Therapeutic Vaccines

Preventive vaccination against infectious diseases is considered one of the most successful health measures of all time. Therapeutic vaccination against established diseases such as persistent infections and cancer has proven much more challenging, because the vaccine intervention must combat an immune system that has been restrained by tolerizing or polarizing mechanisms that sustain the disease in a misguided attempt at self-tolerance. Nevertheless, recent clinical results indicate that the era of successful therapeutic vaccination has arrived. In this Review, we discuss the most attractive preclinical and clinical therapeutic vaccination strategies, as well as opportunities to improve such therapies. With the exception of some forms of premalignant disease, the proportion of patients benefiting from treatment with cancer vaccines, in addition to the mean survival advantages, leaves much to be desired. Better results can most likely be obtained by a better choice of antigens, improvements in vaccine design, and appropriate cotreatments. The latter can alleviate immunosuppressive mechanisms in the cancer microenvironment and boost vaccine performance by appropriate stimulation or modulation of the immune system.

• Clinical Cancer vaccines against non viral antigens

Antigens on nonviral cancers are targeted for immunotherapy, including vaccines, for two main reasons: (a) the antigens can elicit an immune response that selectively attacks cancer cells, and (b) these antigens are (over-)expressed on cancer cells. If such antigens are expressed at all on normal cells, as in the case of differentiation antigens, the immune response to the normal tissues should only cause nonlethal side effects, such as vitiligo in the case of immune responses elicited against melanocyte antigens. In many phase I/II studies, these vaccines have shown clinical benefit, in particular extended overall or disease-free survival, while objective durable regressions of the type associated with targeted or immunomodulatory mAb therapy  or chimeric antigen receptor (CAR)  or adoptive T cell therapy were rarely seen.

Vaccines for nonviral cancers have targeted shared antigens. Vaccines against nonviral cancers have largely utilized target molecules, such as differentiation antigens, cancer testis (CT) antigens, or overexpressed antigens , that are common to a particular cancer type.

Central immunological tolerance mechanisms shape the T cell repertoire that recognizes these antigens; thus, the T cells induced by these vaccines must rely on the T cell repertoire left after the induction of central tolerance, which depletes many, but not all, of the high-avidity T cells directed against such antigens. Indeed, overexpressed CT or differentiation antigens were found in medullary thymic epithelial cells that express virtually all self-molecule, including cancer–associated antigens, although epitope expression failure can occur in the thymus.

Nevertheless, deletional immunological tolerance of the T cell repertoire toward self-antigens is the rule rather than the exception.

Despite the likelihood of elimination through central tolerance mechanisms, adequate T cell repertoires are available to allow clinical benefit. Provenge (sipucleucel-T), which targets the prostate differentiation antigen prostate acid phosphatase (PAP), was the first cancer vaccine to be approved in the US and Europe on the basis of its capacity to prolong overall survival in patients with hormone-resistant prostate cancer by an average of 3 months. This vaccine is a cellular product generated from autologous peripheral blood monocytes (PBMCs) by culturing with a fusion protein of PAP linked to granulocyte-macrophage CSF (GM-CSF). The exact mode of action is not known, because the cultured PBMCs contain both partially activated DCs and T cells as well as other peripheral blood cellular components. At any rate, vaccination increased the number of PAP-specific T cells in the prostate . PROSTVAC-VF (TRICOM, Bavarian Nordic, exclusive option of BMS)  is a cancer vaccine that consists of two recombinant viral vectors. Each vector encodes prostate-specific antigen (PSA) and three costimulatory molecules (CD80, ICAM-1, and LFA-3). Priming is achieved by a vaccinia virus vector, followed by a boost with fowlpox vector. Such a heterologous prime-boost protocol ensures that the response against the tumor-associated antigen (TAA), the only antigen shared between the two viral vectors, is enhanced by the boost. An increase in PSA-doubling time was observed that was associated with a survival benefit of 8.5 months in the vaccinated group versus a control group of patients with hormone-resistant prostate cancer.

