pubs.acs.

org/bc Article

Synthesis and Biological Evaluation of Peptide-Adjuvant Conjugate

Vaccines with Increasing Antigen Content
Taylor R. Cooney, Kathryn Farrand, Sarah L. Draper, Regan J. Anderson, Phillip M. Rendle,
Ian F. Hermans, Benjamin J. Compton,* and Gavin F. Painter*
Cite This: Bioconjugate Chem. 2023, 34, 799−808 Read Online

ACCESS Metrics & More Article Recommendations *

sı Supporting Information
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Downloaded via PEOPLES FRIENDSHIP UNIV OF RUSSIA on June 12, 2023 at 18:45:23 (UTC).

ABSTRACT: Synthetic vaccines that induce T cell responses to

peptide epitopes are a promising immunotherapy for both
communicable and noncommunicable diseases. Stimulating strong
and sustained T cell responses requires antigen delivery to
appropriately activated antigen presenting cells (APCs). One way
this can be accomplished is by chemically conjugating immunogenic
peptide epitopes with α-galactosylceramide (α-GalCer), a glycolipid
that acts as an immune adjuvant by inducing stimulatory interactions
between APCs and type I natural killer T (NKT) cells. Here we
investigate whether increasing the ratio of antigen:adjuvant improves
antigen-specific T cell responses. A series of conjugate vaccines was
prepared in which one, two, four, or eight copies of an immunogenic
peptide were covalently attached to a modified form of α-GalCer via
the poly(ethoxyethylglycinamide) dendron scaffold. Initial attempts
to synthesize these multivalent conjugate vaccines involved attaching the bicyclo[6.1.0]non-4-yne (BCN) group to the adjuvant-
dendron structure followed by strain-promoted azide−alkyne cycloaddition of the peptide. Although this approach was successful for
preparing vaccines with either one or two peptide copies, the synthesis of vaccines requiring attachment of four or eight BCN groups
suffered from low yields due to cyclooctyne degradation. Instead, conjugate vaccines containing up to eight peptide copies were
readily achieved through oxime ligation with adjuvant-dendron constructs decorated with the 8-oxo-nonanoyl group. When
evaluating T cell responses to vaccination in mice, we confirmed a significant advantage to conjugation over admixes of peptide and
α-GalCer, regardless of the peptide to adjuvant ratio, but there was no advantage to increasing the number of peptides attached.
However, it was notable that the higher ratio conjugate vaccines required lower levels of NKT cell activation to be effective, which
could be a safety advantage for future vaccine candidates.

■ INTRODUCTION
Infected or malignant cells can be targeted and eliminated by
activated CD8+ T cells that specifically recognize peptide
epitopes presented via major histocompatibility complex
(MHC) class I proteins on the cell surface. Simple peptide-
based vaccines based on these epitopes can therefore be used
to prime or restimulate CD8+ T cell responses with high
specificity without concerns regarding the immunogenicity of a
delivery vector. The fact that peptides can be readily
synthesized as discrete entities on large scales using automated
technologies with stability and/or manufacturing advantages
compared to antigens based on nucleic acids or whole proteins
adds to the appeal of this vaccine platform. However, peptide
epitopes are poorly immunogenic when administered as single
agents and require coadministration with an immune stimulant
(adjuvant) to support acquisition and/or presentation by
appropriately activated antigen presenting cells (APCs) in vivo.
Strategies to achieve this include oil-in-water emulsions that
aid in delivery,1 cytokines that support the function of APCs,2
and chemical compounds that trigger APCs directly via pattern
recognition receptors such as Toll-like receptors (TLRs), C-
type lectin receptors (CLRs), and nucleotide binding
oligomerization domain (NOD)-like receptors (NLRs).3
Alternatively, APC activation can be achieved through direct
interaction with other immune cells, notably those capable of
producing licensing signals,4 typified by the stimulation of
CD40.5,6 This includes “unconventional” T cells like type I
natural killer T (NKT) cells, which are found in high numbers
in lymphoid tissues and respond rapidly to stimulation with
specific agonistic antigens.7 The glycolipid compound α-
galactosylceramide (α-GalCer) is regarded as a prototypical
Received: March 5, 2023
Revised: March 25, 2023
Published: April 6, 2023

