Photoactivable Glycolipid Antigens Generate Stable Conjugates with CD1d for Invariant Natural Killer T Cell Activation

Article abstract of DOI:10.1021/acs.bioconjchem.8b00484

Activation of invariant natural killer T lymphocytes (iNKT cells) by α-galactosylceramide (α-GC) elicits a range of pro-inflammatory or anti-inflammatory immune responses. We report the synthesis and characterization of a series of α-GC analogues with acyl chains of varying length and a terminal benzophenone. These bound efficiently to the glycolipid antigen presenting protein CD1d, and upon photoactivation formed stable CD1d-glycolipid covalent conjugates. Conjugates of benzophenone α-GCs with soluble or cell-bound CD1d proteins retained potent iNKT cell activating properties, with biologic effects that were modulated by acyl chain length and the resulting affinities of conjugates for iNKT cell antigen receptors. Analysis by mass spectrometry identified a unique covalent attachment site for the glycolipid ligands in the hydrophobic ligand binding pocket of CD1d. The creation of covalent conjugates of CD1d with α-GC provides a new tool for probing the biology of glycolipid antigen presentation, as well as opportunities for developing effective immunotherapeutics.

Full text of DOI:10.1021/acs.bioconjchem.8b00484

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Article
Photoactivable glycolipid antigens generate stable conjugates
with CD1d for invariant Natural Killer T cell activation
Shalu Sharma Kharkwal, Natacha Veerapen, Peter Jervis, Veemal Bhowruth, Amareeta K Besra,
Simon J North, Stuart M. Haslam, Anne Dell, Judith Hobrath, Padraic Quaid, Patrick Moynihan,
Liam Robert Cox, Himanshu Kharkwal, Maurice Zauderer, Gurdyal S. Besra, and Steven A. Porcelli
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00484 • Publication Date (Web): 07 Aug 2018
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Photoactivable glycolipid antigens generate stable conjugates with
CD1d for invariant Natural Killer T cell activation
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Natacha Veerapen1#, Shalu Sharma Kharkwal3#, Peter Jervis1, Veemal Bhowruth1,
Amareeta K. Besra1, Simon J. North6, Stuart M. Haslam6, Anne Dell6, Judith Hobrath7,
Padraic J. Quaid1, Patrick J. Moynihan1, Liam R. Cox2, Himanshu Kharkwal4, Maurice
Zauderer8, Gurdyal S. Besra1*, Steven A. Porcelli.3,5*
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1School of Biosciences, and 2School of Chemistry, University of Birmingham,
Edgbaston, Birmingham, B15 2TT, United Kingdom
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3Department of Microbiology and Immunology, 4Department of Developmental and
Molecular Biology, and 5Department of Medicine, Albert Einstein College of Medicine,
Bronx, NY, 10461, USA
6Department of Life Sciences, Faculty of Natural Sciences, Imperial College
London, South Kensington Campus, London, SW7 2AZ, UK
7Drug Discovery Unit, College of Life Sciences, University of Dundee, Dow Street
Dundee, DD1 5EH, Scotland, UK
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8Vaccinex Inc., 1895 Mount Hope Avenue, Rochester, NY 14620, USA
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#These authors contributed equally to this manuscript
*Joint corresponding authors
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Correspondence to:
Steven A. Porcelli, MD
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Department of Microbiology and Immunology
Forchheimer Building, Room 416
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Albert Einstein College of Medicine
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1300 Morris Park Avenue, Bronx, NY 10461
Phone: 718ꢀ430ꢀ3228. Fax: 718ꢀ430ꢀ8711
Eꢀmail: steven.porcelli@einstein.yu.edu
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Gurdyal S. Besra
School of Biosciences
University of Birmingham
Edgbaston, Birmingham B15 2TT, UK
Phone: +00 44 121 415 8125. Fax: +00 44 121 414 5925
Eꢀmail: g.besra@bham.ac.uk
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Abstract
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Activation of invariant natural killer T lymphocytes (iNKT cells) by αꢀgalactosylceramide
(αꢀGC) elicits a range of proꢀinflammatory or antiꢀinflammatory immune responses. We
report the synthesis and characterization of a series of αꢀGC analogues with acyl chains
of varying length and a terminal benzophenone. These bound efficiently to the
glycolipid antigen presenting protein CD1d, and upon photoactivation formed stable
CD1dꢀglycolipid covalent conjugates. Conjugates of benzophenone αꢀGCs with soluble
or cell bound CD1d proteins retained potent iNKT cell activating properties, with biologic
effects that were modulated by acyl chain length and the resulting affinities of
conjugates for iNKT cell antigen receptors. Analysis by mass spectrometry identified a
unique covalent attachment site for the glycolipid ligands in the hydrophobic ligand
binding pocket of CD1d. The creation of covalent conjugates of CD1d with αꢀGC
provides a new tool for probing the biology of glycolipid antigen presentation, as well as
opportunities for developing effective immunotherapeutics.
