Fig. 5. Mechanism of Lipid A. When TLR4 is activated by lipid A
on the liposome vaccine,and binds to myeloid differentiation factor 2
(MD2) to form A complex, the signal is transduced to the Toll/IL-1R1
homologous region (TIR) domain, followed by two My88: myeloid
differentiation factor 88 (My 88)-dependent and My88-independent or
TIR-domain-containing adaptor (TRIF)-dependent pathways. My 88-dependent
pathway: TLR binds to the TIR domain containing adaptor protein (TIRAP)
of MyD88 and activates MyD88, forming an active TLR4/MyD88 complex that
activates IL-1R-associated kinase (IRAK), which eventually causes
nuclear factor-κB (NF-κB) into the nucleus and triggers subsequent
reactions. My88-independent pathway or TRIF-dependent pathway: complex’s
signal is transmitted to TIRF, the interferon transcription is regulated
by activating TRIF to further activate the NF-κB. Impelling the body to
produce inflammation-related factors.
Optimization of lipid A. It is difficult to obtain
structurally homogeneous lipid A in varied forms from biological
sources. Moreover, lipid A from natural sources may be contaminated by
other components of the bacterial cell wall. Chemical synthesis as an
effective tool can perfectly solve this problem. Previous review, from
the perspective of chemical synthesis of each part in detail elaborated
the lipid A chemical synthesis process and related derivatives, such as
containing saturated phospholipids and double phosphate group of natural
lipids, containing unsaturated double phosphate groups of lipid A,
containing carboxylic acid derivatives, single phosphorus acyl lipid A
and the phosphorylation of lipid A [63].
As the premise, various positions in the disaccharide backbone of lipid
A with the same or different acyl lipid chain, thus lipid A and its
analogues synthesis can be divided into two basic strategies. One is to
build an asymmetric distribution of lipid chains, that is to construct
general disaccharides skeleton and install the same length or different
length of lipid acyl chain in a particular location. Another strategy is
to synthesize a monosaccharide with acyl lipid chain and then stitch
them into a disaccharide structure through glycosylation reaction.
However, the latter is often hard to implement due to the steric
hindrance. Shimoyama et al. synthesized Alcaligenes lipid A (3+3)
(Fig. 4B) by the introduction of benzyl-protected β-hydroxy fatty acid
into the disaccharide with an appropriate acylation reagent and repeated
similar operations [58].
Jiang et al. provided a synthesis
method of pentaerythritol lipid A (PET-LA) (Fig. 4B) which contains an
amine-substituted pentaerythritol instead of a glucosamine unit found in
natural lipid A [59].
After a period of research, Kawther et al. proved that PET-LA had
powerful adjuvant properties in immunogenic mouse tumor models by
loading CD40 and CD86 into polylactic acid-glycolic acid (PLGA)
particles to stimulates the upregulation of CD40 and CD86 [66].
Compared to the glucosamine structure, PET can keep the anomeric
phosphate group well, and because of its simple structure, the synthesis
process is simplified and the molecular stability is also improved.
Niels et al. studied the immune activity of lipid A analogue CRX-527
conjugated with long peptides in different ways to determine whether it
had the potential to be vaccinated. CRX-527 (Fig. 4B), one of aminoalkyl
glucosamine 4-phosphates (AGPs), which contains monosaccharide with a
long-chain acyl group [60]. In vitro , ester-linked compounds
performed better in inducing DC maturation, while ammonia-linked
compounds showed greater activity in presentation. However, according to
the data studied in vivo , it seemed that a conjugate of mounting
the free carboxyl ligand at the N-terminus of the model peptide antigen
can recruit enough protective immune response to effectively kill cancer
cells and exert a powerful immune function for the development of
anti-cancer.
Glucopyranosyl lipid adjuvant (GLA) is a synthetic non-toxic derivative
of lipid A (Fig. 4B). When it is used as adjuvant and co-delivery with
tumor antigen, it plays the role of TLR4 agonist. GLA targeting human DC
and peripheral blood mononuclear cells (PBMC) has 10-100-fold more
activity and safety than semi-synthetic MPLA. The intensity of
immune-induced response is also different due to the change of the type
of preparation. GLA prepared as stable oil-in-water emulsion (GLA-SE)
formulations can induce stronger Th1 reaction than that prepare as an
aqueous(GLA-AF) [61]. In a trial of an advanced solid tumor vaccine
expressing NY-ESO-1, after stimulating the systemic innate immune
response, locally intra-tumoral injection of GLA-SE can recruit
NY-ESO-1-specific CD8+ T cells into the tumor
[67]. In previous dose-increasing trials, GLA-SE at doses of 2-10 mg
showed potential clinical benefits and reliable safety in the
experimental patient population. Six of the eleven patients showed
increased expression of CD4+ T cells after vaccination
and four patients showed increased expression of CD8+T cells, but none of them showed a correlation between GLA-SE dose and
induction of immune [68]. And when the vaccine is injected away from
the tumor, Deepak et al. proved that the addition of GLA led to alters
the kinetics of changes in T cell trafficking, and it can induce a
massive proliferation of vaccine primed antigen-specific T cells in the
spleen [8]. The most fundamental reason is that GLA precociously
matures APC at the injection site, reduces the phagocytic function of
APC and reduces the delivery of antigens to draining lymph nodes. But
the use of GLA adjuvant can lead to a great number of antigen-specific T
cells proliferation induced by vaccine in immune organs, driving more T
cells into tumor tissue and infiltrating tumor T cells, resulting in
stronger cytotoxicity.
