REVIEW

published: 30 March 2021

doi: 10.3389/fimmu.2021.627932

Cancer Vaccines, Adjuvants, and

Delivery Systems
Samantha J. Paston 1, Victoria A. Brentville 1, Peter Symonds 1 and Lindy G. Durrant 2*
1 Biodiscovery Institute, Scancell Limited, Nottingham, United Kingdom, 2 Biodiscovery Institute, University of Nottingham,

Faculty of Medicine and Health Sciences, Nottingham, United Kingdom

Vaccination was first pioneered in the 18th century by Edward Jenner and eventually led to
the development of the smallpox vaccine and subsequently the eradication of smallpox.
The impact of vaccination to prevent infectious diseases has been outstanding with many
infections being prevented and a significant decrease in mortality worldwide. Cancer
vaccines aim to clear active disease instead of aiming to prevent disease, the only
exception being the recently approved vaccine that prevents cancers caused by the
Human Papillomavirus. The development of therapeutic cancer vaccines has been
disappointing with many early cancer vaccines that showed promise in preclinical
models often failing to translate into efficacy in the clinic. In this review we provide an
overview of the current vaccine platforms, adjuvants and delivery systems that are
Edited by:
Pål Johansen, currently being investigated or have been approved. With the advent of immune
University of Zurich, Switzerland checkpoint inhibitors, we also review the potential of these to be used with cancer
Reviewed by: vaccines to improve efficacy and help to overcome the immune suppressive
Kwong Tsang,
Precision Biologics, Inc., United States
tumor microenvironment.
Steve Pascolo,
Keywords: adjuvant, peptide vaccine, DNA vaccine, cancer, vaccine
University of Zurich, Switzerland

*Correspondence:
Lindy G. Durrant
lindy.durrant@nottingham.ac.uk INTRODUCTION
Specialty section: The potential to develop a cancer vaccine has been extensively researched in both humans and
This article was submitted to animal models, the majority of these vaccines are therapeutic vaccines that aim to activate the
Vaccines and Molecular Therapeutics, immune system to recognize and kill established tumors. A prophylactic vaccine that prevents
a section of the journal cancers caused by the Human Papillomavirus [Types 6, 11, 16, 18] has been approved and in the
Frontiers in Immunology
UK, children aged 12-13-years-old are routinely offered this vaccine. Developing a therapeutic
Received: 10 November 2020 cancer vaccine has been more problematic with many encouraging results in preclinical studies not
Accepted: 12 March 2021 translating into the clinic. To date the FDA has only approved one vaccine, sipuleucel-T, that is used
Published: 30 March 2021
to treat metastatic castration-resistant prostate cancer in a limited group of nearly asymptomatic
Citation: patients (1). There are number of reasons for these failures such as the immune suppressive TME,
Paston SJ, Brentville VA,
lack of a robust T cell responses, sub optimal vaccine formulations, delivery, adjuvants and
Symonds P and Durrant LG
(2021) Cancer Vaccines,
identifying the best patients to target. The ideal setting for a cancer vaccine to work is in patients
Adjuvants, and Delivery Systems. following surgical resection, chemotherapy or radiotherapy, all of which stimulate an immune
Front. Immunol. 12:627932. response themselves. Vaccination at this stage and in combination with a checkpoint inhibitor will
doi: 10.3389/fimmu.2021.627932 provide the best setting to induce a potent anti-tumor immune response.

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Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

Over the last couple of decades, a better understanding of the BAGE (9), both T cell and antibody responses to these antigen
tumor microenvironment (TME) and immune suppressive have been detected in patients.
mechanisms have opened a number of new avenues that can The differentiation antigens are expressed on normal and
be explored and has led to the next generation of new cancer tumor cells from the same tissue, targeting such antigens requires
therapies. The immune suppressive TME is a major obstacle to careful consideration to any potential toxicity to the normal
the success of any cancer vaccine with the description of tissue. Differentiation antigens include the melanoma antigens
immunologically “cold” tumors that on their own do not Melan A/MART-1 (10, 11), gp100 (12), tyrosinase (13), the
appear to be immunogenic with an absence of tumor prostate antigen prostate specific antigen (PSA) (14, 15) and
infiltrating lymphocytes (TILs). In contrast “hot” tumors are the colon antigen carcinoembryonic antigen (CEA) (16, 17).
immunogenic and have induced an immune response that has The overexpressed antigens are generally expressed at low
resulted in the infiltration of TILs but are not able to function due levels on normal cells but are over expressed on tumor cells, there
to the presence of various checkpoint molecules such as PD-1, are many antigens that fall into this group including HER2 (18),
CTLA-4, LAG-3, TIM-3 or the presence of immune suppressive hTERT (19, 20), p53 (21), survivin (22–25), MUC1 (26), WT1
cells such as regulatory T cells, myeloid-derived suppressor cells (27), cyclin B (28, 29) and many more. Targeting over expressed
(MDSCs), M2 macrophages, regulatory natural killer (NK) T antigens can be challenging, preclinical studies need to ensure
cells or cytokines such as transforming growth factor-beta that normal low-level expressing cells are not targeted by the
[TGFb], IL-10, and IL-13 (2, 3). The success of any cancer vaccine induced immune response.
vaccine relies on overcoming the immune suppressive TME and The oncogenic viral antigens are expressed on virus infected
converting “cold” tumors into “hot” tumors and therefore cells that have subse quently undergo ne malignant
inducing a robust tumor specific immune response that can transformation. Oncogenic viral antigens have been targeted in
kill cancer cells. both prophylactic vaccines such as HPV but also in therapeutic
vaccines to treat existing malignancies. The most commonly
targeted oncogenic viral antigens in this group include EBV
TARGET ANTIGENS LMP-1 and LMP-2A (30–32), HPV E6/E7 (33), HTLV-1
Tax (34).
The choice of antigen to target in any cancer vaccine is extremely The last group of cancer antigens are those antigens that are
important to the efficacy of the vaccine in the clinic. The ideal mutated, these mutations can be generated at the gene level or as
antigen should be specifically expressed on cancer cells with no a result of post translational modifications leading to the
expression on normal cells, ideally the antigen should be generation of a new peptide. In the last couple of years there
necessary for cell survival and be highly immunogenic. has been a renewed effort in generating vaccines that target
Tumor antigens fall into two broad categories, the tumor mutated antigens, in particular the neoantigens. There are very
associated antigens (TAAs) and tumor-specific antigens (TSAs). few mutated antigens described where the mutated peptide is
Within each category a number of different types of tumor shared across patients or cancer types, the most studied shared
antigens have been described and are summarized in Table 1. mutations are KRAS (35), NRAS (36), epitopes from BCR-ABL
The cancer germline antigens (also called cancer testis antigens) translocation (Chronic myeloid leukemia) (37, 38), ETV6 (acute
are the most studied group of cancer antigens, historically they myeloid leukemia) (39), NPM/ALK (anaplastic large cell
were attractive antigens to target due to their expression only on lymphomas) (40, 41) and ALK (neuroblastoma) (42, 43). A
germ cells of immune-privileged organs and high expression on number of groups are developing personalized vaccines that
tumor cells. The most common cancer germline antigens that are target neoantigens identified from the patient’s tumor, very few
targeted include MAGE (4, 5), NY-ESO1 (6), GAGE (7, 8) and if any of these mutations are shared epitopes and therefore any

TABLE 1 | Different types of tumor antigens.

Class of tumor Description Tumor Example of tumor antigen

antigen specificity

Tumor Specific Cancer Germline Expression on healthy cells limited to testes, fetal ovaries and High MAGE, NY-ESO-1, GAGE, BAGE
Antigens (TSA) antigens trophoblasts.
Expressed on a wide a variety of cancer types.
Tumor specific Mutations resulting in the generation of a new peptide. High KRAS, p53, NRAS, BCR-ABL
mutated antigens Mutations can be generated at the gene level, from chromosome translocation, ETV6, NPM/ALK, ALK.
translocations or due post translational modification.
Oncogenic viral Abnormal expression on cells infected with an oncovirus. High EBV LMP-1/LMP-2A, HPV E6/E7,
antigens HTLV-1 Tax
Tumor associated Tissue Antigen expressed on tumor cells and normal cells. Low Melan A/MART-1, gp100,
Antigens (TAA) Differentiation Tyrosinase, PSA, CEA.
antigens
Overexpressed Antigen over expressed on tumor cells and normal level of expression Low HER2, hTERT, p53, Survivin, MUC1,
antigens on healthy cells. WT1, cyclin B.