Therapeutic vaccines have also been evaluated in patients with breast cancer, lung cancer, melanoma, pancreatic cancer, colorectal cancer, and renal cancer. A survival advantage was seen with some vaccines in phase II trials that was sometimes associated with an immune response to the vaccine in renal cell carcinoma, but no objective cancer regressions were noted. Survival advantage was also noted in a study of an HLA-A2–binding gp100-derived short peptide vaccine together with IL-2, versus treatment with IL-2 alone in patients with advanced melanoma.

Mesothelin is a cell-surface molecule that is overexpressed on a variety of malignancies, including mesothelioma, pancreatic cancer, lung cancer, and ovarian cancer. Cancer regressions without appreciable toxicity were seen with an Ab-based immunotoxin against this target.  In one study of a Listeria-based vaccine incorporating mesothelin together with an allogeneic pancreatic cancer–based G-VAX vaccine in a prime-boost approach, a median survival of 6.1 months was noted in patients with advanced pancreatic cancer versus a median survival of 3.9 months for patients treated with the G-VAX vaccine alone. G-VAX vaccine consists of allogeneic cancer cell lines transduced with GM-CSF; in principle, vaccination with such cells protects against all immunogenic antigens in these lines that are shared with the patient’s cancer, with the exception of unique neoantigens.

Another vaccine approach in which antigen identification is bypassed involves the use of autologous tumor lysates, which, in theory, have the advantage of incorporating the full range of neoantigens resulting from somatic mutations, without having to identify these neoantigens directly. The disadvantage is that one does not know how effective the processing of neoantigen epitopes from the lysate is for effective DC presentation. Also, the lysate, if presented in a highly immunogenic vaccine formulation, may conceivably induce autoimmunity to autoantigens also represented in the lysate.

In hematological malignancies, vaccination against the idiotype of monoclonal surface Ig on malignant B cells has been associated with prolonged disease-free survival in a phase III vaccine trial . However, regular anti–B cell mAbs, bispecific T cell–enhancing (BITE) Abs (39, and CARs targeting CD19 will likely supplant anti-idiotype approaches because of off-the-shelf availability or greater effectiveness.

In considering other vaccine approaches, in particular DC vaccines, it is noteworthy that Wilms tumor-1 (WT-1) peptide vaccination or vaccination with DCs electroporated with WT1 mRNA in patients with acute myelogenous leukemia (AML) has led to occasional leukemia regression,  or conversion from partial to complete remission following vaccination. Therapeutic vaccination with ex vivo–prepared tumor antigen–loaded DCs, particularly in patients with metastatic melanoma, has been practiced by numerous groups, mainly with monocyte-derived, in vitro–cultured DCs. Recently, a shift from such DCs to natural DC subsets occurring in blood and similarly loaded with TAAs has shown a tendency toward better clinical results . Also, whereas ex vivo–generated DCs were mostly loaded with class I MHC epitopes, CD4+ The cell targeting has improved the clinical result. These results were confirmed by a study of in vitro interactions between DCs and CD4+ and CD8+ T cells. A skin test measuring infiltration by antigen-driven T lymphocytes reportedly predicts patient survival . DC vaccination with mRNA-encoding melanocyte-associated antigens electroporated into DCs following complete resection of metastases has shown encouraging survival results . However, apart from the results with sipuleucel-T, which hardly qualifies as a DC vaccine sensu stricto, no successful phase III trial results have been reported for DC-based cancer vaccines.

Emerging neoantigens for therapeutic vaccination against nonviral cancers. In the hierarchy of therapeutic vaccine targets in nonviral cancers, powerful arguments for selecting neoantigens (based on mutation) in addition, or even exclusively, whenever possible, were recently provided by both preclinical and clinical observations. Vaccines against mutation-based neoantigens are unique to each patient, because the mutations induced by carcinogens or UV light are random. Of course, the preparation of personalized vaccines poses new challenges.

