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In Focus

Cancer Vaccines: Steering T Cells Down the Right Path to Eradicate Tumors Patrick A. Ott and Catherine J. Wu

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts. Broad Institute of MIT and Harvard, Cambridge, Massachusetts. Harvard Medical School, Boston, Massachusetts.Corresponding Author: Catherine J. Wu, Dana-Farber Cancer Institute, Dana 520C, 44 Binney Street, Boston, MA 02115. Phone: 617-632-5943; Fax: 617-632-6380; E-mail: cwu@partners.orgdoi: 10.1158/2159-8290.CD-18-1357©2019 American Association for Cancer Research.

Summary: Lack of tumor T-cell infiltration is a barrier to the efficacy of immune checkpoint inhibitors and other immunotherapies in patients with cancer. Because of their ability to activate and augment tumor-specific T cells, vaccines are an attractive approach to direct T-cell responses into the tumor.

Although the capability of the immune system to spe-cifically recognize and eliminate cancer cells was recognized more than a century ago, effective strategies, such as adoptive T-cell transfer and immune checkpoint blockade, to consist-ently direct and enhance tumor-specific immune responses therapeutically in patients have emerged only recently. Adop-tive transfer of autologous T cells expanded in vitro from tumor-infiltrating lymphocytes (TIL) and genetically engi-neered lymphocytes bearing chimeric antigen receptors have demonstrated compelling clinical efficacy in select cancers (1, 2), and targeting inhibitory receptors including PD-1 and CTLA4 with mAbs, a concept termed immune checkpoint blockade (ICB), has demonstrated efficacy in a wide spectrum of solid and hematologic malignancies (3, 4). Approvals of ICB by regulatory agencies have changed the standard of care and improved outcomes for many patients with cancer. The remarkable clinical efficacy of adoptive cell therapy in B-cell malignancies and the broad activity of PD-1/PD-L1 pathway inhibition provide proof-of-concept for cancer immunother-apy in general and the potential of tumor-specific T cells in eradicating tumors. However, with some exceptions (micros-atellite-instable tumors, Merkel cell carcinoma, desmoplastic melanoma, cutaneous squamous cell carcinoma, and Hodg-kin lymphoma), only up to 30% of patients benefit from the objective responses in most tumor types with proven ICB efficacy, and many malignancies, including highly prevalent ones such as colon and prostate cancers, are largely resistant to this approach.

EFFEcTIVE cAncER IMMunoTHERAPY RELIEs on THE PREsEncE oF T-cELL InFILTRATEs In THE TuMoR

There is increasing evidence that PD-1 pathway inhibi-tion is effective primarily in tumors with preexistent T-cell

infiltration. The observation of high-density CD8+ T-cell infiltration at the invasive margins of metastatic melanoma tumors that regressed after PD-1 inhibition, allowing precise prediction of response to anti–PD-1 therapy, provides per-haps the most direct evidence for the importance of tumor T-cell infiltration for effective immunotherapy in humans (5). Additional evidence supporting tumor T-cell infiltration as a prerequisite for the efficacy of ICB is the association of mRNA signatures of IFNγ-related genes and PD-L1 expression on tumor and immune cells with clinical outcomes (6, 7). The marked increase in T-cell numbers and expression of immune-modulatory molecules in early on-treatment tumor samples of patients with advanced melanoma treated with PD-1 inhibi-tion also supports this concept (8). In patients with advanced melanoma, the numbers of Ki67+CD8+ T cells in the periph-eral blood increased after treatment with the PD-1 inhibitor pembrolizumab. These CD8+ T cells were CD45RAloCD27hi and Eomeshi and T-betlo, indicating an antigen-experienced, exhausted phenotype (9). Ki67+PD-1+CD8+ T cells also increased in the peripheral blood of patients with non–small cell lung cancer who had received PD-1–targeted therapies. Similarly, these peripheral CD8+ T cells expressed markers consistent with an antigen-experienced, effector phenotype (HLA-DR+, CD38+, CD28+, CD27+, ICOS+; ref. 10). These observations indicate that the size and specificity of the T-cell repertoire is shaped by the physiologic interaction between the tumor and the host immune response and is consistent with the mechanism of PD-1 blockade affecting antigen-experienced T cells in the tumor. Mechanisms that can limit the generation of tumor-specific memory effector T cells and their infiltration into a tumor are (i) insufficient T-cell prim-ing; (ii) absence of antigens or dysfunctional antigen presen-tation leading to immunologic ignorance; (iii) suppressive soluble factors or inhibitory immune cell populations leading to immune tolerance; and (iv) vascular factors, chemokines, or extracellular matrix conditions posing barriers to migration of T cells into the tumor (11). Recent work in mouse models and patients with cancer has highlighted the critical role of den-dritic cells (DC) and natural killer (NK) cells in priming and recruiting T cells into the tumor in the context of ICB (12–14).