© 2023 American Chemical Society https://doi.org/10.1021/acs.bioconjchem.3c00106

799 Bioconjugate Chem. 2023, 34, 799−808
Bioconjugate Chemistry
Scheme 1. Synthetic Route to the 1:1 Adjuvant:Antigen (G0) GLP Vaccine 1
agonist for NKT cells8 and has been shown to act as an
immune adjuvant when introduced into vaccines in preclinical
studies,9−11 while a variant of this structure has been shown to
have adjuvant activity in humans.12 In order to function as an
immune adjuvant, the NKT cell agonist must be acquired and
presented via the MHC class I-like molecule CD1d on APCs
to NKT cells, which in turn rapidly releases cytokines and
directs molecular signals, including the provision of CD40L,
that transactivate APCs. If these same APCs have acquired and
presented peptide epitopes from the vaccine, peptide-specific T
cell responses can be initiated or boosted.8,9,13,14
The capacity of α-GalCer to adjuvant synthetic peptide-
based vaccines in various disease models including cancer15−17
and infectious disease18−21 has been enhanced by strategies
that encourage presentation of α-GalCer and peptide by the
same APC.20,22−24 This includes encapsulation into a variety of
different synthetic vaccine particles,25−30 employing filamen-
tous bacteriophages or virus-like particles,31 and attachment to
gold nanoparticles.32 Although these strategies to promote
codelivery have proven effective in animal models, controlling
and quantifying the ratio of adjuvant and antigen in these
approaches is challenging. Self-adjuvanting α-GalCer-based
glycolipid-peptide (GLP) conjugate vaccines overcome this
limitation by chemically linking the antigen and adjuvant, thus
providing synthetically accessible, chemically defined con-
structs that are highly manufacturable with a prolonged shelf
life as they can be stored indefinitely as lyophilized powders or
frozen DMSO solutions. Although proven to generate strong T
cell responses in preclinical studies, current α-GalCer-peptide
conjugate vaccines have been restricted to an equimolar ratio
of antigen and adjuvant, which may be suboptimal considering
peptide-based therapeutics have a short circulation half-life due
to proteolytic degradation. Indeed, studies have shown that
800
pubs.acs.org/bc Article
vaccines capable of delivering a higher antigen density, either
through encapsulation30,33,34 or conjugation,35,36 can lead to
stronger antigen-specific responses owing to increased
circulation time and/or acting as an antigen depot.
A synthetically tractable approach to generate well-defined,
multipeptide conjugate vaccines is to employ a dendron
scaffold to link the adjuvant core and the peptide antigen.
Dendrons offer an organized, nanosized branched framework
that can accommodate a vast array of functional groups
attached to the termini, including those suitable for attaching
peptides. Commonly used peptide-presenting dendritic
structures include branched lysine, 37 polyamidoamine
(PAMAM),38 and polyphenylene (PPDs)39 repeating units.
More recently, poly(ethoxyethylglycinamide) (PEE-G) den-
dron has been investigated for multipeptide display.40 Due to
its high stability, high aqueous solubility, low cytotoxicity,
chemical orthogonality, and low immunogenicity,41 PEE-G is
considered a particularly attractive scaffold for pharmaceutical
applications.
To investigate whether PEE-G-based conjugate vaccines
with increasing number of peptide epitopes (i.e., antigen load)
attached to each α-GalCer molecule could induce stronger
peptide-specific T cell responses than those of equal molarity,
vaccines with adjuvant:peptide ratios of 1:1, 1:2, 1:4, and 1:8
(generations (G)0 to G3, respectively) were synthesized. Here,
we adapted our previously reported approach17 that utilizes a
modified α-GalCer compound in the vaccine design. Previous
research has shown that GLP vaccines comprising a
“rearranged” α-GalCer adjuvant, formed by the migration of
the cerotic acid moiety, conjugated to peptide antigen via the
protease sensitive para-aminobenzyl-dipeptide linker are able
to induce strong antigen-specific T cell responses in vivo.42
The activity of these vaccines is contingent on intracellular
https://doi.org/10.1021/acs.bioconjchem.3c00106
Bioconjugate Chem. 2023, 34, 799−808
Bioconjugate Chemistry pubs.acs.org/bc Article

Scheme 2. Synthesis of the G1 GLP Vaccine 14 Comprising an Adjuvant:Antigen Ratio of 1:2

Scheme 3. Synthesis of the tetra-BCN Dendron Core and Its Attempted Reaction with “Rearranged” α-GalCer Derivative

linker degradation leading to the close temporal release of both
the adjuvant and peptide antigen inside the same APC. In the
work presented here, the same α-GalCer-based vaccine design
was employed, except the citrulline residue in the immolative
dipeptide linker was substituted for alanine. This change was
based on previous studies that have shown Val-Ala linkers are
801
stable in circulation but are readily cleaved by proteolytic
cathepsin enzymes.43−45 Also, compared to citrulline, alanine
has improved solubility in organic solvents, thus offering a
more facile synthesis of the α-GalCer-linker vaccine
component. The peptide chosen for this study is an MHC
class I-binding peptide from human papillomavirus (HPV) E7
https://doi.org/10.1021/acs.bioconjchem.3c00106
Bioconjugate Chem. 2023, 34, 799−808
Bioconjugate Chemistry
protein (E749−57; RAHYNIVTF), which has been defined as a
CD8+ T cell epitope in C57BL/6 mice restricted by H2-Db. To
ensure facile release of the attached peptide within APCs, the
known proteolytic cleavage site -FFRK-46 was incorporated at
the peptide N-terminus alongside either the 5-azido-pentanoyl
or aminooxy acetamido (AoA) groups for strain-promoted
azide−alkyne cycloaddition (SPAAC) or oxime ligation,
respectively. Multimerising the E749−57 peptide antigen on
the “migrated” α-GalCer adjuvant core was achieved via the
PEE-G dendron core, terminally modified with either a ring-
strained alkyne or pendant ketone that would permit
chemoselective ligation with an appropriately functionalized
peptide.
The work reported here describes the development and in
vivo evaluation of such a dendron-based vaccine platform with
controlled conjugation of up to eight peptide copies onto α-
GalCer.
■ RESULTS AND DISCUSSION
Synthesis of Dendritic Vaccines Using SPAAC
Ligation. The synthesis of the G0 vaccine (1) comprising
an equimolar ratio of antigen to adjuvant commenced from the
addition of the para-nitrophenol (pNP)-activated dipeptide
linker (2)44 and “migrated” α-GalCer (3)22 to give carbamate
4 (Scheme 1). Removal of the valine Fmoc protecting group
and subsequent capping of the liberated amino group (in 5)
with commercially available pNP-exo-BCN (6) gave cyclo-
octyne 7 in excellent yield. Stirring 7 with 5-azidopentanoyl-
FFRKRAHYNIVTF (8) in DMSO at ambient temperature
afforded the GLP conjugate vaccine 1 in 85% yield and 99%
purity after purification by preparative HPLC.
Increasing the antigenic load from one peptide (as in the G0
vaccine, 1) up to eight copies (G3 vaccine) was considered
best achieved by “growing” the PEE-G dendron core through
simple amide bond formation.41 Through this approach, it was
envisaged that an appropriately functionalized dendron scaffold
could be attached to the adjuvant core which, upon
chemoselective conjugation of antigen, would afford the
desired multigeneration GLP vaccines. Toward this, dendron
synthesis commenced from the commercially available
dialkylated glycine derivative (9) which was hydrogenolyzed
to liberate the carboxylic acid 1041 followed by acid-mediated
removal of the Boc protecting groups to afford bis-amine 11
(Scheme 2). Reacting 11 with pNP-exo-BCN 6 under basic
conditions gave bis-cyclooctyne 12 in 77% yield which was
subsequently coupled with α-GalCer amine 5 in the presence
of HBTU, HOBt, and DIPEA to afford α-GalCer bis-
cyclooctyne 13 in good yield. Subjecting 13 to 3 equiv of
azido-peptide 8 in DMSO at ambient temperature for 18 h
gave, after purification by preparative HPLC, the G1 GLP
vaccine (14) bearing two peptide copies. Although 14 was
obtained in an unusually low yield of 20% compared to that
typically reported for similar GLP conjugates (which range
from 40% to 60% with purities >95% by HPLC), a sufficient
quantity of this vaccine was obtained for immunological
testing.
Synthesis of the G2 GLP vaccine containing four peptide
copies was attempted using a similar to that which gave G1
GLP vaccine 14. Doubling the antigenic load from two peptide
copies (as in 14) to four was considered best achieved by
doubling the branching within the dendron core (Scheme 3).
The G2 dendron scaffold 17 was prepared as reported41 and,
upon removal of the Boc protecting groups to give tetra-amine
802
pubs.acs.org/bc Article
18, was N-capped with the exo-BCN group to give tetra-
cyclooctyne 19. Attempts to react 19 with the rearranged form
of α-GalCer (5) employing HBTU, HOBt, and DIPEA,
conditions that were successful generating the G0 (1) and G1
(14) vaccines, failed to give an appreciable amount of the
desired G2 tetra-cyclooctyne product 20.
To investigate this further, the reaction between α-GalCer
amine 5 and tetracyclooctyne 19 was monitored by LCMS.
After 2 h, approximately 55% of starting material 19 remained,
while the appearance of a new peak, accounting for 45% of the
reaction mixture, with a retention time and m/z of 2574.7
matching that expected for 20 was observed. Allowing the
reaction to progress at ambient temperature revealed that only
20% of purported 20 was present after 14 h, which diminished
to <15% after 24 h. The appearance of a series of new peaks in
the chromatogram were observed (after 24 h) corresponding
to masses 16, 32, 48, and 64 Da higher than that expected for
G2 tetra-cyclooctyne product 20 suggested that these coupling
conditions were promoting oxidative degradation of the
dendron terminal BCN groups. Due to the nature and varying
degrees of degradation, individual compounds were not able to
be isolated from this reaction mixture.
Degradation of the BCN unit was first reported by Gibson et
al.47 who observed the acidic hydration of the alkyne upon
addition of trichloroacetic acid to an BCN functionalized
oligonucleotide. Feng et al.48 observed a similar occurrence
whereby BCN-containing carbamates formed cyclooctanones
under acidic conditions. Because acidic hydration would
produce byproducts of 18 Da higher and not 16 Da as we
observed, we synthesized the model BCN propylcarbamate
(21) (Scheme 4) to further investigate our observations that
Scheme 4. Prolonged Exposure of the BCN
Propylcarbamate 21 to HBTU/HOBt Coupling Reagents
Leads to Enone Formation
similar, but likely different, degradation products can form
using HBTU and HOBt with DIPEA. Reacting pNP-exo-BCN
6 with propylamine afforded carbamate 21 which was
subjected to the same amide coupling conditions that lead to
the degradation of 20. Analyzing this reaction by HPLC
revealed the formation of a major product with a mass 16 Da
greater than 21 which, when isolated, was shown to have
undergone degradation of the alkyne to an enone system (as in
22). A potential mechanism for this observation is proposed to
form via HOBt which, by virtue of its N−O bond, may act as
an oxidant (Supplementary Scheme 1). While the BCN moiety
can survive relatively brief exposure to these conditions, as
exemplified for the preparation of α-GalCer bis-cyclooctyne
13, when couplings are slow or multiple copies of BCN are
present in the molecule, degradation can become a significant
problem, as we observed. For these reasons we investigated
oxime ligation as an alternative chemoselective conjugation
strategy.
https://doi.org/10.1021/acs.bioconjchem.3c00106
Bioconjugate Chem. 2023, 34, 799−808
Bioconjugate Chemistry pubs.acs.org/bc Article