The first evidence of the potential of neo-epitope vaccination was made in the B16 mouse melanoma model, in which synthetic long peptide (SLP) vaccination against two mutant antigens was associated with a marked antitumor effect . More recently, in preclinical mouse experiments with a chemically induced escape variant tumor that had lost a major mutant rejection epitope, tumor rejection could still be achieved by Ab-mediated immune checkpoint blockade (anti–CTLA-4 or anti–PD-1). This effective treatment was demonstrated to act by reactivation of existing T cells against two other mutant CTL epitopes . Interestingly, the same therapeutic effect was also accomplished by vaccination with two SLPs incorporating these two mutant epitopes together with TLR3 ligand polyinosinic poylycitidylic acid (poly I:C). Similar observations were made in an independent mutation–based cancer model Likewise, in a clinical study of patients with metastatic melanoma, clinical benefit from treatment with anti–CTLA-4 was strongly associated with a high mutational load in the patients’ cancer, indicating that the responses to neoantigens that were unleashed by the anti–CTLA-4 treatment were clinically useful. Anti–CTLA-4 treatment of patients with metastatic melanoma reportedly broadens the T cell responses to shared antigens such as overexpressed antigens, differentiation antigens, and CT antigens. The contribution to clinical success of this broadening, next to the arousal of T cell responses against neoantigens, is currently unknown, although evidence is emerging that adoptive T cell therapy with T cells directed against mutations is more effective than that directed against differentiation antigens. In any case, it has now been shown beyond doubt that tumor-infiltrating lymphocytes (TILs) from melanoma patients that successfully eradicate tumors frequently contain CD8+ or CD4+T cells against neoantigens that are likely responsible for, or heavily contribute to, anticancer effects

Antigens of choice in virus-induced cancers. Cancers caused by viruses and other infectious agents such as Helicobacter pylori constitute approximately 20% of all cancers worldwide. Notably, preventative vaccines are available for only two of the human oncogenic virus types: hepatitis B virus (HBV) and HPV.

• Therapeutic Vaccines Against Human Cancer Viruses

HBV and hepatitis C virus vaccines. Despite the fact that preventive vaccines for HBV have been available for approximately 32 years, hundreds of millions of individuals worldwide are still persistently infected with HBV. Thus, therapeutic vaccines will be needed for years to come.

Several types of therapeutic vaccines against persistent HBV infection and its sequelae have been developed. These vaccine platforms include recombinant HBV proteins, DNA vaccines, recombinant virus vaccines, and subviral particles, as well as immune complexes of HBV surface antigen (HBsAg) and IgG anti-HBsAg . Immune complex targeting through Fc receptors facilitates both DC ingestion of antigen and DC activation. Vaccination with these immune complexes consisting of HBsAg and IgG Abs yielded promising results in a phase II trial , but a phase III randomized trial showed no clinical or virological benefit in patients persistently infected with HBV. Therapeutic vaccines against hepatitis C virus (HCV) have utilized by and large the same vaccination platforms as those for vaccines against HBV . So far, good immunogenicity data have been collected for some vaccines, but no efficacy data are available as of this writing . HBV and HCV do not contain oncogenic proteins that need to remain expressed in the transformed cells, but rather cause hepatocellular cancer (HCC) by indirect mechanisms such as inflammatory events. This necessitates targeting persistent viral infection before malignant transformation, because HCC may not necessarily express viral proteins.

Short peptides (<15 amino acids) do not require processing by professional antigen-presenting cells (APCs) and therefore bind exogenously to the HLA class I molecules of all nucleated cells that have surface HLA class I. Thus, most of these injected short peptides will end up in nonphysiologically large numbers, clogging the appropriate HLA class I molecules of nonprofessional APCs in the absence of costimulatory molecules. In the case of short peptide vaccination in incomplete Freund’s adjuvant (IFA), the T cells elicited by vaccination traveled to the vaccination site instead of to the tumor and appeared to die there. Vaccination with SLPs did not lead to such tolerance and was associated with proper antitumor activity, completely in line with our findings with short and long peptides in IFA. Indeed, effector CD8+ T cell induction by short peptides and the associated antitumor effect is much less efficient than what is observed following vaccination with long peptides encompassing the same CD8+ T cell epitope. Also, vaccination with long peptides in IFA or its close relative Montanide ISA-51 supports robust T cell responses to SLPs, but not to most short peptides .Only ex vivo loading of preactivated DCs with short peptides or replacement of the CD4+ helper signal with agonist anti-CD40 Ab, with or without TLR ligand poly or with CpG can circumvent such tolerance induction against short, MHC class I–binding peptides in mice or patients.