THERAPEuTIc APPRoAcHEs To InDucE T-cELL InFLAMMATIon In coLD TuMoRs

Therapies that address one, or possibly more, of these rate-limiting steps should have the ability to convert a non–T

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cell–inflamed “cold” tumor into a T cell–inflamed tumor. It is noteworthy that limiting factors are likely different among individuals, emphasizing the importance of biomarkers that can inform which modality will be most beneficial for an indi-vidual patient. Conceptually, several therapeutic modalities can be envisioned given the multitude of mechanisms that can account for the absence of T-cell inflammation. Strate-gies that induce or enhance innate immune response, thereby providing critical signals to activate specific T-cell immune response (secretion of type I IFNs, activation of DCs leading to augmented antigen presentation and T-cell priming, etc.), have been investigated in preclinical models and are being tested in clinical trials in patients with cancer. Examples include the pharmacologic agonists of Toll-like receptors (TLR) and the STING pathway (15, 16). For instance, intratumoral injection of the TLR9 agonist CMP-001 in combination with PD-1 inhi-bition was recently shown to mediate objective tumor responses in patients with advanced melanoma who were resistant to PD-1 blockade (17), and the combination of a different TLR9 agonist, SD-101, and low-dose radiation demonstrated clini-cal efficacy in patients with low-grade B-cell lymphoma (18). In both studies, an increase in CD8+ T cells and upregu-lation of inflammatory genes were seen in post-treatment tumors. Intratumoral administration of oncolytic viruses such as Talimogene laherparepvec (T-VEC) induces both direct tumor cytolysis and an innate immune response, triggering a systemic effect on noninjected metastatic sites (19). In a study of 21 patients with metastatic melanoma combining T-VEC and the PD-1–blocking antibody pembrolizumab, a complete response rate of 33% and overall response rate of 62% suggested a syn-ergistic effect (20); some of these objective responses occurred in patients whose baseline tumors exhibited low CD8+ T-cell density and low or absent IFNγ signatures. Increases in the CD8+ T-cell density and PD-L1 expression in both injected and uninjected tumors were observed, predominantly in patients who responded to therapy. Blockade of oncogenic pathways such as MAPK, WNT–β-catenin, and PI3K/AKT can reverse defects in T-cell recruitment and priming (21, 22). BRAF and/or combined MEK/BRAF inhibition has been shown to mediate upregulation of melanoma antigens and increased CD8+ T-cell infiltration in patients with BRAFV600-mutant melanoma (23). Preclinical and clinical evidence indicates that a number of other therapeutic approaches including chemo-therapy, radiotherapy, agonistic antibodies directed against costimulatory molecules, or the inhibition of VEGF can gener-ate tumor T-cell infiltration (24). Nevertheless, the majority of treatment modalities that mediate anticancer immunity have unintended consequences, such as a decrease in intratumoral T cells, for example, because they are either inherently unspe-cific (e.g., chemotherapy or radiation) or are directed at targets that are central to many other biological functions (e.g., onco-genic pathways or VEGF).

cAncER VAccInEs: An APPEALInG APPRoAcH To DRIVE cAncER-sPEcIFIc EFFEcToR MEMoRY T cELLs InTo TuMoRs