Scheme 5. Synthetic Route to the 1:1 Adjuvant:Antigen (G0) GLP Vaccine 26

Scheme 6. Synthesis of the G1−G3 GLP Vaccines

803 https://doi.org/10.1021/acs.bioconjchem.3c00106
Bioconjugate Chem. 2023, 34, 799−808
Bioconjugate Chemistry pubs.acs.org/bc Article

Figure 1. Evaluation of GLP vaccines with increasing antigen dose compared to injected admixes. Groups of C57BL/6J mice (n = 5) were
intravenously administered the indicated GLP vaccines with molar dose of conjugated NKT cell agonist at 0.5 nmol; G0 (26, 1:1), G2 (37, 1:4)
and G3 (38, 1:8). Comparator groups of mice were administered the equivalent molar ratios of unconjugated α-GalCer (α-GC) and E749−57
peptide (1:1, 1:4, 1:8), with an additional high ratio mix (1:64); control animals received PBS vehicle. (A) Assessment of cytokines released into
serum in response to treatment at indicated times, with heatmaps showing concentration normalized to sum of all values for each cytokine. (B)
Serum concentrations of indicated cytokines. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with Dunnett’s multiple
comparisons test. (C) Flow cytometric analysis of NKT cells (CD1d tetramer+ cells) in the spleen on day 7, identified by the gating strategy in
Supplementary Figure 1, showing frequency, total number in spleen, percentage of NK1.1-negative cells (as an indication of activation), and
expression of CD69 and PD-1. One-way ANOVA with Tukey’s multiple comparisons. (D) Analysis of splenic E749−57-specific T cell response in
spleen by flow cytometry (left) using gating strategy in Supplementary Figure 2 using H2-Db/peptide pentamers to detect antigen-specific cells or
by ELISpot (right) after restimulation of splenocytes with peptide ex vivo. One-way ANOVA with Tukey’s multiple comparisons.