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Keywords
CD1d, αꢀgalactosylceramide, benzophenone, invariant Natural Killer T cells
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Introduction
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Invariant Natural Killer T (iNKT) cells are a prominent subset of unconventional T cells
that bridge innate and adaptive immunity to contribute to a wide range of immune
responses.1 They recognize and respond to glycolipid antigens presented by CD1d, a
membrane protein specialized for binding and presentation of lipid antigens.2 The most
extensively studied CD1dꢀpresented glycolipid antigens are synthetic forms of αꢀ
galactosylceramide (αꢀGC), which potently stimulate iNKT cell proliferation, expansion
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and cytokine secretion.3 In mice, various structural analogues of
αꢀGC have shown
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impressive antiꢀcancer effects4, as well as a broad range of activities in models of
infection, vaccination, autoimmunity and inflammatory diseases.5 Thus, there has
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been increasing interest in using
αꢀGC analogues to develop new approaches to
vaccination or immunotherapy.4c, 6
Despite the potent immune activating properties of αꢀGC and the
conservation of a CD1dꢀrestricted iNKT cell subset in humans, there has been
limited success so far in translating iNKT cellꢀbased approaches into clinical
applications. This may reflect problems with systemic delivery of glycolipid agonists,
which leads to their uptake and presentation by a wide range of cell types and the
stimulation of unpredictable or antagonistic immune responses.6a, 7
Several approaches to overcoming these problems have been developed that
involve delivery of αꢀGC by antigen presenting cells (APCs) or soluble recombinant
CD1d proteins loaded ex vivo with the glycolipid. 8 These approaches have shown
potential to stimulate more effective antitumor responses compared to injections of
free glycolipids in mouse models, as well as promising preliminary results in
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preclinical and clinical studies.4c, 6a, 8c, 8d, 9 Also of note is the apparent ability of
these methods to induce substantial iNKT cell activation while triggering less of the
longꢀterm unresponsiveness (anergy) or depletion of iNKT cells that has been a
problem with injections of free glycolipids.10
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However, a potential limitation is presented by the readily reversible binding
of glycolipid ligands to CD1d, which is mediated by noncovalent hydrophobic and
hydrogen bonding interactions.2 Rapid dissociation has been reported to result in
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halfꢀlives for αGCꢀCD1d complexes in some in vitro studies as short as a few
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minutes or less, which may be further reduced in vivo by the presence of lipid
exchange and binding proteins.11 The relatively short halfꢀlife and instability of such
complexes limits the duration and potency of their desirable biologic effects, and the
release of free glycolipids in vivo may contribute to unwanted effects such as iNKT
cell anergy or toxicities including liver damage or sensitization to endotoxic shock.12
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Thus, the relative instability of αGCꢀCD1d complexes remains a suboptimal feature
in approaches that involve ex vivo loading of cells or CD1d proteins with glycolipids,
and can compromise the efficacy and precision of such controlled delivery methods.
In the current study, we have developed an approach to circumvent the
problem of glycolipid dissociation from CD1d by the use of photoactivatable forms
of
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ꢀGC to create covalently stabilized and highly active
αGCꢀCD1d conjugates. A
series of analogues of
α
ꢀGC containing a benzophenone group at the terminus of
their Nꢀacyl chains was synthesized and optimized for iNKT cell stimulating activity.
Controlled exposure to UV irradiation generated stable covalent conjugates of these
glycolipids with CD1d, and these retained their ability to potently stimulate iNKT
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cells in vitro and in vivo in mice. Specific immunologic properties such as cytokine
production could be modulated by variations in the size of the aliphatic chain
bearing the benzophenone group, which was correlated with the affinity of the
conjugates for the antigen receptors of iNKT cells. Mass spectrometry identified a
unique site for covalent bond formation in the CD1d protein, enabling detailed
modeling of the structure of the stable conjugates. The development of controlled
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covalent bond formation for stabilization of αGCꢀCD1d conjugates provides a new
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tool for the study of glycolipid antigen presentation, and also forms the basis for
improving immunotherapies based on the targeted delivery of iNKT cell activators.
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Results
Synthesis of BPGCs
Extensive previous work has shown that many modifications of the fatty acyl chain of
αꢀGC can be tolerated without disruption of glycolipid binding to CD1d or loss of iNKT
cell stimulating activity3, consistent with the large volume of the hydrophobic ligand
binding site of CD1d.2, 13 Thus, we developed a synthetic strategy for introduction of a
photoactivatable moiety on the acyl chain terminus of αꢀGC to enable the controlled
formation of a covalent bond between the glycolipid and the CD1d protein (Fig. 1). The
benzophenone group was chosen as it can be activated by UV irradiation to give the
corresponding benzhydril biradical, which we postulated would react with a proximal
CꢀH bond present in the CD1d protein to form a permanent covalent bond. A group of
benzophenoneꢀcontaining derivatives of αꢀGC (BPGCs) bearing acyl chains of various
lengths (compounds 9ꢀ15) were synthesized to determine which would optimally
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associate with CD1d and effectively activate iNKT cells. Based on the resemblance of
benzophenone to a C10 isoprene unit, we synthesized a range of BPGCs which mimic
Nꢀacyl chain lengths from C16 (C6:BP (9)) to C26 (C16:BP (15)), thus spanning the
range found in most highly active αꢀGC analogues.3 These compounds were readily
prepared via acylation of the parent compound 1 with carboxylic acids (2ꢀ8), following
their conversion to the corresponding acid chlorides using oxalyl chloride. Ensuing
acylation of the amine 1 in a 1:1 mixture of THF and saturated sodium acetate solution
afforded the benzophenoneꢀderivatised glycosphingolipids (BPGCs) 9-15 (Fig. 1).