By exposing a patient with cancer to suitable tumor anti-gens in the context of T-cell activation signals, a vaccine

is designed to amplify preexisting tumor-specific T-cell responses and broaden the T-cell repertoire by inducing de novo responses of either naïve or antigen-experienced T cells. Because of the specificity of the approach, vaccination is a particularly attractive tool to achieve infiltration of tumors with the “relevant” T-cell populations, that is, T cells that are specific for the tumor and functional, “armed and ready” to destroy the tumor cell. Ideally, a vaccine-induced tumor-directed immune response will be comprised of long-lasting memory T-cell phenotypes mediating durable clinical tumor responses.

The four components of a cancer vaccine are tumor anti-gens, formulation, immune adjuvants, and delivery vehicles (25), and for decades tumor vaccination was the principal modality in the quest of effective cancer immunotherapy, with much attention on optimizing these components. How-ever, because of the modest clinical efficacy observed in previous large-scale vaccine efforts (26), the overall approach had fallen out of favor, until recently when a new source of tumor antigens became available. The vast majority of previ-ous cancer vaccines have used tumor-associated antigens (TAA), self-antigens which demonstrate tumor-specific or development-specific expression. The development of next-generation sequencing opened up the opportunity to utilize tumor-specific antigens (TSA), such as neoantigens, which arise from somatic cancer mutations. As opposed to TAAs, which also include viral antigens, TSAs are not subject to central tolerance and are therefore more immunogenic.

sYsTEMATIc IDEnTIFIcATIon oF nEoAnTIGEns: sTIMuLATInG THE nEXT GEnERATIon oF cAncER VAccInEs

Neoantigens are generated by cancer mutations and have been implicated as key targets of effective tumor-specific T-cell responses, including the correlation of tumor neoanti-gen load with tumor cytolytic T-cell infiltration and clinical benefit from immunotherapies, the expansion of neoantigen-specific T cells in patients who derive clinical benefit from immunotherapies, and direct evidence of tumor cell killing by neoantigen-specific T cells in preclinical and human studies (27). The vast majority of cancer mutations are unique to an individual tumor. Coupled with the diversity of HLA mol-ecules, an effective neoantigen-directed therapy will therefore require a personal approach. It is also possible to target mul-tiple neoantigens in each patient, given the observed muta-tional loads, which can help meet the challenges of clonal heterogeneity and clonal evolution. Technological advances including rapid and cost-effective next-generation sequenc-ing and improved mass spectrometry–based epitope predic-tion algorithms allow the just-in-time design and production of therapeutic vaccines targeting neoantigens expressed by an individual patient’s tumor. Phase I clinical trials in patients with melanoma have already demonstrated that such a per-sonalized vaccination approach is safe and immunogenic in patients with melanoma, with encouraging signs of clini-cal activity (28–30). Recent studies have demonstrated that tumor mutational burden is not the sole predictive marker of tumor response to ICB and that immune signatures, including

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those of T cells, DCs, and NK cells, are complementary (5, 31, 32). These data support the investigation of vaccine–ICB combinations in tumors with lower mutational burden. Innovative antigen and adjuvant delivery approaches have the potential to substantially enhance the immunogenicity of cancer vaccines. For example, nanoparticle systems can achieve prolonged and controlled release of antigen and adjuvant by exploiting efficient draining of nanocarriers to lymphoid tissues. In a mouse model, codelivery of peptide and adjuvant in a synthetic high-density lipoprotein (LPS) nanodisc induced more than 30-fold higher frequencies of peptide-specific cytotoxic CD8+ T cells compared with the TLR9 agonist combined with Montanide, which had been considered one of the strongest adjuvants currently used in clinical trials (33). Similarly, the delivery of TLRs on polymer scaffolds enhanced DC activation and cytokine production in the draining lymph node and induced up to 100-fold higher numbers of vaccine-specific CD8+ T cells (34). For RNA-based vaccines, the challenge of rapid extracellular degradation of RNA can be overcome by formulating the vaccines in a lipid carrier, thus enabling systemic delivery of a vaccine format that has intrinsic adjuvant properties (35).

