, 13, and 15) and three times with aPD-1 (on days 9, 13, and 17). B, The tumor volume is depicted as mean � SEM (statisticalanalysis two-way ANOVA with Tukey multiple comparisons test). C, The level of na€�ve CD8þ and CD4þ (CD44�CD62Lþ) T cells in tumor draining lymph nodes ofna€�ve or preimmunized mice is reported. Statistical analysis, unpaired Student t test two tailed; � , P < 0.05; ��, P < 0.005; ���, P < 0.001; ���� , P < 0.0001. D, Effectormemory (CD44þCD62L�) CD4þ T cells in tumor draining lymph nodes and tumor is shown. Statistical analysis ordinary one-way ANOVA with Tukey multiplecomparison test. � , P ≤ 0.05; �� , P ≤ 0.01; ��� , P ≤ 0.001.

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immunologic “help” provided by CD4þ T-cell population (12, 30).Indeed, the CD4þ T cells are required to shape the response and thememory of cytotoxic CD8þ T cells (12, 31), “highlighting the potentialof CD4þ T cells as a tool for cancer immunotherapy” (32). Never-theless, only few studies until now have actively exploited the prom-ising interplay between CD4þ and CD8þ cells to elicit a strong andmore effective antitumor response (8).

Here, we investigated the cross-talk between CD8þ and CD4þ

T cells within the TME and evaluated the strategy of exploitingthe memory repertoire of CD4þ T cells to mount a fast and reliableantitumor response. To this end, we describe a new cancer immu-notherapy approach that takes advantage of the preexisting path-ogen-specific immunologic memory present in the worldwidepopulation of vaccinated individuals. To increase the antitumorresponse, we modulated the interplay between CD4þ and CD8þ

T cells within the TME by using our PeptiCRAd platform, whichis based on oncolytic adenovirus coated with MHC-restrictedpeptides. PeptiCRAd platform was further developed here into ahybrid treatment engaging not only the antitumor CTL but also theCD4þ help for an optimally shaped and robust enough antitumorimmune response to occur. The feasibility of our strategy wasdemonstrated and validated in melanoma using tetanus and polio-boostrix vaccines available for humans, highlighting the universalnature of the CD4þ memory in boosting cancer-specific CTLresponses.

One of the most important aspects of the hybrid PeptiCRAdsystem is that when DCs process the virus and the peptides attachedonto its surface, the DCs present not only tumor-specific peptides toCD8þ T cells to trigger the antitumor immune response, butimportantly they also present pathogen-specific peptides to CD4þ

T helper cells that potentiate and sustain the cytotoxic immuneresponse. Following intratumoral therapy with double-coated Pep-tiCRAd, we were able to detect increased levels of TT-specific Th1CD4þ cells expressing CD40L, which has been previously shown toengage its receptor CD40 on APCs (23), leading to licensing andmaturation of these cells. Indeed, we saw an increase in infiltrationand activation of DC within the TME in the mice that had the bestcontrol of the tumor growth, which is in line with the notion thatDCs play a major role in linking the innate and the adaptiveimmune responses and they are capable of stimulating the T cellswithin TME (33). These results are in line with a recent clinicalstudy (34) showing that preconditioning with tetanus–diphtheriatoxoid is sufficient to enhance the DC migration and improveantitumor response in patients with glioblastoma by activatingpathogen-specific CD4 T cells. Moreover, consistently with thehigh DC activation, a significantly higher frequency of effectormemory CD4þ and CD8þ TILs as well as activated CD8þ T cellswith lower expression levels of exhaustion marker TIM3 were seenin responding mice compared with the nonresponders. This high-lights the importance of the interplay between the innate andadaptive arm of the immune system as well as the key role ofeffector memory CD4þ T cells in supporting the ongoing antitumorresponse.

The use of immune checkpoint inhibitors (ICI) to block theinteraction between the immune checkpoint receptors and theirligands is worldwide accepted as the breakthrough of the cancerimmunotherapy field with anti–CTLA-4 (ipilimumab) anti–PD-1(nivolumab and pembrolizumab), and anti–PD-L1 (atezolizumab)agents approved for the treatment of several indications, includingmelanoma (35, 36). The concept has revolutionized the way canceris treated, and in some cases, has completely changed the life

expectancy of cancer patients. It has become very clear that thepresence of CD8þ T lymphocytes within the tumor is a criticalparameter, necessary but not sufficient, to predict patient's positiveoverall response even with ICI therapies (36, 37). However,many cancer types are resistant to ICI treatments or developresistance over time (38). To this end, the use of oncolytic virusesin combination with ICIs has been proposed as an effective strategy,as oncolytic viruses possess a natural ability to induce beneficialchanges in the TME (39). Indeed, T-VEC, the first oncolytic therapyapproved for metastatic melanoma, has been tested in combinationwith ICI (18, 40). In these studies, T-VEC modulated the T-cellinfiltration, promoting increased frequency of CD8þ T cells inpatients that responded to the combination therapy (28). Oncolyticvaccines that actively promote T-cell activation and tumor infil-tration have regained an enormous momentum, leading to theinflux of public and private investments (18, 41). However, simplyinducing a T-cell response without having control over the qualityof this response might not be sufficient in many cases.