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Figure 1: Synthesis of BPGCs. (a) Structure of the prototypical iNKT cell activating
glycolipid, αꢀGC C26:0. (b) General scheme for synthesis of BPGCs with acyl group
spacers of varying length (compounds 9 – 15). (a) NaH, DMF, 0 ºC to rt; (b) PDC, wet
THF, rt; (c) CaCO3, THF, H2O, 100 ºC, 12 h; (d) NaH, DMF, 0 ºC to rt; (e) (COCl)2, 60
ºC, 2 h; (f) NaOAc, THF, rt, 12 h. Incorporation of the benzophenone group into
carboxylic acids was accomplished through the use of a flexible ether linkage to allow
rotational freedom and optimal orientation of the aromatic group in the CD1d ligandꢀ
binding pocket. To synthesize carboxylic acids (2-8), we used a versatile strategy
involving an SN2 displacement of a bromide using various diols and 3ꢀ
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(bromomethyl)benzophenone (16) or through a variety of bromocarboxylic acids by 3ꢀ
(hydroxymethyl)benzophenone (17). The monoalkylation of the diols with 3ꢀ
(bromomethyl)benzophenone (16), which was obtained using published procedures14,
was achieved in reasonable yields by using the diols in excess. Oxidation of the
corresponding alcohols to the acids with pyridinium dichromate (PDC) was sluggish and
only afforded compounds 2-5 in average yields. In contrast, alkylation of the alcohol 17
with the bromocarboxylic acids in DMF and sodium hydride as base proceeded
smoothly to afford the desired carboxylic acids 6-8 in quantitative yields (see
Supplemental Information for further details of synthesis and characterization).
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Stimulation of iNKT cells by BPGC analogs
A variety of in vitro biological assays were performed to assess the iNKT cell activating
properties of the BPGCs upon presentation by CD1d, and to determine the effect of the
varying acyl chain lengths and the presence of a bulky terminal benzophenone group on
their biologic activities. A standard assay using coꢀculture of mouse bone marrowꢀ
derived dendritic cells (BMDCs) and a murine iNKT cell hybridoma was used to assess
the activity and relative potencies of BPGCs (Fig. 2a).15 This showed significant iNKT
cell stimulating activity for all BPGCs tested, with a substantial impact of the length of
fatty acyl chain. Optimal iNKT stimulation in the mouse cell culture system was
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achieved with 13, which along with several other BPGCs was significantly more potent
than the standard αꢀGC (C26:0), which is generally considered a highly potent iNKT cell
activator both in vitro and in vivo.4a, 15 A similar in vitro analysis was carried out using a
canonical human iNKT cell clone coꢀcultured with HeLa cells transfected to express
human CD1d (Fig. 2b).16 This also revealed strong activity of BPGCs as measured by
IFNꢀγ secretion, which was similar to or greater than that stimulated by C26:0. As for
the mouse culture system, compounds 12 and 13 showed the greatest potency in this
assay, although length of the fatty acyl tail had much less apparent impact in the human
system.
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To directly assess activity in vivo, we analyzed serum cytokine levels following
intravenous injection of selected BPGCs. For this analysis, we tested 9 which was a
relatively weak activator of murine iNKT cells in vitro, and 13 and 15 which were more
potent than C26:0 in both mouse and human cell culture assays. Mice were injected
with 4 nmoles of each glycolipid and bled after 2, 10 and 24 hours to quantitate serum
IFNꢀγ and ILꢀ4, as previously described.15 Significant levels above baseline for serum
ILꢀ4 were detected with 13 and 15 at two hours, which declined to undetectable levels
by 10 hours (Fig. 2c). The ILꢀ4 levels were several fold higher for 13 and 15 compared
to C26:0, indicating a rapid and powerful activation of iNKT cells. Consistent with this,
IFNꢀγ levels showed a sustained rise with 13 and 15 with a peak at 10 hours that closely
resembled the response to C26:0, while 9 did not stimulate detectable levels above
background for either cytokine tested. Thus, BPGCs retained their iNKT cell activating
properties in vivo, and the potencies of different analogues varied depending on the
length of their acyl chains.
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Figure 2: iNKT stimulatory activity of BPGCs. (a) Responses of mouse iNKT cell
hybridoma DN3A4ꢀ1.2 cultured with bone marrow derived dendritic cells from C57BL/6
mice and the indicated concentrations of BPGCs or αꢀGC C26:0. ILꢀ2 secretion was
measured in supernatants after 18 hours of culture. Representative doseꢀresponse
curves are shown on the left for three of the seven BPGCs and standard αꢀGC C26:0.
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Concentrations stimulating 50% of the maximum response (EC50) were determined for
each glycolipid, and reciprocal values are plotted on the right. (b) Similar analysis as in
(a) except using human iNKT clone HDE3 and HeLa cells expressing human CD1d with
measurement of secreted IFNγ as the readout for activation. (c) In vivo activities of 9,
13 and 15 were determined by quantitating serum ILꢀ4 and IFNꢀγ 2, 10 and 24 hours
post glycolipid injection. Peak values for serum IL4 (left) and IFNꢀγ (right) were found at
2 and 10 hours respectively.
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All symbols and bars are means for triplicate samples, and error bars are ±1 SD. *P <
0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for comparisons to vehicle treated mice
(twoꢀway ANOVA with Dunnett’s multiple comparison test). All data are representative
of at least three separate experiments.
