Strategies for developing and optimizing cancer vaccines

1. Nucleic acid/DNA/mRNA vaccines

The use of nucleic acids with the hope of developing gene therapies to correct any mutations or deficiencies or vaccines to induce specific antibody or T-cell responses against a pathogen (prophylactic vaccines) or a tumor antigen (cancer vaccines) was one of the highlighted fields in biomedical advances in 1990s. The concept of using non-live vaccines offers an opportunity to induce both humoral and cellular immunity without any concern of introducing infectious organisms that can potentially cause the illness in the vaccine recipients or unintended individuals.

Typically, a DNA vaccine is prepared by inserting the gene sequence of interest into a plasmid backbone that needs to be expanded and purified for administration via intradermal, subcutaneous, or intramuscular routes³⁰. When the plasmid enters the residential APCs or surrounding cells (such as myocytes), transcription occurs, resulting in expression of the protein of interest. The cell machinery provides post-translational modifications to the antigen in a manner similar to that in target cells of the vaccine. APCs play a dominant role in the effect of DNA vaccines by presenting the processed peptides on major histocompatibility complex (MHC) class I molecules either from a direct transfection or through cross-presentation of antigens taken up by phagocytosis of transfected cells³¹. Also, secreted tumor antigens are processed by the endocytic pathway. Then antigen-loaded APCs travel to a draining lymph node (DLN) to present the antigen to naïve T cells with co-stimulatory molecules to induce T-cell expansion, cytokine secretion, or interaction with B cells. If the vaccine is administered intramuscularly, transfected muscle cells can also present the peptide-MHC complexes, increasing the chances of T-cell expansion, although preclinical evidence suggests that the relevant presentation is primarily by professional APCs in the DLNs³². Another approach is to use DNA priming followed by a boost such as modified vaccinia Ankara (MVA) or a canarypox vector, as was researched originally in HIV vaccines^33,34. Variations in delivery methods, including electroporation, that can deliver the plasmid along with other elements (such as toxoids or cytokines) and the use of xenogeneic DNA to enhance immune response have been studied and translated to clinical trials in solid tumors, including melanoma and prostate cancer^35–39. In veterinary medicine, DNA vaccines targeting cancer have been licensed in a number of solid tumors. Accumulated information from the animals may contribute to the development of next-generation vaccine platforms^40,41.

Even though DNA vaccines have shown immune responses in humans, the potency is still limited. The development of novel technologies in the delivery of nucleic acids, such as hydrophobic peptide mix, gene gun, or electroporation, can be incorporated to enhance the efficiency^39,42,43. Large batch manufacturing and a long shelf life make a DNA vaccine attractive to the community practice setting, not limiting the treatment opportunities to cutting-edge research centers, in contrast to some other personalized vaccine strategies. The other significant benefit of a DNA vaccine is the versatile construction that enables the developer to include encoding of optional safety features instead of using any intact viral particles. However, integration to cellular DNA, the theoretical risk of antibiotic resistance due to selection markers in the plasmid, and the development of autoimmune conditions due to autoantibody production have yet to be addressed. Guidelines from the regulatory groups in the European Medicines Agency, the World Health Organization, or the FDA are available to address such safety concerns about developing DNA vaccines but those guidelines are focused on preventive vaccines for infectious organisms^44–46.

In a study of patients with metastatic melanoma (ClinicalTrials.gov identifier: NCT02035956), Sahin et al. analyzed the mutated neoepitopes, prioritized by predicted MHC affinity, and treated the cancers with up to 10 neoepitope-encoding mRNAs⁴⁷. About 60% of the patients developed de novo immune responses and the majority were CD4 responses. Tumor tissue sections showed neoantigen-specific killing by vaccine-induced T cells. The authors also described objective tumor responses, decreased cumulative metastasis, and sustained survival in selected patients. This study supports a newer concept of cancer vaccines using nucleic acids.