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Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

generated vaccine is only specific to the individual. Neoantigens

are immunogenic because they harbor mutations, they have
escaped central tolerance and are recognized as “non self” by
the adaptive immune system (44). Despite the higher
immunogenicity of neoantigen’s only 1-2% of T cells recognize
these antigens (45). The poor immunogenicity of many tumors
means that designing an effective neoantigen tumor vaccine will
need to overcome these challenges.
The post translational modified cancer antigens are another
group of antigens, they are not subject to thymic deletion and are
therefore attractive vaccine candidates. A number of different
post-translational modifications have been described that
generate tumor specific epitopes including glycopeptides (46),
phosphopeptides (47, 48) and citrullinated peptides (49). Cancer
cells often exhibit different phosphorylation patterns leading to
the generation of phosphorylated antigens, these make attractive
vaccine candidates (47, 48, 50). Phosphorylated epitopes can be FIGURE 1 | Schematic of the citrullination or deamidation of arginine.

naturally processed and presented on the cell surface in

association with MHC class I molecules for recognition by within viable cells the calcium concentrations can be high leading
CD8+ T cells (50–52). Unregulated signaling cascades in to the activation of the PADI enzymes. Citrullination can occur
tumor cells often lead to an increase in protein within autophagosomes as a result of autophagy, here high
phosphorylation within the cell which in turn leads to the calcium levels activate PADI enzymes that then citrullinate
generation of phosphopeptides (52). Phosphopeptides have engulfed proteins from the cytoplasm (36, 37), this process is
been identified by mass spectrometry analysis of tumor induced in stressed cells (17) such as cancer cells. During stress
biopsies and cancer cell lines (53). Engelhard et al. (53) induced autophagy and in the presence of inflammation
identified two phosphorylated peptides derived from the citrullinated peptides can be presented on major
insulin receptor substrate 2 (IRS2) protein and breast cancer histocompatibility complex (MHC) class II molecules for
anti-estrogen resistance 3 (BCAR3). The ISR2 protein is recognition by CD4+ T cells (63). During inflammation many
overexpressed in many cancer types and in vivo has been cytokines are produced, the majority are proinflammatory that
shown to enhance metastasis (54–56), BCAR3 is associated result in the upregulation of MHC class II expression that then
cellular migration and resistance to therapeutic anti-estrogens activates CD4+ T cells (Figure 2).
in breast cancer cells (57, 58). Phosphopeptides restricted by A number of studies performed in autoimmune patients have
HLA-*02:01 were identified by mass spectrometry and included demonstrated that CD4+ T cell responses can be detected to
in a phase 1 clinical trial (NCT01846143) in patients with citrullinated proteins such as the intermediate filament protein
resected stage IIA–IV melanoma. All patients had treatment vimentin and the glycolytic enzyme enolase (64–68). In ovarian
related adverse events, but none were grade 3-4, T cell responses cancer patients we have demonstrated the presence of CD4+ T
were induced to the phosphorylated IRS2 (1097-1105) peptide in cell responses to citrullinated peptides derived from a-enolase
5/12 patients and to the phosphorylated BCAR3 (126-134) and vimentin (60). The constitutive expression of MHC class II is
peptide in 2/12 patients. This trial showed that mainly restricted to APCs such as DCs, B cells and macrophages
phosphopeptides are safe and induced an immune response in but other cells such thymic epithelia cells and activated T cells
some patients, however, with the advent of immune checkpoint can also express MHC class II (69). The expression of MHC class
inhibitors future studies will need to define and enhance the II on most other cells can be induced by interferon gamma
immune response induced to these peptides. (IFNg) present in the local vicinity. The expression of MHC class
Our own research has focused on epitopes that are citrullinated II is controlled by the Class II Major Histocompatibility complex
in tumor cells. Citrullination is a post translation modification transactivator (CIITA) which is regulated by four different
where positive charged arginine residues are converted into promoters, promoter I is active in myeloid cells, promoter III
neutrally charged citrulline in a process catalyzed by the Ca2+ in lymphocytes and promoter IV is necessary for responsiveness
dependent peptidyl arginine deaminase (PADI) enzymes (59, 60) to IFNg (70), the function of promoter II is unknown, transcripts
(Figure 1). This modification can impact the protein structure and from this promoter are rare and therefore its function has not
induce changes that result in protein denaturation potentially been pursued. Most tumors in the presence of inflammation and
altering the structure and the function of the protein (61, 62). IFNg will express MHC class II, if citrullinated peptides are then
We have detected T cell responses to citrullinated peptides in generated in response to stress or autophagy these can then be
healthy donors (60) suggesting that the T cells recognizing them loaded onto MHC class II for presentation on the surface of
are positively and not negatively selected in the thymus. In healthy tumor cells.
cells the PADI enzymes are maintained in an inactive state due to We have focused on citrullinated vimentin and a-enolase as
low concentrations of Ca2+ (34), in double membrane vesicles attractive cancer vaccine targets. Vimentin is an intermediate

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Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

FIGURE 2 | During stress induced autophagy and in the presence of inflammation citrullinated peptides can be presented on major histocompatibility complex
(MHC) class II molecules for recognition by CD4+ T cells. During inflammation many cytokines are produced, the majority are proinflammatory that result in the
upregulation of MHC class II expression that then activates CD4+ T cells. Primed killer CD4 T cells enter the tumor and are reactivated by APCs presenting
citrullinated peptides from tumors allowing recognition and lysis by the killer CD4 T cells.

filament protein that is known to be citrullinated and any microbial stimulation induce tolerance instead of a potent
overexpressed in a wide range of cancers (71–76), particularly immune response (88). Adjuvants need to attract immune cells
during EMT (77). The glycolytic enzyme a-Enolase (ENO1) to the site of injection while also promoting cell mediated
catalyzes the final step in glycolysis (78). Many tumors switch to trafficking of antigen to draining lymph nodes and triggering
generating their energy via glycolysis in a process termed the the activation of APCs.
“Warburg effect” and therefore overexpress ENO1, a wide range
of tumors overexpress ENO1 (79–82). Due to its ubiquitous Current Vaccine Adjuvants
expression, ENO1 is often degraded during autophagy; previous The water-in-oil emulsions such as Montanide ISA 720 and
studies have also shown that ENO1 can be citrullinated (65, 83). Montanide ISA-51 have been widely adopted as adjuvants, they
We have shown that these citrullinated peptides are recognized form a depot at the injection site, this results in the trapping of
and presented to CD4+ T cells by both MHC class II HLA-DR4 the soluble antigens preventing their rapid trafficking to local
and HLA-DP4 molecules (49, 84, 85). HLA transgenic mice lymph nodes, this induces inflammation and the gradual release
vaccinated with citrullinated vimentin and a-enolase peptides of the antigen. In a clinical trial Montanide ISA-51 was shown to
linked to an adjuvant (Modi-1 vaccine) can stimulate CD4+ T induce both CD4+ and CD8+ T cell responses in patients
cells (49, 64, 86) and generate potent anti-tumor responses vaccinated with long peptides of the oncoproteins E6 and
resulting in tumor regression and eradication with no E7 (89).
associated toxicity (49, 87). We have also shown that healthy New vaccine adjuvants have been developed that target
donors have a repertoire of T cells that can be detected following specific components of the immune system to generate a more
stimulation with the citrullinated vimentin and a-enolase robust and longer lasting immune response. Newer adjuvants
peptides showing that citrullinated peptides can be presented that consist of Pathogen-associated molecular pattern molecules
in the thymus allowing positive selection and resulting in specific (PAMPs) are now being used, these provide a danger signal that
T cell repertoires capable of recognizing these peptides (87). Our is recognized by pattern recognition receptors (PRRs) inducing
preclinical data shows that citrullinated vimentin and a-enolase an immune response. Innate cells express PPRs, these receptors
are promising candidate vaccine targets and as a vaccine have include the Toll-like receptors (TLRs), nucleotide binding
generated impressive anti-tumor responses in preclinical oligomerization domain like receptors and the mannose
murine models. receptor. TLR agonists are increasingly being used as a vaccine
adjuvant, they mimic microbial stimulation and have been
shown to increase vaccine efficacy (90) particularly for cancers
ADJUVANTS (91). Lymph node targeted TLR agonists have shown a direct
relationship between the magnitude of CD8+ T cell responses
Antigens alone in a vaccine are poor inducers of the adaptive and the amount of TLR agonist accumulated in draining lymph
immune response. In the absence of an adjuvant antigens nodes, demonstrating the importance of providing sufficient
targeted to immature DCs in the absence of inflammation or inflammatory signals during immunization (92). A number of