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Formation of covalent CD1d-glycolipid conjugates by photoactivation
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The known photochemical properties of benzophenones predicted that loading of
BPGCs into CD1d proteins followed by exposure to UV irradiation should form
covalently stabilized proteinꢀglycolipid conjugates. We validated this initially using a 3ꢀ
fold molar excess of 14Cꢀlabelled analogue of 13, compound 26 (Scheme 2, SI) to load
soluble recombinant mCD1d protein. Aliquots of the loaded protein were then exposed
to a 365 nm UV lamp for times ranging from 0 to 90 minutes, followed by denaturation
(1% SDS at 100° C for 5 min) and separation by SDSꢀPAGE. Staining of the gel with
Coomassie blue revealed intact protein in all samples, which ran at the predicted size of
~57 kDa for monomeric soluble CD1d (Fig. 3a). Autoradiography of the same gel
revealed the presence of the 14C label coꢀmigrating with CD1d protein in the samples
exposed to UV light, but not in the unexposed sample (Fig. 3b). Maximum association
of radiolabel, indicating the formation of stable proteinꢀglycolipid conjugates that were
resistant to denaturation, was achieved following 30 to 60 minutes of UV exposure,
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corresponding to a delivered dose range of 400 – 600 mJoules/cm2. Radiometric
analysis showed that approximately 70% of CD1d molecules were conjugated to 14C
labeled 26 after 60 minutes of UV exposure (Supplemental Figure S1).
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Figure 3: Covalent conjugation of BPGCs to mCD1d. (a) Coomassie stained gel
and (b) autoradiograph of denaturing SDSꢀPAGE after exposing mCD1d: 14Cꢀlabelled
analogue of 13, compound 26 (Scheme 2, SI) complexes (~57 kDa) for various lengths
of time. (c) Diagrammatic representation of click reaction of DBCOꢀTAMRA dye with
azideꢀlinkedꢀ25 (SI) either loaded nonꢀcovalently or covalently conjugated to CD1d
protein. (d) Coomassie stained and fluorescent images of denaturing SDSꢀPAGE gel of
fluorescently tagged noncovalentꢀcomplexes (0 min UV exposure) and covalentꢀ
conjugates (60 min UV exposure) of mCD1d fusion protein (~78 kDa). (e) Complexes
(No UV, left) or conjugates (UV, right) loaded with the indicated BPGC or αꢀGCꢀC26:0
were coated on high binding plates and incubated for 3 days at room temperature either
without (white bars) or with (black bars) washing twice per day with PBS + 0.1% Triton
Xꢀ100. Residual glycolipid binding to mCD1d was detected by ELISA using biotinylated
monoclonal antibody L363. *P < 0.001 for multiple t tests of comparisons of washed
versus unwashed samples with each glycolipid. (f) Splenocytes (2 x 105 per well in 0.2
ml complete medium) were stimulated in vitro for 18 hours with complexes (hatched
bars) and conjugates (solid bars) immobilized on high binding plates. Supernatants
were collected and assayed for IFNꢀγ by ELISA. (g) JAWS II cells (5 x 104 per well)
pulsed overnight with 100 nM glycolipids (C26:0 or 13) were either exposed to UV (~400
mJ/cm2) or left untreated. Cells were stained with L363ꢀAlexaFluor647 either directly
(solid black bars) or after multiple washes (hatched bars) to allow dissociation and
analyzed by flow cytometry. ****P < 0.0001, twoꢀway ANOVA with Bonferroni correction
for indicated comparisons. (h) JAWS II cells were exposed to UV (600 mJ/cm2) either
before (black symbols) or after (white symbols) pulsing with vehicle, C26:0 or 13 at 100
nM concentration. Cells were washed thrice during 24 hours of incubation and
adoptively transferred i.v. into mice (3 x 105 cells per mouse, 4 mice per group). Blood
samples were obtained after 2, 12 and 24 hours to quantitate serum ILꢀ4 and IFNꢀγ. **P
< 0.01, twoꢀway ANOVA with Bonferroni correction for indicated comparisons. Data
plotted in (e) – (f) are shown as means for a minimum of three replicates, and error bars
are ± 1 SD. All experiments were performed at least three times.
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Further analysis of conjugate formation was carried out in studies using
compound 25 (synthesis described in SI), an analogue of 13 carrying an azido group at
the 6’ꢀ position of the saccharide head group (Fig. 3c). Complexes formed between this
glycolipid and soluble mCD1d were either irradiated with UV λ365 or not, and then
incubated with fluorescent DBCOꢀTAMRA, which reacts with the azido group of the
glycolipid.17 The samples were then denatured and analyzed by SDSꢀPAGE. While
gels stained with Coomassie blue showed similar CD1d protein in both samples
(migrating at ~78 kDa, consistent with the mCD1d fusion protein used for this
experiment; see Online Methods for details), only the sample exposed to UV had a
fluorescent signal coꢀmigrating with CD1d (Fig. 3d). This confirmed the formation of
stable conjugates following photoactivation of the benzophenone moiety in CD1dꢀ
glycolipid complexes. In addition, the ability of the DBCOꢀTAMRA reagent to couple to
the azido group was consistent with correct orientation of the 13 in the CD1d lipid
binding groove, with the carbohydrate headꢀgroup exposed and accessible at the
surface of the protein.
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To confirm the correct conformation and stability of covalent mCD1dꢀBPGC
conjugates, we tested reactivity with mAb L363, which binds specifically to CD1d loaded
with αꢀGC in a manner that closely mimics the TCRs of iNKT cells.18 Plate immobilized
mCD1d proteins were loaded with BPGCs, then either UV irradiated or not and tested
for binding of L363. This showed binding to levels comparable to that with C26:0
loaded mCD1d (Fig. 3e). Furthermore, after repeated washing with buffer containing
mild detergent to remove reversibly bound glycolipids, we observed significant loss of
L363 binding in samples without UV irradiation. In contrast, UV exposed samples
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loaded with BPGCs showed no significant loss of L363 binding, indicating covalent bond
formation. As expected, L363 binding to mCD1d loaded with C26:0 was reversible
under these conditions with or without UV exposure. To further characterize the
biologic activity of these complexes or conjugates, we also assessed their ability to
stimulate iNKT cell activation in mouse splenocytes (Fig. 3f). The UV treated stable
conjugates retained their iNKT cell stimulating activity at levels comparable to
noncovalent complexes, indicating that UV exposure and covalent bond formation did
not adversely alter TCR recognition. Analysis of all seven BPGCs in this assay showed
an influence of the acyl chain spacer length on potency of stimulation, with 13 generally
showing the strongest stimulation.