804 https://doi.org/10.1021/acs.bioconjchem.3c00106
Bioconjugate Chem. 2023, 34, 799−808
Bioconjugate Chemistry
Revised Synthesis of Dendritic Vaccines Using Oxime
Ligation. Oxime ligation between carbonyl-containing com-
pounds (i.e., aldehydes or ketones) and peptides functionalized
with an aminooxy group is a proven way to efficiently generate
glycolipid-peptide conjugate vaccines.20,21,49 Conditions pro-
moting oxime bond formation include the use of aniline
catalysts under mildly acidic conditions (∼pH 3−5).50 To
investigate this ligation approach, the corresponding G0
vaccine construct was synthesized whereby α-GalCer amine
5 was reacted with the activated ester of 8-oxo-nonanoic acid
(23) to give α-GalCer ketone 24 (Scheme 5). Having
previously shown that a solution of aniline and TFA in
hexafluoroisopropanol (HFIP) can efficiently catalyze the
oxime ligation of an appropriately functionalized glycolipid
and peptide,21 these reaction conditions were examined for
synthesis of the G0 vaccine (Scheme 5). Starting with ketone
24 and aminooxy acetyl (AoA)-FFRKRAHYNIVTF (25), a
series of concentrations of aniline (100, 200, and 400 mM) in
HFIP were assessed for the synthesis of the G0 1:1 conjugate
(26). Monitoring the reactions by LCMS showed that 100
mM aniline and 400 mM aniline reactions were complete
within 24 h, while the 200 mM aniline reaction exhibited a
faster reaction rate and was complete within 12 h likely owing
to this system having a high catalyst loading while operating at
a favorable pH for oxime formation. Because the higher (400
mM) concentration of aniline was not proven to be
detrimental (i.e., there was no observed formation of side
products or degradation products in HPLC chromatogram for
this reaction), the reaction of glycolipid 24 and peptide 25 was
repeated using 400 mM aniline + 130 mM TFA (pH ∼4) and
was shown by LCMS to form vaccine 26 cleanly within 12 h.
Accordingly, this reaction system was selected for the synthesis
of the larger G1−3 conjugate vaccines.
The G1, G2, and G3 prevaccines (27, 28, and 29,
respectively) were prepared using a common strategy. Reacting
acid 23 with either bis-amine 11 or tetra-amine 18 in DMF
with Hünig’s base afforded the G1 (30) and G2 (31) dendrons
in good yield (Scheme 6). Coupling bis-amine 15 and tetra-
Boc acid 17 with HBTU and HOBt in DMF with Hünig’s base
gave the N-Boc, O-Bn protected G3 dendron core motif 32.
Hydrogenolysis (to 33)41 followed by acid-mediated N-Boc
deprotection gave octaamine 34 which was in turn coupled
with 23 to afford the G3 dendron 35. Subsequent coupling of
glycolipid 5 to each G1−3 dendron core (30, 31, and 35,
respectively) gave the ketone-containing adjuvant-dendron
constructs ready for oxime ligation. The G1 and G2 vaccines
were prepared by treating the ketone precursor (30 and 31,
respectively) with 6 equiv of AoA-peptide 25 in 400 mM
aniline + 130 mM TFA at 40 °C overnight and purified by
preparative HPLC to give the G1 (36) and G2 (37) vaccines
in 50% and 45% yields, respectively. Employing these same
conditions for the G3 prevaccine (29) led to incomplete
coupling even when 12 equiv of 25 per ketone was used. The
undercapped vaccines bearing six and seven peptides were
separated from the desired eight peptide product by careful
preparative HPLC chromatography, affording the G3 vaccine
(38) in 50% yield.
Immunological Evaluation of Multipeptide Conju-
gate Vaccines. With the series of conjugate vaccines in hand,
we investigated their ability to activate NKT cells and induce
HPV-specific T cell responses in vivo. The experimental setup
included taking blood at 6 and 18 h to assess cytokine levels in
serum, which typically reflect the cascade of cellular activities
805
pubs.acs.org/bc Article
triggered after NKT cell activation. This was expected to
include (among others) early IFN-γ production by the NKT
cells themselves, and IL-12p70 production by transactivated
APCs, and a later additional IFN-γ burst by natural killer
cells.51,52 Flow cytometry was used to examine NKT cell and
peptide-specific CD8+ T cell responses in spleens harvested 7
days after vaccination. Fluorescent multimeric CD1d/glyco-
lipid complexes were used to identify NKT cells, while
multimeric H-2 Db/E749−57 complexes were used to identify
peptide-specific T cells. Evaluation of T cell response was also
conducted using ELISpot on splenocytes to identify IFN-γ-
producing cells responding to in vitro restimulation with the
E749−57 peptide. The experimental groups included the G0 (26,
1:1), G2 (37, 1:4), and G3 (38, 1:8) dendron conjugates and
admixtures of α-GalCer and E749−57 peptide (ratios of 1:1, 1:4,
1:8, and 1:64). All vaccines were injected by the intravenous
route in C57BL/6 mice, where the dose of NKT agonist was
held constant.
Multiple cytokines were detected in serum after injection of
the admixes as shown as fold changes from PBS controls in
heatmaps (Figure 1A). The changes observed for each
cytokine were reasonably consistent across the different ratios
of peptide to glycolipid, suggesting that the constant molar
dose of α-GalCer is the primary driver of the early immune
response. Injection of the conjugates also induced cytokine
production, but fold changes were generally lower, particularly
in response to the higher generation dendron structures (G2,
37 and G3, 38) at 18 h. Statistical analysis of the key cytokines
IFN-γ and IL-12p70 highlights this trend, with the admixes