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Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

TLR agonists are currently in trial as adjuvants for cancer mediated anti-tumor responses (118). The effect of GM-CSF was
vaccines, one of the most commonly used TLR agonist is also observed in clinical trials where a low dose of GM-CSF
polyinosinic–polycytidylic acid with polylysine and induced the expansion of CD14 positive, HLA-DR low/negative
carboxymethylcellulose (Poly-ICLC) a TLR3 agonist (93), myeloid cells. In another study GM-CSF was used with
others include monophosphoryl lipid A (MPLA) a TLR4 incomplete Freund’s adjuvant and resulted in a low T cell
agonist (94, 95), imiquimod a TLR7 agonist (96, 97), response when compared to vaccine adjuvant without GM-CSF
resiquimod a TLR7 and TLR8 agonist (98, 99), CpG (119). Despite these results a number of clinical trials are
oligodeoxynucleotide (CpG ODN) a TLR9 agonist (90, 100, 101). currently underway using GM-CSF as an adjuvant component.
We have previously described our Modi-1 peptide vaccine
New Emerging Vaccine Adjuvants (60), this vaccine comprises of two citrullinated vimentin
Other newer adjuvants are also being investigated to increase the peptides, as well as a citrullinated peptide from a-enolase, each
efficacy of a cancer vaccine, these include the CD40 agonists, peptide is conjugated to the TLR1/2 ligand adjuvant
these directly target the antigen to the early endosomes of DCs AMPLIVANT ® (ISA Pharmaceuticals BV, Leiden, the
and mediate cross presentation. Although CD40 agonist Netherlands). In preclinical studies we have shown that by
antibodies have not been extensively studied in clinical trials as combining a peptide vaccine with a TLR ligand adjuvant
a vaccine adjuvant, they have been studied independently as promotes a Th1 response that is capable of inducing a potent
monotherapy (102). A number of preclinical mouse models have anti-tumor response in tumor bearing mice (60). The CD4+ but
shown that CD40 agonists can be used in combination with TLR not CD8+ T cells were essential for the generation of the anti-
agonists in a vaccination strategy (103, 104), whether this tumor response, depleting CD4+ T cells abrogated this response
translates into clinical efficacy is still to be determined. and a corresponding increase in CD4+ tumor-infiltrating
Another class of potential adjuvants is the Stimulator of lymphocytes (TILs) was associated with tumor regression (60).
interferon genes protein (STING) agonists. STING is a A comparison of different Toll-like receptor (TLR)-stimulating
transmembrane protein located in the endoplasmic reticulum adjuvants showed that Modi-1 induced strong Th1 responses
(105), its activation triggers a type I interferon response in when combined with GM-CSF, TLR9/TLR4, TLR9, TLR3, TLR1/
response to intracellular DNA (106). STING agonists include 2 and TLR7 agonists. The strongest response was observed with
synthetic cyclic dinucleotide derivatives and cyclic di-guanosine TLR1/2 AMPLIVANT® adjuvant. The AMPLIVANT® adjuvant
monophosphate, these have all shown anti-tumor activity in is already being used in an ongoing study evaluating two HPV-16
mice (107, 108). STING expression is highest on T cells and peptides in patients with head and neck squamous cell
STING activation can lead to T cell apoptosis, such effects are not carcinoma (NCT02821494). These results highlight the
seen with macrophages and DCs (109). To use a STING agonist importance of screening a range of adjuvants and doses to find
in a cancer vaccine it would need to be combined with an the optimal adjuvant and dose to induce a potent immune
adjuvant or delivery system that targets only myeloid cells in response. The Modi-1 vaccine will enter a Phase 1/2 clinical
vivo (110) preventing T cell apoptosis. STING agonists do induce trial in 2021.
some systemic toxicity and to overcome this intratumoral
injection is the preferred route of administration. In addition,
preclinical studies of STING agonists have been complicated by
their differential binding properties in murine and human cells DELIVERY SYSTEMS
(111). The potential toxicity of STING agonists and lack of
specific targeting could limit their use as adjuvants in a Electroporation and Gene Gun Vaccine
cancer vaccine. Delivery
In addition to using pathogen derived molecules as adjuvants There have been significant improvements in optimizing vaccine
a number of cytokines have also been shown to act as an administration routes to overcome poor cellular uptake. Also
adjuvant. Immunostimulatory cytokines such as IL-2 (112, improvements with delivery and plasmid design have improved
113), IFN (114), IL-12 (115, 116) and granulocyte-macrophage the efficacy of DNA vaccines in both pre-clinical and clinical
colony stimulating factor (GM-CSF) (117–119) have all been studies (121). One strategy for improving the uptake of plasmid
investigated, although recent studies have focused mainly on DNA into antigen presenting cells (APCs) is by using
their application in cellular based therapies and vaccines. GM- electroporation. Electroporation delivers small electrical pulses
CSF is the most studied immunostimulatory factor and has been that causes transient pores to form in the cell membrane. During
included in numerous cancer vaccine trials (120). In preclinical the period of membrane destabilization plasmid DNA present in
studies GM-CSF looked a very promising candidate, it helps the extracellular environment around the target cell gains access
recruit DCs to the injection site, it can promote the maturation of to the intracellular compartment (122). Following the transfer of
DCs and antigen presentation resulting in an enhanced adaptive DNA into the cell the membrane then reseals. The transient
immune response (117). However, in clinical trials GM-CSF has increase in the permeability of the target cell membrane
generated disappointing results with only a few trials having enhances the uptake of plasmid DNA (123). Electroporation
shown a clinical benefit, the results across the majority of trials increases DNA uptake by over a 1000-fold and has an adjuvant
have been inconsistent. Preclinical studies indicated that GM- effect due to local tissue damage and the resulting stimulation of
CSF could expand MDSCs resulting in the suppression of cell proinflammatory cytokine in the local vicinity (124, 125). A

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Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