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In addition to the analyses of loading and photoꢀcrosslinking to purified
recombinant CD1d proteins, we also assessed the formation of stabilized mCD1dꢀ
glycolipid conjugates on intact CD1dꢀexpressing antigen presenting cells. We used the
murine immortalized dendritic cell line JAWS II for this, since it expresses mCD1d and is
capable of glycolipid antigen presentation.19 Incubation of these cells with either C26:0
or 13 generated strong surface staining with mAb L363, which for both glycolipids was
greatly reduced following incubation for 1 day in the absence of the glycolipids. In
contrast, exposure of the cells to UV irradiation following culture with the 13 eliminated
any loss of L363 staining with subsequent culture, whereas UV irradiation of C26:0
loaded cells did not prevent decay of L363 binding under the same conditions (Fig. 3g).
This strongly suggested that stabilized covalent CD1dꢀglycolipid conjugates were
produced by UV photoactivation of BPGCs in living cells. This was further evaluated by
testing the ability of JAWS II cells loaded with 13 and UV treated to stimulate iNKT cell
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responses in vivo following adoptive transfer of the cells into mice (Fig. 3h). We
detected strong serum cytokine responses in mice receiving cells bearing the putative
covalently stabilized conjugates, and a reduced level of ILꢀ4 relative to IFNꢀγ was
observed when compared to injection of cells presenting noncovalent complexes (i.e.,
JAWS II cells without UV photoactivation or loaded with C26:0).
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Impact of conjugation on TCR affinity and biologic activity in vivo
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By eliminating dissociation of glycolipid binding to CD1d, we anticipated that conjugation
should increase the overall affinity of cognate interactions with iNKT cell TCRs. To
assess this, fluorescent tetramers of soluble mCD1d loaded with C26:0 or BPGCs were
prepared with and without covalent crosslinking. Binding avidities of tetramers to the
TCRs of mouse iNKT cell hybridoma line DN3A4ꢀ1.2 were quantified by measuring
equilibrium binding to the cells over a range of concentrations, as previously
described.15 Extrapolation of the equilibrium dissociation constant (KD) values showed
maximum avidities for C26:0 and 15 loaded noncovalent complexes, while avidities
declined progressively for BPGCs with shorter acyl tail spacers (Fig. 4a). This trend
was not evident with the binding of covalent conjugate tetramers, as the conjugates with
shorter acyl chain variants of BPGCs showed enhanced and more uniform avidities.
These results indicated that dissociation of the glycolipids with shorter acyl tails had a
major impact in TCR avidity, which was reversed by covalent stabilization of the
glycolipidꢀprotein interaction. One apparent exception was the 9 conjugate, which
showed a distinctly lower TCR avidity than the other conjugates tested despite covalent
stabilization.
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Figure 4: Affinity for iNKT cell TCR and effects on cytokine profiles. (a)
Equilibrium binding of mCD1d tetramers loaded with BPGCs over a range of tetramer
concentrations was used to assess avidities of complexes (not UV irradiated, left) and
conjugates (UV irradiated, right) for TCRs of mouse iNKT cell hybridoma DN3A4ꢀ1.2.
Cells stained with tetramers for 1 hour at room temperature were analyzed by flow
cytometry. Normalized representative binding curves are shown for three BPGC loaded
tetramers, and for standard C26:0 loaded tetramers (not UV irradiated) for comparison
(dashed lines). Bar graphs show reciprocal of KD values (1/EC50) to summarize results
for all tetramers. (b) Serum cytokine levels following i.v. injection of mice with
complexes (open bars) or conjugates (filled bars) loaded with the indicated BPGCs.
Serum levels are shown at 2 hours post injection for ILꢀ2, TNFꢀα and ILꢀ4, and at 10
hours for IFNꢀγ. Background levels of cytokines in sera from mice injected with inert
aqueous vehicle (gray bars), and levels for mice injected with 4 nmoles of free C26:0
glycolipid (horizontal dashed lines) are shown for reference. Bars represent means for
groups of 4 animals each, and error bars show 1 SD. ****P < 0.0001 for conjugate
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versus complex in the indicated comparisons (2ꢀway ANOVA with Bonferroni’s multiple
comparisons test). NS, not significant (P > 0.05). Differences were not significant for
other comparisons shown between complexes and conjugates.
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We next assessed the in vivo activities of soluble mCD1dꢀBPGC complexes and
conjugates using three different BPGCs that varied in their affinities for iNKT cell TCRs.