Derivatives of lipid A. All lipid A contain a highly
similar core structure of β-1,6-linked disaccharide of D-glucosamine.
Specially, subtle changes in chemical structure can result in
dramatically different immune activities. An understanding of
structure–activity relationships help to provide a better choice when
developing a cancer vaccine. In previous studies, many factors such as
the number and length of the acyl chain, the structure and substituent
pattern of the disaccharide scaffold, the length and phosphorylation
state of the acyl chain at positions 1 and 40 of the disaccharide
backbone had a strong effect on the activation of the immune response
[69].
Gram-negative bacteria have
evolved the diversified of their lipid A structures. A typical lipid
containing six acyl chains exhibits severe immunological and endotoxic
activities, such as asymmetrically distributed (4+2) from E. coli[70], or symmetrically (3+3) from Neisseria meningitidis[71] or Alcaligenes [58, 72]. However, removal of the
phosphate group at the O-1-position to generate monophosphoryl lipid A
(MPLA) derived from Salmonella Minnesota (Fig. 4B), which greatly
eliminates the endotoxin properties without affecting its
immune-stimulating activity, has been approved by the FDA for using as
an adjuvant vaccine [62]. Most studies have tried to prove that MPLA
is a favorable candidate for the formulation of different cancer
vaccines with better immune potency.
Combination strategy of lipid A. The construction of a
multi-functional liposome vaccine delivery platform with a variety of
immune stimulants is the focus of the current preclinical research,
researchers are committed to adding vaccine adjuvants and antigen
delivery to achieve preferable anti-tumor effects. In order to improve
the immunogenicity of the vaccine and the survival rate of cancer
patients, the combination strategy of lipid A is a good choice. The
combined use of adjuvants achieved dual effects, and the combination of
cancer drugs expanded the therapeutic effect of drugs.
Currently, mature adjuvant combination formulations have been tested in
preclinical or clinical trials of cancer vaccines. For example, in AS01,
MPLA is combined with QS-21, a triterpenoid glycoside saponin extracted
from the bark of Quillaja Saponaria Molina, which is a powerful adjuvant
[73]. AS02 precipitates MPLA and QS-21 in a stable oil-in-water
emulsion [74]. The TLR9 agonist CpG ODN was added to the existing
formula, and then the whole was wrapped in lipids to form an AS15
combination [75]. Based on these approved formulations, scientists
have developed several new adjuvant coordination strategies. Zhu et al.
synergistically activated the immune response with TLR7/8 agonist
imiquimod and TLR4 agonist MPLA. From immune study in vivo , the
addition of combined adjuvant effectively improved the proliferation and
activation of immune cells, especially antigen-specific T lymphocytes.
Surprisingly, the mice received the tumor challenge again after
vaccination, of which 2/8 mice maintain a tumor-free state to 92 days
[76]. The vaccination greatly prolonged the median survival time of
the mice. Both complex vaccine adjuvants ((HA/MPLA/QS21), HMQ and
(HA/MPLA/R837), HMR) were prepared by Shin, which exerted important
immunostimulatory effects in vitro and essential antitumor
therapy in vivo [77]. With the help of HA, HMQ or HMR were
easily prepared into aqueous solution and freeze-dried in the form of
powder, showing high stability, even when stored at room temperature.
TLR4 agonist MPLA and 9 agonist oligonucleotide CpG combined within vitro stimulating DC secretion IL-12, TNF-α, and proliferation
of the same foreign CD8+ T cells have unique intensive
effects, producing adding adjuvant effect [78]. The combination of
TLR agonists and non-TLR agonists also presented an effective power
[62]. The strong anti-tumor cytotoxic response dependent on natural
killer cells can be induced by α-galactose ceramide (α-GalCer) and when
cooperated with MPLA and DNA antigens, multiple immune targets are
activated at the same time, resulting in more lasting immunotherapy
[79]. The cell killing activity of mice generated by the combined
strategy is more than twice that of the single adjuvant. Not only that,
the use of combined adjuvants can also reduce the amount of antigen in
the vaccine to avoid possible problems and obstacles in the use of high
doses of antigen.
The efficacy can also be amplified
in combination with commonly used cancer drugs. Doxorubicin (DOX), as a
broad-spectrum anti-tumor chemotherapy drug commonly used in clinical
practice, promoted immunogenic cell death by increasing the expression
of tumor antigen in vivo , thus triggering immune response.
However, its anti-tumor effect is
limited, and it is also necessary to maintain high doses of drug
concentrations in the tumor site. This requires increased DOX
administration, which inevitably causes drug toxicity and drug
resistance problems. The addition of MPLA assists in the immune response
triggered by DOX. The joint processing of MPLA-DOX significantly
increases the number of invasive CD8+ or
CD4+ T cells, which basically inhibits tumor growth
[80]. Immune checkpoint inhibitors can inhibit the related receptors
PD-L1 or PD-1, which are a successful treatment for patients with
advanced melanoma. When combined with MPLA, MPLA-induced interferon γ
(IFN-γ) production by T cells can further promote PD-L1 expression,
promote the proliferation and CTL of the infiltrated regulatory T cell,
thereby finally eradicating the primary tumor and inhibiting tumor
transfer and recurrence [81, 82]. Therefore, the combined strategy
can make the tumor treatment effects better, which provides a desired
strategy for research work.