number of DNA vaccines currently in clinical trials are using efficient targeting of RNA to dendritic cells. The optimized
electroporation to delivery DNA plasmids, the ability of these Lipoplex:RNA formulation uses a charge ratio of 1.3:2, this was
plasmids to induce an immune response has been demonstrated found to effectively target RNA to the spleen, form monodisperse
in prostate cancer and melanoma (126). Another similar strategy and stable particles and was fully resistant to degradation by
is using a gene gun to deliver plasmid DNA that is coated with a mouse serum at 37°C (135). In addition to targeting APCs,
heavy metal, typically gold particle are used, APCs at the liposomes can also protect the RNA to be delivered from
injection site are bombarded with plasmid coated particles. extracellular ribonucleases and mediates efficient uptake and
The gene gun strategy reduces the amount of DNA required expression by DCs and macrophages located in various
by 100-1,000 (127); some promising preclinical data has led to lymphoid compartments. Lipoplex complexed with RNA
phase 1 and 2 clinical trials in head and neck squamous cell encoding tumor antigens has also been shown to induce strong
carcinoma and cervical cancer (128). Electroporation is also effector and memory T cells responses and mediate IFNa
being used to deliver plasmid DNA in the infectious disease dependent rejection of progressive tumors (135). Vaccines
field, there are a number of COVID-19 DNA vaccines currently using Lipoplex complexed with RNA induce and mobilize both
in in clinical trial (WHO landscape report Dec 2020) that use the adaptive and innate immune responses mimicking an
DNA plasmids encoding the Spike antigen and using antiviral response.
electroporation as a delivery system (NCT04445389, Sahin et al. (136) recently conducted at phase 1 trial
NCT04447781, NCT04642638, NCT04627675). The main (NCT02410733) in melanoma patients who received
disadvantages of a DNA vaccine is when electroporation is melanoma FixVac (BNT111) an intravenously administered
used as a delivery system, electroporation can cause liposomal RNA (RNA-LPX) vaccine that targets four tumor
considerable pain and anxiety on administration and not associated antigens (NY-ESO-1, MAGE-A3, tyrosinase and
suitable for mass vaccination programs, alternative delivery TPTE) (135). Interim analysis (136) has shown that melanoma
systems are currently being pursued. FixVac when used alone or in combination with the checkpoint
inhibitor PD-1 mediates durable responses in patients with
Nanoparticle Vaccine Delivery Systems unresectable melanoma and with prior experience with a
Nanoparticle based drug delivery platforms offer an alternative checkpoint inhibitor. FixVac induced clinical responses and
vehicle for delivering drugs that have previously suffered from potent CD4+ and CD8+ T cell responses could be detected
pharmacokinetic limitations including poor bioavailability, a with the cytotoxic T cell responses in some patients reaching the
short half-life or poor solubility. A variety of nanoparticles same level as reported for patients on T cell therapy trials. The
have been explored as delivery systems or as adjuvants, such as completion date for this trial is estimated for December 2021, so
polymeric nanoparticles, liposomes, micelles, carbon nanotubes, far 119 patients (as of August 2020) have been enrolled.
mesoporous silica nanoparticles, gold nanoparticles and virus
nanoparticles, that can all be used alone or in combination (129). Self-Assembling Peptides
Liposomes are a popular nanoparticle vaccine delivery system, Self-assembling peptides can also be used as a delivery system to
they are versatile and can be constructed with a variety of deliver antigens to target cells. Self-assembling peptides can
different properties by changing the lipid composition, charge, spontaneously form into ordered structures in response to
size and surface properties (130–132). Nanoparticle based drug changes in pH, solvent, co-assembling molecules, temperature
delivery platforms use well-known lipid carriers to deliver and ionic strength (137, 138). They can have a diverse range of
biotherapeutic encoding tumor antigens directly into APCs properties and can be manufactured to form nanomicelles,
such as dendritic cells. The targeting of APCs in the lymphoid nanovesicles, nanofibers, nanotubes, nanoribbons and
compartments is accomplished by using well-known lipid hydrogels (139). Self-assembling peptide deliver systems have a
carriers such as DOTMA, DOTAP, DOPE and cholesterol and number of advantages over liposomes or nanoparticles including
by adjusting negative net charge of the nanoparticles to provide high drug loading, low drug leakage, biodegradability and are
optimal drug delivery. Cationic liposomes are mainly composed highly permeable to target cell membranes. The particle size is
of the lipids DOTMA and DOPE that form colloidally stable important for vaccine delivery and can impact the efficiency of
nanoparticles of reproducible particle size (200–400 nm) with an uptake by APCs, with smaller particles (20-200 nm) being more
excess of positive charge preventing excessive aggregation (133). immunogenic but there is no optimal size and this should be
Liposomes can increase the immunogenicity of target antigens optimized for each vaccine candidate (140–142). The smaller
for cancer vaccines and have been used to deliver RNA, DNA particle size is thought to improve uptake into DCs and also the
and antigens. Hydrophilic and lipophilic antigens can be loaded lymphatic system, in addition to size the shape, stability and
into liposomes, the hydrophilic antigens are trapped in the ability to display multiple antigen can also improve
aqueous inner space and the lipophilic components are immunogenicity. Self-assembling peptides can be designed to
inserted into the lipid bilayer by adsorption or chemical provide vaccines with the desired properties to enable efficient
attachment. Liposomes have been utilized to improve lymph delivery to the target cell.
node trafficking of small molecule adjuvants to the lymph A delivery system based on modified cell penetrating peptide
node (134) (CPP) based gene vectors, the Glycosaminoglycan (GAG)-binding
BioNTech have developed a lipid-based nanoparticle enhanced transduction (GET) delivery system has been used to
formulation called Lipoplex. Lipoplex has been used to provide enhance delivery of nucleic acids for lung gene therapy and bone

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regeneration in vivo (143, 144). The GET peptides (143–150) are eradicate established tumors in preclinical models (160).
multi-domain sequences comprising of a heparan sulphate (HS) Immunization with ImmunoBody® DNA vectors induces high
cell targeting sequence fused to a CPP for improved membrane frequency and avidity of T cell responses that are superior when
association and synergistically enhanced intracellular delivery of compared to those induced by immunization with DNA
therapeutic cargoes (145). GET peptides can deliver self-reporting encoding full-length antigen or when using peptides or peptide
cargo (monomeric red fluorescent protein; mRFP) into difficult to loaded onto dendritic cells (161–164). The first ImmunoBody®
transduce cell types including mesenchymal stem cells (MSCs), in the SCIB series, SCIB1, targets four epitopes from the
human embryonic stem cells (hESCs) and human induced melanoma-associated antigens TRP2 and gp100, our second
pluripotent stem cells (hiPSCs). Delivery involves a heparin vaccine, SCIB2, incorporates several epitopes derived from the
sulphate (HS) cell targeting system fused to a CPP and an NY-ESO-1 cancer testis antigen. A first-in-human study
endosomal escape peptide and a system which stabilizes performed by Patel et al. used SCIB1 ImmunoBody® that
particles to prevent aggregation and promote diffusion and cell incorporated HLA-A*02:01 restricted epitopes from gp100 and
uptake by PEGylation. The tripeptide complexes the DNA into TRP-2 in addition to HLA-DR*04:01 and HLA-DR7/DR53/DQ6
nanoparticles and can be delivered by simple intramuscular restricted epitopes from gp100. In a cohort of 15 melanoma
injection. This tripeptide formulation has achieved exceptional patients SCIB1 was shown to be safe (165). In this trial 7/15
results in DNA delivery applications in particular in lung, brain, patients had stable disease, 5/20 fully resected patients
and has huge potential in vaccine delivery. experienced disease recurrence and 1 patient had measurable
disease, all patients were still alive at the last observation time of
37 months. A phase 2 study in melanoma patients receiving
pembrolizumab is now recruiting (ClinicalTrials.gov
GENETIC VACCINES Identifier: NCT04079166).

DNA Vaccines RNA Vaccines

DNA vaccines have a number of advantages, they are simple to The RNA vaccine platform has the advantage that RNA does not
design, relatively low production costs, good stability (stable at + integrate into the host cell genome and thus avoids potential
2-8°C) and solubility, can be rapidly modified, it is a versatile safety concerns, it is also quick to manufacture and can encode
platform that can have many applications including in infectious multiple epitopes. RNA is single stranded and therefore has a
diseases and oncology. DNA vaccines were first shown to be built-in adjuvant function through TLR7 and TLR8 stimulation.
immunogenic in the 1990s (151–153), they are an attractive cancer However, RNA is very susceptible to cellular degradation, to
vaccine platform (154) and this led to a flood in preclinical and overcome this in clinical trials it has either been injected directly
clinical trials. Plasmid DNA vaccines can be designed to act as into inguinal lymph nodes or delivered using a nanoparticle
both an antigen and adjuvant (155), unmethylated DNA delivery system that protects the RNA. RNA is particularly
containing cytosine-guanine rich regions can act as an adjuvant susceptible to degradation by RNases, to improve transfection
stimulating an immune response (156). DNA vectors have a and avoid degradation many groups have used delivery systems
negatively charged backbone and have a low molecular weight, such as nanoparticles and liposomes (135, 166–170). RNA has an
therefore naked DNA often suffers from poor cellular uptake advantage over DNA in that it only needs to be delivered to the
resulting in poor antigen production (157–159). With a significant cytoplasm for translational into protein unlike DNA that needs
improvement in our knowledge of cancer immunology particular to enter the nucleus for transcription.
the TME and immune suppression the reasons for the failures of The first clinical trials using RNA was performed by Weide
early DNA vaccines are now better understood. et al. (171, 172) in patients with metastatic melanoma. In a phase 1
In addition to improving the delivery of plasmid DNA the trial in 15 melanoma patients (171) the intradermal
vector itself can also be modified to specifically target epitopes administration of naked mRNA was shown to be safe. In a
directly to APCs. In preclinical models we have previously phase 1/2 trial (172) 21 patients received i.d. injections of
demonstrated that a DNA plasmid encoding T cell epitopes protamine stabilized mRNA coding for Melan-A, Tyrosinase,
within the complementarity determining regions of a human gp100, Mage-A1, Mage-A3 and survivin; GM-CSF was used as
IgG1 antibody (ImmunoBody®) (160), when administered with an adjuvant and half the patients also had keyhole limpet
electroporation (EP) stimulates high avidity T cell responses hemocyanin added to the vaccine. The number of clinical
(161). ImmunoBody® works by the direct uptake of the DNA responses to the vaccine in this trial was low with only 1
into APCs, it is then transcribed, translated and processed, with promising clinical response observed in a patient with
epitopes being presented on the cell surface in combination with measurable disease. In a phase 1/2 trial performed by Rittig
MHC. ImmunoBody® can also be taken up by both antigen et al. in 30 patients with stage IV renal cell cancer (173), naked
presenting cells and non-antigen presenting cells and the mRNA coding for TAA’s was administered intradermally. This
transcribed antibody protein secreted. The secreted antibody is trial demonstrated that vaccination was safe and well tolerated and
internalized via the high affinity FcgR1 receptor (CD64) on induced clinical responses in 16 patients; this trial also
antigen presenting cells, it is then processed, and epitopes cross demonstrated that vaccination induced CD4 and CD8 T cell
presented on MHC class I. The combination of direct and cross responses as determined by IFNg ELISpot and Cr-release assays.
presentation induces T cells with sufficiently high avidity to The results from these trials demonstrated that vaccination with