After a single i.v. injection of mCD1d complexes or conjugates loaded with 9, 13 or 15
(30 ꢁg of mCD1d protein containing ~0.4 nmoles of each glycolipid), serum levels of ILꢀ
2, TNFꢀα, ILꢀ4 and IFNꢀγ were determined at 2, 10 and 24 hours after administration
(Fig. 4b, and Supplemental Fig. S2). Injection of C26:0 as a free glycolipid was used
as a standard which is known to activate iNKT cellꢀdependent release of various
cytokines, such as ILꢀ2, TNFꢀα and ILꢀ4 which peak in serum at approximately 2 hours,
and IFNꢀγ which peaks at 10ꢀ12 hours after the injection.20 The administration of
conjugates formed with 13 or 15 activated secretion of all four cytokines, including
levels of ILꢀ2, TNFꢀα and ILꢀ4 at 2 hours and IFNꢀγ at 10 hours, that equaled or
exceeded those stimulated by free C26:0. Noncovalent complexes gave similar
stimulation as conjugates at 2 hours, but the level of IFNꢀγ at 10 hours was significantly
higher with conjugates for 13, and also for conjugates with 9 which did not stimulate any
detectable serum cytokines at 2 hours. These findings confirmed the iNKT cell
activating properties of soluble complexes and conjugates in vivo, and also were
consistent with the ability of covalent conjugation to stabilize the shorter 9 and 13
glycolipids, extending the duration of their bioactivity. In addition, covalently stabilized 9
conjugates showed a remarkable skewing of the cytokine response such that only IFNꢀγ
was detected among the cytokines measured.
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Figure 5: Mapping of covalent attachment site. (a) Peptide mapping summaries
from nanoꢀLCꢀESꢀMSE analyses are shown for unloaded mCD1d fusion protein (native)
and for proteinꢀglycolipid conjugates containing either 9 or 13, focusing on the lipid
binding region comprising the α1 and α2 regions of CD1d. Amino acid sequences of
mCD1d α1 through α2 domains are shown in single letter code. Bold text indicates
residues forming the lipid binding pocket, blue shading indicates residues mapped with
a high degree of confidence, and grey shading indicates residues mapped with a good
degree of confidence. Residues highlighted in yellow indicate amino acids that were not
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observed in any detected peptides. Peptides containing the four amino acids
underlined in red in both conjugates were detected with high confidence in the native
protein but, were absent in both conjugates. (b) MSE analysis of the doubly charged ion
m/z 661.33 observed at 30.9 min elution time in the analysis of a reduced,
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carbamidomethylated, PNGaseꢀF treated tryptic digest of the 9 conjugate. The
molecular ion is consistent with the tryptic peptide containing the 255YVVR258 residues
modified by a single C6 glycolipid moiety. Labelled ions in the left panel show loss of
water from a cleaved C6 BPGC entity, while the right panel highlights evidence for the
peptide with the y’’1 ion for the Cꢀterminal arginine at m/z 175 and the a1ꢀion for the Nꢀ
terminal tyrosine at m/z 136. The ion at m/z 195 is consistent with loss of the
benzophenyl group from the BPGC molecule. (c) The mCD1d protein structure
previously deduced from Xꢀray crystallography (PDB number 3HE6) is shown as a
ribbon diagram in green, and the two valine residues comprising the proposed covalent
attachment site are shown as bright pink spheres. The view on the left shows the intact
α1 and α2 domains with αꢀGC C26:0 (green and red spheres) bound in the lipid binding
groove. In the view on the right, the αꢀhelices forming the roof of the groove have been
removed, and the bound glycolipid is shown as pink and red spheres.
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Analysis of conjugation site by peptide mapping
Peptide mapping analyses of samples of native mCD1d and mCD1d conjugates with 9
and 13 were performed to identify possible sites of conjugation. Samples with or without
enzymatic deglycosylation were digested with trypsin, or with trypsin plus chymotrypsin.
Digested samples were analysed by online nanoꢀliquid chromatographyꢀelectrospray
tandem mass spectrometry (nanoꢀLCꢀESꢀMSE) (Supplemental Fig.S4). For native
mCD1d samples, peptides covering the entirety of the glycolipid binding domains were
consistently mapped with a high degree of confidence for all but three residues (R156,
M218 and M219) of the putative binding site. Both the 9 and 13 conjugated samples
produced very similar data to that of the native mCD1d, with the notable exception that
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ions covering the unmodified region 255YVVR258 were not observed (Fig. 5a, and
Supplemental Tables S1 – S3). Instead, molecular ions consistent with the presence
of a 13 or 9 modification were detected (Supplemental Figures S4 and S5), and
fragmentation of these modified peptides produced signals corresponding to both the Nꢀ
terminal tyrosine and the Cꢀterminal arginine, as well as y”ꢀ and bꢀions consistent with
modified VVR and YVV (Fig. 5b). Although definitive evidence for covalent modification
of a specific residue was not obtained, these data strongly supported the site of
conjugation to be in the region 255YVVR258, with one or both valine residues at the base
of the F’ pocket of mCD1d being directly involved in the formation of potential
bioconjugation products (Fig. 5C and Supplemental Fig. S6).
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Modelling BPGC analogue binding modes.
To confirm the feasibility of the putative conjugation site identified in mCD1d, the
binding modes of 9 and 13 were modelled into the published crystal structure of the
ternary complex of CD1d with αꢀGC C26:0 and the Vα14ꢀVβ8.2 iNKT cell TCR (PDB
entry code 3HE6) (Fig. 5c and Fig. 6). Briefly, compound 13 was docked without its
benzophenone group, which was deleted prior to docking. The binding of the modified
benzophenone group (i.e., diphenylmethanol, formed by the benzophenone group after
conjugation) was predicted in a separate docking run considering that the flexible linker
in the BPGC analogues would only minimally restrain possible binding conformations of
the end group. The most favorable docking pose showed excellent shape
complementarity and nonꢀpolar contacts in the binding site while linked covalently to the
V257 side chain in the F` pocket. The diphenylmethanol pose and the sugar moiety of
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13 docked without the benzophenone end group were then connected by predicting an
optimal conformation for the flexible linker between them through docking the linker
segment (with constraints). Models of the full 9 and 13 structures were generated
through splicing in the appropriate linkers of the two BPGC analogues, and were then
refined using Prime complex structure refinement in Schrödinger software (Fig. 6).