inducing significantly enhanced IFN-γ levels at 6 and 18 h
compared to PBS-injected controls and enhanced IL-12p70
levels at 6 h, but responses to the higher generation conjugates
failing to reach these same levels (Figure 1B). This likely
reflects lower overall levels of NKT cell stimulation, as both the
percentages and numbers of NKT cells that accumulated in the
spleens of animals injected with the larger conjugates were
lower than in animals that received the admixes, suggesting less
vaccine-induced NKT cell proliferation over the 7 days of the
experiment (Figure 1C, left panels). The NKT cells detected in
all test groups on day 7 were largely NK1.1 negative, which is a
typical outcome after α-GalCer administration, and CD69 was
downregulated compared to PBS (Figure 1C, right panels).
Although the latter is an activation marker, its expression is
known to peak earlier, within days of a strong stimulus, and
then decline for several days. The expression of PD-1 was also
increased on NKT cells in response to activation in all test
groups, but the levels were consistently lower when the
conjugate vaccines were used (Figure 1C, bottom left panel),
consistent with lower level of NKT cell activation.
When assessed by flow cytometry, peptide-specific T cell
responses to the admixes were limited, reaching statistical
significance only over PBS-injected controls when injected at a
ratio of 1:64 (Figure 1D). Analysis by ELISpot was more
sensitive, with responses to all of the admixes increasing over
PBS controls. There were also increased T cell responses
associated with a higher peptide dose. However, this dose
effect was weak, with 64-fold more peptides only achieving a
∼2-fold increase in T cell response. Conjugation significantly
improved T cell responses compared to equivalent admix
doses, highlighting a role for improving codelivery of antigen
and agonist in vivo. However, the impact of increasing peptide
content was again marginal, with no consistent significant
https://doi.org/10.1021/acs.bioconjchem.3c00106
Bioconjugate Chem. 2023, 34, 799−808
Bioconjugate Chemistry
improvement in T cell response between G0 (26) and the
larger structures.
Our results therefore do not provide clear evidence that
conjugating more peptide to an NKT cell agonist improves T
cell responses, but the associated reduction in NKT cell
activity observed with the higher generation structures does
confound interpretation. An alternative delivery method such
as formulation of the individual components in particles may
therefore be better suited for investigating the effect of
increased peptide:agonist ratios. The lower levels of NKT cell
stimulation observed with G2 (37) and G3 (38) suggest that
the higher order structures limit availability of the NKT cell
agonist. It is possible that this is actually a benefit to these
structures, as the corresponding reduction in NKT cell-driven
cytokine production is likely to be a safety advantage. The fact
that high levels of antigen-specific T cell priming were achieved
with less NKT cell activation may be due to more efficient
presentation of the peptide in activated APCs, or that higher
levels of NKT cell activation are simply not required, especially
when increased amounts of peptide are provided. To further
investigate the implications of this, a series of studies probing
rates and efficiency of release rates of the glycolipid and
peptide components would be required.
■ CONCLUSION
Our results support the concept of conjugating peptides to
NKT cell agonists to improve T cell responses and highlight a
novel design for increasing the ratio of adjuvant to peptide
antigen. While increasing the amount of attached peptide does
not significantly improve the size of the T cell response, lower
levels of NKT cell activation are required for these vaccines to
be effective, which could be a safety benefit.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge
athttps://pubs.acs.org/doi/10.1021/acs.bioconj-
chem.3c00106.
Full experimental procedures and compound character-
ization data including supporting schemes, figures, and
tables (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Benjamin J. Compton − Ferrier Research Institute, Victoria
University of Wellington, Lower Hutt 5010, New Zealand;
orcid.org/0000-0002-5245-9767;
Email: Benji.Compton@vuw.ac.nz
Gavin F. Painter − Ferrier Research Institute, Victoria
University of Wellington, Lower Hutt 5010, New Zealand;
Maurice Wilkins Centre for Molecular Biodiscovery,
Auckland 1010, New Zealand; orcid.org/0000-0002-
1310-7769; Email: Gavin.Painter@vuw.ac.nz
Authors
Taylor R. Cooney − Ferrier Research Institute, Victoria
University of Wellington, Lower Hutt 5010, New Zealand;
Maurice Wilkins Centre for Molecular Biodiscovery,
Auckland 1010, New Zealand; orcid.org/0000-0003-
4461-2554
Kathryn Farrand − Malaghan Institute of Medical Research,
Wellington 6012, New Zealand
806
pubs.acs.org/bc Article
Sarah L. Draper − Ferrier Research Institute, Victoria
University of Wellington, Lower Hutt 5010, New Zealand;
Maurice Wilkins Centre for Molecular Biodiscovery,
Auckland 1010, New Zealand; orcid.org/0000-0001-
8330-121X
Regan J. Anderson − Ferrier Research Institute, Victoria
University of Wellington, Lower Hutt 5010, New Zealand;
orcid.org/0000-0003-3467-9202
Phillip M. Rendle − Ferrier Research Institute, Victoria
University of Wellington, Lower Hutt 5010, New Zealand
Ian F. Hermans − Maurice Wilkins Centre for Molecular