2. Peptides and proteins

Synthetic peptides have been used successfully in the induction of tumor-specific T-cell responses in clinical settings against both microbials and cancer⁴⁸. The use of peptides compared with whole cell lysates or protein has an advantage that only the epitopes of interest can be delivered to the immune system instead of overloading immunologic pathways with irrelevant antigens that might compete with the relevant epitopes or might induce autoimmune responses. Also, using only T-cell epitopes can avoid the danger of anaphylaxis associated with antibody responses by avoiding B-cell epitopes. Sometimes, the peptides require modifications to allow enhanced binding to MHC molecules to increase the immunogenicity, a process called epitope enhancement⁴⁹. Often, a few amino acids are added or mutated to enhance the immunogenicity and modulate the amphiphilicity and these changes have been shown to either enhance or inhibit anti-tumor T-cell responses⁵⁰. Vaccines using xenogeneic peptide/protein by themselves or loaded to DCs can be a further extension of enhanced immunogenicity originating from the difference in xenogeneic amino acid sequences while having the majority in common^40,51. Peptides can be encapsulated in particles such as liposomes or conjugated to adjuvants to optimize the antigen uptake by specialized APCs for the MHC class I and II presentation to CD8⁺ cytotoxic or CD4⁺ helper T cells, respectively, and such technologies have been translated to clinical trials in both hematologic (ClinicalTrials.gov identifier: NCT03349450) and solid cancers^52–56. It is relatively easy to add or substitute peptides for other epitopes compared with adoptive cell therapies such as chimeric antigen receptor (CAR) T cells or TCR-transgenic T cells, which are harder to adapt to antigen loss or alteration^57–61. Using synthetic long peptides (SLPs) of 15 to 30 amino acids has been advocated by some groups who propose that professional APC-processed SLP can more efficiently induce anti-tumor responses than direct presentation of shorter peptides to the cytotoxic T cells, that can be mediated by non-professional APCs that lack costimulation^62,63. Melief et al. have demonstrated such a concept in several clinical trials focused on human papilloma virus (HPV)-associated cancers, and vaccination using SLP has been widely applied in various solid tumors^64–70. At the same time, shorter peptide vaccines have also been tested clinically in HPV-related disease and have been shown to be immunogenic⁷¹. Idiotype vaccination in B-cell malignancy is another unique concept of targeting tumor-specific antigens in the form of idiotype produced uniquely by the malignant B cells or plasma cells^72,73. The challenge is the limited knowledge in intracellular processing and methods of tracking it in humans when the limited data are based largely on animal, especially murine, models. MHC class I processing involves endoplasmic reticulum aminopeptidases, cytosolic proteasomes, and peptidases, whereas MHC class II peptide production and processing take place in endolysosomal compartments involving cathepsin-like proteases⁷⁴. The process of cross-presentation allows exogenous antigens, which normally go through the MHC class II pathway, to be processed via the MHC class I pathway to trigger a cytotoxic CD8 T-cell response⁷⁵. Sometimes, endosomal proteases can affect epitope expression during cross-presentation^76,77.

Clinical trials using shared tumor antigens have been safe and immunogenic in many studies but so far have not been successful in showing clinic benefit sufficient for FDA approval. GV1001 against a telomerase peptide (TeloVac) for advanced pancreatic cancer combined with chemotherapy failed to show improved overall survival in a phase III trial⁷⁸. Several other phase III trials, including studies targeting a MUC1 antigen (tecemotide, START trial) and EGFRVIIII in glioblastoma multiforme (rindopepimut, CDX-110, ACT III study), also failed to show improvement in overall survival despite high expectations when they went into advanced clinical studies^79,80. The strategy to include multiple epitopes in one vaccine did not result in any superior results in advanced pancreatic cancer or renal cell carcinoma, indicating the serious need for better strategies to overcome several immunologic hurdles to control the cancer. Personalized peptide vaccines targeting neoantigens are among newer efforts to liberate trammeled immune responses against cancer²⁵.