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Figure 6: Molecular docking of 13 and 9 in mCD1d. (a, b) Two views of the
binding model of 13 and coꢀcrystallized ɑꢀGC (from PDB code 3HE6). The inset
compares the binding models of 13 (C11:BP) and 9 (C6:BP). The modified end in the
F` pocket is covalently attached to V257 (residue V125 in the structure with PDB code
3HE6). (c) Interactions near the sugar moiety of 13. A TCR interaction shared between
αꢀGC C26:0 in the crystal structure and both 9 and 13 models was the hydrogenꢀ
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bonding interaction of the galactose 3’’ꢀOH with N30α. Additional stabilizing interactions
with the sugar moiety of the bound BPGCs included hydrogen bonds with TCR residues
R95α and D29α and with D284 of mCD1d. The binding site that in the crystal structure
is occupied by the vicinal diol of the phytosphingosine chain of αꢀGC was occupied in
the BPGC bound models by a less bulky amide group. This allowed reorientation of the
TCRα R95 side chain (R95α) into the same region, forming a favourable stacking
interaction between the guanidinium of the arginine and the amide of the ligand. The
guanidinium of R95α was also sandwiched between the mCD1d residues D284 and
D211, saltꢀbridging with both aspartates. Other interactions stabilizing the predicted
BPGC binding orientation were apparent, such as hydrogen bonds between the amide
group and residues S207 and T287 of mCD1d, and between the C3 hydroxyl of the
phytosphingosine chain and residue T290 of mCD1d. (d) CD1d surface in the region of
the modified terminal, colored by electrostatic potential (blue indicates nonpolar and red
indicates polar surfaces). The end group forms favourable nonꢀpolar contacts with I229,
V249, F251, W264, W273, L274, I278 and L281 (residue numbers are based on the
mCD1dꢀfusion protein).
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The final refined structures of 9 and 13 glycolipids after docking showed similar
binding modes except for the linker region, as expected (Fig. 6, inset). Compared to
the corresponding sugar moiety of αꢀGC C26:0 in the crystal structure, the
αꢀgalactopyranosyl group in BPGC analogues was positioned closer to the TCR and
slightly shifted (Fig. 6a). The lipid chains occupying the A’ and F’ pockets in the models
with bound BPGCs were switched compared to the orientation of αꢀGC C26:0 in the
crystal structure. Thus, the acyl chain of the covalently bound 9 and 13 was positioned
in the F’ pocket of mCD1d, rather than in the A’ pocket as in the case of the
noncovalently bound αꢀGC C26:0 (Fig. 6a and b). This binding orientation allowed
reorientation of the TCRα R95 side chain (R95α) to form a favourable stacking
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interaction between the guanidinium of the arginine and the amide of the ligand, as well
as a number of other stabilizing interactions (Fig. 6c). The acyl chains of 9 and 13
adopted quite similar orientations in the two BPGC models in spite of their different
lengths, linking the diphenylmethanol moiety covalently bound to the side chain of V257
in a mainly nonꢀpolar pocket (Fig. 6d).
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Discussion
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In this study we synthesized a series of benzophenoneꢀmodified glycolipids that can be
covalently crossꢀlinked to soluble as well as surface expressed CD1d to generate highly
stable conjugates that activate iNKT cells in vitro and in vivo. By appending a
photoactivatable benzophenone group to the end of the amide linked acyl chain of
αꢀGC, we generated a family of BPGCs with varying aliphatic spacer lengths.
Screening for biologic activity using a variety of assays for iNKT cell stimulation showed
that all of these BPGCs could be presented by CD1d, with most experiments showing
13 to be optimal for iNKT cell stimulation. Using relatively brief and low energy UV
irradiation, all of the BPGCs could be activated to form stable conjugates with purified
CD1d proteins. In addition, UV photoactivation could be shown to stabilize BPGC
interactions with CD1d expressed in living cells. By multiple criteria, UV induced
covalent conjugates of BPGCs retained their interactions with iNKT cell TCRs, and
conjugates in either soluble, surface bound or cell associated forms possessed the
ability to activate cytokine secretion by iNKT cells.
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Our primary goal in developing BPGCs was to use conjugate formation with
CD1d as a method for improving delivery of iNKT cell activators as potential agents for
immunotherapy. Particularly in the case of cancer immunotherapy, many studies in
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mouse models have highlighted the potential for iNKT cell activators to deliver striking
antiꢀcancer effects.4c However, administration of αꢀGC as a free glycolipid has the
potential for dose limiting toxicity, and also leads to the rapid development of a longꢀ
lived hyporesponsive state (anergy) that interferes with repeated treatments.10, 21 Most
likely, these issues contribute to the limited efficacy of free αꢀGC injections observed so
far in early phase clinical trials for cancer in humans.9, 22 Approaches to more precisely
deliver αꢀGC to overcome these problems have been developed, including the
administration of ex vivo glycolipid loaded antigen presenting cells or targeted soluble
recombinant CD1d proteins.4c, 8a, 8c, 8d These approaches have shown improved
outcomes in animal models, as well as in limited clinical studies in cancer patients4c, 6b,
23. However, the ability of αꢀGC to dissociate from these delivery vehicles after in vivo
injection remains a suboptimal feature. The use of BPGCs to covalently lock the
glycolipid onto CD1d in an active configuration, as shown in the current study,
represents a method for improving these delivery methods, and achieving more focused
and effective immunotherapy without the limitations or toxic effects of systemic αꢀGC
injections.