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RNA was feasible and safe. With improvements to trial design, vectors are used. A prime boost vaccine regime is commonly
frequency, and route of administration the RNA vaccine platform used, and a number of different strategies have been used to
is progressing through clinical trials in the fields of cancer and overcome the problem with pre-exiting immunity. Strategies
infectious diseases. For COVID-19 two RNA vaccines using a non-human specific virus such as the replication-
(Tozinameran from Pfizer–BioNTech and mRNA-1273 from defective chimpanzee adenovirus (ChAd68 serotype), or using
Moderna) have now been approved by national health regulators. different vectors derived from different viruses for the prime and
In a study performed by Sahin et al. the use of an RNA boost immunization or using different vaccine platforms for the
vaccine encoding neoantigens was explored (174) in melanoma prime and boost immunizations can all avoid problems
patients. In this study neoantigens were identified by associated with pre-existing immunity to the virus vector. A
comparative exome analysis in tumors from thirteen patients common combination is the use of a DNA prime and a viral
with stage III and IV melanoma. Mutations were selected for vector boost. Another commonly used combination is the
incorporation into the vaccine based firstly on the predicted Modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad)
binding score for HLA class II and secondly based on the vectors, both vectors induce potent immune responses that when
predicted binding score for HLA class I. For each patient two used in combination in a prime boost regime these responses are
synthetic RNAs were synthesized incorporating the identified further enhanced (176, 177). These strategies have all been used
mutations. The RNA vaccine was produced within 68 days successfully in the infectious disease field, particularly more
(range 49 to 102 days), following analytical testing they were recently to target SARS-CoV-2, where 40 viral vector vaccines
released within 103 days (range 89 to 160 days). RNA vaccines are currently being assessed in preclinical studies, an additional
work in a similar way to the long peptide vaccines, the RNA is 19 vaccines are currently in clinical trials and another 4 vaccines
translated into protein which is then processed into long peptides have already received approval from regulatory authorities (178).
by APCs, these peptides are then loaded onto MHC class I or A number of different viral vectors have been used for cancer
class II molecules and presented on the cell surface to prime and vaccines (179); with some having progressed into clinical trials.
activate T cells. This study demonstrated the clinical feasibility In clinical trials the efficacy of cancer vaccines using viral vectors
and safety of RNA neo-epitope vaccines. In this study 8/13 have not delivered the same results as those generated in the
patients had no tumors develop during the monitoring period infectious disease field. The immunosuppressive TME and
and neoantigen specific T cells could be detected in the selection of the best cancer antigen to target is problematic and
peripheral blood of these patients. The use of many neoantigen impacts all cancer vaccine platforms. To overcome central
epitopes in a vaccine reduces the risk of single antigen loss tolerance and the immune suppressive TME a cancer vaccine
variants (175), however, in this study the outgrowth of B2M would need multiple boosts in order to induce and sustain a
deficient tumor cells in one patient demonstrates the complexity potent immune response, however, this can be problematic due
of the TME and the selective pressures that drive resistance to anti-vector immunity. In preclinical and clinical studies, a
to therapy. prime-boost approach using a recombinant vaccinia vector and a
recombinant avipox virus have been successfully used, and
multiple boosts using recombinant avipox such as fowlpox is
VIRAL VECTOR VACCINES possible. Preclinical and clinical studies have shown that multiple
booster vaccinations using a fowlpox does not induce host anti-
Viral vectors have been used in both the gene therapy and vector immune responses (180, 181). Both viruses have been
vaccine fields. Viral vectors have the advantage of being shown to be safe, vaccinia was used in the smallpox vaccine that
recognized as foreign by the immune system, inducing potent has been delivered to over 1 billion people worldwide. Avipox is
innate and adaptive immune responses resulting in the induction an avian virus that is unable to replicate in mammals. Both
of strong and durable immune responses. Viral vectors enable vectors do not integrate into DNA and successfully infect APCs
the presentation of intracellular antigens incorporated into the thus stimulate potent immune responses.
vector such as cancer antigens, viral antigens, or a specific gene The TRICOM vaccine platform uses the recombinant
for gene therapy. vaccinia virus (rV-) for the prime and recombinant avipox
The most commonly used viral vectors are derived from (fowlpox, rF-) for multiple booster vaccinations. Each vector
adenoviruses, poxviruses and alpha viruses. The majority of contains one or more TAAs and transgenes for the costimulatory
viral vectors are replication defective or attenuated versions, molecules CD80, ICAM1 and LFA-3. In a phase 2 clinical trial,
these are preferred from a safety point of view. Viral vectors 125 men with metastatic castration-resistant prostate cancer
have a very good safety record with many approved in the received a vaccinia virus encoding PSA in combination with
infectious disease field such a recently approved Ebola vaccine GM-CSF followed by six subsequent boosts using a fowlpox virus
and COVID-19 vaccines that use adenovirus virus vectors. A encoding PSA (PROSTVAC-VF) (182). The results from this
disadvantage of the viral vectors is their ability to also induce phase 2 trial was encouraging with a 10-month improvement in
immune responses that also neutralizes the vector preventing overall survival compared to the empty vector control group
further repeat immunizations. Pre-existing immunity to measles (183). Unfortunately, these results were not seen in a large phase
and adenovirus can be problematic limiting the effectiveness and 3 study and the study was subsequently stopped (184). It is likely
ability to boost responses when adenovirus or measles virus that despite the activation of specific T cells they were either not

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Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