3. Microbial vectors and non-bacterial human pathogens, including viruses and helminths

Microbes can function even in necrotic tissues, and the immune modulatory effect by the microbes can be an additional benefit added to the direct killing of cancer cells. Using bacteria or their products in cancer treatment goes back to 1890s, when Coley described the accidental finding of an inoperable case of sarcoma that improved with erysipelas; that led to treatment with inoculation of Streptococcus and Bacillus prodigiosus, now known as Serratia marcescens⁸¹. In his reports, he described the difficulties in inducing meaningful infection from the inoculation, the clinical course after inoculation of “Coley’s toxin”, and the regression of inoperable tumor that lasted several years in one of the patients. Since then, cancer immunotherapies have used various bacteria or their products, such as Clostridium novyi, Listeria monocytogenes (Lm), and Salmonella typhimurium^82–86. Several organisms have been tested either to serve as a vector purely to express tumor antigens or in combination with DCs or other adjuvants. In addition to those listed above, shigella and bacillus Calmette–Guérin (BCG) among bacteria, and vaccinia, lentivirus, adenovirus, yellow fever, and HPV among viruses are well-known examples of microbial vaccine vectors.

On another front, microbiome research in cancer and immunology has gained momentum in the past decade, leading to versatile approaches in using the microbiome, including the consideration of normal flora such as Lactobacilli as vectors for gynecologic cancer. Besides direct anti-cancer effects of the infection, infection of the tumor tissue with facultative anaerobes can enhance the antigenicity of tumors that otherwise could have been tolerogenic or poorly antigenic while driving the alteration in the immune subsets in a pro-inflammatory microenvironment^87,88.

The changes in immune landscape in the use of bacterial cancer vaccines are noteworthy. MDSCs, tumor-associated macrophages, tumor-entrained neutrophils, and tolerogenic DCs are all myeloid cells that are immune-suppressive and found during tumor growth in both primary and metastatic lesions. Monocytes that are attracted to the tumor generally differentiate into macrophages, typically M2 macrophages that are characterized by arginase 1 (Arg1) expression and IL-10 known as immunosuppressive markers that contribute to immune evasion of cancer, whereas M1 macrophages contributing to anti-tumor activity express nitric oxide synthase (NOS2) and secret tumor necrosis factor-alpha (TNFα). Injection of attenuated S. typhimurium into HER2/neu-expressing CT26 tumors in a mouse model showed the shift to mature phenotype in macrophages in the tumor or spleen. Injection of attenuated Lm to an ID8-Defb29/Vegf-A ovarian carcinoma mouse model led to increased macrophage infiltrates in the tumor, favoring an M1 profile. These findings suggest that bacterial vaccines can reprogram the pro-inflammatory nature of cells and influence M1/M2 phenotype-specific differentiation⁸⁹. Melanoma cells infected with wild-type Lm can differentiate into professional APCs that express mature DC markers such as CD11c, CD40, CD83, and MHC class II⁹⁰. These findings suggest how pathogens can stimulate the immune function via co-stimulatory molecules involving Toll-like receptor (TLR) signaling pathways to activate killer T-cell and NK cell activities to kill tumor cells. On the other hand, tumor-associated neutrophils (TANs) are also of importance in the research of cancer vaccines using bacteria. Vendrell et al. reported increased recruitment of TANs after the injection of Salmonella typhi in a mouse model of breast cancer⁹¹. TANs can alter the tumor microenvironment (TME) by secreting inflammatory cytokines such as IFNγ and TNFα that potentiate the local immune infiltrates and systemic response. Salmonella strains that are engineered to express such inflammatory cytokines have shown enhanced anti-tumor activity in vivo. In regard to T-cell responses, injection of attenuated Salmonella species resulted in increased Th1 phenotype CD4 T cells in tumor infiltrates, slower tumor growth, and improved host survival in mice⁹². These effects were decreased at least in part in a T cell–depleted mouse model, suggesting the requirement for (presumably cytotoxic) T cells in the mechanism of action. Similar observations were reported with Lm and the parasite Toxoplasma gondii⁹³. Bacterial vaccines can further modulate the TME by decreasing the expression of PD-1 in tumor-infiltrating lymphocytes (TILs), inducing central and effector memory T cells, and reducing MDSC immunosuppressive potential. Several strains of attenuated or avirulent Lm strains such as replication-deficient Lm∆dal∆dat (Lmdd) have been tried to activate MHC class I and II pathways and also to induce antigen-specific T lymphocytes⁹⁴. Virulence factor listeriolysin O (LLO) protein is known to boost the immune reaction by inducing IL-12, IL-18, and IFNγ and functions as a naturally occurring adjuvant when delivering the tumor antigens⁹⁵ and can improve ratios of CD8⁺ T cells to regulatory T cells⁹⁶. So far, Lm-based vaccines have been brought into clinical trials for several solid and hematologic cancers, including HPV-associated cervical cancer, melanoma, breast cancer, pancreatic cancer, and lymphoma.