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Detailed analysis of the effects of variation in the length of the acyl chain spacer
in the BPGCs revealed some interesting and potentially important findings. First, we
noted that the 9 and 10 compounds had very low TCR affinity and low stimulatory
activity when not covalently linked to CD1d (Figs. 3f and 4a), presumably because of
their rapid dissociation. However, with conjugate formation these glycolipids showed
improved affinity and enhanced iNKT cell stimulation. This suggests that the BPGCs
with shorter acyl chains, even if released by some mechanism after conjugation, should
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be less likely to induce unwanted effects such as iNKT cell anergy or systemic toxicity.
Another notable finding was that 9, which even after conjugation continued to show a
lower TCR affinity than the other BPGCs, also gave a remarkably biased cytokine
response (i.e., significant IFNꢀγ levels with no detectable ILꢀ4 or other cytokines) when
injected in the form of a conjugate with soluble CD1d protein (Fig. 4b). This type of
“Th1ꢀbiased” cytokine response has been repeatedly associated in previous studies
with analogues of αꢀGC that provide superior antiꢀtumor responses in mouse models.24
Thus, through alterations in the length of the acyl chain spacer, it should be possible to
tune the responses to BPGC conjugates in terms of affinity and biologic response to
optimize desired therapeutic outcomes.
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Our mapping of the site of covalent bond formation in 9 and 13 conjugates
yielded the surprising finding of a single major conjugation site for both glycolipids.
Another surprising aspect of this result was that the specific region of CD1d that was
implicated was located near the base of the F’ pocket, which in all CD1dꢀglycolipid
complexes resolved by Xꢀray crystallography would be predicted to be in greater
proximity to the sphingoid base than the acyl tail of the glycolipid (Fig. 5c).2 However,
this apparent switch in the orientation of the two lipid tails of the glycolipid can be readily
accommodated in energetically favorable poses of the glycolipid in molecular models
(Fig. 6). Thus, our findings suggest the possibility of greater flexibility in the lipid
binding process of CD1d, which has only been occasionally hinted at in atomic level
structure studies.25 Further structural analyses of CD1dꢀBPGC conjugates should
enable new types of analyses to expand our understanding of the unique process of
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glycolipid antigen presentation, as well as opportunities for improving immunotherapies
that target iNKT cells.
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4
EXPERIMENTAL METHODS
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Synthesis and compound characterization. Full experimental details of the synthesis
and characterization of all compounds used in this study are provided in the
Supplementary Information.
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Mice. Female C57BL/6J (B6) mice 6–8 weeks old were obtained from Jackson
Laboratories or Taconic and maintained in pathogenꢀfree conditions. All experiments
requiring mice were conducted in compliance with institutional guidelines and under an
authorization delivered by the Institute of Animal Use and Biosafety Committee at Albert
Einstein College of Medicine.
Cell lines, clones and hybridomas. HeLa cells transfected with human CD1d
(HeLa.hCD1d) were cultured in DMEM supplemented with 10% FBS, and JAWS II cells
were cultured in αꢀMEM supplemented with 10% FBS and 20 ng/ml murine GMꢀCSF.
Mouse splenocytes were maintained in RPMI with 10% FBS. BMDCs were prepared as
described earlier and cultured in RPMI supplemented with 10% FBS. Mouse iNKT
hybridoma DN3A4ꢀ1.2 was maintained in complete RPMI medium containing 10%
decomplemented serum.26 Human iNKT clonal HDE3 was clonally expanded by
stimulation with PHAꢀP in the presence of recombinant human ILꢀ2 at 250 IU/ mL ,
recombinant human ILꢀ7 at 10 ng/mL and allogeneic PBMCs (irradiated at 5000 rad),
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and cultures were fed by addition of fresh medium containing ILꢀ7 and ILꢀ2 every 2ꢀ3
days.8d, 15
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Recombinant CD1d proteins and monoclonal antibody L363. Single chain β2mꢀ
mouse CD1dꢀhexahistidine (MW ~57 kDa) and human CD1dꢀβ2mꢀhexahistidine were
purified from CHO cells stably transfected with respective genes.27 15 Single chain
mCD1dꢀβ2m with a Cꢀterminal single chain immunoglobulin Fv fusion (mCD1dꢀβ2mꢀ
ScFv, MW ~78 kDa) was produced in transiently transfected HEK cells and purified as
previously described.8c, 8d The ScFv moieties of these proteins were specific for the
human tumor associated antigens CEA or C35, although their specific binding
properties were not relevant to experiments in the current study.
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To determine the affinity of mCD1dꢀBPGC tetramers for iNKTꢀcell TCRs, 1 x 104
DN3A4ꢀ1.2 cells were stained with a range of concentrations of tetramers loaded with
the different glycolipids to form complexes. Soluble mCD1d proteins were prepared and
biotinylated following published methods with minor modifications.28 Glycolipids were
prepared and loaded onto soluble mCD1d as described in the following section. In
some cases, the loaded mCD1d complexes were converted to covalent conjugates by
exposure to UV irradiation for 60 minutes in solution (400 mJoules/cm2). Formation of
tetramers, equilibrium binding of tetramers to NKT cells, and measurement of binding by
flow cytometry has been previously described in detail.15
Monoclonal antibody L363, specific for mCD1d with bound αꢀGC C26:0 and other
related forms of αꢀGC, has been previously described15, 18, 29 The antibody was purified
from supernatants of the cultured hybridoma line, and was biotinylated using sulfoꢀNHS
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