potent or unable to overcome the immunosuppressive TME recurrence while 2 patients had progressive disease that was
(184). Trials are now ongoing to see if combining successfully treated with anti-PD-1 therapy.
PROSTVAC-VF/TRICOM with check point inhibitors can A number of early peptide-based cancer vaccine trials
improve clinical responses (NCT02933255, NCT04020094, primarily focused on short peptides from tumor associated
NCT03532217, and NCT03315871). antigens, and did not include any delivery system or longer
peptide formulations (189). The majority of these trials failed
when reaching phase 3 trials, a number of additional reasons for
PEPTIDE VACCINES these failures include adjuvant selection, timing of vaccination
and peptide formulation (120). Peptide vaccines do generate an
The number of peptide vaccines being explored has increased immune response but as a monotherapy they can struggle to
due to the discovery of neoantigens. Targeting neoantigens is a show efficacy in the clinic. Kimura et al. immunized 39 patients
personalized therapy and the rapid synthesis of peptides makes with premalignant colon adenomas with a MUC1 peptide
the peptides vaccines an attractive platform. Almost half of the vaccine, they showed the peptides were immunogenic however
clinical trials currently recruiting (as of August 2020) that target in 22 patients there was a lack of a response which correlated
neoantigens are using peptide vaccines, with the RNA and DNA with a high number of myeloid derived suppressor cells in the
vaccine platforms also represented (Figure 3). Following TME pre vaccination (190). In another two trials in melanoma
administration the peptides included in a vaccine need to be and ovarian cancer patients (191, 192), a mixture of peptides
presented on antigen presenting cells (APCs) in order to trigger were used to immunize patients, vaccination induced an immune
an adaptive immune response. To efficiently prime an immune response which was associated with some favorable outcomes.
response the coadministration of an adjuvant is required to The majority of peptide vaccines did not generate an immune
activate the immune system to kill tumor cells expressing the response that was robust enough to see any significant clinical
peptide (185–187). Tumor antigens need to be processed and the benefit (189). These early studies highlighted the need to further
resulting peptides presented on the cell surface in association optimize peptide vaccines, either by better targeting, better
with MHC class I or class II molecules. Cancer specific T cells in adjuvants or used in combination to overcome the
the TME need to recognize the relevant peptides and kill tumor immunosuppressive TME.
cells expressing them. The key to the success of a peptide vaccine
relies on the correct choice of peptides to include and the best Peptide-Adjuvant Conjugate Vaccines
adjuvant to use to generate a local immune response and The administration of free adjuvant with antigens in a cancer
promote antigen trafficking to local draining lymph nodes. vaccine can result in their dissociation following injection and
Bioinformatic applications and algorithm prediction program subsequently do not enter the lymphatic system. Adjuvants can
are commonly used to define peptides capable of binding MHC I be rapidly degraded (193) reducing the amount that reaches the
or MHC class II molecules. Identification of peptides bound to target cells resulting in suboptimal antigen priming and immune
the MHC molecules on the cell surface can be achieved via mass response. Free adjuvant in the circulation can also induce
spectrometry (MS) analysis. Combining data from MS analysis, autoimmunity (194) or toxicity. The co-delivery of adjuvant
epitope predicting algorithms and gene expression data help to with antigens is required to induce a potent immune response
predict the best candidate peptides to include in a vaccine. while avoiding any autoimmunity or toxicity, as already
A vaccine needs to stimulate both CD4+ and CD8+ specific T described a delivery system can be used to deliver the adjuvant
cells. The majority of peptide vaccines use longer peptides and antigen, alternatively the adjuvant and antigen can be linked
typically 20-30mers, these are likely to contain nested CD8+ T to improve targeting.
cell epitopes in addition to longer CD4 T cell epitopes and The direct conjugation of a peptide to an adjuvant is gaining
therefore are able to stimulate both CD4+ and CD8+ T cells. In increasing attention, particularly from groups targeting
addition, multi-peptide vaccines are often used, incorporating neoantigens. Previous reports have described the direct
many peptides meaning that many antigens can be targeted, conjugation of peptides to TLR ligands can enhance the
increasing the chances of overcoming any antigen loss on the immune response by directly targeting the peptide and adjuvant
tumor cells. A number of peptide vaccines targeting neoantigens to the same APC (195–199). Peptides can be linked to
have been developed by a number of groups, this personalized hydrophobic carriers such as lipids (92), fatty acids (200) and
therapy can target a patient’s individual tumor. Ott et al. (188) TLR agonists (196, 201, 202) for more efficient delivery to APCs
used whole-exome sequencing (WES) and RNA-sequencing and subsequently lymph nodes. A recent preclinical study
(RNA-Seq) to identify neoantigens from six stage III/IV performed by Lynn et al. (203) used a peptide platform based
melanoma patients. Using NetMHCpan (v2.4) a list of peptides on charge modified TLR-7/8a peptide conjugates, these conjugates
that bind to MHC class I was generated, synthesized peptides self-assemble into nanoparticles of uniform size which is
were between 15 to 30 amino acids in length and thus capable of independent of the peptide antigen composition. This platform
stimulating CD4+ and CD8+ T cells. Patients were immunized is used to conjugate identified neoantigen peptides, these peptides
with 30 peptides given in combination with poly-ICLC. This would possess a variety of properties, such a platform would be
vaccine-induced polyfunctional CD4+ and CD8+ T cells able to incorporate peptides with a wide range of characteristics.
targeting 58/97 and 15/97 neoantigens respectively across the Neoantigen peptides were predicted (179 peptides) from three
patients. At 25 months post vaccination 4 patients had no murine tumor models, vaccination of mice induced a CD8+ T cell

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Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

FIGURE 3 | Neoantigens currently in clinical trial. According to clinicaltrials.gov (as of 23rd September 2020) there are currently 33 clinical trials recruiting that target neoantigens.

response with approximately 50% of the peptides being be needed to induce T cell priming (203, 214). Peptide conjugates
recognized, this led to enhanced tumor clearance. used in combination with other therapies have a huge potential.
We have directly conjugated citrullinated peptides from A peptide conjugate vaccine can specifically target APCs and that
vimentin and a-enolase (Vim28-49cit, Vim415-433cit and has the advantage of being dose sparing, in combination with a
Eno241-260cit) to the TLR1/2 ligand AMPLIVANT®. The checkpoint inhibitor to relieve immunosuppression in the TME
direct linkage of a TLR agonist to a peptide can enhances the this would provide the best opportunity for these vaccines to
immunogenicity of the vaccine (196–199). In HLA-DR4 and work in the clinic.
DP4 transgenic mice vaccination with the conjugated peptides
induced a high frequency of specific T cells. The direct
conjugation of the Vim28cit, Vim415cit and Eno241cit VACCINES IN COMBINATION WITH
peptides to the TLR1/2 ligand Amplivant ® reduced the OTHER THERAPIES
peptide-equivalent dose required to induce immune responses
by at least 1 log without the loss anti-tumor responses (60). These Immune checkpoint inhibitors constitute an important
results demonstrate that the linkage of a TLR ligand to a peptide breakthrough positively influencing treatment outcomes in
enhances the immune response and supports the development cancer patients. Cancer vaccines have the potential to induce
and application of these peptide/TLR ligand linked conjugates in potent immune responses but are hampered as tumor cells
a clinical setting. possess a variety of immune evasions mechanisms that interfere
Peptide vaccines have many advantages, they can be with the function of T cells (215–220). Upon activation, T cells
chemically synthesized, manufactured at large scale and cost migrate and accumulate in the TME where they can induce tumor
effective (204) when compared to other cancer therapies. In pre- cell killing, however, tumors have evolved multiple mechanisms
clinical and clinical studies they have been shown to be safe and that can dampen or inhibit T cell mediated killing. Tumor cells can
well tolerated (204, 205). Peptide vaccines should include alter the antigen processing machinery, secrete
peptides that target multiple antigens to generate a polyclonal immunosuppressive factors that kill the T cells or activate
antigen T cell response (206–208). Like DNA and RNA vaccines pathways that induce tolerance rendering any tumor therapy
the use of a delivery system can help improve the targeting and ineffective (221). The identification of the key regulators of the
stability of peptides used in a vaccine which reduces any immune response has led to the generation of new therapies that
potential off target effects (209–212). The production of have the potential to reverse some of the immune suppression in
conjugated peptide vaccines can be problematic for some the TME. Immune checkpoint inhibitors are cell surface receptors
peptides, particularly hydrophobic peptides that tend to form that regulate the immune response, they enable self-tolerance
aggregates that complicate manufacturing and when injected while preventing over activation of the immune system resulting
they form injection site depots leading suboptimal immune in autoimmune disease (222). In the TME the expression of
responses (213). The conjugation of peptides with adjuvants checkpoint receptors suppresses T cell activation and thus
improves the delivery of both components to APCs which may provides the tumor with a growth advantage (223). The

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Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