Aside from bacterial or yeast platforms, the viral platform of cancer vaccines has its own vast ground. Many attenuated or less virulent related viruses targeting as measles, mumps, rubella, smallpox, and polio have been used successfully to prevent or decrease the severity of viral illness by inducing protective immunity in the history of modern medicine. With the advancement of genetic engineering, these viruses can also be modified as vectors to deliver and express the antigens that will elicit immune responses even in tumors that are not immunogenic by themselves during the natural history of the specific cancer type. Viral particles can express in situ molecules that will serve as pathogen-associated molecular patterns to increase the possibility that a tumor-associated antigen (TAA) will be presented to the immune system. Activation of TLRs is believed to play a critical role in increased anti-tumor activity in therapies using viruses^97,98. Viral vectors used in cancer vaccines can deliver the TAA, with or without co-stimulatory molecules or cytokines, that will be processed in the MHC class I pathway and produce epitopes expressed on the surface of the infected cells with MHC I, and/or with MHC class II if an APC is infected. In the case of non-lysing virus, delivery of a TAA alone might not generate enough signals for tumor cell killing. With non-lysing virus, co-delivery of a co-stimulatory molecule or cytokines can increase the chance of recruitment of professional APCs. In either case, replicating viruses at the injection site cause pro-inflammatory responses mimicking the immune response against infecting virus in non-cancerous tissues that can result in cancer cell death.

Adenoviruses, especially replication-incompetent Ad2 and Ad5, have been popular to deliver TAAs with or without co-stimulatory molecules⁹⁹. However, the majority of adults have developed immunity from long-term environmental exposure to adenoviruses, and the immune response and clinical effect were minimal because of pre-existing immunity at least in part. Poxviruses have the advantage as pre-existing immunity against poxvirus exists only in patients who were exposed to vaccinia virus. Viral tropism of the members of this family to directly infect APCs is another advantage¹⁰⁰. MVA is a proprietary agent that was produced by more than 500 culture passages of vaccinia in chicken embryo fibroblasts. During the passages, MVA became highly attenuated as it lost many poxviral genes and replicative potential in most mammalian cells. A recombinant MVA-expressing human 5T4 (MVA-h5T4), called TroVax for metastatic castration-resistant prostate cancer, and a recombinant attenuated vaccinia virus (rVV) vaccine or fowlpox virus (rFV) against human carcinoembryonic antigen (c-VV-CEA) with or without a triad of co-stimulatory adhesion molecules (TRICOM) are great examples that were tested in the clinical setting^101,102. The TRICOM vector expresses three co-stimulatory molecules—CD54 (ICAM-1), CD58 (LFA3), and CD80 (B7.1)—co-expressed in the recombinant virus, and the triple combination was found to be more effective than any single one of these molecules¹⁰³. Examples of intelligently designed heterologous prime-boost strategy can again be found in a vaccinia prime–fowlpox boost, PANVAC encoding CEA and MUC1 alongside TRICOM¹⁰⁴. Because the vaccinia vector induces immunity against itself but not against the fowlpox vector, which also does not induce immunity to itself, it is possible to achieve a better response by priming first with the vaccinia vector and then boosting repeatedly with the fowlpox vector. Thus, a heterologous prime boost can be more effective than a homologous prime boost¹⁰⁵. PROSTVAC used priming with rVV-PSA-TRICOM followed by rFV-PSA-TRICOM to deter any neutralizing antibody responses and facilitate antigen-specific T-cell responses¹⁰⁶. Despite promising phase II results, a phase III randomized trial of PROSTVAC as a single agent did not meet its endpoints to show improved overall survival in planned interim analysis in patients with minimally symptomatic metastatic castration-resistant prostate cancer, and combination trials are ongoing^107,108.