Our hybrid PeptiCRAd treatment is based on an oncolyticadenovirus and thus induces the infiltration of TILs to tumors, anability common to several oncolytic viruses, which have been shownto mount an pro-inflammatory response that can result in thegeneration and infiltration of effector T cells in both virus-injectedand distant tumors (28, 42). However, in contrast to these moretraditional oncolytic virus strategies, our technology also effectivelyexploits the interplay between CD4þ and CD8þ T cells and engagesthe pathogen-specific CD4þ memory effector T cells to the fightagainst cancer. As a clear increase of CD8þ TIL infiltration wasobserved when hybrid PeptiCRAd was used as a single treatment, acombination treatment with ICI was a logical next step in thisresearch. Indeed, irrespective of the identity of the pathogen pep-tides or the tumor peptides loaded onto the hybrid PeptiCRAd, thecombination therapy with ICI clearly showed an advantage over thesingle treatments, resulting in the complete response rate of 75% inthe group treated with anti–PD-1 and TT-TRP2-PeptiCRAd. Thisis a notable improvement in treatment efficacy, especially consid-ering that PeptiCRAd is based on human adenovirus 5, which doesnot replicate in or lyse murine tumor cells, unlike other oncolyticviruses that have been reported to increase response rates to ICI inmouse models (28, 43–45).

The localization of the antitumor T cells to the tumor border hasbeen recognized as a central aspect in successful immunothera-py (38). Further, CD4þ T cells have a role in licensing DCsvia CD40-CD40L pathway to prime tumor-specific CTLs in lym-phoid organs and in establishing a durable antitumor immuni-ty (31, 46). Indeed, upon treatment with hybrid PeptiCRAd, wefound a high level of CD4þ TEM cells in the TME and in the draininglymph nodes that positively correlated with the frequency of TRP2-specific CD8þ TILs in preimmunized mice. These results furthercorroborate that the immunologic profile after preimmunizationand hybrid PeptiCRAd treatment resembles a secondary responserather than a primary one that usually is seen with oncolyticvaccines.

In summary, we described for the first time how to modulate theinterplay between CD4þ and CD8þ T cells by using our PeptiCRAdplatform and demonstrated the importance of the CD4þ T-cellmemory repertoire specific for a pathogen antigen in enhancing theCD8þ T-cell antitumor response in cancer immunotherapy. Finally,our technology significantly increased the antitumor efficacy of anti–PD-1, conferring a rationale for a combination therapy of the hybridPeptiCRAd with checkpoint inhibitors.

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Disclosure of Potential Conflicts of InterestS. T€ahtinen is a postdoctoral research fellow at Genentech. C. Capasso is a

consultant at Valo Therapeutics. V. Cerullo is the co-funder and shareholder at andhas ownership interest (including patents) in Valo Therapeutics. No potentialconflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: S. T€ahtinen, S. Feola, C. Capasso, L. Buonaguro, V. CerulloDevelopment of methodology: S. T€ahtinen, S. FeolaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): S. T€ahtinen, S. Feola, N. Laustio, M. Fusciello, M. MedeotAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. T€ahtinen, S. Feola, C. Capasso, C. Groeneveldt,E.O. Yl€osm€aki, L. Yl€osm€aki, B. Martins, M. Tagliamonte, K. Peltonen, V. CerulloWriting, review, and/or revision of the manuscript: S. T€ahtinen, S. Feola,E.O. Yl€osm€aki, L. Yl€osm€aki, J. Chiaro, F. Hamdan, K. Peltonen, T. Ranki, V. CerulloAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): S. Feola, B. Martins, M. Fusciello, M. Medeot, J. Chiaro,F. HamdanStudy supervision: S. T€ahtinen, S. Feola, V. Cerullo

AcknowledgmentsWe thank all the participants for their support and advice. Moreover, the

flow cytometry analysis was performed at the HiLife Flow Cytometry Unit,University of Helsinki, and the animal experiment carried out at the LaboratoryAnimal Center (LAC) of the University of Helsinki. This work has been supportedby European Research Council under the European Union's Horizon 2020Framework programme (H2020)/ERC-CoG-2015 Grant Agreement n. 681219,Helsinki Institute of Life Science (HiLIFE), Jane and Aatos Erkko Foundation(decision 19072019), Orion Research Foundation, Jalmari and Rauha AhokasFoundation, Maud Kuistila Foundation, and Cancer Society of Finland(Sy€op€aj€arjest€ot).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 5, 2019; revised December 6, 2019; accepted February 24, 2020;published first February 27, 2020.

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