cytotoxic T lymphocyte protein 4 (CTLA-4) and programmed cell combination with another form of therapy is required to
death protein 1 (PD-1) are the best characterised checkpoint improve tumor specific T cell function in the TME.
receptors in the immunotherapy field, others have been In preclinical murine models using the ImmunoBody ®
described that are emerging from preclinical studies and vaccine SCIB2 a synergistic effect was observed when given in
entering the clinic. combination with anti-PD-1 (Figure 4) (237). The synergistic
The checkpoint receptor, PD-1, is expressed on activated T effect was also observed with SCIB1 when given with anti-PD1
cells and overexpressed on exhausted T cells (90). There are two (163, 237). These results demonstrate that a cancer vaccine on its
PD-1 ligands, PD-L1 and PD-L2, PD-L1 is expressed on many own will not achieve the expected results in the clinic without
cells including immune cells, epithelial cells, endothelial cells and combining with other therapies aimed at modifying the TME by
tumor cells (88, 91). PD-L2 is expressed on professional antigen reducing immune suppression and also improving T cell
presenting cells including DCs and macrophages (224). The trafficking into the tumor.
binding of PD-1 on T cells to PD-L1 expressed on a tumor cell The first line treatment for the majority of cancer indications is
or PD-L2 expressed on an APC leads to TCR downregulation, radiotherapy or chemotherapy. These traditional treatments are
resulting in lower secretion of TNF-a, IFN-g, and IL-2 (92). The not targeted therapies but the damage they cause to tumors results
expression of CTLA-4 is induced upon T cell activation and in the release of more antigens from the tumor cells. Damage to
competes with the costimulatory molecule CD28 for its co- tissue surrounding a tumor can also induce the recruitment of T
stimulatory ligands. CTLA-4 suppresses the early activation of cells into the vicinity, this is particularly valuable when the tumor
naïve and memory T cells by competing with CD28 (88–90), PD- mutational burden (TMB) is low (238). A number of clinical trials
1 inhibits T cell function at a later activation stage by down are using either radiotherapy or chemotherapy to enhance the
regulating TCR expression. Monoclonal antibodies that block immune response to a vaccine. Radiotherapy can enhance the
CTLA-4 (223), PD-1 (225) or PD-L1 (226) pathways remove the recruitment of T cells into tumor tissue and increase the intensity
inhibition of T cell function (227) and have made significant of specific anti-tumor immune responses (239). Other studies have
clinical impacts. Antibodies that specifically block the CTLA-4 or shown that some chemotherapeutic drugs can enhance the
PD-1/PD-L1 pathway have the potential to remove T cell antitumor activity of tumor vaccines (140, 141) and adoptively
immune suppression enabling the successful recognition and transferred T cells (137, 138). A vaccine combined with immune
killing of tumor cells. checkpoint inhibitors or traditional treatments can induce
Checkpoint blockade has shown promising results in clinical stronger anti-tumor responses (188), as such the majority of
trials and have gained approval for an increasing number of tumor vaccines in clinical trials are in combination with other
cancers including melanoma, renal-cell carcinoma (RCC), therapies. The majority of cancer patients will be offered first line
advanced non-small-cell lung cancer (NSCLC), classic standard of care treatment prior to being offered alternative
Hodgkin’s lymphoma (HL), bladder carcinoma, Merkel cell therapies or participation in a clinical trial.
carcinoma, head and neck cancer, and more recently, solid
tumors with mismatch repair-deficiency. PD-1 and CTLA-4
inhibit T cell responses at different stages and by different
mechanisms, it is therefore tempting to block both pathways in
order to overcome immune suppression. Clinical trials in
melanoma patients that have combined PD-1 and CTLA-4
blockade have shown improved clinical responses, however,
these have come at a cost with an increase in toxicities being
reported (228–230). The combination of anti CTLA-4 and anti
PD-1 is now approved as the first line therapy for advanced
melanoma patients; however, the toxicities have limited the use
of this combination, trials are ongoing to vary the dose and
interval of dosing to reduce toxicity.
Many cancer vaccines currently in clinical trials are combined
with a checkpoint inhibitor such as CTLA-4, PD-1 or PD-L1
inhibitors, which are offered as standard treatment for an
increasing number of cancers. A couple of comparative studies
have shown that the combination of a tumor vaccine with a
checkpoint inhibitor is more effective than monotherapy (231,
FIGURE 4 | Survival of HHDII mice challenged with 5 x104 tumor cells and
232). Less than 50% of patients respond to checkpoint inhibitors immunized with SCIB2 and anti-PD-1 antibody alone or in combination.
(233–235), there are several other factors that can lead to Control vs SCIB2 (*p = 0.037); Control vs anti-PD-1 antibody (p = 0.111);
immune suppression in the TME, such as the action of T- Control vs SCIB2 and anti-PD-1 antibody (***p = 0.0003); SCIB2 vs SCIB2
regulatory cells, myeloid-derived suppressor cells (MDSCs), and anti-PD-1 (* = 0.0177); anti-PD-1 antibody vs SCIB2 and anti-PD-1
(*P = 0.0177). Lack of survival was defined as tumor size > 528 mm3. Each
tumor associated macrophages and immunosuppressive DCs
curve represents at least 10 mice per group.
(236). For a vaccine to show efficacy in the clinic it is likely a

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TUMOR MICROENVIRONMENT AND tumor associated macrophages (TAMs) and cancer associated
TUMOR INDUCED IMMUNE fibroblasts all contribute to this immune suppression (246).
SUPPRESSION MDSCs are a type of regulatory cell that are found within the
TME (246, 247), they produce nitric oxide, cytokines and reactive
Tumors evolve and change, they are heterogenous and oxygen species that can suppress T cells. MDSCs play a critical role
genetically unstable. Tumors generate many mutations in tumor invasion, metastasis and angiogenesis (248, 249). The
overtime, some are cloned, altered or lost in the tumor presence of MDSCs in the TME correlates with poor overall
genome. Advances in sequencing technology now allows the survival and progression free survival (250). In a murine model
analysis of a resected tumor or biopsy, the data gathered is a of rhabdomyosarcoma, the trafficking of MDSCs was inhibited
snapshot of a single tumor or part of a tumor at a specific time and subsequently an enhanced response to anti-PD-1 therapy was
and does not provide information regarding the overall observed (251). In addition to MDSCs the presence of Tregs in the
heterogeneity in the tumor (240, 241). This is a particular TME in many cancer types is also associated with a poor prognosis
problem with the development of a personalized vaccine (252). Tregs are critical for the maintenance of cell tolerance, they
targeting a neoantigen, the information gained via biopsy or suppress T cell responses by binding IL-2 therefore limiting the
resection may represent a mutated tumor subclone or the amount of free IL-2 available to drive T cell proliferation and
neoantigen may be not expressed in the whole tumor or activation (253). Tregs express CTLA-4 and can produce
metastatic tumors compromising the effectiveness of the immunosuppressive cytokines that further contribute to the
vaccine (242). The ideal mutations to target are driver immune suppressive TME (252). The TAMs, in particular
mutations, these are critical for the growth of the tumor and the M2 macrophages are another cell type that can contribute to
are usually expressed in every tumor cell. However, the number the immune suppressive TME (254). The M2 macrophages can
of driver mutations can be low, for example in melanoma only promote tumor growth by stimulating tumor cell motility,
8% neoantigens are driver mutations (243). The degree of tumor angiogenesis, and immune evasion (255). Murine studies have
heterogeneity will vary between patients, indications, and demonstrated that the depletion of macrophages reduces tumor
tumors. Improving our understanding of tumor heterogeneity growth and also by inhibiting the myeloid growth factor signaling
will help identify the best epitopes to include in a cancer vaccine pathway in macrophages overcome resistance to checkpoint
and targeting more than one antigen with help overcome tumor inhibitors in a pancreatic cancer murine model (256, 257). The
heterogeneity. The cancer vaccines targeting neoantigens address depletion or inhibition of MDSCs, Tregs or TAMs all have the
this heterogeneity by targeting more than one neoantigen and potential to improve anti-tumor responses induced by vaccination
also addressing potential antigen loss. or a cellular therapy, however, the impact of depleting these cells in
Another factor that significantly contributes to the success the periphery as well as the tumor increases the potential to
immunotherapy is the TMB, studies by Rooney et al. induce autoimmunity.
demonstrated that the TMB correlated with immune responses Within the TME the most abundant stromal cells are the
(244). Tumors with high TMB, such as melanoma and NSCLC cancer associated fibroblasts (CAF), these have been shown to play
have a higher response rate to immunotherapy compared to a role in tumorigenesis, angiogenesis, metastasis, drug resistance,
tumors with a low TMB, however, this is not the case with all immunosuppression, extracellular matrix (ECM), remodeling and
tumors. Pediatric tumors generally have fewer somatic maintenance of cancer stemness (258–264). Different subtypes of
mutations, a study performed by Zamora et al. showed that CAFs exist each capable of secreting a number of cytokines and
tumors from children with acute lymphoblastic leukemia had a chemokines such as TGF-b, IL-6, IL-8, IL-13, CXCL12, CXCL14,
low TMB but they could still induce a strong anti-tumor and VEGF that inhibit anti-tumor immune responses. Some CAFs
response (245). A number of clinical trials are underway for also express PD-L1/PD-L2 or produce metabolites or enzymes
cancer indications that have an unmet need, poor survival and such as indoleamine-2,3-dioxygenase (IDO), arginase (Arg),
also have a low TMB e.g. glioblastoma and pancreatic cancer. adenosine, and tryptase that recruit Tregs, mast cells and TAMs.
Hopefully the results from these trials will help with our CAFs can also contribute to the integrity of tumors, they can
understanding of how to target tumors with a low TMB. synthesize components that make up the ECM such as collagen,
The TME consists of many different cell types including fibronectin, matrix metalloproteinases and can contribute to ECM
immune and stromal cells, vasculature, extracellular matrix stiffness and thus prevent T cell infiltration. The role of CAFs in
and a variety of cytokines and chemokines. The extracellular cancer progression makes them a promising target for cancer
matrix is made up of cells from endothelial, mesenchymal and therapy. There have been a few studies looking at anti-CAF based
haematopoietic origins. Changes in the TME impact the therapies, however in a murine CAR T cell-based study targeting
trafficking of TILs and efficacy of cancer vaccines that have the fibroblast marker FAP, toxicity was observed due to expression
induced specific T cells but are unable to traffic into the tumor. of FAP on other tissues (265). Other studies are looking at
Tumors have a number of mechanisms that have evolved to depleting CAFs, blocking their function or altering their function.
suppress anti-tumor immune responses. Apart from checkpoint Cytokines and chemokines within the TME can also induce
mediators such as PD-1 and CTLA-4 a number of cell types have an immune suppressive TME and reduce T cells responses. One
been identified that contribute to immune tolerance and evasion in of the most studied cytokines is transforming growth factor beta
the TME, Myeloid-derived suppressor cells (MDSCs), T regs, (TGF-b). TGF-b signaling has a massive impact in the TME