4. Dendritic cell–based vaccines

DC-based vaccines have several advantages despite their inherent logistic problems^109,110. DC maturation is often defective in patients with cancer and this is due in part to cytokines such as vascular endothelial growth factor (VEGF), which induces STAT3 expression and inhibits maturation^111–113. Thus, antigen presentation can be defective in these patients, resulting in poor induction of T-cell responses. DC vaccines can overcome this problem in patients with cancer by starting from DC precursors, such as elutriated monocytes, which then are matured in vitro, away from the negative influences of the cancer environment^22,114. A second related advantage is that the DCs can be matured in vitro to induce higher levels of co-stimulatory molecules or MHC molecules or both, making them more immunogenic^109,115. A third advantage is that viral vectors can be used to transduce the DCs to express tumor antigens without neutralization by pre-existing anti-viral antibodies¹¹⁶.

Even though DC-based vaccination has been a popular platform to elicit immune responses, there is no consensus on how to optimize the preparation for consistent or durable responses. Also, handling individual patient’s cells can be an obstacle in reducing the cost and simplifying the delivery process. To tackle this issue, understanding the biology of DCs is essential. DCs are a heterogeneous group of specialized APCs from the same bone marrow progenitors as monocytes, called monocyte and DC progenitors, that differentiate into three major subsets of DCs: CD1c DCs, which are predominant; CD141⁺ DCs; and plasmacytoid DCs (pDCs)^117–119. The first two are called conventional DCs (cDCs). pDCs play a major role in viral infections by rapid cross-presentation of antigens and production of up to 1000-fold more type I IFNs (α and β). Human DCs lack lineage markers but express MHC class II. Whereas cDCs can be found in almost all peripheral tissues, including lymphoid organs, pDCs are discovered mostly in T-cell areas of lymphoid organs, such as lymph nodes, spleen, bone marrow, Peyer’s patches, tonsils, thymus, and liver. DCs can activate or negate anti-tumor T-cell responses by affecting killer or regulatory T–cell activation¹²⁰. Mobilized CD34 precursors can be differentiated into monocyte-derived DCs (MDDCs) that yield several different types of myeloid cells, including all three major types of DCs, as a consequence of working with higher levels of progenitor cells. It may be important to manipulate each subset individually to improve immunogenicity¹²¹, and some groups use bead-selected DCs. For example, in some studies, Langerhans-like DCs induced with IL-15 were found to be more effective at eliciting CD8⁺ T-cell responses¹²². Moreover, activation of Langerhans cells with a cytokine combination could induce immunity against HPV that then could be boosted with a therapeutic vaccine¹²³. Similarly, the BATF3-expressing DC subset may be critical for cross-priming and for reactivating a memory anti-tumor response¹²⁴. On the other hand, immature DCs have been found to induce regulatory T cells¹²⁵.