Biodiscovery, Auckland 1010, New Zealand; Malaghan
Institute of Medical Research, Wellington 6012, New Zealand
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.bioconjchem.3c00106
Author Contributions
T.R.C. was primarily responsible for the chemical synthesis
with contributions from B.J.C. and S.L.D. under the guidance
of G.F.P. and P.M.R. T.R.C., I.F.H., and G.F.P. designed the in
vivo studies, which were carried out and interpreted by K.J.F.
and I.F.H. B.J.C. and I.F.H. drafted the manuscript with
editorial contributions from all authors. All authors have given
approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was funded by New Zealand Ministry of Business
Innovation and Employment (RTVU1603), IROF to the
Malaghan Institute, and a Maurice Wilkins Centre PhD
scholarship to TRC. The authors thank the Biomedical
Research Unit of the Malaghan Institute for animal husbandry.
■ REFERENCES
(1) Melssen, M. M.; Fisher, C. T.; Slingluff, C. L.; Melief, C. J. M.
Peptide emulsions in incomplete Freund’s adjuvant create effective
nurseries promoting egress of systemic CD4(+) and CD8(+) T cells
for immunotherapy of cancer. J. Immunother Cancer 2022, 10,
No. e004709.
(2) Disis, M. L.; Bernhard, H.; Shiota, F. M.; Hand, S. L.; Gralow, J.
R.; Huseby, E. S.; Gillis, S.; Cheever, M. A. Granulocyte-macrophage
colony-stimulating factor: An effective adjuvant for protein and
peptide-based vaccines. Blood 1996, 88 (1), 202−210.
(3) Amarante-Mendes, G. P.; Adjemian, S.; Branco, L. M.; Zanetti,
L. C.; Weinlich, R.; Bortoluci, K. R. Pattern Recognition Receptors
and the Host Cell Death Molecular Machinery. Front Immunol 2018,
9, 2379.
(4) Ridge, J. P.; Di Rosa, F.; Matzinger, P. A conditioned dendritic
cell can be a temporal bridge between a CD4(+) T-helper and a T-
killer cell. Nature 1998, 393 (6684), 474−478.
(5) Bennett, S. R. M.; Carbone, F. R.; Karamalis, F.; Flavell, R. A.;
Miller, J. F. A. P.; Heath, W. R. Help for cytotoxic-T-cell responses is
mediated by CD40 signalling. Nature 1998, 393 (6684), 478−480.
(6) Schoenberger, S. P.; Toes, R. E. M.; van der Voort, E. I. H.;
Offringa, R.; Melief, C. J. M. T-cell help for cytotoxic T lymphocytes
is mediated by CD40-CD40L interactions. Nature 1998, 393 (6684),
480−483.
(7) Bendelac, A.; Savage, P. B.; Teyton, L. The biology of NKT cells.
Annu. Rev. Immunol. 2007, 25, 297−336.
(8) Kawano, T.; Cui, J. Q.; Koezuka, Y.; Toura, I.; Kaneko, Y.;
Motoki, K.; Ueno, H.; Nakagawa, R.; Sato, H.; Kondo, E.; et al.
CD1d-restricted and TCR-mediated activation of V(alpha)14 NKT
cells by glycosylceramides. Science 1997, 278 (5343), 1626−1629.
https://doi.org/10.1021/acs.bioconjchem.3c00106
Bioconjugate Chem. 2023, 34, 799−808
Bioconjugate Chemistry
(9) Gonzalez-Aseguinolaza, G.; Van Kaer, L.; Bergmann, C. C.;
Wilson, J. M.; Schmieg, J.; Kronenberg, M.; Nakayama, K.; Taniguchi,
M.; Koezuka, Y.; Tsuji, M. Natural killer T cell ligand alpha-
galactosylceramide enhances protective immunity induced by malaria
vaccines. J. Exp Med. 2002, 195 (5), 617−624.
(10) Singh, S.; Nehete, P. N.; Yang, G.; He, H.; Nehete, B.; Hanley,
P. W.; Barry, M. A.; Sastry, K. J. Enhancement of Mucosal
Immunogenicity of Viral Vectored Vaccines by the NKT Cell Agonist
Alpha-Galactosylceramide as Adjuvant. Vaccines 2014, 2 (4), 686−
706.
(11) Artiaga, B. L.; Whitener, R. L.; Staples, C. R.; Driver, J. P.
Adjuvant effects of therapeutic glycolipids administered to a cohort of
NKT cell-diverse pigs. Vet Immunol Immunop 2014, 162 (1−2), 1−
13.
(12) Tefit, J. N.; Crabe, S.; Orlandini, B.; Nell, H.; Bendelac, A.;
Deng, S. L.; Savage, P. B.; Teyton, L.; Serra, V. Efficacy of ABX196, a
new NKT agonist, in prophylactic human vaccination. Vaccine 2014,
32 (46), 6138−6145.
(13) Fujii, S.; Shimizu, K.; Smith, C.; Bonifaz, L.; Steinman, R. M.
Activation of natural killer T cells by alpha-galactosylceramide rapidly
induces the full maturation of dendritic cells in vivo and thereby acts
as an adjuvant for combined CD4 and CD8 T cell immunity to a
coadministered protein. J. Exp Med. 2003, 198 (2), 267−279.
(14) Hermans, I. F.; Silk, J. D.; Gileadi, U.; Salio, M.; Mathew, B.;
Ritter, G.; Schmidt, R.; Harris, A. L.; Old, L.; Cerundolo, V. NKT
cells enhance CD4(+) and CD8(+) T cell responses to soluble
antigen in vivo through direct interaction with dendritic cells. J.
Immunol 2003, 171 (10), 5140−5147.
(15) Chen, P. G.; Hu, H. G.; Sun, Z. Y.; Li, Q. Q.; Zhang, B. D.; Wu,
J. J.; Li, W. H.; Zhao, Y. F.; Chen, Y. X.; Li, Y. M. Fully Synthetic
Invariant NKT Cell-Dependent Self-Adjuvanting Antitumor Vaccines
Eliciting Potent Immune Response in Mice. Mol. Pharmaceut 2019, 17
(2), 417−425.
(16) Grasso, C.; Field, C. S.; Tang, C. W.; Ferguson, P. M.;
Compton, B. J.; Anderson, R. J.; Painter, G. F.; Weinkove, R.;
Hermans, I. F.; Berridge, M. V. Vaccines adjuvanted with an NKT cell
agonist induce effective T-cell responses in models of CNS
lymphoma. Immunotherapy 2020, 12 (6), 395−406.
(17) Anderson, R. J.; Compton, B. J.; Tang, C. W.; Authier-Hall, A.;
Hayman, C. M.; Swinerd, G. W.; Kowalczyk, R.; Harris, P.; Brimble,
M. A.; Larsen, D. S.; et al. NKT cell-dependent glycolipid-peptide
vaccines with potent anti-tumour activity. Chem. Sci. 2015, 6 (9),
5120−5127.
(18) Miller, D. S.; Finnie, J.; Bowden, T. R.; Scholz, A. C.; Oh, S.;
Kok, T.; Burrell, C. J.; Trinidad, L.; Boyle, D. B.; Li, P. Preclinical
efficacy studies of influenza A haemagglutinin precursor cleavage loop
peptides as a potential vaccine. J. Gen Virol 2011, 92, 1152−1161.