VAccInEs InDucE TRAFFIcKInG oF cAncER-sPEcIFIc T-cELL PoPuLATIons InTo TuMoRs, LEADInG To TuMoR cELL KILLInG In PREcLInIcAL MoDELs

In an established B16 melanoma model, TEGVAX, an auto-logous tumor cell vaccine given in combination with GM-CSF and the TLR agonists glucopyranosyl lipid A (TLR4) and resiquimod (TLR7/8), led to significant increases in tumor infiltration with CD4+ and CD8+ T cells compared with con-trol vehicle, and regression of tumors (36). Notably, when the vaccine was combined with an anti–PD-1 antibody, com-plete responses of all tumors were seen in 50% of the mice. In the adoptive T-cell transfer Pmel melanoma model, a peptide vaccine directed against gp100 given in combination with an anti-CD40 antibody, the TLR7 agonist imiquimod, and IL2 mediated tumor regression and trafficking of gp100-specific T cells into the tumor (37). In the MC38 colon cancer model, tumors of C57BL/6 mice vaccinated with synthetic long pep-tides containing MC38-specific neoantigens together with anti-CD40 antibody and poly(I:C), TILs were enriched for CD8+ T cells specific for 3 immunogenic neoepitopes (measured by MHC-I dexamer staining), suggesting the generation and migra-tion of vaccine-specific T-cell responses into the tumor (38). The neoantigen peptide vaccine also inhibited the growth of MC38 tumors in both the prophylactic and therapeutic setting. In a separate study, neoantigen-directed RNA vaccines targeting MHC Class II neoepitopes mediated tumor regression and infil-tration with both CD8+ and CD4+ T cells in melanoma, colon carcinoma, and breast cancer models (39). Lymphocytes from tumors that regressed after immunization with the dominant Class II B16 melanoma neoantigen M30 were reactive against B16-M30, demonstrating that neoantigen vaccination led to trafficking of vaccine epitope–specific T cells into the tumor. Separately, the T-cell response induced by vaccination with multiple Class II epitopes spread to an immunodominant Class I

epitope that was not contained in the vaccine, indicating that antigen-specific CD4+ T cells may promote cross-priming of cytotoxic T-lymphocytes, thus reshaping the repertoire of tumor- specific T-cell responses in the vaccinated host.

VAccInEs cAn MEDIATE TRAFFIcKInG oF VAccInE-sPEcIFIc T cELLs InTo TuMoRs: EARLY cLInIcAL EVIDEncE In PATIEnTs

Although there is compelling evidence in various mouse models that vaccines can steer cancer-specific T cells into the tumor, evidence in patients has only recently emerged. In one study, in which patients with melanoma were vaccinated with RNA encoding personal neoepitopes, lymphocytes infiltrat-ing a lymph-node metastasis from a patient who had received 4 vaccinations were found to be specific for several vaccine epitopes (29). Reactivity against the neoepitope RETSAT (P546S) was confirmed by MHC multimer staining, and the T-cell receptor (TCR) recognizing RETSAT was characterized by single-cell cloning. Notably, CD8+ T cells transduced with the RETSAT TCR demonstrated killing of autologous mela-noma cells derived from postvaccination tumors. These data demonstrate that vaccination can effectively drive tumor-spe-cific and functional T cells into a metastatic site in a human patient with melanoma.