The first and only FDA-approved cell-based therapy for metastatic prostate cancer, sipuleucel-T, contains a variety of cells, including B cells, monocytes, DCs, and NK cells, that are cultured and processed in the presence of prostatic acid phosphatase (PAP) fused with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 2 days following leukapheresis in designated apheresis centers^1,126. Patients receive up to three doses of freshly prepared products at 2-week intervals. The most commonly used method is to differentiate the monocytes from peripheral blood mononuclear cells (PBMCs) into DCs, so-called MDDCs. PBMCs that are monocyte-elutriated with or without CD14 bead selection are differentiated into immature CD14⁻CD83⁻ DCs by culturing in IL-4– and GM-CSF–supplemented culture media usually for 3 to 5 days followed by antigen loading for 24 to 48 hours. DCs then can be matured with lipopolysaccharide (LPS) and IFNγ or with a cytokine cocktail^22,109. Matured DCs are harvested for the delivery. However, owing to the labor-intensive and costly manufacturing process, aliquoting and freezing individual doses and use of automated closed culture systems are being actively investigated. For allogeneic sources of DCs, cDCs of umbilical cord blood origin can be one option to meet the need for unrelated healthy donors. However, the immune functions of the DCs in umbilical cord are questioned and the use of HLA-half matched allogeneic DCs has not been successfully translated to the clinic yet^127,128.

Several ways have been adopted for antigen loading. As DCs are professional APCs, loading with synthetic peptides, protein, tumor cell lysates, and RNAs and even direct infection with microbes are viable options. APCs fused with tumor cells, whether from an autologous or allogeneic source, were the prototype of cancer vaccine from the 1990s, when the approach was first described^129,130. Despite high production costs due to patient-specific therapies, there have been continued efforts to translate DC vaccines to clinical trials in both solid and hematologic cancers^{114,131–133}. Using whole lysates of either autologous or allogeneic tumor cells can lead to the presentation of multiple epitopes for a prolonged period, enabling longer antigen presentation that will allow both CD4 and CD8 T-cell responses. Wild-type or attenuated pathogens can serve as vectors to carry the genes encoding TAA to be expressed in DCs. Transfection of the nucleic acids encoding TAAs can induce potent TAA-specific T-cell responses at least in vivo. For example, mRNA can be transfected by electroporation (renal cell carcinoma, ClinicalTrials.gov identifier: NCT01582672; advanced melanoma, phase II, ClinicalTrials.gov identifier: NCT01302496) or a cationic lipid such as DOTAP^134–136. Alternatively, retroviral transduction has been used to facilitate and prolong stable TAA expression. Viral vectors such as lentivirus or adenovirus have been employed in several preclinical and clinical studies with DC vaccines because of the advantage of transducing non-dividing cells^137–139. In regard to the issue of adequate involvement of CD4 cells to enhance CD8 cell response in DC-based vaccines, adding other antigens such as keyhole limpet hemocyanin (KLH) as a target for helper T cells that can facilitate the antigen presentation and migration to the DLNs is an option^22,115.

As the manufacturing and quality control of the DC vaccines are highly variable, development of large-scale studies is limited and the interpretation of smaller-scale studies is often difficult^140–142. Also, there is no consensus in measuring the correlatives to evaluate the vaccines. How and what cytokines or markers should be measured and in which samples? For example, is measuring the cytokine from the serum or by intracellular cytokine staining a better indicator of DC functions or activation? Analysis of the activation of vaccinated DCs showed a relationship with clinical benefit, and associated predictive markers of the most effective DCs have been reported¹¹⁵. Given the limited consensus, ground-breaking developments in manufacturing, quality control, and optimized delivery of the vaccine to overcome the hurdle of cross-priming and TME are still needed.

A whole allogeneic pancreatic cell line secreting GM-CSF, called GVAX, is another strategy to use DCs in vivo that stimulate the accrual and function of APCs to the vaccine site. This strategy can potentially involve all DC subsets, not only the subsets in the apheresis product, and result in increased T-cell infiltration and development of tertiary lymphoid structures even in a so-called “non-immunogenic” tumor such as pancreatic cancer or prostate cancer^143,144. This method has the merit of not involving the labor-intensive and expensive ex vivo manufacturing process. However, a phase IIb study of a combination of GVAX and a mesothelin-expressing live-attenuated listeria vaccine CRS-207 did not show improved overall survival^145,146.