(19) Anderson, R. J.; Li, J.; Kedzierski, L.; Compton, B. J.; Hayman,
C. M.; Osmond, T. L.; Tang, C. W.; Farrand, K. J.; Koay, H. F.;
Almeida, C. F. D. S. E.; et al. Augmenting Influenza-Specific T Cell
Memory Generation with a Natural Killer T Cell-Dependent
Glycolipid-Peptide Vaccine. ACS Chem. Biol. 2017, 12 (11), 2898−
2905.
(20) Holz, L. E.; Chua, Y. C.; de Menezes, M. N.; Anderson, R. J.;
Draper, S. L.; Compton, B. J.; Chan, S. T. S.; Mathew, J.; Li, J.;
Kedzierski, L.; Wang, Z. F.; et al. Glycolipid-peptide vaccination
induces liver-resident memory CD8(+) T cells that protect against
rodent malaria. Sci. Immunol 2020, 5 (48), No. eaaz8035.
(21) Meijlink, M. A.; Chua, Y. C.; Chan, S. T. S.; Anderson, R. J.;
Rosenberg, M. W.; Cozijnsen, A.; Mollard, V.; McFadden, G. I.;
Draper, S. L.; Holz, L. E.; et al. 6 “-Modifed alpha-GalCer-peptide
conjugate vaccine candidates protect against liver-stage malaria. RSC
Chem. Biol. 2022, 3, 551−560.
(22) Anderson, R. J.; Tang, C. W.; Daniels, N. J.; Compton, B. J.;
Hayman, C. M.; Johnston, K. A.; Knight, D. A.; Gasser, O.; Poyntz, H.
C.; Ferguson, P. M.; et al. A self-adjuvanting vaccine induces cytotoxic
T lymphocytes that suppress allergy. Nat. Chem. Biol. 2014, 10 (11),
943−949.
807
pubs.acs.org/bc Article
(23) Cavallari, M.; Stallforth, P.; Kalinichenko, A.; Rathwell, D. C.
K.; Gronewold, T. M. A.; Adibekian, A.; Mori, L.; Landmann, R.;
Seeberger, P. H.; De Libero, G. A semisynthetic carbohydrate-lipid
vaccine that protects against S-pneumoniae in mice. Nat. Chem. Biol.
2014, 10 (11), 950−956.
(24) Wang, J.; Wen, Y.; Zhou, S. H.; Zhang, H. W.; Peng, X. Q.;
Zhang, R. Y.; Yin, X. G.; Qiu, H.; Gong, R.; Yang, G. F.; et al. Self-
Adjuvanting Lipoprotein Conjugate alphaGalCer-RBD Induces
Potent Immunity against SARS-CoV-2 and its Variants of Concern.
J. Med. Chem. 2022, 65 (3), 2558−2570.
(25) Ghinnagow, R.; De Meester, J.; Cruz, L. J.; Aspord, C.;
Corgnac, S.; Macho-Fernandez, E.; Soulard, D.; Fontaine, J.;
Chaperot, L.; Charles, J.; et al. Co-delivery of the NKT agonist
alpha-galactosylceramide and tumor antigens to cross-priming
dendritic cells breaks tolerance to self-antigens and promotes
antitumor responses. Oncoimmunology 2017, 6 (9), No. e1339855.
(26) Huang, J.; Zhou, J.; Ghinnagow, R.; Seki, T.; Iketani, S.;
Soulard, D.; Paczkowski, P.; Tsuji, Y.; MacKay, S.; Cruz, L. J.; et al.
Targeted Co-delivery of Tumor Antigen and alpha-Galactosylcer-
amide to CD141(+) Dendritic Cells Induces a Potent Tumor
Antigen-Specific Human CD8(+) T Cell Response in Human
Immune System Mice. Front Immunol 2020, 11, 2043.
(27) Lam, P. Y.; Kobayashi, T.; Soon, M.; Zeng, B. J.; Dolcetti, R.;
Leggatt, G.; Thomas, R.; Mattarollo, S. R. NKT Cell-Driven
Enhancement of Antitumor Immunity Induced by Clec9a-Targeted
Tailorable Nanoemulsion. Cancer Immunol Res. 2019, 7 (6), 952−
962.
(28) Stolk, D. A.; de Haas, A.; Vree, J.; Duinkerken, S.; Lubbers, J.;
van de Ven, R.; Ambrosini, M.; Kalay, H.; Bruijns, S.; van der Vliet, H.
J.; et al. Lipo-Based Vaccines as an Approach to Target Dendritic
Cells for Induction of T- and iNKT Cell Responses. Front Immunol
2020, 11, 990.
(29) Sainz, V.; Moura, L. I. F.; Peres, C.; Matos, A. I.; Viana, A. S.;
Wagner, A. M.; Vela Ramirez, J. E.; Barata, T. S.; Gaspar, M.;
Brocchini, S.; et al. alpha-Galactosylceramide and peptide-based nano-
vaccine synergistically induced a strong tumor suppressive effect in
melanoma. Acta Biomater 2018, 76, 193−207.
(30) Neumann, S.; Young, K.; Compton, B.; Anderson, R.; Painter,
G.; Hook, S. Synthetic TRP2 long-peptide and alpha-galactosylcer-
amide formulated into cationic liposomes elicit CD8(+) T-cell
responses and prevent tumour progression. Vaccine 2015, 33 (43),
5838−5844.
(31) Sartorius, R.; D’Apice, L.; Barba, P.; Cipria, D.; Grauso, L.;
Cutignano, A.; De Berardinis, P. Vectorized Delivery of Alpha-
GalactosylCeramide and Tumor Antigen on Filamentous Bacter-
iophage fd Induces Protective Immunity by Enhancing Tumor-
Specific T Cell Response. Front Immunol 2018, 9, 1496.
(32) Liu, Y. H.; Wang, Z. Y.; Yu, F.; Li, M. J.; Zhu, H. M.; Wang, K.;
Meng, M.; Zhao, W. The Adjuvant of a-Galactosylceramide Presented
by Gold Nanoparticles Enhances Antitumor Immune Responses of
MUC1 Antigen-Based Tumor Vaccines. Int. J. Nanomed 2021, 16,
403−420.
(33) Dolen, Y.; Kreutz, M.; Gileadi, U.; Tel, J.; Vasaturo, A.; van
Dinther, E. A. W.; van Hout-Kuijer, M. A.; Cerundolo, V.; Figdor, C.
G. Co-delivery of PLGA encapsulated invariant NKT cell agonist with
antigenic protein induce strong T cell-mediated antitumor immune
responses. Oncoimmunology 2016, 5 (1), No. e1068493.
(34) Rahimian, S.; Fransen, M. F.; Kleinovink, J. W.; Christensen, J.
R.; Amidi, M.; Hennink, W. E.; Ossendorp, F. Polymeric nano-
particles for co-delivery of synthetic long peptide antigen and poly IC
as therapeutic cancer vaccine formulation. J. Controlled Release 2015,
203, 16−22.
(35) Mascaraque, A.; Kowalczyk, W.; Fernandez, T.; Palomares, F.;
Mayorga, C.; Andreu, D.; Rojo, J. Glycodendropeptides stimulate
dendritic cell maturation and T cell proliferation: a potential influenza
A virus immunotherapy. MedChemComm 2015, 6 (10), 1755−1760.
(36) Cai, H.; Sun, Z. Y.; Chen, M. S.; Zhao, Y. F.; Kunz, H.; Li, Y.
M. Synthetic Multivalent Glycopeptide-Lipopeptide Antitumor
https://doi.org/10.1021/acs.bioconjchem.3c00106
Bioconjugate Chem. 2023, 34, 799−808
Bioconjugate Chemistry pubs.acs.org/bc Article