We recently demonstrated that personal neoantigen vaccines in patients with glioblastoma induce trafficking of vaccine-specific T cells into intracranial tumors (40). Single-cell RNA sequencing (scRNA-seq) of CD3+ T cells present in a relapsed tumor sample revealed cytotoxic CD8+ and CD4+ T cells and a phenotype of antigen experience and potentially exhaustion. Testing single CD3+ T cells from relapsed tumor and circulating T cells reactive to vaccine epitopes revealed that TCR sequences from 4 CD4+ T cells and 2 CD8+ T cells were identical between tumor and peripheral blood. Specificity for vaccine epitopes was confirmed for a subset of the shared TCRs by cloning and expression into TCR-deficient Jurkat T cells. Notably, although both shared CD4+ T cells expressed cytotoxicity markers, one of them also coexpressed the inhibitory receptors PD-1, TIM3, and TIGIT. These data provide definitive evidence that vac-cine-induced T cells can traffic into an intracranial tumor in patients with glioblastoma. In another recent study, patients with glioblastoma received two sets of vaccinations (in addi-tion to standard therapy with irradiation and temozolomide): (i) actively personalized vaccines (APVAC1) composed of shared, nonmutated peptides from a premanufactured library that were selected on the basis of mass spectrometry–defined HLA Class I and HLA-DR (Class II) affinity and (ii) preferentially mutated peptides (APVAC2; ref. 41). Approximately 50% of the unmu-tated APVAC1 peptides induced ex vivo CD8+ T-cell responses, whereas >80% of the mutated APVAC2 peptides triggered CD4+ T-cell responses. CD4+ T cells specific for an APVAC1 unmu-tated peptide were detected in a post-treatment tumor sample.

PERsPEcTIVEAs a tool for cancer immunotherapy, vaccines are unique

given their ability to focus the host’s tumor-specific immune response. By exposing a patient to the relevant

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(i.e., tumor-specific) antigens in the context of activation signals, vaccines restore a fundamental mechanism that prevents the host from mounting an effective adaptive immune response against cancer cells (despite their “for-eignness” given their genetic aberrations). As described, there is now robust evidence that this approach has the potential to activate, amplify, and steer the immune response toward the generation of cancer-specific T cells (Fig. 1, inset). Moreover, recent evidence by us and others, as described above, demonstrates that this approach can drive these tumor antigen–specific T cells into the tumor (Fig. 1). However, there are still many barriers to achiev-ing effective tumor cytolysis. Multiple active suppressive mechanisms in the tumor microenvironment counteract effective tumor-specific immune responses and therefore, particularly for the treatment of advanced metastatic can-cers, combinatorial approaches will likely be required. Our own observations of exhausted CD4+ T-cell pheno-types and coexpression of three inhibitory receptors on a vaccine-specific CD4+ T cell in the tumor of a patient with

glioblastoma provide direct evidence that blocking inhibi-tory pathways may be an essential complementary therapy for successful cancer vaccine approaches.

Indeed, the effectiveness of PD-1/PD-L1–directed mono-therapy in subsets of patients with cancer points to this pathway as a dominant suppressive mechanism. Our and others’ anecdotal clinical observations in patients with mela-noma who experienced disease progression after neoanti-gen vaccination, followed by rapid-onset complete tumor regressions after treatment with PD-1 inhibitors, are early clinical signals that the combination of vaccine and PD-1 inhibition could be a powerful therapeutic strategy. Clini-cal trials partnering RNA-based and long-peptide personal neoantigen vaccines with the PD-L1 inhibitors atezolizumab (ClinicalTrial.gov identifier NCT03289962) and nivolumab (ClinicalTrial.gov identifier NCT02897765), respectively, are already ongoing. In the latter of these two studies, a phase Ib clinical trial combining a personal long peptide neoan-tigen vaccine and nivolumab in patients with metastatic melanoma, non–small cell lung cancer, or urothelial cancer

Figure 1.  Cancer vaccines can activate, amplify, and steer the immune response toward the generation of cancer-specific T cells (inset). As a result, vaccine-induced, tumor antigen–specific T cells are driven into the tumor.