SLPs (>20 amino acids) are pro-drugs in the sense that they are not biologically active by themselves, but need additional processing to allow loading in DC HLA molecules. The antigen presentation resulting from SLP vaccination reflects physiological pathways associated with much lower, and therefore more appropriate, MHC-ligand presentation than the uncontrollable and usually too-high peptide loading resulting from short peptide vaccination. We and others have shown that only DCs are capable of efficiently processing such SLPs for presentation in both MHC class I and class II molecules and that such processing is much more efficient than that of intact proteins. Moreover, SLPs typically harbor both CD4+ and CD8+ T cell epitopes, ensuring that vaccination with SLPs induces a balanced CD4/CD8 response.

Cancer vaccines must utilize an effective route of administration. The preferred routes of cancer vaccine administration must effectively target the antigen to DCs. This is best achieved by s.c. administration or by delivery into DC-rich lymph nodes. Other effective routes include s.c. long peptide delivery in Montanide . DNA vaccines have also been delivered effectively by i.m. injection in combination with electroporation. An important consideration is the extent to which adjuvants and vaccination routes contribute to the proper circulating and tissue-resident CD4+ and CD8+ T cells with a homing preference for the proper cancer-infiltrated tissues. Considerable insights into the different T cell subsets and specific tissue-homing patterns have been obtained from mouse infection models. Recent data show that mucosal cancers are best treated by vaccines that endow T cells with mucosal homing properties, but much more work is needed before solid rules for more efficient homing to cancerous tissues are incorporated into human cancer vaccine designs.

Therapeutic cancer vaccines must activate DCs with adjuvants. A crucial requirement of the proper action of SLP vaccines is the inclusion of appropriate adjuvants, including TLR ligands such as poly I:CLC (TLR3 ligand), CpG (TLR9 ligand), Montanide, or stimulator of IFN genes (STING) agonists. SLP vaccines can be further improved by the covalent coupling of a powerful TLR ligand to the peptide, leading to superior DC targeting and simultaneous DC activation. DNA or RNA vaccines contain more or less powerful built-in DC activators such as TLR ligands and pattern recognition receptors (PRRs). Other popular cancer vaccines, including PROSTVAC, have utilized viral vectors; however, while such vectors contain numerous PRR ligands capable of DC activation, they also contain sequences that compete with the inserted TAAs. Although PROSTVAC extended patient survival, the performance of vaccines incorporating this prostate antigen may conceivably be improved by excluding potentially competing vector sequences and incorporating strong immunostimulants. Heterologous prime boosting overcomes this problem to some extent.

Cancer vaccines based on recombinant protein delivery have also been used extensively, such as in the MAGE-A3 recombinant protein vaccination trials, in patients with metastatic melanoma or metastatic lung cancer. Recombinant protein vaccines suffer from the serious disadvantage that processing of such proteins by DCs for presentation by HLA class I molecules to CD8 T cells is very inefficient, as illustrated in the case of the MAGE-A3 vaccine by the lack of a demonstrable CD8 T cell response. Moreover, the primary endpoint (extension of disease-free survival) was not met in phase III MAGE-A3 protein vaccination trials .

Therapeutic cancer vaccines cannot be expected to act as a monotherapy. Cancer vaccines have been vilified because they do not approach the effectiveness of adoptive T cell transfer. However, the antigen specificity of adoptively transferred T cells appears to determine the therapeutic efficacy of TILs. Recent evidence indicates that in a substantial number of cases, TILs are likely to have been directed against neoantigens, rather than against the shared antigens utilized as targets for most therapeutic vaccines against nonviral cancers. Indeed, persistence of CTL clones targeting melanocyte differentiation antigens was insufficient to mediate significant melanoma regression in patients. On the other hand, in metastatic melanoma patients, immunotherapy with adoptive transfer of T cells transduced with an avidity-enhanced, NY-ESO-1–specific T cell receptor (TCR) produced marked, durable tumor regression, indicating that CT antigens such as NY-ESO-1 may be better targets for immunotherapy than differentiation antigens. Personalized vaccines against immunodominant CD4+ Th and CD8+ CTL epitopes representing neoantigens may indeed have a greater chance of therapeutic success, as was recently shown with SLP vaccines in mouse models. Development of personalized vaccines will create novel logistical and regulatory challenges that will likely be overcome by new technologies for rapid epitope prediction and validation from cancer exome sequences.

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