Vaccines: Impact of the Cluster Effect on the Killing of Tumor Cells. killer cells is essential for the antimetastatic effect of alpha-
Angew. Chem., Int. Ed. Engl. 2014, 53 (6), 1699−1703. galactosylceramide. Blood 2002, 99 (4), 1259−1266.
(37) Tam, J. P. Synthetic Peptide Vaccine Design - Synthesis and
Properties of a High-Density Multiple Antigenic Peptide System. Proc.
Natl. Acad. Sci. U. S. A. 1988, 85 (15), 5409−5413.
(38) Mitchell, J. P.; Roberts, K. D.; Langley, J.; Koentgen, F.;
Lambert, J. N. A direct method for the formation of peptide and
carbohydrate dendrimers. Bioorg. Med. Chem. Lett. 1999, 9 (19),
2785−2788.
(39) Mihov, G.; Grebel-Koehler, D.; Lubbert, A.; Vandermeulen, G.
W. M.; Herrmann, A.; Klok, H. A.; Mullen, K. Polyphenylene
dendrimers as scaffolds for shape-persistent multiple peptide
conjugates. Bioconjugate Chem. 2005, 16 (2), 283−293.
(40) Shrestha, R.; Petley, E. V.; Farrand, K. J.; Jamieson, S. A.; Jiao,
W. T.; Teesdale-Spittle, P. H.; Mace, P. D.; Hermans, I. F.; Rendle, P.
M. The Synthesis and Anti-tumour Properties of Poly Ethoxy Ethyl
Glycinamide (PEE-G) Scaffolds with Multiple PD-1 Peptides
Attached. ChemMedChem. 2020, 15 (13), 1128−1138.
(41) Toms, S.; Carnachan, S. M.; Hermans, I. F.; Johnson, K. D.;
Khan, A. A.; O’Hagan, S. E.; Tang, C. W.; Rendle, P. M. Poly Ethoxy
Ethyl Glycinamide (PEE-G) Dendrimers: Dendrimers Specifically
Designed for Pharmaceutical Applications. ChemMedChem. 2016, 11
(15), 1583−1586.
(42) Speir, M.; Authier-Hall, A.; Brooks, C. R.; Farrand, K. J.;
Compton, B. J.; Anderson, R. J.; Heiser, A.; Osmond, T. L.; Tang, C.
W.; Berzofsky, J. A.; et al. Glycolipid-peptide conjugate vaccines
enhance CD8(+) T cell responses against human viral proteins. Sci.
Rep 2017, 7, 14273.
(43) Dubowchik, G. M.; Firestone, R. A.; Padilla, L.; Willner, D.;
Hofstead, S. J.; Mosure, K.; Knipe, J. O.; Lasch, S. J.; Trail, P. A.
Cathepsin B-labile dipeptide linkers for lysosomal release of
doxorubicin from internalizing immunoconjugates: model studies of
enzymatic drug release and antigen-specific in vitro anticancer activity.
Bioconjugate Chem. 2002, 13 (4), 855−869.
(44) Bryden, F.; Martin, C.; Letast, S.; Lles, E.; Vieitez-Villemin, I.;
Rousseau, A.; Colas, C.; Brachet-Botineau, M.; Allard-Vannier, E.;
Larbouret, C.; et al. Impact of cathepsin B-sensitive triggers and
Recommended by ACS
hydrophilic linkers on in vitro efficacy of novel site-specific antibody-
drug conjugates. Org. Biomol Chem. 2018, 16 (11), 1882−1889. Dual-Responsive Glycopolymers for Intracellular Codelivery
(45) Poreba, M. Protease-activated prodrugs: strategies, challenges, of Antigen and Lipophilic Adjuvants
and future directions. FEBS J. 2020, 287 (10), 1936−1969. Judith De Mel, Thomas A. Werfel, et al.
(46) Flechtner, J. B.; Cohane, K. P.; Mehta, S.; Slusarewicz, P.; NOVEMBER 14, 2022
Leonard, A. K.; Barber, B. H.; Levey, D. L.; Andjelic, S. High-affinity MOLECULAR PHARMACEUTICS READ
interactions between peptides and heat shock protein 70 augment
CD8(+) T lymphocyte immune responses. J. Immunol 2006, 177 (2), Identification of a Novel Small Molecule That Enhances the
1017−1027. Release of Extracellular Vesicles with Immunostimulatory
(47) Gibson, B.; Verkade, J.; Barta, N.; Hodges, J.; van Delft, F. Potency via Induction of Calcium Influx
Bicyclo[6.1.0]nonyne Phoshoramidites for Metal-Free Conjugation of
Yukiya Sako, Tomoko Hayashi, et al.
Oligonucleotides. Chimica Oggi 2013, 31, 18−21.
APRIL 11, 2023
(48) Spangler, B.; Yang, S.; Baxter Rath, C. M.; Reck, F.; Feng, B. Y. ACS CHEMICAL BIOLOGY READ
A Unified Framework for the Incorporation of Bioorthogonal
Compound Exposure Probes within Biological Compartments. ACS
Covalently Conjugated NOD2/TLR7 Agonists Are Potent
Chem. Biol. 2019, 14 (4), 725−734.
and Versatile Immune Potentiators
(49) Burn, O. K.; Farrand, K.; Pritchard, T.; Draper, S.; Tang, C. W.;
Mooney, A. H.; Schmidt, A. J.; Yang, S. H.; Williams, G. M.; Brimble, Samo Guzelj, Žiga Jakopin, et al.
M. A.; et al. Glycolipid-peptide conjugate vaccines elicit CD8(+) T- NOVEMBER 06, 2022
JOURNAL OF MEDICINAL CHEMISTRY READ
cell responses and prevent breast cancer metastasis. Clin Transl
Immunology 2022, 11 (7), No. e1401.
(50) Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Nucleophilic Nanovaccine Based on a Biepitope Antigen to Potentiate the
catalysis of oxime ligation. Angew. Chem., Int. Ed. Engl. 2006, 45 (45), Immunogenicity of a Neoantigen
7581−7584. Shuyu Chen, Guosong Chen, et al.
(51) Carnaud, C.; Lee, D.; Donnars, O.; Park, S. H.; Beavis, A.; FEBRUARY 08, 2023
Koezuka, Y.; Bendelac, A. Cutting edge: Cross-talk between cells of ACS MACRO LETTERS READ
the innate immune system: NKT cells rapidly activate NK cells. J.
Immunol 1999, 163 (9), 4647−4650.
(52) Smyth, M. J.; Crowe, N. Y.; Pellicci, D. G.; Kyparissoudis, K.;
Get More Suggestions >
Kelly, J. M.; Takeda, K.; Yagita, H.; Godfrey, D. I. Sequential
production of interferon-gamma by NK1.1(+) T cells and natural

808 https://doi.org/10.1021/acs.bioconjchem.3c00106
Bioconjugate Chem. 2023, 34, 799−808