Cytolysisof tumor

Migrationto site oftumor Tumor

Vaccines: steering the immune responseto focus on specific tumor antigens

Cancervaccine

PD-1CTLA4

Brake

Steer

Speed

Adjuvantdelivery

Activation andproliferation oftumor-specific

T cells

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(patients receive nivolumab while vaccines are generated dur-ing the first 12 weeks of the trial), objective responses after vaccination were seen in subsets of patients who had not responded to nivolumab alone (42). Circulating immune responses including ex vivo responses directed at the major-ity of vaccine epitopes were detected in all 16 patients who had completed vaccination and underwent immune analyses. Spreading of circulating T-cell responses to epitopes that were expressed by the tumor but not included in the vaccine, suggesting tumor cell killing (thereby releasing the additional epitopes for recognition by T cells), were detected in 7 of 9 patients tested. Moreover, in a patient with melanoma with durable stable disease after nivolumab and neoantigen vac-cine, the postvaccine tumor was specifically enriched for vac-cine neoepitope–specific TCR transcripts (not evident prior to nivolumab or vaccination), indicating that the vaccine led to trafficking of vaccine-specific T cells into the tumor in this patient. In future studies, it will be important to further define the specificity, frequencies, and functional states of T cells in tumors after vaccination and whether this infor-mation can be linked to clinical benefit from vaccine-based combinatorial therapies.

Although anti–PD-1 or anti–PD-L1 antibodies are an obvi-ous combination partner for a cancer vaccine, recent preclini-cal data suggest that optimal sequencing of these therapies may be critical for achieving maximal antitumor activity. In the lymphocytic choriomeningitis virus (LCMV) model, abrogation of the PD-1 pathway led to impaired formation of LCMV-specific memory T cells while the addition of PD-1 inhibition to a regimen of combined OX-40 agonistic and a peptide vaccine in the human papillomavirus–associated tumor model TC-1 compromised efficacy and led to apopto-sis of tumor-infiltrating vaccine-specific T cells (43, 44). These data indicate that a cancer vaccine may be most effective if administered prior to initiation of PD-1 pathway inhibition. Of note, given the ability of CTLA4 inhibition to enhance T-cell priming in the draining lymph node, there is a rationale to administer vaccine concurrently with a CTLA4 inhibitor (45, 46); PD-1 inhibition could then be given after a series of priming vaccines plus CTLA4 inhibition. These studies also highlight the potential utility of suitable preclinical models to guide the design of clinical studies; a combinatorial vac-cine approach could conceivably fail in the clinic simply because of suboptimal dosing and/or scheduling. Given the large number of therapies available for combination with a vaccine and the likely need for comparative studies to defini-tively prove clinical efficacy, clinical trials must be informed as much as possible by preclinical data and appropriate bio-markers.

concLuDInG REMARKsInfiltration of tumors with specific and functional memory

T-cell responses is critical for durable anticancer immunity. Because of their ability to focus stimulation of tumor-specific T cells, vaccines are arguably the most precise therapeutic approach to achieve effective tumor T-cell infiltration. By tar-geting neoantigens, delivered in conjunction with enhanced immune adjuvants and partnered with complementary immune therapies that address immune-suppressive circuits

in the tumor microenvironment, cancer vaccines may finally fulfill their promise as effective therapy for patients with cancer.

Disclosure of Potential Conflicts of InterestC.J. Wu is a founder, equity holder, member of the scientific

advisory board, and consultant for Neon Therapeutics, Inc. P.A. Ott reports receiving commercial research grants from BMS, Merck, Neon Therapeutics, Celldex, ArmoBiosciences, AstraZeneca/Med-Immune, Novartis, Pfizer, CytomX, and Genentech and is a consult-ant/advisory board member for Merck, BMS, Genentech, Novartis, Pfizer, Neon Therapeutics, Celldex, CytomX, and Array.

AcknowledgmentsThe authors gratefully acknowledge support from the Francis and

Adele Kittredge Family Immuno-Oncology and Melanoma Research Fund (to P.A. Ott), the Faircloth Family Research Fund (to P.A. Ott), the Bender Family Research Fund (to P.A. Ott), the Mathers Family Foundation (to C.J. Wu), and the Blavatnik Family Foundation (to C.J. Wu). C.J. Wu is a Scholar of the Leukemia and Lymphoma Society.

Published first April 1, 2019.

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