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Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

where it can influence cell growth, differentiation and apoptosis CD8+ T cell responses. Targeting non self or mutated tumor
while also inhibiting T cells responses and upregulating Tregs antigens will induce high avidity T cells responses when
(266). In patients with colon cancer TGF-b tends to suppress cell delivered with optimal adjuvant and delivery systems, as such
growth but in advanced stages of the disease the presence of cells the neoantigens and post translational modified antigens
expressing members of TGF-b superfamily tend to have a poor currently show the most promise.
prognosis. In murine models the inhibition of TGF-b reverses its Generating a neoantigen vaccine can be costly both in terms
immunosuppressive effects and improves the activity of T cells of time and money, the peptide vaccines are personalized and
also rendering tumors suspectable to treatment with checkpoint require a significant amount of bioinformatics input to generate
blockade (267, 268). In addition to cytokines in the TME, the best candidate neoepitopes. The Modi-1 vaccine is not a
chemokines such as CXCR2 and CXCR4 that bind to MDSCs personalized therapy and is broadly applicable to many patients
and Tregs respectively contribute to tumor immune evasion. In and cancer types, it is cheaper to manufacture when compared to
murine models the inhibition of CXCR2 and CXCR4 in other platforms and in addition has no time delay constraints
combination with anti-PD-1 reversed immune evasion (251, that is associated with the production of neoepitope vaccines.
269). Targeting cytokines and chemokines in TME could be a The vaccine platform and the delivery systems used have
good cancer immunotherapy strategy that will help change the undergone a huge number of improvements over the last decade.
immune suppressive environment by preventing the recruitment Many new adjuvants have emerged or are currently being
and activation of Tregs, TAMs and MDSCs. investigated in order to improve the immune response at the
Cancer cells can also lose surface antigens as an immune injection site while also increasing antigen trafficking to the lymph
evasion mechanism following natural or therapy induced nodes. The majority of cancer vaccines are currently using TLR
selective pressure. Antigen loss has been observed for CD19 in agonists as adjuvants, these have also been conjugated to peptides
acute lymphocytic leukemia and CD20 in chronic lymphocytic or included in nanoparticles to improve targeting. Other adjuvants
leukemia. Antigen loss is a common reason for resistance to such as STING, CD40 agonist and GM-CSF are currently being
therapy and subsequent relapse. To address the problem with investigated in clinical trials. The correct selection of an adjuvant is
potential antigen loss we have targeted two antigens in our Modi- key to the ability of the vaccine to induce a robust immune
1 and SCIB1 vaccines, with SCIB1 also inducing high avidity T response. We have previously screened a number of different
cells that are capable of responding to a lower number of MHC: potential adjuvants to use in our Modi-1 vaccine, this included
peptide complexes on tumor cells. In addition to antigen loss, CpG (TLR9), MPLA (TLR4), CpG/MPLA (TLR9/TLR4), GM-
tumors can also decrease MHC class I expression rendering the CSF, imiquimod (TLR7), Poly I:C (TLR3) or TLR1/2
immune response powerless. The downregulation of MHC class (AMPLIVANT®). Preclinical studies have shown that when
I has been observed in both human and murine tumors (270– AMPLIVANT® is given in combination with the Modi-1
272). The majority of early primary tumors express MHC class I peptides it induced the strongest anti-tumor response (60). This
but this profile often changes as the tumor progresses and escape highlights the importance of determining the best delivery and
immune surveillance (273). The percentage of cancers that have targeting approach for a vaccine that generates the strongest
HLA class I loss, total loss, haplotype loss or allelic loss can range immune response while reducing any possible toxicity.
from 65-90% (274, 275). We have addressed MHC class I loss by With our ImmunoBody® platform we have modified a DNA
incorporating both MHC class I and class II peptides in our vector by engineering T cell epitopes into the IgG1 CDR regions
SCIB1 vaccine, our Modi-1 vaccine only includes MHC class II (163), the Fc region of the antibody targets the high affinity Fc
restricted peptides. receptor CD64 that is expressed on activated APCs. The SCIB DNA
vaccines allow both direct- and cross-presentation of epitopes by
targeting dendritic cells, and are able to generate high avidity CD8+
CONCLUSIONS T cells that efficiently eradicate tumors.. Vaccination with SCIB1
induces high frequency and high avidity specific T cells (165).
A large number of cancer vaccines have failed to show clinical Improvement with vaccine delivery systems has led to the
efficacy, this can be due to the tumor’s own mechanisms of generation of nanoparticles, self-assembling peptides, and needle
immune evasion and escape that have evolved including antigen free delivery systems. Electroporation was used to administer
loss, MHC loss, the presence of immune suppressive cells or SCIB1 in our phase 1 clinical trial, and has been used to deliver
soluble factors in the TME and lack of a robust anti-tumor other cancer and infectious disease vaccines. However, the pain on
immune response (276–279) and also due to the inability of the administration using electroporation and the requirement for
cancer vaccines to induce sufficiently high avidity T cell specialized vaccine delivery instrument have limited their use,
responses to efficiently destroy tumors. Early cancer vaccines these newer delivery systems provide better alternatives.
were primarily focused on stimulating CD8+ T cell responses Liposomes are increasingly being used as delivery system, they
against tumor associated antigens often using short minimal are versatile, incorporating small drug candidates or antigens in the
epitope sequences, T cells recognizing these antigens are highly form of RNA or peptides, and have a good safety profile.
tolerized and subsequently these vaccines fail in the clinic. The Liposomes do require optimization in order to determine the
incorporation of CD4+ T cell epitopes into peptides, RNA or optimal charge/size of the particle to incorporate their cargo and
DNA vaccine platforms is essential to induce specific CD4+ and deliver it across the cell membrane. The targeting of cancer

Frontiers in Immunology | www.frontiersin.org 13 March 2021 | Volume 12 | Article 627932

Paston et al. Cancer Vaccines, Adjuvants, and Delivery Systems

antigens to improve the local immune response and trafficking to cancer vaccine field and a better knowledge of the TME with
lymph nodes can be achieved through either the linking of peptide time cancer vaccines will start to show good clinical efficacy.
to adjuvant, incorporating the antigen into nanoparticles or by
modifying genetic vectors eg ImmunoBody® platform.
There are a number of challenges to address in the
development of a successful cancer vaccine, we have tried to AUTHOR CONTRIBUTIONS
address a number of these challenges with our SCIB1, SCIB2 and
SP wrote the review article. VB and PS contributed and analyzed
Modi-1 vaccines. The SCIB1 vaccine is currently in phase 2 trials
the data. LD proof read the review and conceived the ideas and
in combination with anti-PD-1 therapy, and the Modi-1 vaccine
work around the Modi-1 and Immunobody vaccine platforms.
will be entering clinical trials in 2021. The success of any cancer
All authors contributed to the article and approved the
vaccine does not rely only on the ability of the vaccine to induce a
submitted version.
robust immune response but also on the modification of the
immune suppressive TME to enable the successful trafficking of
T cells and the ability of these T cells to recognize and kill tumor
cells. The tumor size, TMB and previous treatments will all FUNDING
influence the success of a cancer vaccine and this will vary among
patients and cancer types. With huge improvements in the This work was funded by Scancell Ltd.

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Immune targeting of fibroblast activation protein triggers recognition of
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